Pharmacy

Biotechnology
Part IV
Recombinant blood products. Cytokines.
 Topic 9
Haemostasis.
Anticoagulants and thrombolytic agent.
Haemostasis
Blood plays various vital roles within the body and it is not surprising that a number of processes have evolved
capable of effectively maintaining haemostasis.
Haemostasis - the rapid arrest of blood loss upon vascular damage, in order to
maintain a relatively constant blood volume
In humans, three main mechanisms underline the haemostatic process:
 The congregation and clumping of blood platelets at the site of vascular injury, thus effectively plugging
the site of blood leakage.
 Localized constriction of the blood vessel, which minimizes further blood flow through the area.
 Induction of the blood coagulation cascade. This culminates in the conversion of a soluble serum protein,
fibrinogen, into insoluble fibrin. Fibrin monomers then aggregate at the site of damage, thus forming a
clot (thrombus).

These mechanisms are effective in dealing with small vessel injuries (e.g. capillaries and arterioles), although
they are ineffective when the damage relates to large veins/arteries.
 Table The coagulation factors that promote the blood clotting process. Note that the factor

The coagulation pathway

originally designated as VI was later shown to be factor Va

Factor Common Pathway in

number which it functions Function
name
Fibrinogen
The process of blood coagulation is dependent I Both Forms structural basis of clot after its conversion
to fi brin
upon a large number of blood clotting factors, which II Prothrombin Both Precursor of thrombin, which activates factors I,
V, VII, VIII and XIII
act in a sequential manner. At least 12 distinct III Tissue factor Extrinsic Accessory tissue protein which initiates extrinsic
factors participate in the coagulation cascade, (thromboplastin) pathway
IV Calcium ions Both Required for activation of factor XIII and stabilizes
along with several macromolecular cofactors. The some factors
Proaccelerin
clotting factors are all designated by Roman numerals V Both Accessory protein, enhances rate of activation of X
VII Proconvertin Extrinsic Precursor of convertin (VIIa) which activates X
(see Table) and, with the exception of factor IV, all (extrinsic system)
Antihaemophilic
are proteins. Most factors are proteolytic VIII
factor
Intrinsic Accessory protein, enhances activation of X
(intrinsic system)
zymogens, which become sequentially activated. IX Christmas Intrinsic Activated IX directly activates X (intrinsic system)
factor
An activated factor is indicated by inclusion of a X Stuart factor Both Activated form (Xa) converts prothrombin to
thrombin
subscript ”a” (e.g. factor XIIa is activated factor XII). XI Plasma Intrinsic Activated form (XIa) serves to activate IX
thromboplasti
n antecedent
XII Hageman Intrinsic Activated by surface contact or the kallikrenin
factor system. XIIa helps initiate intrinsic system
XIII Fibrin-stabilizing Both Activated form cross-links fi brin, forming a hard
factor clot
The coagulation pathway
Although the final steps of the
blood clotting cascade are identical,
the initial steps can occur via two
distinct pathways: extrinsic and
intrinsic.

Both pathways are initiated when specific clotting

proteins make contact with specific surface molecules
exposed only upon damage to a blood vessel.

Clotting occurs much more rapidly when

initiated via the extrinsic pathway.
 Extrinsic pathway
Two coagulation factors function uniquely in the extrinsic pathway:
factor III (tissue factor) and factor VII.
Factor VII contains a number of
γ-carboxyglutamate residues (as do factors II, IX and X),
Tissue factor is an integral which play an essential role in facilitating their

membrane protein present in a wide binding of Ca2 ions. The initial events initiating the
extrinsic pathway entail the interaction of factor VII
variety of tissue types (particularly lung with Ca2 and Tissue factor. In this associated form,
and brain). This protein is exposed to factor VII becomes proteolytically active. It
blood constituents only upon rupture displays both binding affinity for, and catalytic activity
against, factor X. It thus activates factor X by
of a blood vessel, and it initiates the proteolytic processing, and factor Xa, which
extrinsic coagulation cascade at the site initiates the terminal stages of clot formation, remains
of damage. attached to the tissue factor–Ca2 complex at the site of
damage. This ensures that clot formation only occurs
at the point where it is needed

Figure. Schematic diagram of the initial steps of the extrinsic

blood coagulation pathway.
Intrinsic pathway
The initial steps of the intrinsic pathway are somewhat more complicated. This system requires the presence of clotting
factors VIII, IX, XI and XII, all of which, except for factor VIII, are endo-acting proteases.

As in the case of the

extrinsic pathway, the
intrinsic pathway is triggered
upon of the clotting factors
to proteins present on the
surface of body tissue The intrinsic pathway
exposed by vascular injury. appears to be initiated when factor
XII is activated by contact with
These protein binding/activation sites probably surface proteins exposed at the site
include collagen. of damage. High molecular mass
kininogen also appears to form part
of this initial activating complex.
Additional protein constituents of the intrinsic cascade include
prekallikrein, an 88 kDa protein zymogen of the protease kallikrein, and
high molecular mass kininogen, a 150 kDa plasma glycoprotein that serves
as an accessory factor.
Intrinsic pathway
Factor XIIa can proteolytically cleave and, hence, activate two substrates:
Factor XIa, in turn,
activates factor IX. Factor
IXa then promotes the  prekallikrein, yielding
activation of factor X, but kallikrein (which, in turn, can
only when it (i.e. IXa) is directly activate more XII to
XIIa).
associated with factor VIIIa.
 factor XI, forming XIa.

