HEMOSTASIS

 Hemostasis- is the combination of cellular and biological events that function in harmony to
keep blood liquid within the veins and arteries, prevent blood loss from injuries by the
formation of thrombi, and reestablish blood flow during the healing process.
 When hemostasis systems are out of balance, thrombosis or hemorrhage threatens life.

Overview of Hemostasis
 Systems involved – Vascular intima; blood platelets, erythrocytes, neutrophils, lymphocytes
and monocytes; the plasma coagulation system; and fibrinolysis.
 Primary hemostasis – mechanisms are triggered by small injuries to blood vessels, such as pin
pricks or by the common place destruction of dying or damaged endothelial cells.
 The blood vessel contracts to seal the wound and platelets fill the open space to form a plug.
 Secondary hemostasis-is triggered by the primary hemostasis mechanisms and is necessary to
control bleeding from large wounds incurred through trauma, surgery, or dental procedures.
 The plasma coagulation system, a series of protein enzymes and enzyme cofactors, accomplish
secondary hemostasis by producing a fibrin thrombus.
 The Vascular intima and platelets are associated with primary hemostasis, and coagulation &
fibrinolysis with secondary hemostasis; all interact in both early & late hemostatic events.

Coagulation Mechanism
 The coagulation mechanism may be thought of as a complex series of cascading reactions involving
development of enzymes from their precursor (zymogens, procoagulants, proenzymes).
 In addition to the zymogens, protein cofactors, membrane phospholipid surfaces, and calcium ions
play an active role in the final development of the fibrin clot.

Nomenclature of Procoagulants
 There are 16 procoagulants (coagulation factors or clotting factors).
 Nearly all are glycoproteins synthesized in the liver; a few are made by monocytes, endothelial cells,
and megakaryocytes.
 Eight are enzymes that circulate in an inactive form –zymogens.
 Six other are cofactors that bind and stabilize their respective enzymes.
 During the thrombosis process, the procoagulants become activated and produce a localized
thrombus.
 At least six plasma glycoproteins serve as anticoagulants to regulate the coagulation process.
 Because of early confusion with the names of the coagulation factors, in 1954 the International
Committee for Standards of the Nomeclature of the Blood Clotting factors established a numbering
system.
 Roman numerals were given in order of their initial description or discovery.
 When a procoagulant is activated, a lower-case “a” appears behind the numeral. For example,
activated factor X = factor Xa.
 Most of the factors, except fibrinogen (I), prothrombin (II), tissue factor (III), and calcium (IV), are
referred to by their number.
 VI – was assigned to what is activated Factor V; it was later withdrawn.
 Prekallikrein and HMWK – have never received Roman numerals because they belong to the
Kallikrein and Kinin systems, respectively, and their primary functions are within these systems.
 Platelet phospholipids are essential to the coagulation process but the group is too heterogeneous for
numerals.

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 Factor Name
I Fibrinogen
II Prothrombin
III Tissue factor
IV Calcium
V Proaccelerin, labile factor
VII Proconvertin, stable factor
VIII Antihemophilic A factor (AHF), antihemophilic globulin (AHG)
IX Antihemophilic B factor (AHB), plasma thromboplastin component
(PTC), Christmas factor
X Stuart factor/Stuart-Prower factor
XI Plasma Thromboplastin antecedent (PTA)
XII Hageman factor, contact factor
XIII Fibrin stabilizing factor
- Fletcher factor, prekallikrein
- High molecular weight kininogen, HMWK, Fitzgerald factor

 Basic cascading reactions of blood coagulation occur in a simplistic manner.

 It is important to understand these simple reactions in the order in which they occur, before trying to
understand some of the more complex inter-reactions that are thought to actually occur in vivo
(within a living organism).
 Coagulation has been divided into two systems: the intrinsic system, in which all substances
necessary for clotting are present in the blood, and the extrinsic system, in which tissue factor (from
injured tissues) is necessary for coagulation.
 There is also the common pathway, where these two systems come together after activating factor X.
 The coagulation mechanism may be thought of as occurring in four phases: (1) contact phase, (2)
activation of factor X, (3) conversion of prothrombin to thrombin, and (4) formation of a fibrin clot.

