Blood CoagulationJolyon Jesty, State University of New York, Stony Brook, New York, USA

The blood coagulation system acts in concert with the platelets to seal damaged blood

vessels by the formation of a clot that consists of aggregated platelets interwoven with

fibrin. Coagulation is initiated by the subendothelial membrane protein tissue factor and

proceeds through a highly regulated proteolytic cascade to the production of thrombin

and the fibrin clot.

Introduction

In higher animals blood is pumped through the vascularsystem under pressure. It is the job of the haemostaticsystem to maintain this system in the face of injury. Whendamage occurs, three systems cooperate to limit blood loss.First, the smooth muscle of the vessel wall immediatelycontracts under sympathetic nervous control, reducingflow. Upon injury to the endothelium, which lines all bloodvessels, the two other parts of haemostasis are initiatedtogether. One is the activation and subsequent aggregationof the blood platelets to form a platelet ‘plug’, which isinitiated by the interaction of platelets with materials –chiefly collagen – present in the vessel wall behind theendothelium. The other is the coagulation system, which isinitiated by a specific membrane protein of cells in thevessel wall, again behind the endothelium, called tissuefactor (TF). The products of coagulation are the enzymethrombin and the insoluble gelatinous protein of the finalclot, called fibrin. Platelet function and coagulation areoften separated for didactic reasons, but the two systemsare closely intertwined, each requiring the other for itsfunction; in fact, a normal clot consists of an interlockedstructure of aggregated platelets and fibrin.

In its general form, the collection of clotting proteins andplatelets present in the plasma can be viewed as primedsystem, set to generate a rapid and highly amplifiedresponse to a TF stimulus. TF is not expressed atsignificant levels in quiescent endothelial cells or bloodcells, and is therefore not normally in contact with theblood. Endothelial cells form a continuous layer, one cellthick, which lines the entire vascular system, amounting inadults to some 700 m2. Much richer in TF are thefibroblasts and smooth muscle cells of the vessel wall,behind the endothelium, and these two cell types areprobably the major source of TF. Additionally, particu-larly in pathological situations such as infection, inflam-mation and atherosclerosis, macrophages and their plasmaprecursors, the monocytes, are capable of significant TFexpression.

The initiating event of coagulation is the formation of acomplex between TF and a plasma zymogen, factor VII.This initiates a cascade of proteolytic reactions involving

the generation and action of a series of highly specificproteolytic enzymes, or proteases. Like other complexproteolytic systems, coagulation is subject to numerousintermeshed controls, including regulation by cofactorproteins of plasma and the vessel wall, positive andnegative feedback controls, and the action of proteaseinhibitors. The result is a system that, in concert withplatelets, can seal areas of endothelial damage veryquickly, and moreover localize this response in the faceof flow. The only important situation in which haemostasisis ineffective is major damage to arteries, where thecombination of high pressure (of the order of 1

6 bar(17 kPa) in adults) and high flow is more than the systemcan cope with. Fortunately most arteries are locatedanatomically much deeper than veins and capillaries.

The majority of clinical defects in haemostasis result inthrombosis rather than bleeding, and the majority of theseare due to pathological changes in blood vessel wall,particularly in areas of the arterial vasculature wheresclerotic plaque formation is common, such as thecoronary and carotid arteries. Apart from the fact thatplaques narrow the arteries, they also contain high levels ofTF, and its release upon rupture is probably the dominantcause of thrombus formation in acute arterial thrombosis.Much is known of risk factors for the development of life-threatening arteriosclerosis, which include the importanceof lipid metabolism and methionine metabolism. Defectsin the latter produce an increase in homocysteineconcentration, which is a significant contributor tovascular pathology, particularly in children. The majoreffect is seen in the arteries, but hyperhomocysteinaemia isalso a contributory risk factor for the development ofvenous thrombosis (McCully, 1996).