Factor VIIIa is formed by the direct action

of thrombin on factor VIII. The thrombin
will be present at this stage because of prior
activation of the intrinsic pathway.
Figure. The steps unique to the intrinsic coagulation pathway.
Factor XIIa can also convert prekallikrein to kallikrein by proteolysis
 Terminal steps of coagulation pathway
Both intrinsic and extrinsic pathways generate activated factor X.
Prothrombin (factor II) is a 582 amino acid, 72.5 kDa
This protease, in turn, catalyses the glycoprotein, which represents the circulating zymogen of
thrombin (IIa). It contains up to six γ-carboxyglutamate
proteolytic conversion of
residues towards its N-terminal end, via which it binds
prothrombin (factor II) into several Ca2 ions. Binding of Ca2 facilitates prothrombin
thrombin (IIa). Thrombin, in turn, binding to factor Xa at the site of vascular injury.
catalyses the proteolytic conversion of
fibrinogen (I) into fibrin (Ia).
Individual fibrin molecules aggregate Fibrinogen (factor I) is a large (340 kDa)
glycoprotein consisting of two identical tri-
to form a soft clot. Factor XIIIa polypeptide units, α, β and γ. Its overall
catalyses the formation of covalent structural composition may thus be
crosslinks between individual fibrin represented as (α β γ)2.
The N-terminal regions of the α and β
molecules, forming a hard clot fibrinogen chains are rich in charged amino
acids, which, via charge repulsion, play an
important role in preventing aggregation of
individual fibrinogen molecules.
 Terminal steps of coagulation pathway Figure 1.

The factor Xa complex then proteolytically

cleaves prothrombin at two sites (Arg274–Thr275 and Arg323–Ble324),
yielding active thrombin and an inactive polypeptide fragment, as depicted
in Figure 1.
Thrombin, which catalyses the proteolytic
activation of fibrinogen, hydrolyses
fibrinogens N-terminal peptides.

This renders individual fibrin molecules more conducive to aggregation, therefore promoting soft clot formation. The soft
clot is stabilized by the subsequent introduction of covalent cross-linkages between individual participating fibrin
molecules. This reaction is catalysed by factor XIIIa.

Figure 2.
Figure 2. Terminal steps of a blood clot (haemostatic
plug): cross-linked fi brin molecules bind together
platelets and red blood cells congregated at the site of
damage, thus preventing loss of any more blood
 Clotting disorders
Genetic defects characterized by Up to 90% of these, however, relate to a
(a) lack of expression or defi ciency in factor VIII, and much of
(b) an altered amino acid sequence of any clotting factor the remainder is due to a defi ciency in
factor IX.
can have serious clinical consequences. Product (tradename) Company Indication
In order to promote effective clotting, both
Advate (rhFactor VIII) Baxter Haemophilia A
intrinsic and extrinsic coagulation pathways
Bioclate (rhFactor VIII) Aventis Haemophilia A
must be functional, and the inhibition of even
Benefi x (rhFactor IX) Genetics Institute Haemophilia B
one of these pathways will result in severely
Kogenate (rhFactor VIII) Bayer Haemophilia A
retarded coagulation ability.
Helixate NexGen (rhFactor VIII) Bayer Haemophilia A
Such clotting disorders are generally treated by ongoing NovoSeven (rhFactor VIIa) Novo-Nordisk Some forms of
haemophilia
administration of whole blood or, more usually,
concentrates of the relevant coagulation factor purified from Recombinate (rhFactor VIII) BaxterHealthcare/ Haemophilia A
whole blood. This entails significant risk of accidental Genetics Institute

transmission of blood-borne disease, particularly hepatitis and
AIDS. In turn, this has hastened the development of blood
coagulation factors produced by genetic engineering, several of
which are now approved for general medical use.
ReFacto (B-domain deleted rhFactor VIII) Genetics Institute Haemophilia A
Table. Recombinant blood coagulation factors that have been approved
for the management of coagulation disorders
 Clotting
disorders
Coagulation disorders are disorders which affect the
coagulation cascade and can either cause excessive or
inadequate clotting. Coagulation disorders usually involve
a deficiency in at least one clotting factor, and the most
common disorders include von Willebrand disease,
hemophilia, and vitamin k deficiency.

Von Willebrand disease is the most common bleeding disorder The

von Willebrand factor involved in primary hemostasis where it helps
platelets stick together also plays a role in secondary hemostasis by
helping stabilize factor VIII.
There are three types of hemophilia: hemophilia A is a deficiency
in factor VIII, hemophilia B is a deficiency in factor IX, and hemophilia
C is a deficiency in factor XI. All types of hemophilia affect the intrinsic
pathway.
Vitamin K is a cofactor required to make factors II, VII, IX, and X
functional. Therefore, vitamin K deficiency affects all pathways.
Factor VIII
 Factor VIII and
haemophilia
Haemophilia A (classical haemophilia, often simply termed haemophilia) is an X-linked recessive disorder
caused by a defi ciency of factor VIII. Von Willebrand disease is a related disorder, also caused by a defect in
the factor VIII complex, as discussed below.

Intact factor VIII, as usually purified from

the blood, consists of two distinct gene
products: factor VIII and (multiple copies
of) von Willebrand’s factor (see Figure).

Figure. (a) Synthesis of factor VIII complex as occurs

in healthy individuals. (b) In the case of persons
suffering from haemophilia A, synthesis of factor VIII:C
is blocked, thus preventing constitution of an active
factor VIII complex in plasma.

(c) Persons suffering from von Willebrand’s disease fail to synthesize vWF. Although they can synthesize VIII:C, this is rapidly
degraded upon entering the blood due to lack of its vWF stabilizing factor
Structure
• Intact factor VIII displays a molecular mass ranging from 1 to 2 MDa, of which up to 15 per cent is
carbohydrate.
• The factor VIII polypeptide portion of the factor VIII complex is coded for by an unusually long gene (289 kb).
Transcription and processing of the mRNA generates a shorter, mature, mRNA that codes for a 300 kDa protein.
• Upon its synthesis, this polypeptide precursor is subsequently proteolytically processed, with removal of a signifi
cant portion of its mid region. This yields two fragments: an amino terminal 90 kDa polypeptide and an 80 kDa
carboxyl terminal polypeptide. These associate non-covalently (a process requiring Ca2 ions) to produce mature
factor VIII (sometimes called factor VIII:C).
• This mature factor VIII is then released into the plasma where it associates with multiple copies of vWF forming
the biologically active factor VIII complex. vWF stabilizes factor VIII in plasma (particularly against proteolytic
degradation).
• It also can associate with platelets at the site of vascular damage and, hence, presumably plays a role in docking
the factor VIII complex in an appropriate position where it can participate in the coagulation cascade.
 Recombinant Factor VIII
Recombinant factor VIII (rFVIII) is available from three sources: Baxter Hyland, Bayer Corporation, and
Wyeth.
rFVIII products may be divided into three classes based on the use of human or mammalian-derived raw materials (Table). rFVIII from
Baxter Hyland (Advate, Recombinate) and Wyeth (Refacto) is produced using transfected Chinese hamster ovary (CHO) cells, whereas that
from Bayer (Kogenate) is produced using trans- fected baby hamster kidney cells.
A major difference between rFVIII from Bayer and
Baxter Hyland is the presence of a Gal 1->3Gal
carbohydrate moiety in the Baxter product. The
recombinant product from Baxter and Bayer consists of
full-length factor VIII which, like plasma-derived factor
VIII, consists of a dimer of the 80-kDa light chain and a
heterogeneous heavy chain of 90 to 210 kDa. The
Wyeth product (Moroctocog alfa, ReFacto) is a deletion
mutant in which the heavy chain lacks nearly the entire
B- domain, which is not needed for clotting activity.
After proteolytic clea- vage by thrombin, the activated
B-domain-depleted molecule is essentially identical to
the activated full- length native rFVIII.
 Pharmaceutical Considerations
Recombinant factor VIII (rFVIII) is available from three sources: Baxter Hyland, Bayer Corporation, and
Wyeth.

