Snake Venoms and The Hemostatic System

Toxicon Vol. 36, No. 12, pp.
1749±1800, 1998
# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0041-0101(98)00126-3 0041-0101/98 $19.00 + 0.00
REVIEW PAPER
SNAKE VENOMS AND THE HEMOSTATIC SYSTEM
FRANCIS S. MARKLAND
Cancer Research Laboratory #106, University of Southern California, School of Medicine, 1303
N. Mission Road, Los Angeles, CA 90033, U.S.A.
(Received 9 December 1997; accepted 16 January 1998)
Francis S. Markland. Snake venoms and the hemostatic system. Toxicon 36,
1749±1800, 1997.ÐSnake venoms are complex mixtures containing many
dierent biologically active proteins and peptides. A number of these pro-
teins interact with components of the human hemostatic system. This review
is focused on those venom constituents which aect the blood coagulation
pathway, endothelial cells, and platelets. Only highly puri®ed and well
characterized snake venom proteins will be discussed in this review. Hemos-
tatically active components are distributed widely in the venom of many
dierent snake species, particularly from pit viper, viper and elapid venoms.
The venom components can be grouped into a number of dierent categories
depending on their hemostatic action. The following groups are discussed
in this review: (i) enzymes that clot ®brinogen; (ii) enzymes that degrade
®brin(ogen); (iii) plasminogen activators; (iv) prothrombin activators; (v)
factor V activators; (vi) factor X activators; (vii) anticoagulant activities
including inhibitors of prothrombinase complex formation, inhibitors of
thrombin, phospholipases, and protein C activators; (viii) enzymes with
hemorrhagic activity; (ix) enzymes that degrade plasma serine proteinase
inhibitors; (x) platelet aggregation inducers including direct acting
enzymes, direct acting non-enzymatic components, and agents that require
a cofactor; (xi) platelet aggregation inhibitors including: a-®brinogenases,
5'-nucleotidases, phospholipases, and disintegrins. Although many snake
venoms contain a number of hemostatically active components, it is safe
to say that no single venom contains all the hemostatically active com-
ponents described here. Several venom enzymes have been used clinically
as anticoagulants and other venom components are being used in pre-
clinical research to examine their possible therapeutic potential. The disin-
tegrins are an interesting group of peptides that contain a cell adhesion
recognition motif, Arg±Gly±Asp (RGD), in the carboxy-terminal half of
their amino acid sequence. These agents act as ®brinogen receptor (integ-
rin GPIIb/IIIa) antagonists. Since this integrin is believed to serve as the
®nal common pathway leading to the formation of platelet±platelet
bridges and platelet aggregation, blockage of this integrin leads to inhi-
bition of platelet aggregation regardless of the stimulating agent. Clinical
trials suggest that platelet GPIIb/IIIa blockade is an eective therapy for
the thrombotic events and restenosis frequently accompanying cardiovascu-
lar and cerebrovascular disease. Therefore, because of their clinical poten-
1749
1750 F. S. MARKLAND Jr
tial, a large number of disintegrins have been isolated and characterized.

# 1998 Elsevier Science Ltd. All rights reserved
INTRODUCTION
The venom producing apparatus of snakes is composed of modi®ed exocrine glands

which produce the toxic venom. Venoms are complex mixtures of proteins and peptides
possessing a variety of biological activities. Proteins and peptides comprise about 90±
95% of the dry weight of the venom. Other components in the venom are metallic cat-
ions, carbohydrates, nucleosides, biogenic amines and very low levels of free amino
acids and lipids (Russell, 1980; Tu, 1988a). Sodium is the major cation in the venom,
but its role is unknown. Zinc and calcium are present in many metalloproteinases found
in snake venom. Carbohydrates are mainly present in the form of glycoproteins
(Russell, 1980; Tu, 1988a).
Snake venoms contain many diverse enzymatic activities including, but not limited to,
phospholipase, phosphodiesterase, phosphomonoesterase, L-amino acid oxidase, acetyl-
cholinesterase, proteolytic enzymes of the serine proteinase and metalloproteinase
classes, arginine esterase, 5'-nucleotidase, hyaluronidase and NAD nucleosidase
(Russell, 1980; Tu, 1988a; Meier, 1990; Stocker, 1990a). Not all of these enzymes are
present in all venoms. Among the peptides found in snake venoms are presynaptic and
postsynaptic neurotoxins, potassium channel-binding neurotoxins, cytotoxins, myotox-
ins, cardiotoxins, and platelet aggregation inhibitors (disintegrins) (Russell, 1980; Tu,
1988a,b; Stocker, 1990a; Niewiarowski et al., 1994).
A number of snake venom proteins are active on the hemostatic system and form the
major focus of this review. Table 1 lists the dierent hemostatically active factors pre-
sent in snake venoms. This area has been previously reviewed by a number of authors
(Stocker, 1990b; Meier and Stocker, 1991; Ouyang et al., 1992; Hutton and Warrel,
1993; Marsh, 1994).
Fontana, an Italian, in the late 1700's was the ®rst to observe the coagulant eects of
snake venoms. He noted that following injection of viper venom into the jugular vein of
rabbits, the blood of the animals coagulated rapidly and they died immediately
(Fontana, 1787). In the 1890's Mitchell and Reichert in Philadelphia made a series of
Table 1. Snake venom proteins active on the hemostatic system

General functional activity Speci®c biological activity
Procoagulant factor V activating
factor X activating
factor IX activating
prothrombin activating
®brinogen clotting
Anticoagulant protein C activating
factor IX/factor X-binding protein
thrombin inhibitor
phospholipase A
Fibrinolytic ®brin(ogen) degradation
plasminogen activation
Vessel wall interactive hemorrhagic
Platelet active platelet aggregation inducers
inhibitors of platelet aggregation
Plasma protein inactivators inhibitors of SERPINS
Snake Venoms and Hemostasis 1751
interesting observations on the coagulant and anticoagulant eects of rattlesnake

venoms (Mitchell and Reichert, 1886). At the present time over 100 dierent snake
venoms are known to aect the hemostatic system by a variety of mechanisms. In this
review discussion will be limited to the aect of venom proteins on the human hemo-
static system. Although rodents and other small animal species are the natural prey of
snakes, snake venom proteins and peptides are highly active on proteins and cellular el-
ements in human blood. The human hemostatic system is based on a number of compli-
cated interactions involving blood proteins, platelets, endothelial cells, and
subendothelial structures. Snake venoms proteins and peptides are known to produce
activating or inactivating aects on many of the various interactions of the hemostatic
system. Some of these venom proteins have potential clinical usefulness for the treat-
ment of human diseases. One group of therapeutically interesting venom proteins are
the thrombin-like (de®brinogenating) enzymes found in the viper and pit viper families
(Pirkle and Theodor, in press). Paradoxically puri®ed snake venom thrombin-like
enzymes have coagulant action in vitro and anticoagulant action in vivo. This has led to
some confusion in the literature regarding the classi®cation of venoms as being either
coagulant or anticoagulant (Rosenfeld et al., 1968).
In the following review a brief glimpse of the hemostatically active venom com-
ponents will be given. The reader should refer to Fig. 1 for an overview of the coagu-
lation and ®brinolytic systems and to determine where the various venom components
interact within these systems.
HEMOSTATICALLY ACTIVE VENOM PROTEINS: COAGULANT ACTIVITIES
Snake venoms, particularly from the pit viper, viper and elapid families, contain a
number of components that interact with proteins of the coagulation cascade and the
®brinolytic pathway. Not every snake species contains all of the following activities in
the venom, but the activities listed below have been described in the venom of at least
one snake species.
Factor V activator
Russell's viper (Daboia russellii formerly Vipera russellii) venom contains a factor V
activating serine proteinase (Kahn and Hemker, 1972) which could be separated from a
factor X activating protein also present in this venom (Schiman et al., 1969). The
enzyme (RVV-V) is a single chain glycoprotein with a molecular weight of 26 100 pos-
sessing one glycosylation site near the carboxy-terminus. The enzyme cleaves a single
peptide bond to convert factor V to factor Va (the activated clotting protein is desig-
nated by the subscript as in Va) (Tokunaga et al., 1988). The bond cleaved by RVV-V
is one of the thrombin susceptible bonds in factor V. There is extensive sequence iden-
tity (62%) between RVV-V and batroxobin, the thrombin-like enzyme in fer-de-lance
(Bothrops atrox) venom (Tokunaga et al., 1988). RVV-V is not inhibited by antithrom-
bin III either in the presence or absence of heparin (Kisiel and Can®eld, 1981). A single
chain glycoprotein with a molecular weight of 36 000 and serine proteinase activity has
been isolated from Bothrops atrox venom (Kirby et al., 1979). The enzyme, called
thrombocytin, is a platelet aggregation inducer which also activates factor V.
Additionally, this enzyme activates factors VIII and XIII and possesses very weak ®bri-
nogen clotting activity (Niewiarowski et al., 1979).
Fig. 1. The ®gure presents a schematic overview of the coagulation and ®brinolytic pathways
and indicates where phospholipids on the platelet surface interact with coagulation pathway in-
termediates. Arrows are not shown from platelets to phospholipids involved in the tissue factor-
VIIa and the factor IXa±VIIIa interactions to avoid confusion. Interactions of the venom pro-
teins are indicated in the black boxes. Inhibitory interactions produced by the venom proteins
are shown by the wide dashed lines; activation or conversion interactions are shown by the wide
solid black lines. The narrow solid lines indicate the normal interactions of the coagulation and
®brinolytic proteins. The solid black ovals in the thrombin and plasmin activity lines indicate in-
hibition by SERPINS. The diagram is not complete with reference to the multiple sites of inter-
action of the SERPINS to avoid overcrowding.
Factor X activator
Russell's viper venom also contains a potent activator of human blood coagulation
factor X (RVV-X) that has been well characterized (Kisiel et al., 1976; Furie and Furie,
1976). Factor X activators have also been isolated from Bothrops atrox (Hofman and
Bon, 1987) and several other snake species (Stocker, 1994; Lee et al., 1995; Zhang et al.,
1995a). RVV-X is a disul®de-linked two chain metalloproteinase composed of a heavy
chain of 59 kDa and a light chain of Mr approximately 19.5 kDa (Takeya et al., 1992).
The heavy chain of RVV-X is composed of metalloproteinase, disintegrin-like and
cysteine-rich domains. The light chain resembles the C-type lectins (calcium-dependent).
RVV-X is a glycoprotein. It activates human factor X by cleaving a speci®c peptide
bond in the amino-terminal region of the heavy chain of the clotting factor. The mech-
anism is identical to that catalyzed by proteolytic enzymes of the blood coagulation
pathway (Di Scipio et al., 1977). Interestingly, the factor X activators from venom of
the elapids, king cobra (Ophiophagus hannah) and banded krait (Bungarus fasciatus),
have been reported to be serine proteinases (Lee et al., 1995; Zhang et al., 1995a) unlike
RVV-X which, as noted, is a metalloproteinase.
Factor IX activator
RVV-X appears to be identical to a protein in Russell's viper venom which activates
factor IX by cleavage of a single peptide bond. This results in the formation of factor
IXa without a change in molecular weight (Lindquist et al., 1978). This contrasts with
the activation of factor IX by factor XIa in the blood coagulation pathway which
involves the release of a peptide and a reduction in molecular weight of the activated
factor IXa.
Prothrombin activators
Prothrombin (also known as factor II) is a single chain glycoprotein with a molecular
weight of 72 000 (Rosing et al., 1988; Rosing and Tans, 1991, 1992). Prothrombin con-
tains 10 g-carboxylated glutamic acid (Gla) residues in its amino-terminal calcium bind-
ing prodomain; the catalytic domain is located in the carboxyl-terminal half of the
molecule. When prothrombin is activated by factor Xa in the presence of factor Va,
phospholipid and CaCl2, two peptide bonds are cleaved. In step 1 the prodomain is
cleaved away (cleavage of the Arg273±Thr274 bond) leaving prethrombin 2, and in step 2
the residual carboxyl-terminal half (prethrombin 2) is cleaved to generate active throm-
bin (cleavage of the Arg322±Ile323 bond), a two-chain molecule (Rosing et al., 1988).
There are several dierent types of activators of prothrombin in snake venoms.
Excellent reviews of these enzymes have appeared (Rosing et al., 1988; Rosing and
Tans, 1991, 1992). The venom prothrombin activators have been divided into four
groups based on their structural properties and their mechanism of prothrombin acti-
vation (Rosing and Tans, 1991, 1992). Group I activators convert prothrombin directly
into meizothrombin (by cleavage of the Arg322±Ile323 bond), which then is autocatalyti-
cally converted to thrombin. Meizothrombin is a two-chain cleaved intermediate with
the same molecular weight as prothrombin, but it has enzymatic activity against low
molecular weight substrates. Members of Group I are metalloproteinases with molecular
weights in the 50 000 to 70 000 range. Activity of members of this group is not in¯u-
enced by components of the prothrombin activator complex (factor Va, CaCl2 and
phospholipid). Ecarin, the prothrombin activator from saw scaled viper (Echis carinatus)
venom is the most well studied member of this group (Morita and Iwanaga, 1978;
Rosing and Tans, 1992). Ecarin cDNA has recently been cloned and the complete
amino acid sequence deduced (Nishida et al., 1995). The open reading frame of 616
amino acids shows remarkable sequence homology with the precursor protein of trigra-
min the disintegrin from green tree viper (Trimeresurus gramineus) venom and jararha-
gin the hemorrhagic protein from jararaca (Bothrops jararaca) venom. The ecarin
proprotein appears to contain a `cysteine switch' sequence, involved in generation of the
active enzyme, in common with other venom metalloproteinases and the mammalian
matrix degrading metalloproteinases. The processed mature protein contains 426 amino
acids and shows strong sequence identity to the factor X activator from Russell's viper
venom (RVV-X). Like RVV-X, ecarin possesses metalloproteinase, disintegrin and
cysteine-rich domains. However, the normal Arg±Gly±Asp sequence in the disintegrins
is Arg±Asp±Asp in the disintegrin domain of ecarin. Thus, ecarin and RVV-X represent
members of the multidomain snake venom metalloproteinase family which contain a
disintegrin domain.
Prothrombin activators in Group II can cleave both bonds in prothrombin leading to
active two chain thrombin. Members of this group are inactive against prothrombin in
the absence of cofactors, but activity is strongly stimulated by phospholipids and factor
Va in the presence of CaCl2. These enzymes are found exclusively in venoms of

Australian elapid and the best studied is from tiger snake (Notechis scutatus scutatus)
venom (Rosing and Tans, 1992). The tiger snake activator is very similar to human fac-
tor Xa and is a two-chain serine proteinase with a molecular weight of 54 kDa. The
enzyme contains a number of Gla residues which probably form a calcium binding site.
It cleaves the Arg273±Thr274 and the Arg322±Ile323 bonds interchangeably to generate
thrombin and meizothrombin. By contrast, activators in Group III only require phos-
pholipid and CaCl2 for the activation of prothrombin. They do not require factor Va
and appear to possess a cofactor (molecular weight of approximately 220 kDa) that is
tightly bound to the catalytic subunit and plays the role of factor Va in prothrombin ac-
tivation. The catalytic subunit has a molecular weight of 57 kDa and is composed of
two peptide chains. This class of activator is also found in Australian elapids and is rep-
resented by the high molecular weight activator from Taipan venom (Oxyuranus scutel-
latus scutellatus) venom. These activators also contain Gla residues and are factor Xa-
like serine proteinases similar to the group 2 activators. The group IV activators are not
able to generate active thrombin, but convert prothrombin into modi®ed forms which
are enzymatically inactive. There is some question as to whether these are speci®c pro-
teinases or broad-acting venom proteinases (Rosing and Tans, 1992; Marsh, 1994).
Recently, a novel prothrombin activator was reported in E. carinatus venom that dif-
fered from ecarin and all other categories of prothrombin activators previously reported
(Yamada et al., 1996). The enzyme is composed of two noncovalently associated subu-
nits, a catalytic metalloproteinase of 62 kDa homologous to the single chain ecarin and
a 25 kDa regulatory subunit with two chains of 17- and 14 kDa. The lower molecular
weight subunit is similar to the IX/X-binding protein, an anticoagulant that binds to
the Gla (g-carboxy glutamic acid) domain of coagulation factors IX and X. The authors
hypothesize that the enzyme complex recognizes the Ca++ bound form of the Gla
domain in prothrombin through the 25 kDa subunit and then prothrombin is converted
to thrombin by the catalytic 62 kDa subunit.
Fibrinogen clotting enzymes