Contact phase.
 The initial method of activation of factor XII is not completely understood.
 One theory is that small amounts of factor XII automatically activate when coming in contact with
negatively charged surfaces (such as collagen)
 (a). Prekallikrein and factor XI circulate in the blood bound to high molecular weight kininogen
(HMWK), which binds to appropriate surfaces at the site of injury. When a small amount of activated
factor XII (XIIa) because available, it converts prekallikrein to kallikrein
 (b). The kallikrein formed, together with HMWK, activates more factor XII
 (c). Factor XI is activated by factor XIIa
 (d). This sequence of events probably occurs as follows:

Negatively charged
a) XII XIIa (small amounts)
Surface

XIIa
b) Prekallikrein kallikrein

kallikrein
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 c) XII XIIa (larger amounts)
HMWK

XIIa
d) XI XIa

 Step “c” allows the coagulation process to proceed more rapidly by making larger amounts of factor
XIIa available.
 Activation of the clotting sequence in vitro (in the test tube) is brought about by surface contact with
glass, celite, kaolin, or other negatively charged surfaces.

Activation of factor X via the intrinsic pathway.

 Factor IX is activated by factor Xia in the presence of calcium ions
 (a). Conversion of factor X to factor Xa most probably takes place on the surface of the platelet and is
catalyzed by a complex composed of factor IXa, factor VIII, calcium ions, and phospholipid (from
the platelet)
 (b). In this reaction factor XIII acts as a cofactor. (Activation of factor VIII markedly increases its
activity. Both factor Xa and thrombin, once formed, will activate factor VIII.) Activation of factor X
proceeds as shown below.

XIa
a) IX IXa
Ca

IXa -VIII
b) X Xa
P -Ca
1

Activation of factor X via the Extrinsic System

 All cells, with the possible exception of those in the blood, contain tissue factor. When injury occurs,
tissue factor is released and acts as a cofactor in initiating coagulation.
 Factor VII binds to tissue factor and to calcium ions to activate factor X
 (a). This complex (factor VII, tissue factor, and calcium ions) is also capable of activating small
amounts of factor IX
 (b). As factor Xa is formed it, in turn, will activate factor VII. Factor IXa will also activate factor VII
but not as efficiently as factor Xa. Factor XIIa is much more active and will speed up the activation of
factor X. A summary of these reactions is given below.

VII –Tissue factor

a) X Xa
P1 – Ca

VII –Tissue factor

b) IX IXa (small amounts)
P1 - Ca

 To summarize the intrinsic and extrinsic systems thus far, the intrinsic coagulation mechanism
requires a contact phase, followed by activation of factors IX and X.
 The extrinsic mechanism’s primary action is the activation of factor X.

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 From this point on, the two systems combine into what is termed the common pathway. It is most
likely that both systems operate simultaneously in vivo.

Conversion of prothrombin to thrombin

 Conversion of prothrombin to thrombin marks the beginning of the common pathway.
 Calcium ions are bound to prothrombin, which adheres to platelet surfaces along with factor Xa and
factor V (factor acts as a cofactor).
 This complex attacks the prothrombin molecule, forming fragment 2 and fragment 1.2
 (a). In the presence of this same complex, thrombin is formed from fragment 2
 (b). The resultant thrombin is capable of activating factor V, thereby increasing its activity.

Xa –V Fragment 2+
a) Prothrombin
P1 –Ca Fragment 1.2

Xa -V
b) Fragment 2 Thrombin
P -Ca
1

Formation of the fibrin clot

 Fibrin formation occurs by the action of thrombin on fibrinogen.
 The fibrinogen molecule consists three polypeptide chains.
 Thrombin splits off two small peptides on each side of the fibrinogen molecule.
 Fibrinopeptide A is released from the α chain first, and the fibrinopetide B is released from the β
chain
 (a). The resultant fibrin monomers will polymerize end to end and laterally to form fibrin polymers
(fibrin strands), which are soluble in 5 M urea
 (b).Fibrinopeptides A and B are negatively charged and must be removed from the fibrinogen
molecule for polymerization to take place; otherwise, the molecules would repel each other. Once
fibrinopeptide A is removed, the molecules are able to join laterally.) Factor XIII, activated by
thrombin and calcium ions
 (c), converts the fibrin polymer to a covalent cross-linked fibrin clot (d). This stable fibrin clot is
insoluble in 5 M urea. In the presence of activated platelets the formed clot will retract (e).