• Advate recombinant antihemophilic factor-plasma/ albumin free method is formulated as a sterile, non-pyrogenic,
lyophilized cake for intravenous injection and is provided in single-dose vials, containing nominally 250, 500,
1000, or 1500 international units (IU).
• Biological potency is deter- mined using an in vitro assay that employs a factor VIII concentrate standard that is
referenced to the World Health Organization (WHO) International Standard for factor VIII:C concentrates.
The specific activity is 4000 to 10,000 IU per milligram of protein.
• The final product contains no preservative nor added human or animal components in the formulation.
• Recombinant antihemophilic factor is administered only by intravenous infusion following
reconstitution with 5mL sterile water for injection. The product contains mannitol, trehalose, sodium, histidine,
Tris, calcium, polysorbate 80, and glutathione. Plastic syringes must be used since the protein can adhere to
glass syringes.
• rFVIII is supplied as sterile, single-dose vials containing 250 to 1000IU of factor VIII. The preparation is
lyophilized and stabilized with human albumin or polysorbate 80.
Storage and use

● The products contain no preservatives and should be stored at 2 0C to 8 0C. The

lyophilized powder may be stored at room temperature (up to 25 0C) for up to 3 months
without loss of biological activity. Freezing should be avoided.

● Factor VIII should be reconstituted with the diluent provided. The reconstituted
product must be administered intravenously by direct syringe injection or drip infusion
within 3 hours of reconstitution.
 Clinical usage
The increase in factor VIII
concentration is dose proportional
and the disposition is similar following
single and chronic dosing. The mean
biological half-life was similar for
recombinant and plasma-derived factor
VIII. Table. Clinical pharmacokinetic profile of rFVIII or rFIX following
intravenous administration of 50 IU/kg.
The incidence of inhibitors to
recombinant and plasma-derived
Dosage of rFVIII (in IU) must be individualized to the needs of the
factor VIII in previously untreated
patient, the severity of the deficiency and of the hemorrhage, the presence
patients is similar, about 20% and
of inhibitors, and to the desired increase in factor VIII activity (in IU/dL, or
10% to 15%, respectively
percentage of normal).

The in vivo percent elevation in factor VIII level may be estimated as

follows:
Dosage required (IU) 1⁄4 Body wt (kg) - Desired increase in factor VIII (%) 0.5 IU/kg %
Safety concerns

● Trace amounts of mouse or hamster protein may be present in rFVIII as

contaminants from the expression system. Therefore, caution should be exercised
when administering rFVIII to individuals with known hypersensitivity to plasma-
derived antihemophilic factor, or with hypersensitivity to biological preparations
with trace amounts of murine or hamster proteins.

● The risk of developing antibodies to rFVIII correlates with severity of disease

and the intensity of exposure to factor VIII.
Factor VIIa
Factor VIIa and
haemophilia
● Development of recombinant factor VIIa was motivated by the fact that a small
fraction of patients with hemophilia (15–20% of patients with hemophilia A and 2–
5% of patients with hemophilia B) develop antibodies (inhibitors) to factor VIII
or factor IX.

● High titers of inhibitors make it impossible to give sufficient coagulation factor to

overcome the inhibitor, and therapy is ineffective or is associated with unaccep-
table side effects. Factor VIIa can be valuable in these instances since in the
absence of Tissue factor, factor VII has very low proteolytic activity.
Structure
● Factor VII is a vitamin K-dependent glycosylated
serine protease proenzyme that is synthesized in the liver. It
has 406 amino acids and a molecular weight
is ∼50 kDa.
● The protein is not functionally active unless it is
γ-carboxylated. There are two sites of N-linked
glycosylation on factor VII. Factor VII is synthesized as a
proenzyme that becomes activated and cleaved upon
hydrolysis of Arg-152 and Ile-153.
 Recombinant Factor VIIa
● Recombinant factor VII is expressed in baby hamster kidney cells as a single-
chain form and is spontaneously activated to factor VIIa during purification.
Characterization of the protein indicates that it is very similar to plasma-derived
factor VIIa with regard to amino acid sequence, carbohydrate composition, and γ-
carboxylation.