Although thrombin has many activities, the ability of a group of snake venom
enzymes to clot ®brinogen has resulted in these enzymes being called thrombin-like
(Ouyang et al., 1992; Hutton and Warrel, 1993; Marsh, 1994; Pirkle and Theodor,
1998). These enzymes are widely distributed, primarily in venom of snakes from true
vipers (Bitis gabonica, Cerastes vipera, etc.) and pit vipers (Agkistrodon contortrix con-
tortrix, Crotalus adamanteus, Bothrops atrox, etc.). There are several groups of snake
venom ®brinogen clotting enzymes based on the rates of release of ®brinopeptides A
and B from ®brinogen. One group releases ®brinopeptide A preferentially (the venom-
bin A group including ancrod from venom of the Malayan pit viper, Calloselasma rho-
dostoma); another group releases both ®brinopeptides A and B (the venombin AB
group including gabonase from venom of the Gaboon viper, Bitis gabonica); and the
third group releases ®brinopeptide B preferentially (the venombin B group including
venzyne from venom of the southern copperhead, Agkistrodon contortrix contortrix).
Thrombin-like snake venom enzymes, particularly those in the venombin A group, have
been puri®ed from close to 40 dierent snake species and the topic is the subject of a
number of excellent reviews (Stocker et al., 1982; Pirkle and Theodor, 1990, 1998;
Pirkle, 1998).
Venombin A group. The class of enzymes releasing ®brinopeptide A is represented by

three well characterized proteins with similar, but distinct properties: ancrod from
Malayan pit viper (Calloselasma rhodostoma formerly Agkistrodon rhodostoma) venom
(Nolan et al., 1976), batroxobin from Bothrops atrox venom (Stocker and Barlow, 1976;
Stocker and Meier, 1988), and crotalase from eastern diamondback rattlesnake (Crota-
lus adamanteus) venom (Markland, 1976). In vivo the enzymes act as benign de®brino-
genating agents to remove ®brinogen from the blood. In the test tube they are
procoagulant and form a ®brin clot.
One common feature all three venom enzymes possess is cleavage of the Arg16±Gly17
peptide bond in the Aa-chain of ®brinogen leading to the release of ®brinopeptide A
and the conversion of ®brinogen to a ®brin clot (Ouyang et al., 1992; Hutton and
Warrel, 1993; Marsh, 1994). These venom enzymes dier from thrombin in a number of
ways, one of which is that they only cleave ®brinopeptide A and not both ®brinopep-
tides A and B from ®brinogen. Further, the venom enzymes do not activate factor XIII
or other coagulation factors, nor do they cause platelet aggregation (Markland and
Damus, 1971; Aronson, 1976; Soutar and Ginsberg, 1993). The three enzymes are serine
proteinases and are all single chain glycoproteins with molecular masses of approxi-
mately 30- to 35 kDa. The carbohydrate content varies considerably between the dier-
ent enzymes. Dierences in the masses of the dierent enzymes is most likely due to
their diering carbohydrate content. Ancrod (35.4 kDa) contains 36% carbohydrate
and the isoelectric point varies from 4.2 to 6.2 for several dierent electrophoretic forms
(Nolan et al., 1976). Dierent forms of batroxobin have been isolated from venoms of
Bothrops atrox (41.6 kDa), which contains 10.2% neutral carbohydrate, and B. moojeni
(35.8 kDa), which contains 5.8% neutral sugar (Stocker and Barlow, 1976; Stocker and
Meier, 1988). Crotalase (32.7 kDa) contains 8.3% carbohydrate content (Pirkle, unpub-
lished). Crotalase was shown to possess kallikrein-like activity based on the cleavage of
kallikrein-susceptible bonds in high molecular weight kininogen with the liberation of
kinin activity and by its inhibition by a kallikrein-speci®c tripeptide chloromethyl
ketone (Markland et al., 1982). Microheterogeneity of the venombin A enzymes has
been observed and is most likely due to varying sialic acid content. These enzymes have
esterase activity with small basic ester substrates such as benzoyl-L-arginine ethyl ester.
Coagulant and esterase activities of the enzymes were inhibited simultaneously by serine
proteinase inhibitors such as diisopropyl¯uorophosphate. The enzymes are also inhib-
ited by histidine reactive inhibitors (Nolan et al., 1976; Stocker and Barlow, 1976).
Ancrod and batroxobin are inactivated by a2-macroglobulin (Nolan et al., 1976;
Stocker and Barlow, 1976). In view of similarities in structure, it is assumed that crota-
lase is also inhibited by a2-macroglobulin.
The primary structure of all three enzymes has been determined (Itoh et al., 1987;
Burkhart et al., 1992; Pirkle et al., 1996) (Fig. 2). The sequence of crotalase reveals one
N-glycosylation site in the 237 amino acid sequence (Pirkle et al., 1996). The 12
cysteines in crotalase are most likely involved in six disul®de bonds since early investi-
gations indicated that there are no free sulfhydryls (Markland and Damus, 1971). The
amino acid sequence of ancrod indicates that it contains 234 amino acids and the align-
ment of the cysteine residues is identical to that in crotalase (Burkhart et al., 1992).
Glycosylation of all ®ve N-glycosylation sites in anrod is suspected. Batroxobin contains
231 amino acids and its 12 cysteine residues are aligned with those in crotalase and
ancrod (Itoh et al., 1987). There are two potential glycosylation sites in batroxobin. On
the basis of homology with mammalian serine proteinases, there is general agreement
Fig. 2. Alignment of sequences of ancrod (234 amino acids, de®brinogenating enzyme from
Calloselasma rhodostoma venom) (Burkhart et al., 1992), batroxobin (231 amino acids, de®brino-
genating enzyme from Bothrops atrox venom) (Itoh et al., 1987), crotalase (237 amino acids,
de®brinogenating enzyme from Crotalus adamanteus venom) (Pirkle et al., 1996), TSV-PA (234
amino acids, plasminogen activator from T. stejnegeri venom) (Zhang et al., 1995b) and TM-
VIG (233 amino acids, b-chain ®brinogenase from Trimeresurus mucrosquamatus venom) (Hung
et al., 1994). Sequences of the proteases were aligned by the positioning of the 12 cysteine resi-
dues. Spaces were inserted as needed for proper alignment. Catalytic triad residues histidine 43,
aspartic acid 88, and serine 182 (ancrod numbering) shown by *. Residues underlined are identi-
cal in all three enzymes (92/231 or 039.8%).
on the positioning of the three active site residues (using the ancrod numbering) includ-
ing serine (residue 182), histidine (residue 43) and aspartic acid (residue 88).
Ancrod and batroxobin have been used as de®brinogenating agents for a number of
clinical conditions including deep vein thrombosis, myocardial infarction, pulmonary
embolus, central retinal vein occlusion, peripheral vascular disease, acute ischemic
stroke, angina pectoris, glomerulonephritis, priapism, sickle cell crises, and renal trans-
plant rejection (Bell, 1988; Stocker, 1988; Bell, 1990; Furukawa and Ishimaru, 1990;
Pollack et al., 1990; Soutar and Ginsberg, 1993; Sherman and the Ancrod Stroke Study
Investigators, 1994; Eschenfelder, 1996). Ancrod (Cercek et al., 1987) and batroxobin
(Tomaru et al., 1988) have also been used in combination with thrombolytic agents in
canine models of arterial thrombosis. Batroxobin acted to prevent coronary artery reoc-
clusion, while ancrod enhanced the eect of thrombolytic agents in a carotid arterial
thrombosis model, probably by depleting ®brinogen and preventing propagation of

existing thrombi. Ancrod was also shown to decrease cyclic ¯ow variations and to cause
thrombolysis in a coronary artery model in the canine (Apprill et al., 1987). A bene®cial
eect of ancrod infusion results from the decrease in blood viscosity due to ®brinogen
depletion. This improves blood ¯ow characteristics and may aid the therapeutic eec-
tiveness of ancrod (Ehrly, 1976).
As already indicated, the venombin A enzymes are all serine proteinases that rapidly
cleave ®brinopeptide A from ®brinogen forming a ®brin clot in vivo. It would appear
that the mechanism whereby the venombin A enzymes produce a benign state of de®bri-
nogenation involves the formation of an abnormal clot that is soluble in 5 M urea or
1% monochloroacetic acid. This is in contrast to thrombin clots which are cross-linked
by factor XIII and are insoluble in these solvents. Within minutes of administration of
the venom enzymes in vivo there is a depression in the ®brinogen concentration in
plasma and within an hour or two the ®brinogen levels become very low and remain so.
Fibrinogen can be maintained at the depressed level by repeated infusions on a daily or
twice daily basis (Bell, 1990). The low levels have been maintained in some patients for
up to seven weeks. Upon termination of venom enzyme infusion there is a slow recov-
ery of the ®brinogen levels. There is a dramatic increase in the levels of ®brin(ogen)
degradation products in the plasma upon venom-induced de®brinogenation. These
degradation products appear to be derived by plasmin digestion of the noncross-linked
®brin clot, which is highly susceptible to the action of the ®brinolytic enzyme. It has
been reported that ancrod (Soszka et al., 1985) and batroxobin (KloÈcking et al., 1987)
appear to induce the release of plasminogen activator from vascular endothelial cells,
thereby activating the ®brinolytic response. However, a recent study in six humans indi-
cated that although there is a massive rise in ®brin(ogen) degradation products and de-
pletion of plasminogen following ancrod infusion, there is no rise in tissue or urokinase-
type plasminogen activator levels (Prentice et al., 1993). These authors reported that
®brinopeptide A was removed from ®brinogen to produce soluble ®brin which was then
removed from the circulation with no evidence of increase in plasminogen activator
levels, but with signi®cant activation and consumption of plasminogen and depletion of
a2-antiplasmin. Further, the ®brin degradation products formed were identical to those
formed by plasmin degradation of soluble (non-crosslinked) ®brin, suggesting that plas-
minogen activation by the ®brinolytic system accounts for the degradation of ®brin.
The importance of the ®brinolytic system in the action of ancrod has been demonstrated
by the use of inhibitors of ®brinolysis in animals following ancrod infusion. This treat-
ment led to signi®cant clot formation in vivo and death.
If the venom de®brinogenating enzymes are used for a prolonged time or if there is a
necessity for repeated usage, there is a loss of enzyme activity due to the development
of immunological resistance by the patient (Pitney and Regoeczi, 1970; Thomson et al.,
1977). Interestingly, there is no immunological cross-reaction between ancrod and
batroxobin as shown by studies with speci®c antibodies (Barlow et al., 1973). This
means that these enzymes should be able to replace one another in successive courses of
long term anticoagulant therapy without loss of activity due to immunological cross-
reactivity. Although crotalase has not been used in humans, there is a similar depression
of ®brinogen with concomitant increase in ®brin(ogen) degradation products in animals
following intravenous administration of the enzyme (Damus et al., 1972).
The danger of therapy with the de®brinogenating enzymes consists of bleeding.
However, the side eects and complications can be controlled by knowledge of contra-
indications, careful control of dosage and daily monitoring of ®brinogen levels (Latallo,
1983; Bell, 1990; Soutar and Ginsberg, 1993).
Venombin AB group. The second class of thrombin-like enzymes releases both ®brino-
peptides A and B is represented by the enzyme from venom of the Gaboon viper (Bitis
gabonica, Ganey et al., 1973). The enzyme was puri®ed to homogeneity by Pirkle and
colleagues (Pirkle et al., 1986). The name gabonase was proposed by Marsh and Whaler
(1984). Gabonase is a serine proteinase and a glycoprotein with a molecular weight of
30.6 kDa. The coagulant action of gabonase is triggered primarily by the cleavage of
the Arg16±Gly17 bond nearest to the amino terminus of the Aa chain of ®brinogen,
releasing FPA. FPB is released more slowly by cleavage of the Arg15±Gly16 bond near-
est to the amino terminus of the Bb chain. No other cleavages could be detected by
polyacrylamide gel electrophoresis of ®brinogen following incubation with gabonase
(Pirkle et al., 1986). Gabonase, like thrombin, activates factor XIII added to ®brinogen-
clotting mixtures, resulting in the formation of g-chain dimers and a-chain polymers of
®brin. Gabonase exhibits strong tosyl-L-arginine methyl esterase (Pirkle et al., 1986).
The activity of the enzyme is stabilized by calcium ions. Gabonase is inactivated by phe-
nylmethanesulfonyl ¯uoride and by tosyl-L-lysyl chloromethane but not by hirudin or
heparin (Pirkle et al., 1986).
Venombin B group. The third class of thrombin-like enzymes releases preferentially

®brinopeptide B and is represented by the enzyme from venom of the southern copper-
head (Agkistrodon contortrix contortrix) (Herzig et al., 1970) The enzyme, called ven-
zyme, releases ®brinopeptide B much more rapidly than ®brinopeptide A and clots
®brinogen only after prolonged incubation. The enzyme is a serine proteinase with a
molecular weight of 64 kDa. The highly puri®ed enzyme does not cause platelet aggre-
gation nor does it activate factor XIII. The enzyme hydrolyses small arginine ester sub-
strates. An enzyme of molecular weight 43 kDa, with similar kinetics of ®brinopeptide
release, has been puri®ed from venom of the Chinese pit viper (Agkistrodon halys pallas)
(Guan et al., 1984).
Table 2 compares the properties of the ®ve ®brinogen clotting enzymes described.
HEMOSTATICALLY ACTIVE VENOM PROTEINS: ANTICOAGULANTS
Anticoagulant activity has been reported in dierent snake venoms and the respon-
sible proteins have been puri®ed in a number of cases. Anticoagulant action of snake
venom proteins is attributed to: (i) the activation of protein C, (ii) the inhibition of
blood coagulation factors IX and X by a venom protein that binds to either or both
clotting proteins, (iii) a thrombin inhibitor, and (iv) phospholipases that degrades phos-
pholipids involved in the formation of complexes critical to the activation of the coagu-
lation pathway.
Protein C activator
Protein C is a vitamin K-dependent, two-chain zymogen that is activated by thrombin
on the endothelial surface in the presence of thrombomodulin (Suzuki, 1995). Activated
protein C degrades factors Va and VIIIa and therefore has anticoagulant activity.
Stocker and colleagues isolated a protein C activator from southern copperhead
Table 2. Comparison of characteristics of ®brinogen clotting enzymes
Property Enzyme
a b
ancrod batroxobin crotalasec gabonased venzymee
Agkistrodon contortrix
Venom source Calloselasma rhodostoma Bothrops atrox (moojeni) Crotalus adamanteus Bitis gabonica contortrix
Molecular weight 35 400 36 000 32 700 30 600 64 000
Isoelectric point pI = 4.2±6.2 pI = 6.6 pI = 4.6 after desialylation 5.3 N.D.
Carbohydrate content 36% 5.8% 8.3% 20.6% N.D.
Fibrinopeptides released A A A A, B B>>A
Additional ®brinogen
degradation Aa-chain degradation None Bb-chain degradation None N.D.
Low mol. wt. inhibitors DFPf DFP DFP PMSF PMSF
Protein inhibitors a2-macroglobulin a2-macroglobulin N.D. N.D. SBTI (partial inhibition)
Esterase activity basic AA esters basic AA esters basic AA esters arginine esters arginine esters
Active site serine serine serine serine serine
Amino-terminus valine valine valine valine N.D.
Factor XIII activation no no no yes no
Snake Venoms and Hemostasis
Stability stable stable stable stable in Ca++ unstable to heat (608C)

a
References: Nolan et al., 1976; Burkhart et al., 1992; Collins and Jones, 1974; Pfeier et al., 1992; Pfeier et al., 1993.
b
References: Stocker and Barlow, 1976; Itoh et al., 1987; Itoh et al., 1988; Lochnit and Geyer, 1995; Tanaka et al., 1992.
c
References: Markland, 1976; Pirkle et al., 1996; Markland and Damus, 1971; Bajwa and Markland, 1979.
d
References: Pirkle et al., 1986; Pirkle and Markland, 1997.
e
References: Herzig et al., 1970; Shaino and Welches, 1988.
f
Abbreviations: DFP, diisopropyl¯uorophosphate; PMSF, phenylmethanesulfonyl ¯uoride; SBTI, soybean trypsin inhibitor; N.D., not determined; AA, amino acid.
1759
(Agkistrodon contortrix contortrix) venom (Stocker et al., 1987). The ability of the
southern copperhead venom fraction to activate protein C, indicates that this venom
has anticoagulant activity. The southern copperhead venom protein C activator is called
Protac1 and is a serine proteinase of molecular weight 37 kDa. The enzyme is a single
chain glycoprotein possessing 16±20% carbohydrate (Stocker et al., 1987). Similar pro-
tein C activating activity has been reported in a number of other American Agkistrodon
species (Stocker et al., 1986; Stocker and Meier, 1988).
Factor IX/X inhibitor