Thrombin
a) Fibrinogen Fibrinopeptides A δ B +
Fibrin monomer

Fibrin polymer
b) Fibrin monomer
(soluble in 5 M urea)

Thrombin
c) XIII XIIIa
Ca

Stable fibrin clot

d) Fibrin polymer
Ca insoluble in 5 M urea

Activated
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 e) Stable fibrin clot Retracted clot
Platelets

Feedback Mechanisms
 Fibrin formation at the site of small wounds takes place in minutes.
 Because the plasma concentration of many procoagulant is measured in micrograms/mL it would take
hours, not minutes, for fibrin to form were it not for feedback mechanisms to increase the velocity of
the cascade reactions.
 In these feedback loops a clotting factor is activated and in turn activates both the next procoagulant
as well as the factor that acted upon it originally.
 For example, factor XIIa can activate both X and IX. In turn, Xa can activate both II and more factor
VII. Meanwhile, IXa activated additional X and VII.
 A positive feedback loop is one that increases the reaction rate.
 A negative feedback loop is one that dampens the reaction rate. For example, the
thrombin/thrombomodulin activation of protein C ultimately stops the formation of new thrombin by
inactivating Va and VIIIa.

The Coagulation Factors

 The coagulation factors may be divided into three groups based on their properties.
 The thrombin sensitive group consists of fibrinogen (factor I) and factors V, VIII, and XIII. They are
consumed during the process of coagulation and are absent in serum and present in plasma.
 These factors are not adsorbed out by barium sulfate or aluminum hydroxide (because citrate is
anticoagulation of choice).
 Factors V and VIII are susceptible to denaturation and reduced in quantity in stored plasma. Vitamin
k is not necessary for synthesis of these factors.
 There is an increased concentration of factors in this group during an inflammatory response and in
pregnancy.
 The vitamin K dependent group includes prothrombin (factor II) and factors VII, IX and X.
 Vitamin K is necessary for their synthesis, which takes place in the liver.
 Coumarin drugs, which compete with vitamin K, cause a functional decrease in these factors by
producing abnormal proteins that cannot bind calcium and so are unable to function normally.
 These abnormal proteins are known collectively as PIVKAs (proteins induced by vitamin k
antagonists) and if measured by an immunologic rather than a functional test will be present in
normal amounts.
 Factors VII, IX, and X are not consumed during the coagulation process and are, therefore, present in
serum as well as plasma. All four factors are adsorbed out of plasma by barium sulfate or aluminum
hydroxide. They are stable and well preserved in stored plasma.
 The contact group is composed of factors XI and XII, prekallikrein, and high molecular weight
kininogen (HMWK).
 These factors are not consumed during coagulation, do not depend on vitamin K for their synthesis,
and are not adsorbed out of the plasma by barium sulfate or aluminum hydroxide. They are relatively
stable.
 Prekallikrein and HMWK also function in the activation of plasminogen (in the fibrinolytic process).
 Fibrinogen (factor I) is synthesized in the liver. It is made up of three pair of peptide chains named α,
β, and δ. When acted on by thrombin, the α chain yields fibrinopeptide A, and the β chain yields
fibrinopeptide B. The normal plasma concentration of fibrinogen is approximately 200 to 400 mg/dL.
A minimum of 60 to 100 mg/dL are required for normal coagulation. Fibrinogen has a half-life of 3 to
4 days. It is relatively stable to heat and storage and may be irreversibly precipitated at 56 0C. A
fraction of fibrinogen, cryofibrinogen, may precipitate at 4 0C but goes back into solution upon
warming.