● Based on the results of several pharmacokinetic/pharmacodynamics

studies, it is necessary to maintain plasma levels above 5 to 6U/mL for adequate hemostasis.
This may be done by administering sufficiently high initial concentrations to ensure this, or
by maintaining a strict 2-hour dosing interval following doses of 70 to 90 μg/kg
Pharmaceutical considerations
● NovoSeven is supplied as a white lyophilized powder in single-use glass vials formulated
with sodium chloride, calcium chloride dihydrate, glycylglycine, polysorbate 80, and mannitol.
The pH is adjusted to 5.3 to 6.3. The product does not contain any stabilizing protein.
● Before reconstitution, NovoSeven should be stored refrigerated (2–8􏳐 0C) avoiding
exposure to direct sunlight.
● NovoSeven should be reconstituted with sterile water for injection, USP (2.2mL for the 1.2-
mg vial and 8.5mL for the 4.8-mg vial).
● After reconstitution with the appropriate volume of diluent, each vial contains approximately
0.6 mg/mL.
● Following recon- stitution, NovoSeven may be stored refrigerated or at room temperature for
up to 3 hours.
● NovoSeven is intended for intravenous bolus injection and should not be mixed with infusion
solutions.
Clinical usage
● Recombinant factor VIIa is indicated for the treatment of bleeding episodes or the
prevention of bleeding in surgical intervention or invasive procedures in patients with
hemophilia A or B with inhibitors to factor VIII or factor IX. It is also indicated for treatment
of bleeding episodes or the prevention of bleeding in surgical intervention or invasive
procedures in patients with congenital factor VII deficiency.
● The recommended dose of recombinant factor VIIa for patients with hemophilia A or B
with inhibitors is 90 μg/kg every 2 hours by bolus infusion until hemostasis is achieved or
until treatment is judged to be inadequate. The minimum effective dose has not been
established. Doses between 35 and 120μg/kg have been used successfully in clinical trials.
Both the dose and administration interval may
be adjusted based on the severity and degree of hemostasis.
Safety
● Based on the clinical safety database of 1939 treatment episodes in 298
patients with hemophilia A or B with inhibitors, adverse events reported at rates of
more than 2% of patients treated included fever, hemorrhage, decreased fibrinogen,
hemarthrosis, and hypertension.
● Recombinant factor VIIa should not be administered to patients with
known hypersensitivity to recombinant factor VIIa or any of the components of
recombinant factor VIIa.
● Recombinant factor VIIa is contraindicated in patients with known
hypersensitivity to mouse, hamster, or bovine proteins.
Anticoagulant
s
Anticoagulants are substances that can prevent blood from clotting and,
hence, are of therapeutic use in cases where a high risk of coagulation is diagnosed.

Although blood clot formation is essential to maintaining haemostasis,

inappropriate clotting can give rise to serious, sometimes fatal medical conditions.

● Thrombus formation in a coronary artery (the arteries that supply the heart
muscle itself with oxygen and nutrients) is termed coronary thrombosis. This results
in a heart attack, characterized by the death (infarction) of oxygen-deprived heart
muscle; hence the term myocardial infarction.
● The development of a thrombus in a vessel supplying blood to the brain can
result in development of a stroke.
Major anticoagulants
Anticoagulants often administered to Table. Anticoagulants that are used therapeutically
patients with coronary heart disease and to or display therapeutic potential
patients who have experienced a heart attack Anticoagulant Structure Source Molecular mass (Da)

or stroke (in an effort to prevent recurrent

episodes). The major anticoagulants used for
therapeutic purposes are listed in Table. Heparin Glycosaminoglycan Beef lung, 3 000–40 000
pig gastric mucosa

Dicoumarol Coumarin-based Chemical 336.3

manufacture

Dicoumarol and related molecules Warfarin Coumarin-based Chemical 308.4

manufacture
are generally used over prolonged periods,
whereas heparin is used over shorter Hirudin Polypeptide Leech saliva, genetic 7 000
engineering
periods. Hirudin has recently been approved
for general medical use, while ancrod Ancrod Polypeptide Snake venom, genetic 35 000
engineering
remains under clinical investigation.
Protein C Glycoprotein Human plasma 62 000
Heparin
Heparin is a carbohydrate-based (glycosaminoglycan) anticoagulant
associated with many tissues, but mainly found stored intracellularly as granules in
mast cells that line the endothelium of blood vessels.

Upon release into the bloodstream, heparin

binds to and thereby activates an additional plasma
protein, namely antithrombin. The heparin–
antithrombin complex then binds a number of
activated clotting factors (including IIa, IXa, Xa, XIa
and XIIa), thereby inactivating them. The heparin now
disassociates from the complex and combines with
another antithrombin molecule, thereby initiating
another turn of this inhibitory cycle.
Heparin
● Heparin was originally extracted from liver (hence its name), but
commercial preparations are now obtained by extraction from beef lung or
porcine gastric mucosa.
● Although the product has proven to be an effective (and relatively
inexpensive) anticoagulant, it does suffer from a number of clinical
disadvantages, including the need for a cofactor (antithrombin III) and
poorly predictable dose responses.
● Despite such disadvantages, however, heparin still enjoys widespread
clinical use.
 Vitamin K antimetabolites
The vitamin K antimetabolites dicoumarol and warfarin are related coumarin-
based anticoagulants which, unlike heparin, may be administered orally. These
compounds induce their anticoagulant effect by preventing the vitamin K-dependent
γ-carboxylation of certain blood factors, specifically factors II, VII, IX and X.

Upon initial hepatic synthesis of these coagulation

factors, a specific carboxylase catalyses the γ-carboxylation
of several of their glutamate residues (for example, 10 of the
first 33 residues present in prothrombin are γ-
carboxyglutamate). This post-translational modification is
required in order to allow these factors to bind Ca2 ions,
which is a prerequisite to their effective functioning.
Vitamin K antimetabolites
Vitamin K is an essential cofactor for
the carboxylase enzyme, and its replacement
with the antimetabolite dicoumarol renders this
enzyme inactive. As a consequence, defective
blood factors are produced that hinder effective
functioning of the coagulation cascade. The only
major side effect of these oral anticoagulants is
prolonged bleeding; thus, the dosage levels are
chosen with care. Dicoumarol was first isolated
from spoiled sweet clover hay, as the agent that
promoted haemorrhage disease in cattle.

sweet clover
(Melilotus officinalis)
Hirudin
Hirudin is a leech-derived anticoagulant that functions by directly inhibiting thrombin.

The presence of an anticoagulant in the saliva of the

leech, Hirudo medicinalis, was first described in 1884.
However, it was not until 1957 that the major anticoagulant
activity present was purified and named hirudin. Hirudin is a
short (65 amino acid) polypeptide, of molecular mass 7000
Da. The tyrosine residue at position 63 is unusual in that it
contains a sulfate group. The molecule appears to have two
domains. The globular N-terminal domain
is stabilized by three disulfide linkages,
whereas the C-terminal domain is more
elongated and exhibits a high content
of acidic amino acids.