Although arterial thrombosis is more common, venousthrombosis is also of major clinical importance. Withthe recent discovery of factor V Leiden and commonpolymorphisms that lead to abnormal prothrombin andfactor VII synthesis, the evidence is increasingly strong thatthe majority of venous thrombosis is caused by defects inthe plasma proteins of the clotting system rather thandefects in the vasculature. These may be either quantitative(e.g. abnormal plasma levels of inhibitors or clotting

Article Contents

Secondary article

. Introduction

. Zymogens, Proteases, Cofactors, Inhibitors

. The Cascade

. Localization

. Positive Feedback Controls

. Negative Feedback Controls

. Inhibitors

1ENCYCLOPEDIA OF LIFE SCIENCES © 2001, John Wiley & Sons, Ltd. www.els.net

factors) or qualitative (exemplified by the genetic defect infactor V Leiden that leads to increased risk of thrombosis).

This article focuses on the mechanisms of initiation andpropagation of the coagulation response.

Zymogens, Proteases, Cofactors,Inhibitors

Table 1 lists the major proteins involved in bloodcoagulation and its control.

A zymogen is the precursor of an enzyme: conversion tothe enzyme involves proteolytic cleavage of the zymogenprotein chain by a protease at just one or two sites (peptidebonds). Conversion of a zymogen to an enzyme is alsocalled activation. (Zymogen activation by specific, regu-lated, proteolytic cleavage is by no means restricted tocoagulation: almost all complex proteolytic systems havethis as a central activation mechanism; examples includecomplement, fibrinolysis, blood pressure regulation andapoptosis.) All but one of the clotting zymogens are

precursors of proteases, the exception being factor XIII,which is the precursor of a transglutaminase that catalysesthe crosslinking of fibrin polymers and the linking of fibrinto cell matrix proteins like fibronectin.

Proteases produced by the activation of the otherzymogens (XII, XI, X, IX, VII and prothrombin) are allof the trypsin-like family, i.e. they are serine proteases thatpreferentially cleave at basic amino acid residues. How-ever, all are much more specific than trypsin: they cleavetheir substrates only at arginyl (never lysyl) bonds, andeven then they specifically cleave just one or two of themany arginyl bonds in their target molecules (Bode et al.,1997). Clotting proteases are denoted by adding ‘a’ to theRoman-numeral factor name; for example, activation ofthe zymogen factor X produces the active protease factorXa. Similarly, the conversion of prothrombin to itsprotease, thrombin, can be written (and often is) as theconversion of factor II to factor IIa.

In contrast to the family characteristics of the clottingzymogens, the cofactors are a diverse group. A cofactor is aprotein that itself has no active catalytic site, but is requiredfor and thereby regulates the activity of an accompanying

Table 1 Names, functions and locations of blood coagulation proteins

Common name Common alternative name Infrequent or archaic name Function (location)

Tissue factor Thromboplastin CD142, factor III Cofactor, initiator (subendothe-lium)

Factor XII Hageman factor Protease zymogen (plasma)Factor XI Plasma thromboplastin

antecedent (PTA)Protease zymogen (plasma)

Factor X* Stuart factor Protease zymogen (plasma)Factor IX* Christmas factor Protease zymogen (plasma)Factor VIII Antihaemophilic factor Cofactor for factor IXa in factor X

activation (plasma)Factor VII* Proaccelerin Protease zymogen (plasma)Factor V Labile factor Cofactor for factor Xa in pro-

thrombin activation (platelets,plasma)

Prothrombin* Factor II Protease zymogen (plasma)Fibrinogen Factor I Fibrin precursor (plasma)Factor XIII Fibrin-stabilizing factor Zymogen of transglutaminase

(platelets, plasma)Thrombomodulin Cofactor for thrombin in protein C

activation (endothelial surface)Protein C* Protease zymogen (plasma)Protein S* Cofactor for activated protein C

(plasma)Antithrombin III Antithrombin Heparin cofactor Protease inhibitor (plasma)Tissue factor pathway

inhibitor (TFPI)Extrinsic pathway inhibitor

(EPI); lipoprotein-associ-ated coagulation inhibitor(LACI)

Protease inhibitor (platelets,plasma, endothelial surface)

*Vitamin K-dependent proteins.