An inhibitor of factor X was isolated from the hundred-pace snake (Deinagkistrodon
acutus) venom. The protein is an acidic glycopeptide of molecular weight approximately
20.6 kDa. The protein possesses two peptide chains of approximately 10- and 12 kDa.
The protein has no detectable enzymatic activity and binds to factor Xa in the presence
of Ca++ and prevents its involvement in the prothrombin activation complex (Ouyang
and Teng, 1972). When injected into rabbits there was a signi®cant but short-lived pro-
longation of the whole blood coagulation time with no aect on plasma ®brinogen
levels. The inhibitor did not aect platelet aggregation nor alter physiological par-
ameters in the rabbits (Teng and Seegers, 1991; Ouyang et al., 1992).
Similar anticoagulant proteins have also been isolated from other venoms. A protein
that binds with 1:1 stoichiometry to either factor IX or X in the presence of Ca++ and
prevents their involvement in the coagulation cascade was isolated from habu
(Trimeresurus ¯avoviridis) venom (Atoda et al., 1991). The protein has a molecular
weight of approximately 27 kDa under non-reducing conditions and is composed of two
non-identical chains each of approximately 14.5 kDa. The A chain contains 129 amino
acids and the B chain has 123 residues. The two chains have 47% sequence identity and
are structurally related to C-type lectins (Atoda et al., 1991). The chains are joined by a
single disul®de bond (Atoda and Morita, 1992). Recently Matsuzaki et al. (1996) cloned
cDNAs encoding the sequences of the two peptide chains of the IX/X-binding protein.
It appears that the gene for each chain is transcribed and translated separately. The IX/
X-binding protein binds to the g-carboxy glutamic acid residues at the amino-termini of
factors IX and X (Atoda et al., 1994). Binding of Ca++ by the IX/X-binding protein
appears to be at two sites with diering anities and binding induces a conformational
change in the anticoagulant protein (Sekiya et al., 1995). The sequence of the IX/X
binding anticoagulant (Atoda et al., 1991) is very similar to botrocetin, a two-chain pro-
tein isolated from Bothrops jararaca venom, that has von Willebrand factor-dependent
platelet coagglutinin activity. Interestingly, an anticoagulant protein was also isolated
from B. jararaca venom that was distinct from botrocetin (Sekiya et al., 1993). Thus,
despite a high degree of structural similarity, the anticoagulant protein and botrocetin
have distinct biological activities.
A factor IX/X-binding protein was also isolated from venom of the viper, Echis cari-
natus leucogaster (Chen and Tsai, 1996). The protein is a heterodimer with one subunit
of 131 amino acids and the other of 125 amino acids. There are three intrachain disul-
®de bonds in each of the two chains and one interchain disul®de bond. The apparent
dissociation constants (Kd) for binding of the IX/X binding protein to factors IX and X
were determined to be 6.6 and 125 nM, respectively. The reduced and S-pyridylethylated
subunits no longer had binding activity. Binding to deglycosylated factor IX was not
dierent from binding to native factor IX and various sugars did not eect binding of
IX/X binding protein, ruling out a lectin-like mechanism for IX/X binding protein.
Thrombin inhibitor
A unique thrombin inhibitor was puri®ed from Bothrops jararaca venom by Zingali
et al. (1993). This is the only report to date of a snake venom inhibitor of this type.
The inhibitor, named bothrojaracin, forms a noncovalent equimolar complex with a-
thrombin. Bothrojaracin is composed of two polypeptide chains of 15- and 13 kDa
linked by disul®de bridge(s); the intact protein has a molecular weight of 27 kDa. The
amino-terminal sequences of the two chains of bothrojaracin show considerable simi-
larity to the two chains in botrocetin, the platelet agglutinating agent from B. jararaca
venom (Fujimura et al., 1991). Monteiro et al. (1997) reported that isoforms of the in-
hibitor are found in venom obtained from the milking of a single snake. Bothrojaracin
is a speci®c inhibitor of thrombin-induced platelet aggregation and secretion, it prolongs
®brinogen clotting time by competitively inhibiting the binding of a-thrombin to ®bri-
n(ogen), and it inhibits a-thrombin binding to thrombomodulin and decreases the rate
of protein C activation by a-thrombin. These results indicate that the inhibitor acts as a
very potent ligand for the ®brinogen binding exosite of a-thrombin, but does not inter-
act with the catalytic site of thrombin. More recently Arocas et al. (1996) showed that
bothrojaracin binds both to exosite 1 (ABE I) and exosite 2 (ABE II) on thrombin.
This was concluded from ®ndings that the rate of inhibition of thrombin by the antith-
rombin III-heparin complex is signi®cantly depressed by bothrojaracin (competition
between bothrojaracin and heparin for ABE II binding) and that the binding of throm-
bin to ®brinogen is blocked (ABE I involvement). Bothrojaracin binds to prothrombin
and it inhibits platelet activation by clot-bound thrombin. Bothrojaracin also slowly
releases thrombin from ®brin clots, suggesting that the inhibitor can inhibit both soluble
and clot-bound thrombin (Arocas et al., 1996). Recently Arocas et al. (1997) have
reported the cloning and expression of bothrojaracin in COS cells. The A and B chains
are composed of 132 and 127 amino acid residues, respectively, and there is a high
degree (47%) of identity between the two chains.
Phospholipase A2
The prothrombinase complex is composed of factors Va, Xa, phospholipid and cal-
cium ions. Snake venom phospholipases appear to inhibit formation of the prothrombi-
nase complex by degrading phospholipids involved in this complex. Phospholipases
have been isolated from a number of snake venoms and have a number of pharmaco-
logical actions including eects on blood coagulation (Ouyang et al., 1992). It has been
suggested that the anticoagulant action results from the formation of a hydrolytic com-
plex between the phospholipase and phosphatidylserine on the platelet surface (Boa
and Boa, 1976). It has further been reported that enzymatic activity can be separated
from anticoagulant activity, suggesting that binding to phospholipids rather than their
hydrolysis per se may account for the anticoagulant eect of the phospholipases
(Condrea et al., 1981). Based on the potency of their action the phospholipases have
been classi®ed as strong, weak or non-anticoagulant. The strong anticoagulants act to
inhibit both the extrinsic factor X and the prothrombin activation complexes. The weak
anticoagulants, by comparison, only inhibit the extrinsic factor X activation complex
(Subburaju and Kini, 1997).
An example of an anticoagulant phospholipase is the one isolated from Formosan
habu (Trimeresurus mucrosquamatus) venom (Ouyang et al., 1981). The protein is a
basic phospholipase A2 with a molecular weight of 11.7 kDa. Anticoagulant action is
due to inhibition of the factor X and prothrombin activation complexes by the venom
enzyme. This action is mediated both through binding to phospholipids and by phos-
pholipid hydrolysis by the venom enzyme.
HEMOSTATICALLY ACTIVE VENOM PROTEINS: FIBRINOLYTIC PROTEINASES
Direct-acting ®brinolytic enzymes have been isolated from venom of a number of

North and South American snakes including rattlesnakes and copperheads. Fibrinolytic
enzymes have also been puri®ed from elapid venoms including cobras, and European
vipers (Markland, 1991).
Fibrinolytic proteinases
The substrate for the ®brin(ogen)olytic enzymes, ®brinogen, appears as a large trinod-
ular protein by electron microscopy. The protein contains two symmetric half-molecules
which are disul®de-linked. Each half contains three chains designated as Aa, Bb and g
with molecular weights of 63 500, 56 000 and 47 000, respectively. The ®brinogen mol-
ecule has a molecular weight of 340 kDa (Greenberg, 1994). Fibrinogen contains long
stretches of amino acids which are exposed to proteolytic enzymes including the snake
venom proteinases. Fibrin, however, has a cross-linked structure and is much less sus-
ceptible to proteolysis (Doolittle, 1994).
As indicated previously (Markland, 1991), venom ®brinolytic enzymes may be classi-
®ed as being either a- or b-chain ®brin(ogen)ases. Thus far there have been virtually no
reports of a ®brin(ogen)olytic snake venom enzyme with cleavage speci®city directed
solely to the g-chain of ®brin(ogen). Speci®city for the a- or b-chains is not absolute
since there is substantial degradation of the alternate chain with increasing time. The
venom enzymes dier substantially from plasmin which is a serine proteinase and is
readily inactivated by plasma serine proteinase inhibitors (SERPINS). Further, plasmin
cleaves peptide bonds at the carboxy-terminal side of lysine residues in the a-, b- and g-
chains of ®brin(ogen), sites dierent than those cleaved by the venom ®brin(ogen)olytic
enzymes.
Several recent reviews have appeared on snake venom ®brin(ogen)olytic enzymes
(Markland, 1988; Stocker, 1990b; Markland, 1991; Meier and Stocker, 1991; Ouyang et
al., 1992; Siigur and Siigur, 1992; Hutton and Warrel, 1993; Marsh, 1994). In a recent
inventory, Markland (1998) described 67 puri®ed venom ®brin(ogen)olytic enzymes. All
of these enzymes are direct acting and do not rely on enzymatic components in the
blood for activity. The majority of the ®brin(ogen)olytic enzymes are puri®ed from
snakes of the Asian (22/67), North American (23/67), and Central and South American
(10/67) Crotalid family. The majority of the ®brin(ogen)olytic enzymes are metallopro-
teinases (46/67) with speci®city directed preferentially towards the Aa-chain and with
somewhat lower activity towards the Bb-chain. However, generalizations about chain
speci®city are not always applicable since there are at least three reports of ®brinogen-
olytic metalloproteinases whose preference is directed to the Bb-chain. Most of the
metalloproteinases are ®brinolytic. By contrast, serine proteinases with ®brin(ogen)olytic
activity preferentially cleave the Bb-chain with lower activity towards the Aa-chain,
although there are a number of exceptions to this generalization. Many of the serine
proteinases are both ®brinogenolytic and ®brinolytic. However, a number of them are
not ®brinolytic.
Many of the venom ®brinolytic enzymes that have been characterized in detail
recently are zinc metalloproteinases. They are members of the metzincin family
described by Stocker et al. (1995). Members of this family are so named by virtue of
their being zinc-containing metalloproteinases and having a common methionine turn
below and carboxy-terminal to a helical segment containing two of the three histidine
residues involved in the zinc-binding site. The methionine turn forms a hydrophobic
basement beneath the central active site helix and the substrate binding cavity.
Members of this family include mammalian matrix-degrading metalloproteinases (the
matrixins), bacterial metalloproteinases (the serralysins), and the astacins (including
astacin a collagenolytic enzyme from the cray®sh digestive system), as well as the
venom metalloproteinases (adamalysins). The zinc-binding site has a common amino
acid sequence in the dierent members of this family of metalloproteinases,
HEBXHXBGBXHZ, where H is histidine, E is glutamic acid, G is glycine, B is a bulky
hydrophobic residue, X is any amino acid, and Z is dierent in all four subfamilies but
is conserved in any given subfamily. Further interest in the venom class of enzymes was
generated when Bode determined the three-dimensional structure of the ®rst venom
member of this family, adamalysin (Gomis-Ruth et al., 1993, 1994). Adamalysin is a
24 kDa metalloproteinase from eastern diamondback rattlesnake (Crotalus adamanteus)
venom that contains one zinc and one calcium atom per molecule. The enzyme has
been shown by Kress (Kurecki et al., 1978) to degrade proteinase inhibitors in human
blood including antithrombin III and a1-antiproteinase. Although it does not possess
®brinolytic activity, it serves as a three-dimensional structural prototype for the 22- to
26 kDa venom ®brin(ogen)olytic and hemorrhagic enzymes (to be described later) with
which it shares extensive sequence identity. For example ®brolase, the ®brinolytic
enzyme from southern copperhead venom (Randolph et al., 1992), exhibits approxi-
mately 59% sequence identity with adamalysin.
The ®brin(ogen)olytic metalloproteinases are apparently stored in the venom gland as
inactive zymogens and activated by a cysteine switch-like mechanism similar to that
described for the closely related hemorrhagic metalloproteinase from Western diamond-
back rattlesnake (Crotalus atrox) venom, hemorrhagic toxin e (Ht-e) (Hite et al., 1992),
and adamalysin (Grams et al., 1993). In this mechanism a conserved cysteine thiol in
the prosequence of the inactive zymogen binds to the active site zinc atom, thereby
blocking enzymatic function. Following proteolytic processing, by an as yet undeter-
mined mechanism, but which may be autolytic, the thiol is displaced and the active
enzyme is generated.
Studies carried out thus far on the metalloproteinase class of venom ®brin(ogen)olytic
enzymes reveal that their cleavage preference is commonly directed to the amino-term-
inal side of hydrophobic amino acid residues. This is true in general for venom metallo-
proteinases that are members of the adamalysin family (Gomis-Ruth et al., 1993). This
group of venom metalloproteinases has also been referred to as the reprolysins by
Bjarnason and Fox (1995).
The venom ®brin(ogen)olytic serine proteinases oer an interesting dilemma with
respect to enzyme classi®cation, much as the hemorrhagic and ®brinolytic metalloprotei-
nases do. These proteinases, as well as the venom plasminogen activator, share extensive
sequence homology with the thrombin-like venom serine proteinases ancrod (Nolan et
al., 1976; Burkhart et al., 1992), batroxobin (Stocker and Barlow, 1976; Itoh et al.,
1987) and crotalase (Markland and Damus, 1971; Markland, 1976) (Fig. 2), and with
other serine proteinases such as the kallikrein-like enzyme from C. atrox (Bjarnason et
al., 1983) and the protein C activator from A. c. contortrix (Stocker et al., 1987)
venoms. Clearly there are subtle dierences between these homologous enzymes that
determine whether they exhibit ®brin(ogen)olytic, plasminogen activator, thrombin-like,

or other speci®c enzymatic activities.
The ability to degrade ®brin clots and the lack of susceptibility to SERPINS has gen-
erated interest in the potential therapeutic use of venom ®brinolytic enzymes for the
treatment of patients with occlusive arterial or venous thrombotic diseases. The poten-
tial of venom ®brinolytic enzymes for clinical use was ®rst pointed out by Didisheim
and Lewis (1956) who studied ®brinolytic activity in a number of snake venoms and
suggested that if the ®brinolytic enzymes could be separated from the other venom com-
ponents, they may have clinical applicability since they would not be inhibited by pro-
teinase inhibitors in human blood.
Of the puri®ed ®brin(ogen)ases from snake venoms which are devoid of hemorrhagic
activity, atroxase from western diamondback rattlesnake venom (Willis and Tu, 1988)
and ®brolase from southern copperhead venom (Retzios and Markland, 1988) degrade
the a-chains of ®brin and ®brinogen more rapidly than the b-chains; the ®brin(ogen)oly-
tic enzyme from northern copperhead (Agkistrodon contortrix mokasen) venom (Moran
and Geren, 1981) degrades only the Aa-chains of ®brinogen and presumably ®brin; two
®brin(ogen)olytic enzymes from Mexican west coast rattlesnake (C. b. basiliscus) venom,
degrade the Aa- and Bb-chains of ®brinogen at approximately the same rate, although
with ®brin the rate of degradation of the a-chain is faster than that of the b-chain
(Retzios and Markland, 1992). The ®brinogenase from spitting cobra (Naja nigricolis)
appears to degrade only the a-chain or a-polymer of ®brinogen and ®brin (Evans, 1981,
1984). Cleavage appears to be in the carboxy-terminal region (Evans and Barrett, 1988)
and ®brinogen so digested is still clottable by thrombin. None of these enzymes exhibits
activity with the g-chain of ®brin or ®brinogen. However, cerastase the 22.5 kDa metal-
loproteinase with anticoaguant activity from Egyptian sand viper (Cerastes cerastes)
venom does appear to degrade the g-chain following 48 h of incubation (Daoud et al.,
1987, 1988). This enzyme also degrades the a-chain of ®brin(ogen) followed by the b-
chain. Despite the presumed similarity of all of these enzymes both structurally (all are
presumably members of the reprolysin/adamalysin family) and functionally, they display
distinct and unique cleavage characteristics with the presumably natural substrate ®bri-
n(ogen).
Studies with ®brolase reveal that this enzyme neither activates nor degrades plasmino-
gen (Retzios and Markland, 1988). Further, ®brolase does not activate protein C and
does not possess thrombin-like coagulant activity. Fibrolase is inactivated by a2-macro-
globulin from human and other plasmas (Guan et al., 1991; Pretzer et al., 1993). The
rate of inactivation of the enzyme by plasma is species speci®c, the fastest rate of inacti-
vation being with rat plasma followed by rabbit, then dog and ®nally human.
Interestingly, the ®brinogenase from N. nigricolis venom was not inhibited by a2-macro-
globulin, but inactivated the proteinase inhibitor by cleaving a bond in the bait region
(Evans, 1984). This proteinase diers from the other ®brin(ogen)olytic enzymes in that
its molecular weight is 58 kDa, which may account for its failure to be inhibited by a2-
macroglobulin. The other ®brin(ogen)olytic enzymes have molecular weights in the 22-
to 26 kDa range.
Siigur and Siigur (1991) reported the isolation of lebetase, a ®brinolytic metalloprotei-
nase of molecular weight 23.7 kDa, from desert adder (Vipera lebetina) venom. The
enzyme is direct acting, degrading the ®brin and ®brinogen a-chain somewhat faster
than the b-chain, and it does not activate plasminogen. The amino acid sequence of
lebetase, derived from the cDNA sequence (Siigur et al., 1996), shows signi®cant simi-
larity to ®brolase (Randolph et al., 1992) (Fig. 3). Sequence analysis revealed that the
Fig. 3. Alignment of sequences of ®brolase (203 amino acids) (Randolph et al., 1992), atroxase
(199 amino acids from the translated amino acid sequence) (Baker et al., 1995), lebetase (203
amino acids) (Siigur et al., 1996), ACLF-I (deduced amino acid sequence from cDNA sequence,
222 amino acids) (Selistre-de-Araujo and Ownby, 1995), and LHF-II (a hemorrhagic metallopro-
teinase with ®brinolytic activity containing 200 amino acids) (Sanchez et al., 1991). Sequences
were aligned through the positioning of the invariant Cys 118 and the active site residues 141±
151, which includes two of the three zinc binding histidine residues. The third histidine involved
in zinc binding is residue 153. Underlined residues are identical in all ®ve enzymes (84/197 or
042.6%).
lebetase proprotein contains a cysteine switch motif (Siigur et al., 1996) suggesting acti-
vation by a mechanism similar to that employed by the matrix metalloproteinases and
the hemorrhagic venom metalloproteinases (Grams et al., 1993; Hite et al., 1992), as
already indicated. The amino acid sequences of several ®brinolytic enzymes have been
determined recently and all are members of the adamalysin subclass of the metzincin
family; they all possess extensive sequence similarity (Fig. 3).
A separate Aa, Bb-®brin(ogen)olytic enzyme from Vipera lebetina venom does not
activate plasminogen or prothrombin but does degrade them both slightly (Gasmi et al.,
1991, 1993). In contrast to lebetase, this direct-acting ®brinolytic metalloproteinase of
molecular weight 26 kDa degrades the Bb-chain of ®brinogen somewhat more rapidly
than the Aa-chain. The enzyme degrades the a- and b-chains of ®brin rapidly, and also
appears to degrade the g-chain after prolonged (24 h) incubation. The enzyme inhibited
platelet aggregation in human platelet rich plasma and has been used as a thrombolytic
agent in a rat venous thrombosis model (Gasmi et al., 1997).
Hemorrhagic activity of the ®brin(ogen)olytic enzymes is of concern with respect to
potential clinical utilization of these enzymes. In this regard ®brolase does not possess
hemorrhagic activity by either in vitro (Guan et al., 1991) or in vivo analysis (Markland
et al., 1994). Lebentase possesses weak hemorrhagic activity (Siigur and Siigur, 1991).
The other ®brin(ogen)olytic enzyme from V. lebetina venom is not hemorrhagic (Gasmi
et al., 1993). Cerastase has weak hemorrhagic activity (Daoud et al., 1986). Atroxase is
devoid of in vitro and in vivo hemorrhagic activity (Willis and Tu, 1988; Willis et al.,
1989). The a-®brin(ogen)ases of Asian pit vipers by comparison appear to possess
hemorrhagic activity (Ouyang and Huang, 1977; Teng et al., 1985). Properties of several
of the ®brinolytic snake venom enzymes are summarized in Table 3.
In collaboration with Lucchesi, University of Michigan, thrombolytic activity of
recombinant (rÿ) ®brolase was examined in a canine reoccluding carotid arterial throm-
bosis model (Markland et al., 1994). The model was developed by Lucchesi as a coron-
ary artery thrombosis model (Romson et al., 1980) and has been applied more recently
to the carotid artery (Rote et al., 1993). In this model, dogs had both carotid arteries
subjected to electrolytic injury leading to occlusive thrombus formation. Each dog was
anesthetized and a catheter was inserted into the jugular vein for blood sampling and
administration of the test drug. Arterial blood pressure was monitored from the cannu-
lated femoral artery and heart rate was recorded throughout the experimental protocol.
A doppler ¯ow probe was placed on each common carotid artery proximal to both the
point of insertion of the intraarterial electrode and a mechanical constrictor. The con-
strictor is adjusted to produce a regional stenosis so that the pulsatile ¯ow pattern is
reduced by 25±30% without altering mean ¯ow. Blood ¯ow velocity in each carotid
vessel was monitored continuously. Blood pressure, heart rate and carotid artery ¯ow
velocity were monitored for 2 h after achieving successful thrombolysis.
r-Fibrolase (4 mg/kg, in 3 ml) was infused over 5 min proximal to the thrombus in
the left carotid artery only. Physiological saline was infused proximal to the thrombus
in the right carotid artery, simultaneously. If lysis and reperfusion was achieved,
0.8 mg/kg of 7E3 F(ab')2, a monoclonal antibody to platelet GPIIb/IIIa that acts as a
®brinogen receptor antagonist (Coller et al., 1989), was administered intravenously, ®ve
minutes after thrombolysis. The administration of 0.9% NaCl (3.0 ml over 5 min) proxi-
Table 3. Properties of snake venom ®brinolytic enzymes