Hirudo medicinalis
 Hirudin
Hirudin exhibits its anticoagulant effect by tightly binding thrombin, thus inactivating it. In addition to its critical role
in the production of a fi brin clot, thrombin displays several other (nonenzymatic) biological activities important in sustaining
haemostasis. These include:
 it is a potent inducer of platelet activation and aggregation;
 it functions as a chemoattractant for monocytes and neutrophils;
 it stimulates endothelial transport.

One molecule of hirudin binds a single molecule of

thrombin with very high affinity. Binding and inactivation
occur as a two-step mechanism. The C-terminal region of
hirudin first binds along a groove on the surface of thrombin,
resulting in a small conformational change of the enzyme.
This then facilitates binding of the N-terminal region to the
active site area (See Figure).

Binding of hirudin inhibits all the major functions of thrombin. Fragments of hirudin can also bind thrombin, but will
generally only inhibit some of thrombin’s range of activities. For example, binding of
an N-terminal hirudin fragment to thrombin inhibits only the thrombin catalytic activity.
 Recombinant hirudin
The hirudin gene was cloned in the 1980s, and it has
subsequently been expressed in a number of recombinant
systems, including E. coli, Bacillis subtilis and
Saccharomyces cerevisiae.
A recombinant hirudin (tradename Refludan) was first
approved for general medical use in 1997. The recombinant
production system was constructed by insertion of a
synthetic hirudin gene into a strain of S. cerevisiae. The
yeast cells secrete the product, which is then purified by
various fractionation techniques (See Figure).
The recombinant molecule displays a slightly altered
amino acid sequence compared with the native product. Its
first two amino acids, leucine and threonine, replace two
valines of native hirudin. It is also devoid of the sulfate
group normally present on tyrosine63. Figure. Outline of the production and purification of antithrombin
from the milk of transgenic goats. Purification achieves an overall
product yield in excess of 50 per cent, with a purity greater than
99 per cent
Clinical trials

Clinical trials, however, have proven this slightly altered product to be both
safe and effective.
The final product is presented in freeze-dried form with the sugar mannitol
representing the major added excipient.
The product, which displays a useful shel flife of 2 years when stored at room
temperature, is reconstituted with saline or WFI immediately prior to its i.v.
administration. A second recombinant product (tradename Revasc, also produced in
S. cerevisiae) has also been approved.

A second recombinant product (tradename

Revasc, also produced in S. cerevisiae) has
also been approved.
Antithrombi
n Antithrombin, already mentioned in
the context of heparin, is the most abundantly
occurring natural inhibitor of coagulation. It
is a single-chain 432 amino acid glycoprotein
displaying four oligosaccharide side chains
and an approximate molecular mass of 58
kDa. It is present in plasma at concentrations
of 150 µg ml1 and is a potent inhibitor of
thrombin (factor IIa), as well as of factors IXa
and Xa.
Structural elements of antithrombin in its native state. The b-sheets A, B and
It inhibits thrombin by binding directly to it in C are colored yellow, green and purple, respectively. Helical secondary structural
a 1:1 stoichiometric complex. elements are shown in orange, except for helix D that is shown in black. Helix D
plays a crucial role in heparin penta saccharide binding. The P3-P14 portion of the
reactive center loop consisting of P1-P17 and P1’-P17’ residues is colored dark blue.
The Arg393 (P1) residue is shown by a ball-and-stick representation.
Recombinant antithrombin
● Recombinant antithrombin has been expressed in the milk of transgenic goats, and this
product (tradename Atryn) was approved for general medical use in Europe in 2006. The
recombinant product displays an identical amino acid sequence to that of native human
antithrombin, although its oligosaccharide composition does vary somewhat from the native
protein.
● A related product (tradename Xigiris, also known as Drotrecogin alfa) has also been
approved for medical use. Xigiris is a recombinant human activated protein C, a molecule that
plays an important role in controlling coagulation in vivo.
● The recombinant product is produced in an engineered mammalian cell line and, like several
other blood proteins, is characterized by the presence of several γ-carboxyglutamate and β-
hydroxylated residues.
● Activated protein C is indicated for the treatment of severe sepsis, largely in order to prevent
multiple organ failure that can be triggered by sepsis-associated blood clot formation.
Thrombolytic
agents
 Thrombosis In situations where inappropriate clot
formation results in the blockage of a blood
vessel, the tissue damage that ensues depends, to
a point, upon how long the clot blocks blood flow.
Rapid removal of the clot can often minimize
the severity of tissue damage. Early thrombolytic
therapy can decrease mortality and improve
coronary artery patency in patients with acute
myocardial infarction (AMI).
The market for an effective thrombolytic
agent is substantial. In the USA alone, it is
estimated that 1.5 million people suffer acute
Deposition of fibrin and platelets in the myocardial infarction each year, and there are
vasculature leads to thromboembolic diseases that another 0.5 million suffer strokes.
are responsible for considerable mortality and
morbidity.
Table. Thrombolytic agents approved for general medical use
Producta Company

Activase (rh-tPA) Genentech

Ecokinase (rtPA; differs from human tPA in that three of its fi ve Galenus Mannheim
domains have been deleted)

Retavase (rtPA; see Ecokinase) Boehringer Manheim/Centocor

Rapilysin (rtPA; see Ecokinase) Boehringer Manheim

Tenecteplase (also marketed as Metalyse) (TNK-tPA, modifi ed rtPA) Boehringer Ingelheim
TNKase (tenecteplase; modifi ed rtPA; see Tenecteplase) Genentech
Streptokinase (produced by Streptokinase haemolyticus) Various
Urokinase (extracted from human urine) Various
Staphylokinase (extracted from Staphylococcus aureus and produced in Various
various recombinant systems

a: recombinant; rh: recombinant human.