Blood Coagulation

2

protease, which is ultimately responsible for catalysis. TFis a cofactor that binds factor VII in the initiating event ofclotting, and is required for the proteolytic activity of theformed protease factor VIIa. It is a relatively small integralmembrane protein possessing a short C-terminal cytoplas-mic domain, one transmembrane domain and a largerextracellular domain. It is expressed in many cell types, butnot normally in blood cells or endothelial cells. Anothercritical regulatory membrane protein cofactor is thrombo-modulin, which resides on the vascular surface ofendothelial cells. It functions as a special cofactor tothrombin, diminishing the enzyme’s procoagulant actionon fibrinogen or platelets, and greatly augmenting itsactivity on protein C, which initiates a negative, antic-oagulant, pathway. The other two cofactors, factors VIIIand V, are found in the plasma and platelets, and they playcentral regulatory roles in controlling the activity of theirrespective proteases (factors IXa and Xa). They are closelyrelated, very large molecules, and are synthesized asinactive, or almost inactive, precursors. They are convertedto the active cofactors (VIIIa and Va) by the proteolyticaction of thrombin.

Although a number of protease inhibitors of the plasmacan inhibit clotting enzymes, just two are known to beimportant in regulating coagulation. Antithrombin III(ATIII) is a member of the serpin class of inhibitors, and itschief target enzymes are thrombin and factor Xa. Hetero-zygous, partial, deficiency greatly increases the risk ofthrombosis, and homozygous deficiency in humans isalmost certainly lethal, having never been described. Theother inhibitor, tissue factor pathway inhibitor (TFPI), isprobably also a major regulator, and homozygous expres-sion of a mutated defective inhibitor in mice is lethal to theembryo. TFPI is not an ordinary serpin, and has separateinhibitory domains for two clotting enzymes, factor Xaand the TF–VIIa complex (Broze, 1995).

Synthesis of plasma clotting proteins

The hepatocytes of the liver are the major site of synthesisfor probably all the plasma clotting proteins. Some areknown to be synthesized to some extent in other tissues –particularly prothrombin, and possibly factor VIII. FactorVIII is also complicated in its being transported in theblood bound to von Willebrand factor. This protein issynthesized mainly in the endothelial cells, and it too has acentral function in haemostasis, playing a major role in theinitial adhesion of platelets to proteins of the damagedvessel wall. The platelet a granules also contain varyingamounts of some plasma coagulation proteins: factors XI,VIII, V, fibrinogen and TFPI. However, only in the case offactor V has the platelet protein been clearly demonstratedto play a key role beyond that performed by the plasmaprotein. The plasma levels of the clotting proteins remain

essentially constant, with no evidence for significantinductive or repressive control of expression levels.

The Cascade

Initiation

TF is the physiological initiator of blood coagulation.However, an alternative pathway of initiation can bedemonstrated experimentally that involves factor XII andits interaction with negatively charged, often nonphysio-logical, surfaces. Examples are glass and other silicates,ellagic acid, sulfated lipids and sulfated polysaccharides.Regardless of whether or not such initiation occurs to someextent in vivo, the key observation is that people who lackfactor XII are clinically normal. Thus if this pathway,sometimes called the intrinsic pathway, does normallyfunction, its contribution to haemostasis is certainly notmajor (but see Colman and Schmaier, 1997). In contrast, adeficiency of TF has never been observed in humans, and isprobably lethal. In studies of transgenic mice, the largemajority (more than 80%) of TF-/- mouse embryos diefrom haemorrhage into the yolk sac around day 10 ofembryogenesis, at the time the vascular system begins toform and circulation starts (Bugge et al., 1996; Toomeyet al., 1996).

Figure 1 shows the major features of system initiation.The initiating complex (Banner et al., 1996), formed whenthe blood comes into contact with TF (TF–VII), is azymogen complex and has no measurable proteaseactivity, but physiologically a trace of factor VIIa (lessthan 1% of the factor VII level) is always present in plasma(Morrissey et al., 1993). This enables a trace of TF–VIIa tobe formed upon the appearance of TF, sufficient to start thefeedback loop of factor Xa generation and TF–VIIactivation.

TF + VII

TF – VII

TF – VIIa

IXa

X Xa

IX

VIIIa PL– PL–

Figure 1 The initiation of clotting by tissue factor (TF): parallel pathwaysto factor Xa formation, and the feedback role of factor Xa in TF–VIIageneration. Blue arrows denote the action of an enzyme (e.g. TF–VIIa, IXa)in catalysing the reaction pointed to; green arrows indicate the proteolyticreaction being catalysed. Species shown beside an arrow (here, VIIIa andanionic phospholipid, PL2 ) denote required cofactors.