Snake speciesa
Properties and activities Vipera lebetina Crotalus atrox Agkistrodon c. contortrix
Common name lebetase atroxase ®brolase
Mol. wt. 23 700 23 500 23 000
pI 4.6±5.4 9.6 6.8
Carbohydrate content 2.4% ÿ ÿ
Hemorrhagic activity +at high dose ÿ ÿ
Fibrinogenolytic activity Aa>Bb Aa>Bb Aa>Bb
Fibrinolytic activity a and b a and b a>b
Inhibitors EDTA EDTA EDTA
no ability to induce or no plt agg. or inhibit.
Platelet activity N.D. inhibit agg. activity in vitro
Guan et al., 1991; Retzios
References Siigur and Siigur, 1991 Willis and Tu, 1988 and Markland, 1988
a
Abbreviations: N.D., not determined; EDTA, ethylenediaminetetraacetic acid; +, activity is present; ÿ, ac-
tivity not detected.
mal to the thrombus in the right carotid artery failed to achieve clot lysis in each of the
5 animals. The administration of r-®brolase proximal to the thrombus in the left carotid
artery achieved thrombolysis in all animals within a mean time of 6 2 1 min.
Subsequent administration of 7E3 F(ab')2 maintained left carotid artery patency in four
of the ®ve animals treated with ®brolase. When comparing blood ¯ow velocity in the
left carotid arteries (infused with r-®brolase plus 7E3) vs the right carotid arteries
(infused with saline), r-®brolase resulted in a restoration of blood ¯ow (70% of the pre-
injury state), whereas saline-treated arteries remained occluded. There was a signi®cant
(40%) decrease in the weight of residual thrombi in the arteries treated with r-®brolase
plus 7E3 compared to the weight of thrombi retrieved from arteries treated with saline.
There were minimal changes in blood cell counts and in the mean arterial blood press-
ure and heart rate. Administration of r-®brolase did not alter the activated partial
thromboplastin time. Ex vivo platelet aggregation in response to arachidonic acid or
ADP was decreased by approximately 60% after ®brolase administration (this was
interesting in view of our in vitro ®ndings indicating a lack of platelet reactivity of ®bro-
lase). Complete inhibition of ex vivo platelet reactivity was achieved after the adminis-
tration of 7E3. There was no evidence of hemorrhage in the r-®brolase treated dogs.
In summary, the model studies revealed that highly puri®ed ®brolase produced rapid
and consistent thrombolysis. The lack of physiologic alterations attributable to ®brolase
demonstrates that the enzyme holds promise for clinical use. This direct-acting enzyme
functions independently of the native ®brinolytic system and oers a safe, eective,
rapid, and speci®c mechanism for clot dissolution and may prove useful as an alterna-
tive to, or for use in synergistic combination with, presently used thrombolytic agents.
Several other in vivo studies with ®brinolytic enzymes have provided promising results
(Willis et al., 1989; Ahmed et al., 1990; Mao et al., 1995; Gasmi et al., 1997). These stu-
dies revealed that highly puri®ed ®brinolytic snake venom enzymes produced consistent
thrombolysis. The venom enzymes act by a completely dierent mechanism than the
plasminogen activators, the only agents presently approved for clinical use (Collen and
Lijnen, 1995; Sherry, 1990), and may have certain advantages over the plasminogen
activators: (i) they are not inhibited by the blood serine proteinase inhibitors
(SERPINS); (ii) since they do not activate plasmin, secondary eects such as platelet ac-
tivation related to plasmin formation would be avoided. The lack of pathologic altera-
tions and the absence of demonstrable histologic changes, further suggest the
therapeutic potential of these enzymes.
Recently a chimeric derivative of ®brolase has been prepared by covalently coupling
the enzyme to a cyclic Arg±Gly±Asp-containing peptide (provided by Diatide Inc.,
Londonderry, NH) with antiplatelet activity (®brinogen receptor antagonist) (Sanchez et
al., 1997). The chimera has both ®brinolytic and platelet aggregation inhibitory activi-
ties in vitro. In vivo studies are currently in progress to determine the clinical potential
of this interesting molecule.
Plasminogen activators
Snake venoms have been reported to stimulate the release of plasminogen activators
from endothelial cells. This activity was most pronounced in the venoms of the rattle-
snakes Crotalus atrox and C. adamanteus (Kirschbaum et al., 1988). Enzymatically
active batroxobin, the thrombin-like enzyme from Bothrops moojeni venom, has been
reported to cause a dose-dependent increase in the release of tissue plasminogen activa-
tor from an isolated perfused pig ear preparation (KloÈcking et al., 1987). Additionally,
habutoxin, the thrombin-like enzyme from Trimeresurus ¯avoviridis venom, stimulated

bovine pulmonary artery endothelial cells to release increased levels of urokinase (u-PA)
and plasminogen activator inhibitor (PAI) (Sunagawa et al., 1996). Although these scat-
tered reports indicated that snake venoms contained proteins that stimulate the release
of plasminogen activator activity from endothelial cells, there were no reports until
recently of a direct acting plasminogen activator in snake venom. However, the ®rst
report of what appears to be a true plasminogen activator in snake venom appeared not
long ago (Zhang et al., 1993, 1995b). The enzyme, isolated from the Chinese green tree
viper (Trimeresurus stejnegeri) venom, is a single chain glycoprotein with a molecular
weight of 33 kDa and an isoelectric point of pH 5.2; it represents about 0.5% of the
total protein of the pooled venom. The enzyme is a serine proteinase and is inhibited by
phenylmethanesulfonyl ¯uoride. It activates plasminogen by an enzymatic action that
leads to plasmin generation. However, unlike plasminogen activation by tissue-type
plasminogen activator (t-PA), activation by the venom enzyme was not stimulated by
®brin fragments. Furthermore, the venom enzyme was much less active as a plasmino-
gen activator than ®brin fragment stimulated t-PA. With human Glu-plasminogen, clea-
vage by the venom enzyme occurs at a single Arg561±Val562 peptide bond; the same
bond cleaved by the physiological plasminogen activators urokinase (u-PA) and t-PA.
The enzyme does not activate nor degrade prothrombin, factor X or protein C, and it
does not clot ®brinogen or show ®brin(ogen)olytic activity in the absence of plasmino-
gen. A full-length cDNA encoding the protein has been cloned and the deduced com-
plete amino acid sequence was determined. The protein, called TSV-PA, contains 234
amino acids with a single N-glycosylation site. It shows extensive sequence similarity
with other snake venom serine proteinases including 66% identity with the protein C
activator from A. c. contortrix venom, 63% with batroxobin the thrombin-like enzyme
from B. atrox venom and 60% with the factor V activator from Russell's viper venom.
By contrast, TSV-PA exhibits only 21±23% sequence similarity with the catalytic
domains of u-PA and t-PA. Importantly, the venom enzyme only contains the catalytic
domain of the mammalian plasminogen activators and is missing the kringle and other
amino-terminal domains. Further, it lacks the sequence that is responsible for the inter-
action of the mammalian plasminogen activators with plasminogen activator inhibitor-1
(PAI-1). Interestingly, Braud et al. (1997), using recombinant methodology, inserted a
positively charged sequence in TSV-PA that is similar to a sequence that endows u-PA
with PAI-1 susceptibility. When inserted in a position corresponding to that in u-PA,
the TSV-PA mutant was sensitive to PAI-1. The thrombolytic potential of this enzyme
Table 4. Properties of snake venom plasminogen activator

Properties and activities Snake species
Trimeresurus stejnegeri
Common name TSV-PA
Molecular weight 33 000
pI 5.2
Carbohydrate content single glycosylation site
cleaves single peptide bond in plasminogen, the
Direct plasminogen activation plasminogen activator susceptible Arg561±Val562 bond
does not clot ®brinogen or activate or degrade factors II,
Other activities X or protein C
Inhibitors phenylmethylsulfonyl ¯uoride
Platelet activity not determined
Fibrin(ogen)olytic activity not detected
References Zhang et al., 1993; Zhang et al., 1995b
or recombinant mutants is intriguing, but yet to be explored. Properties of TSV-PA are

summarized in Table 4.
HEMOSTATICALLY ACTIVE VENOM PROTEINS: PLATELET INTERACTIVE COMPONENTS
Snake venoms have been reported to contain a number of platelet active components
including those that cause platelet aggregation and inhibit platelet aggregation. This
area has been the subject of several recent reviews (Kini and Evans, 1990; Smith and
Brinkous, 1991; Teng and Huang, 1991). Table 5 lists various snake venom platelet
aggregation inducers and inhibitors.
Activators of platelet aggregation

Smith and Brinkous (1991) divided the platelet-aggregating agents derived from snake
venoms into several groups including enzymatic components with direct action on plate-
lets, non-enzymatic components with direct action on platelets, and those components
requiring a plasma cofactor. Only those components which have been puri®ed and well
characterized will be brie¯y described here.
Enzymes with direct action on platelets. One group of enzymes with direct action on
platelets are serine proteinases including crotalocytin, thrombocytin and some throm-
bin-like enzymes (Teng and Ko, 1988). Aggregating activity of the venom thrombin-like
enzymes was determined to be mainly due to ADP (a platelet agonist released by plate-
lets) and dierent from that caused by thrombin. Crotalocytin was puri®ed from timber
rattlesnake venom and shown to be a single chain polypeptide of molecular weight
55 kDa (Schmaier et al., 1980). The enzyme induced simultaneous platelet aggregation
and secretion of ATP from platelets (Schmaier and Colman, 1980). Another related
enzyme, thrombocytin, was puri®ed from the South American pit viper Bothrops atrox
(Niewiarowski et al., 1977). This enzyme is a glycoprotein of molecular weight 36 kDa.
It aggregates platelets directly and induces the platelet release reaction. Both enzymes
are typical thrombin-like serine proteinases and are inhibited by diisopropyl¯uoropho-
sphate. However, neither clotted ®brinogen and they dier in their mechanisms of indu-
cing platelet aggregation. Thrombocytin appears to release platelet ADP, which itself is
a platelet aggregating agent, whereas crotalocytin induces ADP release and 14 C-seroto-
nin secretion and to some extent resembles the action of low dose thrombin.
Among the thrombin-like serine proteinases, cerastocytin, from Tunisian viper
(Cerastes cerastes) venom (Marrakchi et al., 1995, 1997a), and cerastobin, from
Table 5. Snake venom proteins active on platelets

Functional activity Speci®c biological role or requirement
Platelet aggregating agents
Direct enzymatic serine proteinases
phospholipases A2
Direct non-enzymatic lectins
GPIb binding proteins
Co-factor required von Willebrand factor
Platelet aggregation inhibitors a-®brinogenases
phospholipases A2
5'-nucleotidases
disintegrins
Egyptian sand viper (Cerastes vipera) venom (Farid et al., 1990), induce platelet aggre-
gation. Blocking receptor sites for ®brinogen or thrombin binding (integrins GPIIb/IIIa
and GPIb, respectively), prevents aggregation induced by cerastocytin (Marrakchi et al.,
1997a).
A separate group of venom enzymes that induce platelet aggregation are the phos-
pholipases A2. Mounier et al. (1994) demonstrated that phospholipase A2 from cobra
(Naja m. mossambica) venom induced platelet aggregation and secretion of ATP. In
general these agents act by cleaving platelet membrane phospholipids resulting in the
release of arachidonic acid. Aspirin, a cyclooxygenase inhibitor, blocked the eect of
the venom phospholipase suggesting that the eect is most likely due to the formation
of arachidonic acid metabolites such as thromboxane A2 (Mounier et al., 1994).
Interestingly, some other venom phospholipases inhibit platelet aggregation while others
have a biphasic eect, initiating platelet aggregation at low concentrations, and inhibit-
ing at high concentrations or following longer incubations (Kini and Evans, 1989,
1990). An example of a speci®c phospholipase A2 which induces platelet aggregation is
the protein isolated from southern copperhead venom (Takagi et al., 1988). Aggregation
induced by the enzyme was lost, as was phospholipase activity, after treatment of the
enzyme with the histidine modifying agent p-bromphenacyl bromide. Enzyme induced
aggregation was blocked by prostacyclin but not by aspirin, suggesting released arachi-
donic acid had no role in the aggregation mechanism.
Cobra (Teng et al., 1986) and Russell's viper (Teng et al., 1984) venom phospho-
lipases induced a biphasic eect on washed rabbit platelets. The ®rst phase was a revers-
ible aggregation and the second phase was an inhibitory eect on platelet aggregation
induced by arachidonic acid, ADP or collagen, but not induced by thrombin. The
authors concluded that the aggregating eect was due to thromboxane formation and
the antiplatelet eect might be due to the inhibitory action of split products produced
by the phospholipases.
Non-enzymatic components with direct action on platelets. A number of other venom