Tissue plasminogen activator
tPA (also known as fibrinokinase) represents the most important physiological activator of plasminogen. tPA is a 527
amino acid serine protease with a molecular mass of 64 kDa. It is synthesized predominantly in vascular endothelial cells
(cells lining the inside of blood vessels) and displays five structural domains, each of which has a specific function (Table).

tPA domain Function

tPA displays four potential
glycosylation sites, three of which are
normally glycosylated (residues 117, 184 and
448). The carbohydrate moieties play an
important role in mediating hepatic uptake of
tPA and, hence, its clearance from plasma. It
is normally found in the blood in two forms:
a single-chain polypeptide (type I tPA) and a domain (EGF domain)
two-chain structure (type II) proteolytically
derived from the single-chain structure. The
two-chain form is the one predominantly
associated with clots undergoing lysis, but
both forms display fibrinolytic activity.
Finger domain (F domain)
Protease domain (P domain)
Epidermal growth factor
Kringle-1 domain (K1 domain)
Kringle-2 domain (K2 domain)
Promotes tPA binding to fi brin
with high affi nity
Displays plasminogen-specific
proteolytic activity
Binds hepatic receptors, thereby
mediating hepatic clearance of
tPA from blood
Associated with binding to the
hepatic receptor
Facilitates stimulation of tPA’s
proteolytic activity by fibrin
 Structure
Approximately 6% to 8% of the molecular mass consists of carbohydrate. A schematic of the
primary structure of human t-PA is shown in Figure
Two forms of t-PA that differ by the
There are 17 disulfide bridges absence or presence of a carbohydrate at
and an additional free cysteine at Asp184 have been characterized: Type I t-
position 83, and 4 putative N-linked PA is glycosylated at asparagine 117, 184,
glycosylation sites recognized by the and 448. Type II t-PA lacks a glycosylation
consensus sequence Asn-X-Ser/Thr at at asparagine 184. The asparagine at amino
residues 117, 184 and 448. In addition, acid 218 is normally not occupied in either
form of t-PA. Asparagine 117 contains a
the presence of a fucose attached to
Thr61 via an high-mannose oligosac- charide whereas
O-glycosidic linkage has been Asn184 and Asn448 are of the complex
reported. carbohydrate type.

Complex N-linked glycan structures contain a disaccharide and terminate in sialic acid residues,
while an oligomannose (high mannose)-type glycan contains only mannose in the outer arms.
 Fibrinolysis
Fibrin contains binding sites for both
plasminogen and tPA, thus bringing these into
close proximity. This facilitates direct
activation of the plasminogen at the clot
surface (Figure). This activation process is
potentiated by the fact that binding of tPA to fi
brin (a) enhances the subsequent binding of
plasminogen and (b) increases tPA’s activity
towards plasminogen by up to 600-fold.
Overall therefore, activation of the thrombolytic
cascade occurs exactly where it is needed, i.e. on the
surface of the clot. This is important, as the substrate
specificity of plasmin is poor, and circulating plasmin
displays the catalytic potential to proteolyse fibrinogen,
factor V and factor VIII.
Figure. (a) The fibrinolytic system, in which tPA proteolytically converts the zymogen plasminogen into active plasmin, which in turn
degrades the fibrin strands, thus dissolving the clot. tPA and plasminogen both bind to the surface of fibrin strands (b), thus ensuring rapid and
efficient activation of the thrombolytic process
Although soluble serum tPA displays a much reduced
activity towards plasminogen, some free-circulating plasmin
is produced by this reaction. If uncontrolled, this could
increase the risk of subsequent haemorrhage.
This scenario is usually averted
because circulating plasmin is rapidly
neutralized by anther plasma protein, α2-
antiplasmin (a 70 kDa single-chain
glycoprotein that binds plasmin very tightly in
a 1:1 complex). In contrast to free plasmin,
plasmin present on a clot surface is very
slowly inactivated by α1-antiplasmin. The
thrombolytic system has thus evolved in a
self-regulating fashion, which facilitates
efficient clot degradation with minimal
potential disruption to other elements of the
haemostatic mechanism.
 Figure. Generational classification and features of thrombolytic drugs.

Although tPA was first studied in the late 1940s, its extensive characterization was hampered by the low
levels at which it is normally synthesized. Detailed studies were facilitated in the 1980s after the discovery that the
Bowes melanoma cell line produces and secretes large quantities of this protein. This also facilitated its initial
clinical appraisal. The tPA gene was cloned from the melanoma cell line in 1983, and this facilitated subsequent
large-scale production in CHO cell lines by recombinant DNA technology. The tPA cDNA contains 2530
nucleotides and encodes a mature protein of 527 amino acids. The glycosylation pattern was similar, though not
identical, to the native human molecule.
 First-generation tissue plasminogen activator
A marketing licence for the product was first issued in the USA to Genentech in 1987 (under the tradename Alteplase). The
therapeutic indication was for the treatment of acute myocardial infarction. The production process entails an initial fermentation
step, during which the cultured CHO cells produce and secrete tPA into the fermentation medium. After removal of the cells by sub-
micrometre filtration and initial concentration, the product is purified by a combination of several chromatographic steps. The final
product has been shown to be greater than 99 per cent pure by several analytical techniques, including HPLC, SDS-PAGE, tryptic
mapping and N-terminal sequencing.

Like melanoma-derived t-PA, rt-PA lacks glycosylation at Asn218 and exists in two forms that differ by the absence or presence of
a carbohydrate at residue Asn184 . Type II t-PA has a slightly higher specific activity in vitro compared with Type I t-PA.