Blood Coagulation

3

The early stages are complicated because TF–VIIaactivates two plasma zymogens, factors X and IX,producing parallel pathways for the formation of factorXa. At high concentrations of TF such as those used inclinical screening tests like the prothrombin time, sufficientTF–VIIa is rapidly formed to allow direct activation offactor X to Xa. Under these conditions adequate factor Xageneration does not depend on factors VIII and IX.However, at low TF levels, direct factor X activation byTF–VIIa is slower, and under these conditions, whichprobably correspond more closely with the situation invivo, the parallel pathway becomes the major route forfactor Xa generation, dependent on factor IX and itscofactor, factor VIII. (The requirement for the activatedcofactor form of factor VIII is considered in more detailbelow, under Positive Feedback Controls.) From thesevere bleeding seen in haemophilia A and B, which arisefrom deficiencies of factors VIII and IX, it is clear that thesecofactors are critical to normal haemostasis.

Thrombin

Once formed, factor Xa goes on to activate prothrombin,generating the enzyme thrombin. Just as factor IXarequires activated factor VIII (VIIIa) as a cofactor in theactivation of factor X, so factor Xa requires a cofactor inprothrombin activation – in this case, factor Va. Bothreactions also require anionic phospholipid. Factors VIIIand V are closely related in both structure and function,and both are subject to essential regulatory feedbackcontrol (both positive and negative) by thrombin. Inthe normal physiological setting, prothrombin activa-tion occurs on activated platelets, which provide both

anionic phospholipid and factor Va (Figure 2) (see alsoLocalization).

Thrombin is the final protease produced in clotting andhas diverse functions, some of which have been mentioned:

1. It is the major feedback activator of factors VIII and V,converting the inactive, or near-inactive, plasmaspecies of these proteins to the active cofactors thatregulate their respective proteases.

2. It forms fibrin by removing small peptides proteoly-tically from the peptide chains of fibrinogen.

3. In concert with the endothelial protein thrombomo-dulin, it initiates the anticoagulant protein C pathway(see Negative Feedback Controls).

4. It is a major agonist, or activator, of platelets and thusplays a central role in linking the platelet and clottingsystems in haemostasis.

5. Numerous reports exist of thrombin affecting fibrino-lysis function, both positively and negatively, and theoverall physiological picture is particularly confused.Examples include a thrombin-activatable fibrinolysisinhibitor (Nesheim et al., 1997) and the thrombin-inducible expression in endothelial cells of tissueplasminogen activator (Emeis et al., 1997), urokinase(Shatos et al., 1995) and plasminogen activatorinhibitors (Wojta et al., 1993).

An additional role for thrombin has also been proposed:the activation of factor XI. It used to be thought that thisprotein was activated during clotting by factor XIIa (seeInitiation). However, whereas factor XII has no majorfunction in haemostasis, factor XI does: most peopledeficient in this protein do bleed to some extent, althoughthe clinical picture is rather less severe than in haemophiliaA and B. For this reason alternative mechanisms ofactivation of factor XI have been sought (Broze andGailani, 1993). Although thrombin does activate factor XIin pure experimental systems, the physiological signifi-cance is unknown.

Fibrin

Thrombin converts the soluble plasma protein fibrinogento a molecule that polymerizes to form the fibrin gel, showndiagramatically in Figure 3. Fibrinogen is a dimer of atrimer, containing two Aa chains, two Bb chains and two gchains, with the N-termini of all six disulfide-linked in acentral domain. Thrombin (‘IIa’ in Figure 3) proteolyticallycleaves small peptides (fibrinopeptides A and B) from theN-termini of the Aa and Bb chains, forming the fibrinmonomer unit. Polymerization involves noncovalent,mainly ionic, interactions between the central domain ofthe molecule and the larger terminal domains of twoneighbouring molecules, forming an initial two-strandpolymer with a half-staggered overlap (Figure 3). Initially

Prothrombin Thrombin

–

– –

–

–– –

–

–

–

–

–

–

–– – –

––––

–

–

––

–

–

–

–

–

–

––

––

–

VaXaXaVa

XaVa VaXa

VaXa

VaXa

VaXaVaXa

VaXa

VaXa

XaVa

XaVa

XaVa

VaXa

P

–

Figure 2 Prothrombin activation. Activated platelets (P) provide thenecessary cofactors, anionic phospholipid (2 ) and factor Va, required formaximum efficiency of the proteolytic action of factor Xa on prothrombin.