platelet aggregation inducers have been reported, among these are non-enzymatic com-
ponents with direct action on platelets (Kini and Evans, 1990; Smith and Brinkous,
1991). In this group are lectins isolated from the bushmaster (Lachesis muta) and other
snake venoms, which bind to platelets and induce a conformational change in the ®bri-
nogen receptor (platelet glycoprotein IIb/IIIa) allowing ®brinogen binding and sub-
sequent aggregation.
Convuluxin from the South American rattlesnake (Crotalus durissus terri®cus) and
other related species represents another type of non-coagulant, non-enzymatic venom
platelet aggregating agent. This protein is a 300 kDa glycoprotein complex containing
multiple copies of two subunits. It is a very potent platelet aggregating agent (Smith
and Brinkous, 1991). Convuluxin appears to share a similar mechanism of platelet
aggregation with collagen (Kini and Evans, 1990). More recent studies (Faili et al.,
1994) suggest that platelet activation mediated by convuluxin involves a phosphoinosi-
tide-speci®c phospholipase C as well as other mechanisms. Convuluxin belongs to the
C-type lectin family (Polgar et al., 1997a) as shown by sequence analysis and the pre-
sence of two subunits obtained by sodium dodecylsulfate polyacrylamide gel electro-
phoresis (SDS±PAGE). Unlike other snake venom C-type lectins, convuluxin does not
bind to platelet membrane glycoprotein Ib. Convuluxin induces signal transduction in
part like collagen and it binds to a 62 kDa glycosylated membrane component in plate-
let lysates. Other ®ndings suggest that convuluxin does not use the a2b1 integrin recep-
tor of the collagen signaling pathway to activate platelets. Thus, it would appear that
convuluxin induces platelet aggregation primarily through one of the collagen receptors,
the 62 kDa glycoprotein. Signal transduction by convuluxin shows many similarities to
signaling pathways induced by collagen.
A potent platelet aggregation inducer, platelet aggregoserpentin, was puri®ed from
green tree viper (Trimeresurus gramineus) venom (Ouyang and Huang, 1983a). It is a
single chain glycoprotein of 43.4 kDa. The protein appeared to activate platelets by low-
ering cAMP levels or by activating an endogenous phospholipase A2, resulting in the
formation of platelet activating factor. The mechanism of platelet activation did not
appear to involve prostaglandins. Ca++ was required for activity of the venom protein.
Triwaglerin is a potent inducer of platelet aggregation that was puri®ed from
Wagler's pit viper (Trimeresurus wagleri) venom (Teng et al., 1989). It has a molecular
weight of 68 kDa and no detectable enzymatic activity. Triwaglerin appears to activate
platelets by a unique mechanism that is independent of formation of thromboxane A2
and platelet activating factor, or of released ADP.
Trimucytin is another potent platelet aggregation inducer isolated from Chinese habu
(Trimeresurus mucrosquamatus) venom (Teng et al., 1993). It is a 68 kDa glycoprotein
with a run of Gly±Pro-X repeats, like collagen, at its amino-terminus. Trimucytin
induces ATP release and thromboxane formation in rabbit platelets, but its aggregating
action is not due to released ADP. An antibody against platelet membrane glycoprotein
Ia, inhibited trimucytin- and collagen-induced platelet aggregation and ATP release,
suggesting this as the binding site for trimucytin.
Aggretin, from Malayan pit viper venom, is another potent platelet aggregation indu-
cer (Huang et al., 1995). It is a heterodimer of 29 kDa. A monoclonal antibody directed
against glycoprotein Ia/IIa inhibited platelet shape change and aggregation induced by
aggretin. Aggretin binds to platelets and may act as a glycoprotein Ia/IIa agonist to eli-
cit platelet aggregation through the activation of endogenous phospholipase C, leading
to hydrolysis of phosphoinositides and subsequent mobilization of intracellular Ca++.
Trimucytin appears to act through a similar mechanism (Teng et al., 1993).
Platelet aggregation inducers (or inhibitors) requiring von Willebrand factor or aecting
von Willebrand factor±glycoprotein Ib (GPIb) interactions. The binding of plasma von
Willebrand factor (vWF) to platelet glycoprotein Ib (GPIb) plays a key role in one of
the earliest phases of hemostasis, the anchoring of platelets to the injured vessel wall.
vWF is a heterogeneous multimeric glycoprotein of molecular weight between 500 kDa
to 20 000 kDa. It is composed of subunits of 275 kDa linked by disul®de bonds. It has
been shown that under high shear stress, platelet aggregation is initiated by interaction
between multimeric vWF and GPIb. This condition may mimic that in stenosed coron-
ary arteries in vivo. A number of snake venom proteins have been isolated that speci®-
cally bind to and modulate the interaction of vWF and GPIb. This area has been
recently reviewed by Fujimura et al. (1996). Botrocetin or venom coagglutinin, puri®ed
from several snake venoms particularly those of the Bothrops genus, acts via vWF to
cause platelet aggregation and agglutination (Read et al., 1978; Smith and Brinkous,
1991). Unlike aggregation, agglutination is a passive process that only involves the
physical interaction of a protein or glycoprotein ligand, which has two binding sites,
with the platelet surface. Aggregation on the other hand is an active transmembrane
process which requires metabolically active platelets. Thus, formaldehyde ®xed platelets
will agglutinate, but will not aggregate. Botrocetin is a 26.5 kDa disul®de-linked hetero-
dimeric protein that has no known enzymatic activity. It acts by a two-step process to
cause platelet agglutination. In the ®rst step, botrocetin binds to vWF forming an active
agglutinating complex. In the second step, this complex binds to integrin GPIb, the
vWF receptor, on the platelet membrane and serves as a bridging agent resulting in pla-
telet agglutination or aggregation. Sugimoto et al. (1991) identi®ed a discontinuous site
in vWF, within residues 539±643, to which botrocetin may bind to modulate the inter-
action with GPIb.
A separate protein, bitiscetin, isolated from pu adder (Bitis arietans) venom also
speci®cally interacts with vWF and induces platelet agglutination via interaction with
platelet GPIb (Hamako et al., 1996). Bitiscetin is a heterodimer composed of 16 kDa
and 13 kDa subunits. It appears that bitiscetin interacts directly with vWF, but the
mechanism of the interaction is dierent from that of botrocetin.
Interestingly, several GPIb binding proteins have been isolated from snake venoms
and shown to be heterodimers structurally related to botrocetin and to C-type lectins
(Fujimura et al., 1996). One group of these, the alboaggregins, from white-lipped tree
viper (Trimeresurus albolabris) venom were characterized in detail and shown to directly
agglutinate formalin-®xed platelets and aggregate washed human platelets by binding to
GPIb (Peng et al., 1991, 1992; Yoshida et al., 1993). Platelet release or shape change
did not accompany this interaction. The binding of alboaggregin to GPIb does not lead
to activation of the ®brinogen receptor (GPIIb/IIIa). Alboaggregin binding to GPIb
inhibited botrocetin-induced human vWF binding to GPIb. A novel 50 kDa form of
alboaggregin was recently isolated from Trimeresurus albolabris venom. This protein
potently induced platelet activation and activated protein kinase C and tyrosine
kinase(s). These results suggest that the 50 kDa form of alboaggregin induces cyto-
plasmic signaling following its binding to GPIb (Andrews et al., 1996). GPIb binding
proteins have also been isolated from the timber rattlesnake (Crotalus horridus horridus)
(Scarborough et al., 1991a). These proteins, named CHH-A and CHH-B, are potent in-
hibitors of vWF binding to GPIb. Both proteins are heterodimers of molecular weights
23- to 25 kDa with two disul®de-linked 12- to 15 kDa chains. The proteins blocked
botrocetin-induced agglutination of platelets. The sequence of the two chains of CHH-B
were determined and found to be highly homologous to each other, to the C-type snake
venom lectins, and to botrocetin. CHH-B appeared to bind directly to GPIb. A similar
protein was isolated from Bothrops jararaca venom and called GPIb-BP (Fujimura et
al., 1995). The protein inhibited botrocetin-induced vWF binding to GPIb. Amino acid
sequences of the two chains were determined (Kawasaki et al., 1996). The GPIb binding
site is on the 123-amino acid b-subunit. The protein is a member of the C-type lectin
snake venom family and shows extensive sequence homology with botrocetin and
alboaggregin.
Another platelet agonist that binds to platelet GPIb was isolated from Asian lance-
head viper (Trimeresurus tokarensis) venom. The 29 kDa protein, called tokaracetin, is
another member of the C-type lectin family containing 16- and 15 kDa chains held
together by disul®de bond(s) (Kawasaki et al., 1995). Tokaracetin inhibited the binding
of bovine vWF, and human vWF in the presence of botrocetin, to ®xed human plate-
lets. The venom protein also abolished vWF-dependent sheer-induced platelet aggrega-
tion.
Finally, two high molecular weight GPIb binding proteins were isolated from
Trimeresurus ¯avoviridis venom (Taniuchi et al., 1995). The proteins, called ¯avocetin-A
and -B had molecular weights of 149- and 139 kDa, respectively. On reduction, chains
of 17- and 14 kDa were observed in both proteins, but a third chain of 15 kDa was also
found in ¯avocetin-B. There is a high degree of amino-terminal sequence homology
with other venom C-type lectins including botrocetin and alboaggregin. Both ¯avocetin-
A and -B inhibited vWF-dependent aggregation of ®xed human platelets and inhibited
sheer-induced platelet aggregation at high sheer stress.
Platelet aggregation inhibitors

The snake venom inhibitors of platelet aggregation have been subdivided into four
groups on the basis of their mechanism of action (Teng and Huang, 1991). Recently,
however, other nonenzymatic inhibitors of platelet aggregation have been identi®ed
with mechanisms of action dierent from those of these four groups. These will be con-
sidered after the discussion of the fourth group, the disintegrins.
a-®brinogenases. The ®rst group includes the a-®brinogenases found in some South-
east Asian venoms. These enzymes degrade preferentially the Aa-chain of ®brinogen.
The a-®brinogenase from Calloselasma rhodostoma venom had an inhibitory eect on
aggregation of washed rabbit platelets (Ouyang et al., 1985). This eect was reversed by
treating the enzyme with EDTA, which inhibited ®brin(ogen)olytic activity. The platelet
aggregation inhibitory eect of the enzyme was also reversed by adding ®brinogen. The
authors concluded that the inhibitory eect of the a-®brinogenase on platelet aggrega-
tion was due to its action on ®brinogen which is required for platelet aggregation via
binding to the ®brinogen receptor GPIIb/IIIa. By contrast, the a-®brinogenase from
spitting cobra (Naja nigricollis) venom inhibits platelet aggregation, but it does so in the
absence of plasma ®brinogen (Kini and Evans, 1991). Therefore, it is not a general
phenomenon that all snake venom a-®brinogenases that inhibit platelet aggregation do
so via an eect on plasma ®brinogen.
Phospholipases A2. Another group of platelet aggregation inhibitors includes the phos-
pholipases A2, which may either inhibit platelet aggregation or induce aggregation.
Some phospholipases A2 have a biphasic action which involves an aggregatory eect
after short incubations or at low enzyme concentrations, or on long term incubation or
at high concentrations an inhibitory eect. Inhibition may be due to the formation of
cleaved products derived from arachidonic acid metabolites. A number of snake venom
phospholipases that act as inhibitors of platelet aggregation are listed by Teng and
Huang (1991). Some more recent examples of snake venom phospholipases A2 that are
potent inhibitors of platelet aggregation are given here. One of these is the enzyme iso-
lated from the Australian copperhead snake (Austrelaps superba) venom (Yuan et al.,
1993). The enzyme has a molecular weight of 15 kDa and inhibits platelet aggregation
and serotonin release induced by a variety of platelet agonists. Similarly, a phospho-
lipase A2 isoform from Russell's viper venom inhibited ADP-induced platelet aggrega-
tion dose-dependently (Prasad et al., 1996). Another phospholipase A2 with potent
platelet aggregation inhibitory activity was isolated from king cobra (Ophiophagus han-
nah) venom (Huang et al., 1997). This enzyme inhibited aggregation induced by a num-
ber of agonists. When phospholipase activity of the enzyme was inhibited by p-
bromphenacyl bromide, platelet aggregation inhibitory activity was only partially inhib-
ited. These ®ndings suggest that the antiplatelet eects may be separate from enzymatic
activity. The antiplatelet activity appears to be caused in part by a dramatic change in
the platelet cytoskeleton induced by the phospholipase, and hence the loss of the release
reaction. A separate phospholipase A2 of 15 kDa was puri®ed from Papaun black snake
(Pseudechis papuanus) venom (Laing et al., 1995). This enzyme was a potent platelet
aggregation inhibitor and it also induced a change in platelet morphology including a
disruption of the cytoskeleton. Laing et al. (1995) also found that p-bromphenacyl bro-
mide treatment of the enzyme did not completely inhibit platelet aggregation inhibitory
activity. The eect of phospholipases on the cytoskeleton suggests that this may be a
general mechanism for inhibition of platelet aggregation. Whether this is related to the
enzymatic action on phospholipids of the platelet membrane remains to be determined.
5'-nucleotidases. A third group of inhibitors of platelet aggregation includes the 5'-

nucleotidases from snakes of the viper and pit viper families. These enzymes cause the
degradation of ADP, a platelet aggregation inducer released from platelet dense gran-
ules by various agonists. One of these enzymes was puri®ed from Trimeresurus grami-
neus venom (Ouyang and Huang, 1983b). It is a thermostable, single chain glycoprotein
of 74 kDa. Activity was inhibited by EDTA but reversed by Zn++ or Co++. The
enzyme completely inhibited sodium arachidonate and ADP-induced platelet aggrega-
tion in rabbit platelet-rich plasma. Since the enzyme cleaves ADP as well as AMP, it
was concluded that removal of ADP (which is released by platelet agonists) and poss-
ibly the generation of adenosine, was responsible for the enzyme's inhibitory eect on
platelet aggregation.
Fibrinogen receptor antagonists. The fourth and probably the most interesting group
of platelet aggregation inhibitors are the disintegrins, which act as ®brinogen receptor
antagonists. Because of their potential as therapeutic agents, this group of snake
venom-derived peptides has received a great deal of attention (Table 6). Several excel-
lent reviews on the structure and function of these small molecules have been published
recently (Gould et al., 1990; Teng and Huang, 1991; Williams, 1992; Niewiarowski et
al., 1994; Perutelli, 1995). Disintegrins are a family of small, disul®de-rich, polypeptides
that bind to integrin receptors (cell surface adhesion receptors) (Gould et al., 1990; Wil-
liams, 1992; Niewiarowski et al., 1994). Disintegrins are speci®c inhibitors of integrins
of the b1 and b3 subfamilies including the ®brinogen receptor GPIIb/IIIa (aIIbb3), the
vitronectin receptor (avb3) and the ®bronectin receptor (a5b1). Although initially
reported as inhibitors of platelet aggregation, disintegrins bind to integrins on the sur-
face of many cell types, including malignant cells. Close to 50 disintegrins and their iso-
forms have been puri®ed from the venom of dierent snake species from the viper or
pit viper families (Dennis et al., 1989; Scarborough et al., 1991b, 1993; Niewiarowski et
al., 1994). Additionally, disintegrins have been cloned and expressed (Gan et al., 1989;
Jacobson et al., 1989) and chemically synthesized (Garsky et al., 1989). Since disinte-
grins inhibit ®brinogen binding to platelets, they act as ®brinogen receptor antagonists.
Fibrinogen binds to the exposed integrin GPIIb/IIIa on the platelet surface and causes
platelet±platelet interaction. Since this is the ®nal common pathway for platelet aggre-
gation, disintegrins inhibit aggregation caused by a number of agonists including ADP,
thrombin, collagen and arachidonic acid. Concentrations of disintegrins that cause 50%
inhibition of human platelet aggregation (IC50) vary from 30 to 300 nM. There is both
in vitro (Huang et al., 1989; Clark et al., 1994) and in vivo (Cook et al., 1989) evidence
that there are subtle dierences in biological activities between members of several
dierent structural subgroups of the disintegrins. There appears to be three dierent
Table 6. Potential clinical uses of snake venom disintegrins