The pharmacokinetics of rt-PA have been studied in mice, rats, rabbits, primates, and humans. After intravenous
administration, the plasma concentrations decline rapidly with an initial dominant half-life of less than
5 minutes in all species. rt-PA exhibits non-linear pharmacokinetics at high plasma concentrations. The pharmacokinetics
are essentially linear in cases where plasma concentrations do not exceed 10% to 20% of Km . Data show that rt-PA has an initial
volume of distribution approximating plasma volume, and a rapid plasma clearance. The initial half life was less than 5 minutes.
There was no difference in the pharmacokinetics following the different infusion regimens. A lower plasma clearance was
noted following intravenous bolus injection, possibly suggesting saturation of clearance mechanisms. The primary route of alteplase
clearance is via receptor-mediated clearance mechanisms in the liver. Data suggest that this carbohydrate-independent clearance is
mediated by the low-density lipoprotein receptor related protein.
 Clinical usage
Recombinant human t-PA (alteplase t-PA) is indicated for use in the management of AMI in adults for the
improvement of ventricular function following AMI, reduction of the incidence of congestive heart failure,
reduction of mortality associated with AMI, and for the management of acute massive pulmonary embolism in
adults. It is also indicated for the management of acute ischemic stroke if therapy is initiated within 3 hours after
the onset of stroke symptoms and after exclusion of intracranial hemorrhage by cranial computerized tomography
(CT) scan.
For acute ischemic stroke, the recommended dose is 0.9
mg/kg not to exceed 90 mg infused over 60 minutes, with 10% of the Safety
total dose administered as an initial intravenous bolus over 1 minute. Since thrombolytic therapy
The safety and efficacy of this regimen with concomitant use of heparin
and aspirin during the first 24 hours has not been investigated.
concerns
increases the risk of bleeding, alteplase is
contraindicated in patients with a history
of cerebrovascular accidents, or patients
The recommended dose for treatment of pulmonary who have any kind of active internal
embolism is 100 mg administered by intravenous infusion over 2 bleeding, intracranial neoplasm,
hours. Heparin therapy should be instituted or reinstituted near the end arteriovenous malformation, or aneurism
of or immediately following alteplase infusion when the partial or who have had recent intracranial or
thromboplastin time or thrombin time returns to twice normal or less. intraspinal surgery or trauma.
 Pharmaceutical considerations
Recombinant human t-PA (Alteplase; Activase; Actilyse) is supplied as a sterile, white to off-white
lyophilized powder. rt-PA is practically insoluble in water, and arginine is included in the formulation to increase
aqueous solubility. Phosphoric acid and/or sodium hydroxide may be used to adjust the pH.
The sterile lyophilized powder should be stored at controlled room temperatures not to exceed 30 0C, or
refrigerated at 2 0C to 8 0C, and it should be protected from excessive light.
The powder is reconstituted by adding the accompanying sterile water for injection, USP to the vial, resulting in
a colorless to pale yellow transparent solution containing 1mg/mL rt-PA, with a pH of approximately 7.3 and an
osmolality of approximately 215 mOs/kg. rt-PA is stable in solution over a pH range of 5 to 7.5.
Since the reconstituted solution does not contain any preservatives, it should be used within
8 hours of preparation and should be refrigerated before use. The solution is incompatible with bacterio- static water
for injection.
Other solutions such as sterile water for injection or preservative-containing solutions should not be used for further
dilution. The 1-mg/mL solution can be diluted further with an equal volume of 0.9% sodium chloride for injection, USP
or 5% dextrose injection, USP to yield a solution with a concentration of 0.5 mg/mL. This solution is compatible with
glass bottles and polyvinyl chloride bags.
 Microbes in thrombolytic therapy
Many microbial-derived thrombolytic
enzymes have been discovered and characterized.

Bacteria are the first-line sources

because bacterial proteins are suitable for oral
administration and facilitate large-scale
production. Bacillus sp. are among the most preferred
sources; various strains have been reported to have
fibrinolytic activity (Figure). In addition, several
other strains with fibrinolytic properties have been
reported, although their mode of action is yet to be
elucidated, such as Bacillus sp. DJ–2.
B. subtilis A26-derived subtilisin BSF1 and BAF1
obtained from B. amyloliquefaciens
An6, enzyme URAK produced by B. cereus NK, B.
cereus GD 55-derived protease, B. cereus

IND1 and B. halodurans IND18 are the sources of proteolytic enzymes that exhibit both
thrombolytic activity and PLG activation properties, while
a fibrinolytic protease with absolute clot dissolution ability in a short span of time (within 4 h) in in vitro conditions was obtained
from Bacillus sp. IND12, B. pseudomycoides strain MA02, and B. cereus RSA1.
 Streptomyces sp. are the largest
fibrinolytic enzyme-producing genus. Figure 3B
summarizes the fibrinolytic agents derived from
different strains of Streptomyces sp
 Streptokinase
Streptokinase (SK) is the most commonly used first-generation thrombolytic agent. It is a 48 kDa
extracellular bacterial protein produced by several strains of Streptococcus haemolyticus of Lancefield group A, C,
and G. Its ability to induce lysis of blood clots was first demonstrated in 1933. Modern chromatographically pure
streptokinase preparations are usually supplied in freeze-dried form. These preparations (still obtained by non-
recombinant means) often contain albumin as an excipient. The albumin prevents flocculation of the active
ingredient upon its reconstitution.
Streptokinase is a widely employed thrombolytic agent. It is administered to treat a variety of thrombo-
embolic disorders, including:
 pulmonary embolism (blockage of the pulmonary artery which carries blood from the heart to the lungs for
oxygenation by an embolism), which can cause acute heart failure and sudden death;
 deep-vein thrombosis (thrombus formation in deep veins, usually in the legs);
 arterial occlusions (obstruction of an artery);
 acute myocardial infarction.
Streptokinase induces its thrombolytic effect by binding specifically and tightly to plasminogen. This induces a
conformational change in the plasminogen molecule that renders it proteolytically active. In this way, the streptokinase–
plasminogen complex catalyses the proteolytic conversion of plasminogen to active plasmin.
 Safety
concerns
Tillette and Sherry reported accompanying adverse
effects of SK, which causes pyrogenic reactions with
symptoms like arthralgia, occasionally nausea, and
headache, etc., ergo, limiting the administration of
multiple doses of SK. Nevertheless, continued efforts have
been made to achieve reduced pyrogenic reaction of SK
by using various approaches such as structural and
chemical modifications, liposomal entrapment or
encapsulation, and domain fusion (Table); the
recombinant SK (rSK) thus obtained possessed less
antigenicity compared to wild-type SK. Cloning the SK
gene in non-pathogenic microbes permits the
production of fortified rSK that eliminates the risk of
infection by potentially pathogenic Streptococci.

Table. Summary of strategies used for SK modifications.