Blood Coagulation

4

quite weak, fibrin polymer is later crosslinked by the actionof factor XIIIa to form a stronger and more stablestructure. Crosslinking also makes fibrin more resistantto the fibrinolytic enzyme plasmin. Factor XIIIa is itselfformed from a zymogen by the action of thrombin.

Localization

The most important regulatory feature of the clottingsystem is that clot formation is confined to sites of bloodvessel damage. Only in pathological states is systemicactivation and fibrin formation seen. This is calleddisseminated intravascular coagulation (DIC), and is oftencaused by the systemic release of TF into the circulation,by a wide variety of mechanisms that include major organinjury.

Activated platelets

The chief agent in localization is the activated platelet.Remember that platelet adhesion and activation areinitiated by components of the subendothelium at thesame time as the clotting system is initiated by TF. Plateletsadhere to several proteins of the damaged vessel wall,where they are activated concomitantly, and then adhere toand activate one another to produce platelet aggregates. Asfar as clotting is concerned, the key event in plateletactivation is the translocation of anionic phospholipidfrom the inner leaflet of the platelet cell membrane to theoutside. Quiescent cells, including platelets, activelyrestrict aminophospholipids, including the key anionicphospholipid, phosphatidylserine (PS), to the inner leafletof their cell membranes by means of an adenosinetriphosphate-dependent aminophospholipid translocase(Bevers et al., 1996), but this polarized distribution is lost

upon cell activation and PS appears on the outside. PS ofthe activated cell surface binds Ca21 (present in theplasma), and this enables the binding of the vitamin K-dependent proteins of clotting.

Vitamin K-dependent factors, coumarinanticoagulants

Six coagulation proteins require vitamin K for an essentialposttranslational modification that is closely related totheir binding to PS: factors VII, IX, X and prothrombin inthe cascade leading to thrombin generation, and proteins Cand S in the anticoagulant protein C pathway. Theendoplasmic reticulum of hepatocytes and many othercells contains a vitamin K-dependent carboxylase whichmodifies all the glutamic acid residues in the immediate N-terminal domains of these proteins, up to about amino acidresidue 50. Depending on the protein, there are 9 to 12modified glutamic acids in this domain, and they tend tocome in pairs. All subsequent glutamic acid residuesremain unmodified through to the C-terminus.

The product of carboxylation is g-carboxyglutamic acid,denoted by the abbreviation Gla. The Gla domains of theseproteins bind Ca21 , which probably serves as a bridge toanionic phospholipids (Figure 4). As normal cells do nothave significant amounts of PS on their outer surfaces, inthe vasculature this binding should be restricted almostexclusively to activated platelets. (Other mechanisms arealso probably involved in the binding of Gla domains tophospholipid, and additionally several of these proteinscontain other Ca21 -binding sites, but these are not withinthe scope of this article.)

Vitamin K is a fat-soluble quinone produced by thebacteria of the gut and by many green vegetables. The daily

IIa

Fibrinogen

IIa

A A

B B

IIaIIa

α

βγ

α

βγ

Fibrin monomer

+ Fibrinopeptides A and BFibrin

Figure 3 Fibrin formation and initial polymerization. Thrombin (IIa)cleaves fibrinopeptides from the central N-termini of the Aa and Bb chainsof fibrinogen. The ‘fibrin monomer’ formed polymerizes to form an initialhalf-staggered two-chain protofibril of fibrin.