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 Prothrombin (factor II) is synthesized in the liver and is almost entirely consumed in the coagulation
process so that little remains in the serum. It is an α-2 globulin with a molecular weight of
approximately 69,000. Prothrombin is heat stable and has a half-life of about 60 hours.
 Tissue factor (factor III) is a lipoprotein found in most of the body tissues, with increased
concentrations in the lungs and brain. It has no enzymatic activity and acts as cofactor in activating
extrinsic coagulation.
 Calcium (factor IV), in the ionized state, is necessary for coagulation. The fact that it is essential for
coagulation makes possible the use of anticoagulants, which bind the calcium and, therefore inhibit
coagulation. It is unlikely that a bleeding tendency is ever caused by a deficiency of calcium because
clinical tetany occurs with higher levels of calcium than are necessary for coagulation.
 Factor V (proaccelerin) is synthesized in the liver but does not need vitamin k for its production. It is
a globulin with a molecular weight of over 300,000. Factor V is the most unstable of the coagulation
factors and deteriorates rapidly at room temperature. It is more stable in citrated plasma than in
oxalated plasma. Little, if any, factor V is present in serum. It has a half-life in the plasma of
approximately 12 to 14 hours.
 Factor VII (proconvertin) is synthesized in the liver and requires vitamin K for its production. It is a β
globulin with a molecular weight of 60,000. Although it is stable at 4 0C for 2 or more weeks, it is
slightly heat labile. It has an in-vivo half-life of any 4 to 6 hours, which means that it disappears
rapidly from the blood when production is halted (as in therapy with coumarin drugs).
 Factor VIII circulated in the blood bound to von Willebrand factor (vWF). This unit is called the
factor VIII complex.it was originally thought that factor VIII and vWf were the same molecule. This,
however, has been found not to be the case. The site of synthesis for factor VIII is not completely
understood and it may be produced in multiple sites, including the liver. Factor VIII is a single chain
glycoprotein. It is heat labile, has a half-life of about 12 hours, and is unstable in citrated plasma.
During coagulation, it functions as a cofactor to enhance the activation of factor X by IXa with
phospholipid and calcium ions. Based on its characteristics, factor VIII may be symbolized as
follows: (1) factor VIII, factor VIIIC, and factor VIII: C stands for the coagulant property of the
factor, that portion of the molecule that is measured by standard factor VIII assays, and it is markedly
decreased in classic hemophilia (hemophilia A); (2) factor VIII antigen (factor VIII: Ag) represents
the antigenic properties of factor VIII measured by immunoassays.
 Von Willebrand factor functions in primary hemostasis, acts as carrier for the coagulant portion of the
factor VIII complex, and constitutes greater than 90% of this complex. It is synthesized in the
megakaryocytes and endothelial cells and is also present in the α granules of the platelets. Based on
its characteristics von Willebrand factor may be symbolized as follows: (1) vWf or von Willebrand
factor, which represents that portion of the factor responsible for a normal bleeding time, or normal
platelet adhesiveness in-vitro; (2) von Willebrand factor antigen (vWf:Ag) was previously termed the
factor VIII related antigen (VIIIR: Ag) and is measured by immunoassays; (3) ristocetin co-factor
activity, which is that property of the factor which causes platelet aggregation in the presence of
ristocetin.
 Factor IX (antihemophilic B factor) is a single chain glycoprotein synthesized in the liver, and
requires vitamin K for its production. It is decreased in the plasma of patients with Christmas disease
(hemophilia B). It stable at 40C for several weeks. It has a half-life of approximately 24 hours and is
present in serum.
 Factor X (Stuart factor) is a glycoprotein. It is synthesized in the liver and requires vitamin K for its
production. It is relatively heat stable and may be stored for up to 2 months at 4 0C. It has a half-life of
approximately 40 hours. It may be activated by both the intrinsic and extrinsic coagulation system.
 Factor XI (plasma Thromboplastin antecedent) is a β-2 globulin that is thought to circulate in plasma
in a complex with high molecular weight kininogen. It is probably synthesized in the liver, it
relatively stable at room temperature, and has a half-life of approximately 45 hours.
 Factor XII (Hageman factor) is a single chain polypeptide.

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 Fibrin (crosslinked)
(urea insoluble)

Plasmin

Complex DD/E Complex YD/DY Complex YY/DXD

Degradation of cross-linked fibrin plasmin

 The actual site of production is not known. It is stable in that it can be stored (in oxalated plasma) at
40C for almost 3 months. It is relatively heat stable and remains in serum after 30 minutes at 60 0C.
 Factor XIII (fibrin stabilizing factor) is heat stable and has a half-life of 3 to 12 days. Although its
site of production is not known, it is thought that the liver may play a role.
 Prekallikrein (Fletcher factor) is a single chain γ globulin. It is produced in the liver but is not
dependent on vitamin K for its production. This factor is present in serum and is not adsorbed out of
the plasma by barium sulfate and aluminum hydroxide.
 High molecular weight kininogen (HMWK) (Fitzgerald Factor) is a single chain glycoprotein, has a
half-life of 6.5 days, and is present in serum. It is produced in the liver, is not vitamin K dependent,
and is present in barium sulfate and aluminum hydroxide adsorbed plasma.