Activity Disintegrin (snake species) References
Prolonged time to occlusion and reduced

size of ischemic damage in rat middle
cerebral artery thrombosis model tri¯avin (Trimeresurus ¯avoviridis) Kaku et al., 1997
Imaging of deep venous thrombosis and
123
pulmonary embolus I-bitistatin (Bitis arietans) Knight et al., 1996
Antithrombotic activity in canine carotid contortrostatin (Agkistrodon c.
and coronary artery thrombosis models contortrix) Markland et al., 1995
bitistatin (Bitis arietans) Shebuski et al., 1990
kistrin (Calloselasma rhodostoma) Yasuda et al., 1991
echistatin (Echis carinatus) Holahan et al., 1991
Protects platelets during extracorporeal
circulation echistatin (Echis carinatus) Bernabei et al., 1995
Inhibits experimental metastasis of contortrostatin (Agkistrodon c.
human and murine melanoma contortrix) Trikha et al., 1994a,b
albolabrin (Trimeresurus albolabris) Soszka et al., 1991
Inhibits breast cancer growth and contortrostatin (Agkistrodon c.
spontaneous metastasis contortrix) Zhou et al., 1996a
Inhibits breast cancer angiogenesis contortrostatin (Agkistrodon c.
(CAM) contortrix) Zhou et al., 1996a
Inhibits retinal pigment epithelial cell-
induced tractional retinal detachment echistatin (Echis carinatus) Yang et al., 1996
Inhibits sperm±egg fusion processes echistatin (Echis carinatus) Bronson et al., 1995
Inhibits bone resorption in vivo echistatin (Echis carinatus) Fisher et al., 1993
subgroups of disintegrins based on size. The short disintegrins with four disul®de bonds
and a single polypeptide chain of 49±51 amino acids, the medium subgroup with six dis-
ul®de bonds and a polypeptide chain of 68±73 amino acids and the long subgroup with
seven disul®de bonds and a polypeptide chain of 83 amino acids. Interestingly, there are
three reports of multimeric platelet aggregation inhibitors that appear to bind to the
®brinogen receptor. Gabonin, from Gaboon viper (Bitis gabonica) venom was reported
to exist as a disul®de-linked dimer of 21.1 kDa (Huang et al., 1992). Upon reduction
the protein was converted to an 11 kDa monomer. Gabonin dose-dependently inhibited
platelet aggregation by various agonists including ADP, collagen and thrombin. Ceras-
tatin is a potent platelet aggregation inhibitor isolated from the venom of the Tunisian
viper (Cerastes cerastes) (Marrakchi et al., 1997a,b). The protein is made up of at least
three subunits and exists as a 32 kDa protein under non-reducing conditions. The pro-
tein inhibited aggregation of washed human platelets induced by thrombin, collagen
and other agonists with an IC50 of 40 nM. It appears to be a ®brinogen receptor antag-
onist. However, there is little or no sequence information for these multimeric proteins.
By contrast, contortrostatin, the disintegrin isolated from southern copperhead venom
appears to be a homodimer with a molecular weight of 13.5 kDa under non-reducing
conditions and 7.0 kDa under reducing conditions (Trikha et al., 1994a). The cDNA
sequence of contortrostatin has recently been determined. The deduced protein sequence
reveals an RGD motif in the proper location relative to other known disintegrins, but
there is a 12-residue truncation at the amino-terminus (Zhou and Markland, unpub-
lished ®ndings). Since there are two cysteine residues in the missing region, it is possible
that the 61-residue mature protein dimerizes due to the abnormal cysteine pairing result-
ing from the amino-terminal truncation. Since the amino acid sequences of the other
two multimeric proteins are unknown, it is unclear whether a similar situation may per-
tain, or whether these proteins are even disintegrins.
All disintegrins contain an Arg±Gly±Asp (RGD) sequence in the carboxyl-terminal

half of the molecule which is essential to their ability to block integrin interaction with
ligands (Dennis et al., 1989; Scarborough et al., 1993). One disintegrin, barbourin, from
southeastern pigmy rattlesnake (Sistrurus m. barbouri) venom contains a Lys±Gly±Asp
(KGD) sequence which imbues this disintegrin with high anity for the ®brinogen
receptor, GPIIb/IIIa (Scarborough et al., 1991b). The positioning of the disul®de bonds
and of the RGD/KGD sequence is invariant in all disintegrins thus far reported.
However, in the short disintegrins, but not the other two subgroups, a cysteine residue
is uniquely positioned on the carboxy-terminal side of the RGD sequence. This is most
likely done to preserve disul®de pairing in the short disintegrins. There are no free sulf-
hydryls in the disintegrins, all cysteine residues are involved in disul®de bonds
(Niewiarowski et al., 1994). Disintegrins appear to be synthesized in the snake venom
gland as multidomain proteins containing metalloproteinase, disintegrin, and cysteine-
rich domains. Interestingly, there appears to be dierential proteolytic processing at
both the amino- and carboxyl-terminal ends. This probably leads to the production of
the isoforms observed with many puri®ed disintegrins (Dennis et al., 1989; Scaloni et
al., 1996).
Nuclear magnetic resonance has been employed to determine the three-dimensional
solution structure of the disintegrins albolabrin, from Trimeresurus albolabris venom
(Jaseja et al., 1993; Smith et al., 1996); kistrin, from Calloselasma rhodostoma venom
(Adler et al., 1991); ¯avoviridin, from Trimeresurus ¯avoviridis venom (Senn and Klaus,
1993); and echistatin, from Echis carinatus venom (Chen et al., 1991; Saudek et al.,
1991a,b; Cooke et al., 1992; Atkinson et al., 1994). These studies revealed that disinte-
grins display no regular secondary structure (Saudek et al., 1991a,b; Adler et al., 1991).
They are characterized by a number of irregular turns and loops to form a rigid core
stabilized by hydrogen bonds and held together by disul®de bonds. Pairing of the four
disul®de bonds has been determined for the 49-residue disintegrin echistatin as Cys 2 to
Cys 11, Cys 7 to Cys 32, Cys 8 to Cys 37, and Cys 20 to Cys 39 (Atkinson et al., 1994).
In the 68-residue disintegrin kistrin, which has six disul®de bonds, pairing is Cys 4 to
Cys 19, Cys 6 to Cys 14, Cys 13 to Cys 36, Cys 27 to Cys 33, Cys 32 to Cys 57, Cys 45
to Cys 64 (Adler et al., 1993). The disul®de bond pairing in ¯avoviridin, which has 70-
residues, is identical to that in the homologous disintegrin kistrin (Calvete et al., 1992;
Klaus et al., 1993). Interestingly, the disul®de pairing for the 73-residue albolabrin is
Cys 6 to Cys 15 or 16, Cys 8 to Cys 16 or 15, Cys 21 to Cys 34 or 35, Cys 29 to Cys
59, Cys 35 or 34 to Cys 38, Cys 47 to Cys 66 (Jaseja et al., 1993). This is dierent than
the pairing in the homologous disintegrins kistrin and ¯avoviridin and raises questions
about the validity of the disul®de bond assignments or the signi®cance of the dier-
ences, if they are real. Nonetheless, the RGD sequence in all four disintegrins of known
three-dimensional structure is in the identical location at the tip of a ¯exible 13-residue
hairpin loop stabilized by two disul®de bonds at its base (Niewiarowski et al., 1994).
There is high mobility in the RGD sequence, thereby allowing rapid recognition and
binding into the integrin binding site. Further, the loop protrudes from the central core
making it readily available to interact with integrins.
An interesting mutant of echistatin was recently constructed by converting the normal
ARGDD sequence to a CRGDC sequence (Yamada and Kidera, 1996). This mutation
introduced an additional disul®de bond into the protein, without altering the four
already present, and introduced an additional conformational constraint, sterically
freezing the RGD in a type II' beta-turn. The mutant was twice as eective as native
echistatin in inhibiting ®brinogen binding to GP IIb/IIIa. Interestingly, the disul®de-
linked dimeric disintegrin, contortrostatin, had a dierential eect on platelet signal

transduction than a monomeric disintegrin, multisquamatin, puri®ed from the viper
Echis multisquamatus venom (Clark et al., 1994). Contortrostatin, but not multisquama-
tin, mediated signaling events typically triggered by ®brinogen cross-linking of GPIIb/
IIIa, as demonstrated by tyrosine phosphorylation of tyrosine kinase pp72syk and a
140 kDa platelet protein. However, both disintegrins inhibited platelet aggregation and
aggregation-dependent tyrosine phosphorylation of a number of proteins including
pp125FAK, the focal adhesion kinase. Thus, structurally distinct disintegrins have dier-
ing eects on tyrosine phosphorylation.
The site of disintegrin interaction with GPIIb/IIIa has been examined by Calvete et
al. (1994) using a heterobifunctional cross-linking agent bound to the disintegrins
through a lysine residue(s). Using puri®ed platelet GPIIb/IIIa, the cross-linking site of
four 125 I-labeled disintegrins, albolabrin, echistatin, bitistatin and eristostatin, was ident-
i®ed. For all disintegrins, which includes members of the short (echistatin and eristosta-
tin from venoms of Echis carinatus, and the leaf-nosed viper, Eristocophis macmahoni,
respectively), medium (albolabrin from venom of Trimeresurus albolabris) and long
(bitistatin from venom of the pu adder, Bitis arietans) subclasses, the attachment site
was identi®ed within GPIIIa residues 217 to 302. This positions the disintegrin attach-
ment site within 15 to 20 AÊ from the RGD binding site and at the interface between the
heterodimeric integrin subunits. However, there may be additional contact sites not
identi®ed by the strategy employed which relied on photoactivation for cross-linking.
Also, since the integrin was puri®ed from platelets, its structure may not resemble that
of native platelet associated integrin, and an artifactual association site may, therefore,
be created. Further along these lines, it has been shown that two dierent, but closely
related disintegrins, echistatin and eristostatin bind to dierent epitopes on the integrin
avb3 and bind to dierent, but overlapping sites, on aIIbb3 (Marcinkiewicz et al., 1996).
Similarly, Rahman et al. (1995) have shown that members of the medium disintegrin
subclass, kistrin and elegantin, disintegrins from the venoms of Calloselasma rhodostoma
and the Asian lancehead (Trimeresurus elegans), respectively, appear to interact with
two distinct, but possibly interacting sites on platelet aIIbb3. Thus, even closely related
disintegrins within the same subclass have somewhat diering speci®cities. This may be
related to the dierences around the RGD sequence in the disintegrins. Echistatin has
the sequence ARGDD around the RGD site whereas eristostatin has the sequence
ARGDW. When a synthetic echistatin was prepared in which the sequence around the
RGD site was mutated to ARGDW, like eristostatin, the resulting mutant had the
properties of eristostatin with respect to increased platelet aggregation inhibitory prop-
erties and increased speci®city for aIIbb3 and decreased anity to avb3 (McLane et al.,
1996). A molecular model of eristostatin indicated that the tryptophan adjacent to the
RGD sequence caused the RGD loop of this disintegrin to be wider than that of echis-
tatin. Thus, it was proposed that the width and shape of the RGD loop may be an im-
portant structural feature as related to ®tting of the ligand into the binding pockets of
integrins aIIbb3 and avb3. Based on an examination of dierences in inhibition by the
disintegrins kistrin, elegantin and albolabrin, of platelet adhesion via integrin aIIbb3 to
dierent ligands, similar conclusions were drawn by Lu et al. (1994) concerning the im-
portance of amino acid residues surrounding the RGD sequence. All three of these dis-
integrins have dierent sequences around the RGD site but possess signi®cant overall
amino acid sequence similarity.
Separate structural features, in addition to the RGD loop, have also been suggested
to be important to the selectivity and anity of disintegrins. Thus Pfa et al. (1994)
suggested after comparing binding anities of six disintegrins of known sequences to

dierent integrins, that more remote regions of the structure than the RGD loop and
the alignment of the disul®de bridges may be important structurally. Additionally,
Wright et al. (1993) studied the importance of the carboxy-terminal region of echistatin
to GP IIb/IIIa binding. Using a synthetic decapeptide containing the carboxy-terminal
10 residues of echistatin, these investigators found that the peptide: (i) inhibited binding
of GP IIb/IIIa to immobilized echistatin but not to ®brinogen, (ii) activated GPIIb/IIIa
binding to ®bronectin, vitronectin and collagens types I and IV, and (iii) stimulated
125
I-®brinogen binding to human platelets. The authors concluded that this non-RGD
peptide interacts with GPIIb/IIIa leading to activation of the integrin and extension of
its binding speci®city to include proteins (collagen types I and IV) not usually regarded
as ligands. In contrast to these ®ndings, site-directed mutagenesis of the RGD site in
kistrin (Dennis et al., 1993) suggested that a favorable conformation of the RGD
sequence alone is largely responsible for the high anity binding of this disintegrin to
GP IIb/IIIa.
In vivo studies carried out by several groups have shown that disintegrins are safe at
clinically eective doses in animals and can be infused without detrimental eect on vas-
cular or endothelial function (Shebuski et al., 1990; Yasuda et al., 1991; Holahan et al.,
1991). Although disintegrins appear to have a half-life in the circulation in the order of
minutes (Cook et al., 1989), their biological activity span appears to be several hours.
This may be attributed to binding to platelet integrins which protect the disintegrins
and prolong their biological half-lives (Markland and Lucchesi, unpublished).
The eect of contortrostastin, the disintegrin puri®ed from southern copperhead
venom, on reocclusion after thrombolysis in a canine reoccluding carotid arterial throm-
bosis model was examined in a collaborative eort between the author and Lucchesi at
the University of Michigan (Markland et al., 1995). Contortrostatin blocks integrins of
the b1 and b3 subclasses and inhibits platelet aggregation by binding to the ®brinogen
receptor (integrin GPIIb/IIIa) (Trikha et al., 1994a; Trikha et al., 1994b). The canine
model is similar to the one described earlier (Markland et al., 1994). Following 30 min
of continuous occlusion of the carotid artery, acylated plasminogen-streptokinase acti-
vator complex (APSAC) was administered locally to induce thrombolysis. Immediately
after APSAC, saline (n = 6) or contortrostatin (0.15 mg/kg, n = 7) was administered
intravenously. Saline-treated animals reoccluded within 412 8 min. None of the contor-
trostatin-treated animals reoccluded. Heart rate and blood coagulation parameters were
not changed in the contortrostatin-treated animals. A transient decrease in platelet
count was observed. Ex vivo platelet aggregation in response to ADP or arachidonic
acid was inhibited by contortrostatin and bleeding time was increased. However, the
bleeding time returned almost to the baseline value upon completion of the experiment
and the platelet count returned to normal. Thrombus weight was signi®cantly reduced
in the contortrostatin group. In conclusion, contortrostatin eectively prevents reocclu-
sion in the canine carotid arterial thrombosis model. Similar results in coronary artery
models had been previously reported. Bitistatin, the disintegrin from the viper Bitis arie-
tans was shown to be a potent inhibitor of platelet aggregation in vitro, and in an in
vivo coronary thrombosis model it accelerated thrombolysis and prevented reocclusion
(Shebuski et al., 1990). Kistrin also demonstrated eective antithrombotic activity in
vivo in a canine coronary artery thrombosis model system (Yasuda et al., 1991), as did
echistatin (Holahan et al., 1991). Further, in a rat middle cerebral artery thrombosis
model, the disintegrin tri¯avin, a 7.5 kDa polypeptide puri®ed from Trimeresurus ¯avo-
viridis snake venom, dose-dependently prolonged the time to occlusive thrombus for-
mation and also reduced the size of ischemic cerebral damage 24 h after thrombus in-
itiation (Kaku et al., 1997). These actions were thought to be due to blocking of the
platelet ®brinogen receptor, integrin GP IIb/IIIa, by tri¯avin. It has also been reported
that echistatin in combination with iloprost (an analogue of prostacyclin) at doses that
are too low for any clinical side eects, completely inhibited platelet aggregation and
preserved platelet function during extracorporeal circulation (Bernabei et al., 1995).
These ®ndings supported earlier studies by Musial et al. (1990) indicating that a number
of dierent disintegrins protected platelets in stimulated extracorporeal circulation by
preventing their adhesion to surfaces and, therefore, protecting platelets from the shear
stress of ¯owing blood that fragments adherent platelets.
In addition to their potential use as antithrombotic agents, disintegrins have found a
number of other interesting applications. Thus, 123 I-bitistatin has been used to image
deep venous thrombosis (DVT) and pulmonary embolus (PE) (Knight et al., 1996). It
was found that 123 I-bitistatin had a higher uptake in DVT and a higher DVT to blood
ratio than any other disintegrin tested, than 125 I-®brinogen, and than 99 Tc-labeled plate-
lets. Similarly, the uptake and embolus to blood ratio in PE was superior for 123 I-bitis-
tatin to any other compound tested. These ®ndings suggest that labeled disintegrins
may be useful agents for rapid and eective imaging of both thrombi and emboli.
Echistatin has been studied in an egg±sperm fusion assay (Bronson et al., 1995).
Motile capacitated human spermatozoa and zona-free hamster eggs were coincubated in
the presence or absence of echistatin and the numbers of spermatozoa adhering to the
oolemma and penetrating the oocyte were measured. Echistatin at micromolar concen-
trations signi®cantly reduced the number of spermatozoa adhering to the oolemma in a
concentration dependent manner. However, echistatin did not inhibit the penetration of
oocytes by the sperm.
Echistatin has also been used in a rabbit retinal pigment epithelial (RPE) cell detach-
ment assay (Yang et al., 1996). Echistatin inhibited RPE cell attachment to the extra-
cellular matrix and also inhibited RPE cell-induced vitreous contraction in a dose-
dependent manner. In an in vivo model, rabbit eyes were injected intravitreously with
either rabbit RPE cells alone or with disintegrin to induce tractional retinal detachment.
Echistatin signi®cantly inhibited RPE cell-induced tractional retinal detachment com-
pared with the control group at two and four weeks after surgery.
The interaction of disintegrins with osteoclasts has also been examined. Echistatin at
nanomolar concentrations appears to be a potent inhibitor of bone resorption by iso-
lated mammalian osteoclasts in culture (Sato et al., 1990, 1994). Further, it was shown
that echistatin was an eective inhibitor of osteoclast-mediated bone resorption in vivo
(Fisher et al., 1993). The interaction of echistatin with osteoclasts is most likely
mediated via the vitronectin (avb3) receptor, the primary integrin on osteoclasts interact-
ing with RGD-containing peptides and with the bone extracellular matrix. Kistrin was
also shown to be an eective inhibitor of bone resorption in vitro and in vivo (King et
al., 1994). These studies suggest a potential use for disintegrins in the treatment of
osteoporosis or other disorders involving bone loss.
Disintegrins have also been found to be eective anti-adhesion and anti-invasion
agents for a number of dierent tumor systems. Thus, echistatin has been shown to
inhibit in a dose-dependent manner the in vitro attachment of B16-BL6 mouse mela-
noma cells to ®bronectin, vitronectin and laminin (Staiano et al., 1995). Soszka et al.
(1991) showed that albolabrin, a 7.5 kDa peptide isolated from Trimeresurus albolabris
venom, inhibited the adhesion of B16F10 mouse melanoma cells to extracellular matrix
(ECM) proteins. In an in vivo experimental metastasis system, albolabrin at 300±
600 nM inhibited lung colonization following tail vein injection of the mouse melanoma
cells and was at least 2000-times more active than a linear RGD tetrapeptide, RGDS.
In an extension of these studies it was reported that three additional disintegrins, eri-
stostatin, a short disintegrin puri®ed from the venom of Eristocophis macmahoni, echis-
tatin and barbourin, injected intravenously with B16F10 mouse melanoma cells into
C57BL/6 mice, inhibited experimental lung metastasis (Beviglia et al., 1995). The eect
of tri¯avin, the disintegrin from T. ¯avoviridis venom, on hepatoma cell adhesion to
ECM proteins has also been studied (Sheu et al., 1994a; Sheu et al., 1996). It appears
that tri¯avin binds via its RGD sequence to multiple integrin receptors (a5b1, a3b1 and
avb3) on the surface of the hepatoma cells and inhibits adhesion of the cells to ECM
components such as ®bronectin, ®brinogen, collagen type I and vitronectin in a dose-
dependent manner. It inhibits binding to laminin and collagen type IV poorly. Tri¯avin
also inhibits dose-dependently the adhesion of human cervical carcinoma (HeLa) cells
to ®bronectin, ®brinogen and vitronectin, but it exerted a limited eect on adhesion to
laminin and collagen (Sheu et al., 1994b). Rhodostomin, the disintegrin from
Calloselasma rhodostoma venom which contains 68 amino acids, inhibited adhesion of
human colon adenocarcinoma cells to ®bronectin and inhibited the binding of several
monoclonal anti-integrin antibodies to the surface of the colon cancer cells. These ®nd-
ings suggest that rhodostomin binds via its RGD sequence to integrins (possibly aIIbb3,
avb3 or a5b1) on the surface of the colon adenocarcinoma cells (Chiang et al., 1994a,
1996). Rhodostomin also inhibited adhesion of human osteosarcoma cells to ECM pro-
teins (Chiang et al., 1995a). Huang has reported that disintegrins, such as trigramin and
rhodostomin, inhibit platelet aggregation induced by dierent human cancers including
breast (Chiang et al., 1995b), colon (Chiang et al., 1994b) and osteosarcoma (Chiang et
al., 1995a). Trikha et al. (1994a,b) reported that contortrostatin is an eective inhibitor
of M24met, a human metastatic melanoma cell line, attachment to immobilized extra-
cellular matrix proteins including vitronectin, ®bronectin and collagen type 1, but inhi-
bits binding to laminin poorly. Scatchard analysis indicates that contortrostatin binds
to at least two binding sites on the M24met cells, one of which is most likely a5b1
(®bronectin receptor). In an in vivo experimental metastasis model in nude mice, contor-
trostatin inhibited lung colonization of tail vein injected M24met cells. In summary,
these studies reveal that disintegrins bind via their RGD sequences to integrins on the
surface of a number of dierent tumor cells and inhibit cell adhesion and tumor metas-
tasis.
Morris et al. (1995) in an elegant study examined the eect of the disintegrin eristos-
tatin on experimental metastasis to the liver of mouse B16F1 melanoma cells, after their
injection into a mesenteric vein. The authors examined at which step in the hematogen-
ous metastatic process the disintegrin exerted its action. In vivo videomicroscopy was
used to examine in real-time the microcirculation within intact livers of anesthetized
mice. Eristostatin dramatically reduced the mean number of liver metastases at 11 d
postinjection. Using control and disintegrin-treated ¯uorescently labeled cells, somewhat
surprisingly, it was found that the disintegrin did not inhibit either cell arrest, cell extra-
vasation, or migration after extravasation. The authors concluded, therefore, that the
disintegrin exerted its eect by regulating the number of individual cancer cells that
grew after extravasation. Since eristostatin did not eect growth of the B16F1 cells in
vitro, the disintegrin may be aecting the ability of the cells to be activated by other in
vivo factors.
Ongoing investigations in the authors laboratory have shown that contortrostatin
inhibits adhesion of highly metastatic human breast cancer cells, MDA-MB-435, to
both immobilized human ®bronectin and vitronectin in a dose dependent manner.