 Second generation recombinant thrombolytic
developagents
Modified forms of tPA have also been generated in an effort to
a product with an improved therapeutic profile.
Reteplase is the international non-proprietary name given to one such
modified human tPA produced in recombinant E. coli cells and is sold under
the tradenames Ecokinase, Retavase and Rapilysin. This product’s
development was based upon the generation of a synthetic nucleotide
sequence encoding a shortened (355 amino acid) tPA molecule. This
analogue contained only the tPA domains responsible for fibrin
selectivity and catalytic activity. The nucleotide sequence was integrated
into an expression vector subsequently introduced into E. coli (strain
K12) by treatment with calcium chloride. The protein is expressed
intracellularly, where it accumulates in the form of an inclusion body.
Owing to the prokaryotic production system, the product is non-
glycosylated. The final sterile freeze-dried product exhibits a 2-year
shelf life when stored at temperatures below 25 0C. An overview of the
production process is presented in Figure.
Figure. Production of Ecokinase, a modified tPA molecule that
gained regulatory approval in Europe in 1996.
 The lack of glycosylation, as well as the absence of the EGF and K1 domains, confers an extended serum half-life upon
the engineered molecule. Reteplase-based products display a serum half-life of up to 20 min, facilitating its administration as a
single bolus injection as opposed to continuous infusion. Absence of the molecule’s F1 domain also reduces the product’s
fibrin-binding affinity. It is theorized that this may further enhance clot degradation, as it facilitates more extensive diffusion of
the thrombolytic agent into the interior of the clot.
tPA domain Function
Finger domain (F domain) Promotes tPA binding to fi brin Tenecteplase is yet an additional engineered
with high affi nity tPA now on the market. Produced in a CHO cell
line, this glycosylated variant differs in
Protease domain (P domain) Displays plasminogen-specific
proteolytic activity sequence to native tPA by six amino acids (Thr
103 converted to Asn; Asn 117 converted to Gln
Epidermal growth factor Binds hepatic receptors, thereby and the Lys–His–Arg–Arg sequence at position
domain (EGF domain) mediating hepatic clearance of
296–299 converted to Ala–Ala–Ala–Ala).
tPA from blood
Collectively, these modifications result in a
Kringle-1 domain (K1 domain) Associated with binding to the prolonged plasma half-life (to between 15 and
hepatic receptor 19 min), as well as an increased resistance to
PAI-1 (plasminogen activator inhibitor 1, a natural
Kringle-2 domain (K2 domain) Facilitates stimulation of tPA’s
proteolytic activity by fibrin tPA inhibitor).
 Staphylokina
se Staphylokinase (SAK) is a third-generation plasminogen activator obtained from
Staphylococcus aureus GH38 which exerts its anti-thrombin activity by converting passive
plasminogen to active plasmin.
The staphylokinase gene has been cloned in E. coli, as well as various other recombinant
systems. The protein is expressed intracellularly in E. coli at high levels, representing 10–15 %
of total cellular protein. It can be purified directly from the clarified cellular homogenate by a
combination of ion-exchange and hydrophobic interaction chromatography.

Structure SAK is a 136 amino acid monomer of 15.5 kDa and comprises two
equal-sized domains with flexible dumbbell shapes. Structurally, it has been
reported that amino acid at the 26th position (methionine-26) is useful for
PLG activation by SAK. Notably, the functional activity is lost if this amino
acid is replaced with arginine or valine, whereas little or no effect on
functional activity was observed by replacing it with leucine or cysteine.
 Action mechanism clinical trials This complex (via the
plasmin) then to catalyse the
Although staphylokinase shows no conversion of free plasminogen
significant homology with streptokinase, it to plasmin, and it may even
induces a thrombolytic effect by a somewhat accelerate the process of
similar mechanism: it also forms a 1:1 conversion of other
stoichiometric complex with plasminogen. The staphylokinase–plasminogen
proposed mechanism by which staphylokinase complexes into
induces plasminogen activation is outlined in staphylokinase–plasmin
Figure. complexes.

SK-PLG complex is a highly specific protease

that further cleaves other circulating PLG molecules The net effect is
and is capable of converting them to active serine generation of active plasmin,
protease, plasmin, that can degrade the fibrin clot via which displays a direct
its specific lysine binding site. thrombolytic effect by
degrading clot-based fi brin,
Complex formation induces subsequent as described previously .
proteolytic cleavage of the bound plasminogen to
form plasmin, which remains complexed to the
staphylokinase Figure. Schematic representation of the mechanism by which staphylokinase appears to activate the thrombolytic
process via the generation of plasmin.
Clinical trials
The thrombolytic ability of (recombinant) staphylokinase has been evaluated in initial
clinical trials with encouraging results. Some 80 per cent of patients suffering from acute
myocardial infarction who received staphylokinase responded positively (10 mg
staphylokinase was administered by infusion over 30 min).
The native molecule displays a relatively short serum half-life (of 6.3 min), although
covalent attachment of PEG reduces the rate of serum clearance, hence effectively increasing
the molecule’s half-life significantly. As with streptokinase, patients administered
staphylokinase develop neutralizing antibodies. A number of engineered (domain-deleted)
variants have been generated that display significantly reduced immunogenicity.
Conclusions
Vascular occlusion remains a major cause of morbidity and mortality worldwide. Although
numerous thrombolytic agents have been identified and characterized from diverse sources, promising
scientific data available from in vitro and in vivo studies have failed to translate into clinical trials
successfully. Therefore, continuous efforts are needed in the search for more efficacious, safer, and cost-
effective thrombolytic drugs. Microbial-derived thrombolytic agents represent a step towards a potent
approach in the prevention and treatment of vascular diseases such as CVDs, stroke, transient ischemic
attack, venous thromboembolism, etc..

Several thrombolytic enzymes have been reported to be isolated from microbial sources with
therapeutic application in vascular diseases and have been shown to possess the following advantages over
currently available treatment strategies:
1) extended plasma half-life,
2) increased fibrin specificity,
3) high therapeutic index, Recombinant technology has brought about
4) lower allergic response, and significant advances in the treatment of coagulation
5) reduced risk of bleeding complications. disorders and in the availability of thrombolytic agents
for the treatment of thrombotic disorders.
Thank you for
attention!