Fibrinolysis
 The primary purpose of fibrinolysis is to digest fibrin clots as they are formed in order to keep the
vascular system free of deposited fibrin and fibrin clots. Fibrinolysis occurs when plasminogen is
converted into plasmin, which dissolves the fibrin (or fibrinogen) into smaller fragments termed fibrin
(ogen) degradation products
 Plasminogen. Plasminogen is a single chain glycoprotein found in the plasma in a concentration of 20
to 40 mg/dL and in all other body fluids in lesser amounts. It is produced by the liver.
 During the clotting process, it forms complexes with fibrin and is absorbed into the clot.

Fibrinogen
or
Fibrin (urea soluble)

Plasmin

Fragment X

Plasmin

Fragment Y Fragment D

Plasmin

Fragment D Fragment D

Degradation of fibrinogen and non-cross-linked fibrin by plasmin

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Plasminogen activators
 Activation of plasminogen is carried out by plasminogen activators. Intrinsic activators are present in
the blood and include factor XIIa, kallikrein, and HMWK.
 Tissue-type plasminogen activator (t-PA), secreted by the endothelial cells, and urokinase-like
plasminogen activator (u-KA), produced by the kidney, are extrinsic activators.
 There are three therapeutic activators that are used for treatment of thromboemboli: streptokinase,
urokinase, and tissue-like plasminogen activator (t-PA). These drugs will induce a high degree of
fibrin (ogen)-olysis.

Fibrinolysis mechanism
 During formation of a clot, small amounts of plasminogen become bound to the fibrin and are
incorporated into the fibrin clot.
 Plasminogen activator (t-PA) is released from surrounding endothelial cells as a result of various
stimuli (such as thrombin).
 The t-PA has a high affinity for fibrin and thereby absorbs onto the fibrin clot and activates the
plasminogen present to form plasmin.
 As the clot begins to dissolve the partially degraded fibrin binds additional plasminogen, which is
activated to plasmin so that fibrinolysis continues.

Degradation of fibrin
 Degradation of non-cross-linked fibrin produces almost the same degradation products as those
formed from fibrinogen.
 Degradation of cross-linked fibrin, however, is slower than that of fibrinogen (and non-cross-linked
fibrin), and the products formed are different because of the bonding present in the cross-linked
fibrin.
 The smallest complex formed (DD/E) is composed of fragment DD (termed a D-dimer) and fragment
E. Other complexes formed are YD/DY and YY/DXD. Further breakdown of these complexes may
be prevented in-vivo by fibrinolytic inhibotors.
Inhibitors of fibrinolysis
 The primary inhibitor of plasmin is α2 antiplasmin. It is present in the plasma and also in platelets.
 Its inhibitory effect on free plasmin in the blood is very fast. However, plasmin is inactivated by this
inhibitor relatively slowly when the plasmin is bound to fibrin.
 α2 Macroglobulin is also an inactivator of plasmin.
 Thrombospondin, released by the platelets, inhibits activation of fibrin-bound plasminogen.
 There are also naturally occurring inhibitors to plasminogen activators: plasminogen activator
inhibitor-I (PAI-1) and Plasminogen activator inhibitor-2 (PAI-2).
 PAI-1 is secreted into the blood by the endothelial cells and is also present in platelets. It inactivates
t-PA and urokinase.
 Antistreptokinase antibodies, when present, will inactivate therapeutic streptokinase.
Control of fibrinolysis
 The fibrinolytic system must be regulated so that unwanted clots are dissolved but not broken down
prematurely before bleeding has ceased and the healing process begun. Because of its location in the
clot, plasmin is inaccessible to the α2 antiplasmin. However, any plasmin that escapes from the clot
into the plasma will be immediately neutralized by this inhibitor. PAI-1, stored in platelets in the area
of the clot, may be released and thereby inhibit the action of t-PA, preventing premature lysis of the
platelets following stimulation by thrombin, inhibits activation of the fibrin-bound plasminogen.