Further, immobilized contortrostatin supports binding of MDA-MB-435 cells in a dose
dependent manner, suggesting that the binding involves integrins on the tumor cell sur-
face. This binding is blocked by an RGD peptide and by EDTA. These ®ndings indicate
that CN binds to integrin receptors solely via an RGD-dependent mechanism. The in-
hibitory eect of contortrostatin on invasion of a synthetic basement membrane
(Matrigel) by the MDA-MB-435 cells was also demonstrated (Fry et al., 1996). A spon-
taneous (orthotopic) metastatic model was established by implantation of MDA-MB-
435 cells in the mammary fat pads of nude mice. Palpable tumors appeared by the 10th
day post-implantation. Daily injections of contortrostatin into the tumor masses of each
of the groups were carried out, starting at the 14th day post-implantation. By the 8th
week post-implantation, tumors were removed. The animals were allowed to survive for
2 more weeks without contortrostatin administration. The animals were then sacri®ced
and lung metastases were carefully examined. Local injection of contortrostatin was
found to substantially inhibit the growth rate of the tumor. Further, metastatic spread
in the control (saline treated) group was much more extensive than the high dose con-
tortrostatin group which showed >90% inhibition of metastasis (Zhou et al., 1996a).
The eect of contortrostatin on angiogenesis in the MDA-MB-435 tumor was then
examined. In the ®rst study, basic ®broblast growth factor (bFGF) and vascular endo-
thelial growth factor (VEGF), well established angiogenic factors, were employed.
Angiogenesis induced by these growth factors can be blocked by antagonists of integrins
avb3 and a5b1, respectively. Filter discs containing the individual growth factors were
put on the chorioallantoic membrane (CAM) of 10-day old chick embryos.
Contortrostatin was applied topically on the ®lter discs for three days. Contortrostatin
eectively blocked angiogenesis induced by both growth factors. Next, MDA-MB-435
tumor masses were inoculated on CAM of 10-day old chick embryos. Contortrostatin
was injected intravenously into the CAM on day 2 post-inoculation. Tumor induced
angiogenesis and the inhibitory eect of contortrostatin on angiogenesis can be easily
observed in the CAM after 3 days of incubation. In control samples (saline treated),
vessels are distributed in a convergent manner with the tumor mass in the center in con-
trol embryos. In the contortrostatin treated embryo, vessels were thinner and less dense
than control and the convergent distribution pattern disappeared completely; tumor
masses were smaller than that on control CAMs. These data suggest that contortrosta-
tin has an eective anti-angiogenic activity.
The eect of disintegrins on human umbilical vein endothelial cells (HUVEC) ad-
hesion to immobilized vitronectin and ®bronectin has been examined (Juliano et al.,
1996). It was observed that several disintegrins eectively inhibited HUVEC adhesion
to vitronectin and that these disintegrins also bound with high anity to immobilized
avb3 (vitronectin receptor). HUVEC also adhered to immobilized disintegrins. The
authors suggested that disintegrins interact with HUVEC selectively through vitronectin
receptors and this interaction results in the expression of a ligand induced binding site
epitope that may be involved in the regulation of binding anity and signaling path-
ways.
Disintegrin-like proteins are now known to be widely distributed throughout the ani-
mal kingdom and there appears to be a superfamily of cellular proteins containing a
disintegrin-like domain (Weskamp and Blobel, 1994). These proteins may function as
integrin ligands in important cell±cell and cell±matrix adhesive interactions (Blobel et
al., 1992).
Other inhibitors of platelet aggregation

Aside from the ®brinogen receptor antagonists (disintegrins), several dierent inhibi-
tors of platelet aggregation which interfere with collagen interaction with the platelet
receptor have been isolated from snake venoms recently. Included in this group is jarar-
hagin, a 53 kDa multidomain hemorrhagic protein from Bothrops jararaca venom,
which is composed of an amino-terminal metalloproteinase domain, a disintegrin-like
domain and a carboxy-terminal cysteine-rich domain (Paine et al., 1992). The enzyme
acts as a collagen receptor (integrin a2b1) antagonist and blocks collagen-induced plate-
let aggregation. Kamiguti et al. (1996a) proposed that jararhagin binds to the a2-subunit
via its disintegrin-like domain and the proteinase domain then cleaves the b1-subunit
with loss of the integrin structure necessary for binding of collagen. Interestingly, it was
also reported that jararhagin inhibits the collagen-induced release of 5-hydroxytrypta-
mine by platelets (Kamiguti et al., 1997). A 28 kDa protein (jararhagin-C) from B. jar-
araca venom has the identical sequence to the carboxy-terminal region of jararhagin
containing the disintegrin-like and cysteine-rich domains (Usami et al., 1994). This pro-
tein inhibits ADP- and collagen-induced platelet aggregation. Catrocollastatin is another
venom protein that interferes with collagen-mediated platelet adhesion and aggregation.
Catrocollastatin is a 50 kDa multidomain protein isolated from western diamondback
rattlesnake venom (Zhou et al., 1995) that is similar to jararhagin. However, catrocol-
lastatin binds to collagen directly and inhibits platelet aggregation rather than acting
via the collagen receptor as is the case with jararhagin (Zhou et al., 1996b). A 23.6 kDa
protein, catrocollastatin-C containing the carboxyl-terminal disintegrin-like and
cysteine-rich domains of catrocollastatin has been isolated from C. atrox venom recently
(Shimokawa et al., 1997). This protein, like the parent molecule, is an inhibitor of col-
lagen-stimulated platelet aggregation. A synthetic, cyclic peptide whose design was
based on the binding region of the disintegrin-like domain of catrocollastatin-C, has
potent platelet aggregation inhibitory activity suggesting that the same region in the
parent molecule is at least in part responsible for inhibition of platelet aggregation
(Shimokawa et al., 1997). Finally, crovidisin, a 53 kDa protein with selective inhibitory
activity of collagen-induced platelet aggregation has been puri®ed from venom of the
rattlesnake Crotalus viridis (Liu and Huang, 1997). This is a multidomain protein simi-
lar to catrocollastatin. It appears that crovidisin binds directly to collagen and blocks
the interaction between platelets and collagen. This results in the blockage of collagen-
induced adhesion, release reaction, thromboxane formation and aggregation, but with-
out aecting the GPIIb/IIIa-mediated platelet interaction with ®brinogen. All three of
the proteins with disintegrin-like domains mentioned here have an Ser±Glu±Cys±Asp
(SECD) sequence in place of the RGD motif found in the disintegrins (the complete
sequence of crovidisin is not available, but the sequence that has been determined is
identical to catrocollastatin). Presumably the SECD substitution in the disintegrin-like
domain of these proteins changes their binding speci®city from GPIIb/IIIa to some
other binding site that accounts for their distinct action on platelets.
An interesting protein that inhibits binding of von Willebrand factor to platelet glyco-
protein Ib has been puri®ed from venom of Echis carinatus (Peng et al., 1993). The pro-
tein, called echicetin, inhibits agglutination of ®xed platelets induced by several platelet
GPIb agonists including bovine vWF and snake venom alboaggregins. Unlike alboag-
gregins, echicetin bound to GPIb but did not induce agglutination. Echicetin inhibited
platelet aggregation and secretion induced by low concentrations of thrombin.
Apparently this eect was achieved by blocking the high anity thrombin binding site
on GPIb (Peng et al., 1995). Interestingly, in vivo echicetin signi®cantly prolonged the
bleeding time in mice, suggesting that it may inhibit vWF binding to GP Ib in vivo as
well as in vitro (Peng et al., 1993). Echicetin has a molecular weight of 26 kDa and
appears to be a heterodimer composed of two subunits of 16- and 14 kDa. The amino
acid sequences of the b-chain has been determined and contains 123 amino acids (Peng
et al., 1994). There is sequence similarity with botrocetin and the factor IXa/Xa-binding
protein. The sequence of the a-chain has also been determined (Polgar et al., 1997b).
Thus, echicetin is another member of the recently described snake venom subclass of
the C-type lectin protein family.
HEMOSTATICALLY ACTIVE VENOM PROTEINS: HEMORRHAGIC PROTEINASES
Hemorrhagic activity is caused by venom metalloproteinases that degrade proteins of

the basement membrane in the blood vessel wall. This action leads to the loss of capil-
lary integrity with resultant hemorrhage at the local site. Leakage of blood through the
endothelial barrier surrounding the vessel walls can be easily assayed by a skin test
involving intradermal injection of a hemorrhagic snake venom fraction under the depi-
lated skin on the back of an experimental animal. This causes readily observable hemor-
rhagic necrosis (Kondo et al., 1960). Readers are referred to the excellent reviews on
snake venom hemorrhagic metalloproteinases (Bjarnason and Fox, 1994; Takeya and
Iwanaga, 1994).
Hemorrhagic toxins have been isolated from the venom of many snake of the crotalid
and viper families. The hemorrhagic toxins that have been studied in detail are metallo-
proteinases possessing a single mole of zinc per molecule of proteinase. Removal of the
zinc by chelating agents such as EDTA completely eliminates hemorrhagic activity.
There appears to be microheterogeneity among the hemorrhagic proteinases with mul-
tiple forms found in several venoms (Bjarnason and Fox, 1994). This is most evident in
venom from the western diamondback rattlesnake (Crotalus atrox) in which no less
than seven hemorrhagic components have been isolated diering in molecular size and
amino acid composition. Bjarnason and Fox (1994) have divided the hemorrhagic
metalloproteinases into three classes based on the molecular size of the proteinase. The
class P-I proteinases are small toxins with relatively weak hemorrhagic activity and with
molecular masses of 20- to 30 kDa; the class P-II proteinases are medium size toxins
with molecular masses of 30- to 60 kDa; and the class P-III hemorrhagic proteinases are
large toxins with the most potent activity and having molecular masses of 60- to
100 kDa. As of 1994, 65 hemorrhagic toxins from 24 dierent species of snake had been
reported, almost half of them (31 out of 65) are members of class I and virtually all of
the 65 are metalloproteinases. The hemorrhagic toxins are capable of degrading proteins
of the basement membrane including ®bronectin, laminin, and type IV collagen. They
also degrade gelatin and ®brinogen. From studies on small molecular weight substrates,
it appears that the hemorrhagic proteinases prefer to cleave at the amino-terminal side
of a hydrophobic amino acid. Using western diamondback rattlesnake venom hemor-
rhagic proteinases to study substrate speci®city, it was found that there are signi®cant
dierences between the diverse hemorrhagic proteinases (Bjarnason and Fox, 1994).
From the amino acid sequences of the hemorrhagic proteinases that have been deter-
mined to date, it is obvious that the class P-I proteinases are structurally related. They
contain between 200±205 amino acids and they possess the consensus zinc-binding
sequence noted in the description of the ®brin(ogen)olytic enzymes. Further, the venom
metalloproteinases appear to be activated by a cysteine switch mechanism similar to
that used by the mammalian matrix-degrading metalloproteinases (Hite et al., 1992). As

described above, the thiol of a cysteine residue in a conserved sequence in the pro-
region of the venom proteinase binds to the active site zinc and inactivates the catalytic
domain. During activation the conserved cysteine is processed away from the catalytic
domain and catalytic activity is generated (Grams et al., 1993). Interestingly, although
the higher molecular weight hemorrhagic metalloproteinases of class P-II and -III are
multidomain proteins, their proteinase domain is structurally similar to that in the class
P-I proteinases. In addition, the higher molecular weight class P-II and -III hemorrhagic
metalloproteinases contain a disintegrin-like domain at the carboxyl terminus of the
metalloproteinase domain (Kini and Evans, 1992; Bjarnason and Fox, 1994). However,
unlike the disintegrins, most of the disintegrin-like domains of the hemorrhagic snake
venom metalloproteinases have an SECD or related sequence replacing the RGD
sequence that enables disintegrins to bind to integrin receptors on cells. This substi-
tution changes the 13-residue loop structure of the disintegrins (with the RGD sequence
at the tip of the loop) to a 14-residue loop stabilized by disul®de bonds in the disinte-
grin-like proteins. Nonetheless, there is evidence that mammalian disintegrin-like pro-
teins bind to integrins (Almeida et al., 1995). In the case of the snake venom
disintegrin-like proteins, Kamiguti et al. (1996a, 1997) showed that jararhagin binds to
the collagen receptor, integrin a2b1, via its disintegrin-like domain.
Processing of a class P-II hemorrhagic metalloproteinase from Crotalus atrox venom
with a molecular weight of 51.7 kDa, pro-atrolysin E, was recently described by Fox
(Shimokawa et al., 1996). The recombinant protein was expressed in the pro-form and
its processing observed in vitro. It appears that processing of pro-atrolysin E is per-
formed by another metalloproteinase in C. atrox venom, probably not by an autolytic
event. The mechanism involves a two-step procedure in which the pro-region is ®rst
released by proteolytic cleavage and then the disintegrin domain is processed proteolyti-
cally. The mature processed protein has molecular weights of 25- and 27 kDa as seen
on SDS±PAGE. Presumably these two forms represent the processed metalloproteinase
domain with (27 kDa) and without the 18-residue spacer region (25 kDa). The protein-
ase responsible for the processing has yet to be identi®ed and the detailed mechanism is
still being studied.
In addition to the disintegrin-like domain, the higher molecular weight hemorrhagic
toxins of class P-III also contain a cysteine-rich domain of unknown function, carboxyl-
terminal to the disintegrin-like domain (Bjarnason and Fox, 1994). Substrate speci®city
of the larger hemorrhagic proteinases may be modulated by their multidomain struc-
ture. Recent studies by Fox (Jia et al., 1997), suggest that the disintegrin-like domain of
the class P-III hemorrhagic metalloproteinases my contribute to the overall biological
potency of these enzymes. These authors expressed the disintegrin-like/cysteine-rich
domains (A/DC) of the Crotalus atrox hemorrhagic metalloproteinase atrolysin A, the
most potent hemorrhagic proteinase in this venom, in insect cells. Interestingly, atroly-
sin A is a potent inhibitor of platelet aggregation. The authors demonstrated that the
expressed protein (A/DC) also inhibited platelet aggregation induced by collagen and
ADP. The loop structure containing the ±Arg±Ser±Glu±Cys±Asp± (RSECD) sequence,
analogous to the disintegrin RGD sequence, was shown to be the region responsible for
the antiplatelet action. Further, the authors suggested that the cysteine residue in the
RSECD sequence was involved in a disul®de bond that was critical to biological ac-
tivity. They also presented evidence that the two acidic residues ¯anking this cysteine
were critical to biopotency. These ®ndings support the hypothesis that the extra
domains in the class P-III hemorrhagic metalloproteinases contribute to their much
greater hemorrhagic activity, by inhibiting platelet activation. In separate studies jarar-

hagin-C, a protein from Bothrops jararaca venom, which contains a disintegrin-like and
cysteine-rich domains has been isolated and characterized (Usami et al., 1994). This pro-
tein, which is a proteolytically processed form of the high molecular weight hemorrhagic
metalloproteinase jararhagin (Paine et al., 1992), is similar to the A/DC expressed pro-
tein from C. atrox venom. Jararhagin-C also inhibits platelet aggregation induced by
collagen and ADP. In separate studies, Kamiguti et al. (1996b) reported a systemic
hemorrhagic activity induced by jararhagin, the metalloproteinase-domain containing
parent protein of jararhagin-C. Jararhagin is not inhibited by plasma proteinase inhibi-
tors including a2-macroglobulin and is, therefore, able to degrade the adhesive plasma
protein von Willebrand factor as well as the platelet collagen receptor (integrin a2b1).
Degradation of these hemostatic components may lead to systemic bleeding and
suggests this could be another mechanism for inducing hemorrhage, in addition to local
bleeding, that the large multidomain venom hemorrhagic metalloproteinases employ. In
summary, these ®ndings suggest that the large molecular weight hemorrhagic toxins of
the P-III class exert their hemorrhagic activity by a dual mechanism that involves both
direct proteolysis of capillary basement membrane proteins, and inhibition of platelet
aggregation by the disintegrin-like domain and proteolysis of integrins and plasma ad-
hesive proteins by the metalloproteinase domain. Further, it has been reported that jar-
arhagin enhances plasma ®brinolysis in vitro by increasing tissue-type plasminogen
activator (t-PA) activity via dissociating the complex of t-PA and plasminogen activator
inhibitor type 1 (PAI-1) (Sugiki et al., 1995). Additionally, jararhagin decreases the ac-
tivity of a2-plasmin inhibitor, the main inhibitor of the ®brinolytic pathway. Thus, these
actions of jararhagin to stimulate the ®brinolytic pathway may also contribute to the
systemic hemorrhage observed in victims of B. jararaca envenomation.
HEMOSTATICALLY ACTIVE VENOM PROTEINS: VENOM INACTIVATORS OF PLASMA SERINE

PROTEINASE INHIBITORS
Plasma proteinase inhibitors represent about 10% of the total plasma proteins
(Kress, 1988) and provide an eective mechanism to inhibit proteolytic activity in
blood. The ®rst description of plasma proteinase inhibitor inactivating activity in snake
venoms appeared when Kress and Paroski (1978) reported that human serum lost anti-
tryptic and antichymotryptic activity following in vitro incubation with crotalid, viperid,
or colubrid venoms. The authors suggested that following envenomation by snakes from
these families, proteinase inactivating activity of the host blood was altered or severely
compromised. Elapid (cobras) and hydrophid (sea snake) venoms by contrast were with-
out eect on the plasma serine proteinase inhibitors (SERPINS) (Kress and Paroski,
1978). Subsequent reports by Kress (Kress, 1988; Kurecki et al., 1978) characterized the
responsible venom enzyme that inactivated a1-proteinase inhibitor (Kress, 1986) and
antithrombin III (Kress and Catanese, 1981). The enzyme was shown to be a metallo-
proteinase as it was inhibited by EDTA but not by PMSF. Further studies by Kress
(Kress et al., 1983; Catanese and Kress, 1985) and by others (Janssen et al., 1992;
Svoboda et al., 1995) have described venom inactivating enzymes for a number of the
plasma proteinase inhibitors. However, in many cases the responsible venom enzymes
have not been puri®ed and characterized. This area has been previously reviewed
(Kress, 1986; Kress, 1988). In this section only three well characterized venom SERPIN
inactivating enzymes will be described.
Crotalus adamanteus venom proteinase II (adamalysin II)

Proteinase II is a 23 kDa metalloproteinase puri®ed from eastern diamondback rattle-
snake venom (Kress, 1988; Kurecki et al., 1978). Proteinase II has recently been
renamed adamalysin II. As mentioned previously the three-dimensional structure of
adamalysin II has been determined (Gomis-Ruth et al., 1993, 1994) and this protein
serves as the structural prototype for other members of the snake venom metalloprotei-
nase subclass of the metzincins. Adamalysin degrades and inactivates SERPINS such as
a1-proteinase inhibitor and antithrombin III by cleaving peptide bond(s) at their car-
boxyl-termini. EDTA inhibited this action indicating the metalloproteinase nature of
the venom proteinase. Dierent peptide bonds in antithrombin III (which contains 432
amino acids) are cleaved by adamalysin in the presence or absence of heparin. In the
absence of heparin a slow inactivating cleavage occurs at Ala384±Ser385. In the presence
of heparin rapid inactivation occurs with cleavage ®rst at the amino-terminus (between
residues Glu37±Gln38), followed by inactivating cleavage at the Ala387±Val388 bond near
the carboxy-terminus (Kress and Catanese, 1981). Thus, the conformational change
induced in antithrombin III by interaction with heparin must signi®cantly alter the in-
teraction of the proteinase inhibitor not only with thrombin, but also with adamalysin.
During the inhibition of thrombin by antithrombin III, cleavage occurs at the Arg393 ±
Ser394 bond in the plasma proteinase inhibitor (Kress and Catanese, 1981; Kress, 1988).
In a1-proteinase inhibitor, which contains 394 amino acids, adamalysin cleaves a single
peptide bond Ala350±Met351 leading to inactivation of the proteinase inhibitor (Kress,
1986; Kress et al., 1979). The bond cleaved by trypsin in this plasma proteinase inhibi-
tor is Met358±Ser359. In the case of both of these SERPINS, cleavage by the venom pro-
teinase is in the loop region of the SERPIN thereby releasing the strained
conformation; this leads to inactivation. That the plasma proteinase inhibitor inactivat-
ing activity is not a general property of snake venom metalloproteinases has been
shown by studies with ®brolase, the 23 kDa ®brinolytic metalloproteinase from southern
copperhead venom (Guan et al., 1991). Fibrolase does not inactivate a1-proteinase in-
hibitor as shown by direct assay of inhibitory activity of the SERPIN after incubation
with the ®brinolytic venom enzyme (Johnson and Markland, unpublished).
Antithrombin III inactivating enzyme from Causas rhombeatus venom

Janssen et al. (1992) have isolated a protein from African night adder (Causas rhom-
beatus) venom that inactivates antithrombin III in a heparin-dependent reaction. The
protein, called CR-serpinase, is a glycoprotein of molecular weight 45.5 kDa. It cleaves
antithrombin III at the active site (Arg393±Ser394). Interestingly in contrast to other
venom proteinase inhibitor inactivating enzymes which appear to be metalloproteinases,
CR-serpinase is a serine proteinase. However, it is not inactivated by antithrombin III
or by incubation with human plasma.
C. atrox venom a-protease

Western diamondback rattlesnake venom contains a non-glycosylated metalloprotei-
nase of molecular weight 26.7 kDa that was isolated and characterized by Kruzel and
Kress (1985). The enzyme, called a-proteinase, enzymatically inactivates a1-proteinase
inhibitor and a1-antichymotrypsin by limited proteolysis similar to adamalysin II. a-
Proteinase cleaves peptide bonds in inter-a-trypsin inhibitor and C1 inhibitor (near its
amino-terminus), but does not lead to inactivation of either inhibitor. a2-Macroglobulin
forms a complex with a-proteinase that inhibits proteolytic activity of the venom inhibi-
tor (Kruzel and Kress, 1985).
CONCLUSIONS
In this review an attempt has been made to show the broad diversity of proteins and
peptides in snake venoms that interact with components of the human hemostatic sys-
tem. The author apologizes to the many investigators whose excellent work was not
cited in this review because of limitations of time and space. Snake venoms are rich
reservoirs of proteins which, when puri®ed, may be employed to aid our understanding
of basic biological mechanisms involved in hemostasis. It is interesting that in the evol-
ution of snake venom enzymes several repeating themes occur over and over. Thus,
many of the venom serine proteinases including thrombin-like enzymes, protein C acti-
vators, plasminogen activator and the factor V activator from Russell's viper venom
(and probably many more), with diverse activities seem to have been derived from some
common ancestral precursor and comprise a multigene family. In support of this,
Deshimara et al. (1996) studying the cDNA sequences of a number of serine proteinases
from Trimeresurus ¯avoviridis and T. gramineus venom glands found that the serine pro-
teinases from these sources have diversi®ed their amino acid sequences in an accelerat-
ing manner. These authors observed that the introns and untranslated regions are
highly conserved, but the protein-coding regions have diverged at an unusually high
rate. Thus, it would appear that crotalid serine proteinases have diverged by an acceler-
ated rate of evolution possibly to gain functional diversity.
The multigene family concept appears to hold true for the venom metalloproteinases
as well. Even the high molecular weight, multidomain metalloproteinases bear striking
resemblance in the metalloproteinase domain to the low molecular weight metalloprotei-
nases with hemorrhagic, ®brinolytic or other activities. Moura-da-Silva et al. (1996)
recently compared the structures of the metalloproteinase±disintegrin±cysteine-rich
(MDC) gene family from snake venom and mammalian sources with that of the matrix-
degrading metalloproteinases (MMPs). Based on their observations a common precur-
sor is suggested for the snake venom and mammalian MDCs. It is possible that gene
duplication of the assembled domain structure followed by divergent evolution may
account for the diverse activities of members of the MDC family. Further, the authors
analysis suggested that due to the structural resemblance of the zinc-binding motifs of
the venom MDCs and the MMPs, their relation may best be explained by a common
ancestry with conservation of the proteolytic motifs during divergent evolution. This ex-
planation is favored rather than postulating a mechanism of convergent evolution to
explain the similarity in the zinc-binding motifs but lack of homology throughout the
remainder of the protein structure. These examples of the venom serine proteinases and
the venom and mammalian metalloproteinases represent striking occurrences of conser-
vation of structure in biological evolution.
Some of the venom proteins hold clinical promise or are already being used clinically.
A recent example is provided by the disintegrins. Disintegrins are an interesting group
of peptides that act as ®brinogen receptor (integrin GPIIb/IIIa) antagonists. Since this
integrin is believed to serve as the ®nal common pathway leading to the formation of
platelet±platelet bridges and platelet aggregation, blockage of this integrin inhibits plate-
let aggregation regardless of the stimulating agent. Based on clinical trials it appears
that platelet GPIIb/IIIa blockade is an eective therapy for the thrombotic events and
restenosis frequently accompanying cardiovascular and cerebrovascular disease (Tcheng,

1996). Therefore, disinterins have clinical potential for the development of eective
antithrombotic therapy. Disintegrins have already served as structural prototypes for
the design and development of peptide and non-peptide mimetics for antithrombotic
therapy. Amongst these small molecule antithrombotic agents which are based on disin-
tegrins structure are integrilin which has been used for enhancement of reperfusion in
patients undergoing thrombolytic therapy for acute myocardial infarction (Ohman et
al., 1997), and tiro®ban which serves as an adjunctive therapy for high risk patients
undergoing coronary angioplasty (Kereiakes et al., 1996). Peptide and non-peptide
mimetics of the RGD motif have been shown to be inexpensive and eective agents
with clinical potential for antithrombotic therapy.
Our appreciation of the roles of many of the venom components in the pathology of
envenomation is not yet fully understood. Hopefully, in the coming years as we learn
more about these interesting molecules, both at the structural and gene levels, there will
be a greater understanding of their complicated mechanisms of action. Further, it is
hoped that we will develop eective applications for a number of these agents in the
treatment of human disorders including cardiovascular disease and other illnesses such
as cancer and AIDS.
Acknowledgements ÐThe author would like to acknowledge the excellent collaborative interactions with Dr
Benedict Lucchesi, Department of Pharmacology, University of Michigan Medical School. Also, thanks go to
Dr Patricio Riquelme and Dr Pablo Valenzuela, Chiron Corporation, Emeryville, CA, for providing r-®bro-
lase. Partial support for the ®brolase work was provided by the Heart, Lung and Blood Institute, NIH, Grant
HL-31389 and for the contortrostatin work partial support was provided by the American Heart Association-
Greater Los Angeles Aliate, Grant 938 GI. The author would like to thank Dr Steve Swenson for help in
assembling Figs 2 and 3.
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Snake Venoms and The Hemostatic System

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Toxicon Vol. 36, No. 12, pp.

SNAKE VENOMS AND THE HEMOSTATIC SYSTEM

(Received 9 December 1997; accepted 16 January 1998)

tial, a large number of disintegrins have been isolated and characterized.

The venom producing apparatus of snakes is composed of modi®ed exocrine glands

Table 1. Snake venom proteins active on the hemostatic system

interesting observations on the coagulant and anticoagulant eects of rattlesnake

HEMOSTATICALLY ACTIVE VENOM PROTEINS: COAGULANT ACTIVITIES

Va in the presence of CaCl2. These enzymes are found exclusively in venoms of

Fibrinogen clotting enzymes

Venombin A group. The class of enzymes releasing ®brinopeptide A is represented by

thrombosis model, probably by depleting ®brinogen and preventing propagation of

Venombin B group. The third class of thrombin-like enzymes releases preferentially

HEMOSTATICALLY ACTIVE VENOM PROTEINS: ANTICOAGULANTS

Stability stable stable stable stable in Ca++ unstable to heat (608C)

Factor IX/X inhibitor

HEMOSTATICALLY ACTIVE VENOM PROTEINS: FIBRINOLYTIC PROTEINASES

Direct-acting ®brinolytic enzymes have been isolated from venom of a number of

determine whether they exhibit ®brin(ogen)olytic, plasminogen activator, thrombin-like,

Table 3. Properties of snake venom ®brinolytic enzymes

habutoxin, the thrombin-like enzyme from Trimeresurus ¯avoviridis venom, stimulated

Table 4. Properties of snake venom plasminogen activator

or recombinant mutants is intriguing, but yet to be explored. Properties of TSV-PA are

HEMOSTATICALLY ACTIVE VENOM PROTEINS: PLATELET INTERACTIVE COMPONENTS

Activators of platelet aggregation

Table 5. Snake venom proteins active on platelets

Non-enzymatic components with direct action on platelets. A number of other venom

Platelet aggregation inhibitors

5'-nucleotidases. A third group of inhibitors of platelet aggregation includes the 5'-

Table 6. Potential clinical uses of snake venom disintegrins

Prolonged time to occlusion and reduced

All disintegrins contain an Arg±Gly±Asp (RGD) sequence in the carboxyl-terminal

linked dimeric disintegrin, contortrostatin, had a dierential eect on platelet signal

suggested after comparing binding anities of six disintegrins of known sequences to

both immobilized human ®bronectin and vitronectin in a dose dependent manner.

Other inhibitors of platelet aggregation

HEMOSTATICALLY ACTIVE VENOM PROTEINS: HEMORRHAGIC PROTEINASES

Hemorrhagic activity is caused by venom metalloproteinases that degrade proteins of

that used by the mammalian matrix-degrading metalloproteinases (Hite et al., 1992). As

greater hemorrhagic activity, by inhibiting platelet activation. In separate studies jarar-

HEMOSTATICALLY ACTIVE VENOM PROTEINS: VENOM INACTIVATORS OF PLASMA SERINE

Crotalus adamanteus venom proteinase II (adamalysin II)

Antithrombin III inactivating enzyme from Causas rhombeatus venom

C. atrox venom a-protease

restenosis frequently accompanying cardiovascular and cerebrovascular disease (Tcheng,

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interesting observations on the coagulant and anticoagulant eects of rattlesnake

linked dimeric disintegrin, contortrostatin, had a dierential eect on platelet signal

suggested after comparing binding anities of six disintegrins of known sequences to