Gla

–

–

CH2CHCH

NH

COCH

CH2CH–OOC

Ca2+

–OOCCOO–

COO–

CO

NH

Protein chain

Outer leafletof membrane

Gla

–

–

–

–

–

–

Figure 4 Role of g-carboxyglutamic acid (Gla) in the Ca21 -dependentbinding of vitamin K-dependent factors to anionic phospholipid. Anionicheadgroups (e.g. phosphatidylserine) are shown as red circles (2 ), neutralheadgroups (e.g. phosphatidylcholine) as pink. The protein’s peptidechain, on the right, has a pair of Gla residues in sequence.

Blood Coagulation

5

requirement is extremely low and deficiency is rare.However, antagonists to vitamin K – the coumarins, orcoumadins – are used frequently for long-term antic-oagulant therapy after thrombotic episodes such as deepvein thrombosis, pulmonary embolism, strokes and heartattacks. They include warfarin and dicoumarol. Inaddition to therapeutic use they are commonly used asmouse and rat poisons.

Positive Feedback Controls

The clotting system includes many positive feedbacks, inwhich an enzyme formed later in the cascade (factor Xa orthrombin) feeds back and proteolytically activates anearlier zymogen or cofactor precursor. Some are ofdoubtful importance, but the following ones are major:(1) factor Xa activates the TF–VII zymogen complex toTF–VIIa (Figure 1); (2) thrombin activates the cofactorprecursors VIII and V to their active states; (3) thrombinactivates platelets, providing anionic phospholipid andplatelet factor Va (Figure 2).

There is no immediately obvious reason for the existenceof several positive feedback mechanisms. Why, forinstance, are factors VIII, VII and V not synthesized asthe already active molecules, ready to perform theirfunction immediately upon activation of the system? Oneproposal is that positive feedback, in concert withinhibition of the feedback enzymes, provides thresholdbehaviour. In such systems, a certain threshold stimulussize should exist that depends on the balance of the kineticsof feedback action and enzyme inhibition. For a stimulusbelow the threshold, no significant feedback will occur andno clotting response will be generated, whereas above thethreshold the feedback will occur and a fully amplifiedresponse will be generated. Although the theoreticalbehaviour is clear from mathematical analysis (Beltramiand Jesty, 1995), the proposal has not yet been testableexperimentally.

Negative Feedback Controls

Negative feedback mechanisms are responsible for theinactivation of three activated species generated duringclotting. Two major ones are known – one inhibitory, oneenzymic – and are shown in Figure 5.

Tissue factor pathway inhibitor

TFPI inhibits factor VIIa in the TF–VIIa complex by atwo-step mechanism which requires the initial combina-tion of TFPI with factor Xa. The resulting TFPI–Xacomplex is the species responsible for the inhibition of TF–VIIa. The requirement of TFPI for initial reaction with

factor Xa (the negative feedback) means that TF–VIIamust be present for a finite time before it is inactivated.Thus this inhibitor allows a significant pulse of TF–VIIaactivity (Broze, 1995).

Protein C

In contrast to TFPI control, which is inhibitor based, theprotein C pathway relies on the enzyme-catalysed inactiva-tion of cofactors. Thrombin, in addition to activatingplatelets and forming fibrin, forms a complex withthrombomodulin on the surface of the endothelium andthereby initiates a critical anticoagulant pathway (Esmon,1995). The thrombin–thrombomodulin complex activatesprotein C to an active protease (activated protein C, orAPC), which in company with its cofactor protein Sinactivates the key regulatory cofactors, factors VIIIa andVa (Figure 5). Like TFPI control, because this mechanismrequires thrombin to be formed first it ensures a pulse ofcoagulant response over the period during which thecofactors are in their active state. The importance of theprotein C pathway is clear from the often fatal systemicthrombosis seen in newborns with homozygous protein Cdeficiency. Recently, a much more common genetic defecthas been discovered in one of the target cofactors, factor V,in which the factor Va generated, while having normalcofactor activity, is resistant to inactivation by APC andcauses a significantly increased risk of thrombosis (Dahl-back, 1997). This mutation, called FV(R506Q) or factor VLeiden, is caused by a single base change in the factor Vgene that changes Arg to Gln at one of the sites in factor Vathat are cleaved by APC. It occurred very recently inevolutionary time and is essentially restricted to cauca-

TFPI+Xa

TF + VII

TF – VII

TF – VIIa

IXa

X

IX

TFPI – Xa

APC

VIIIa

Protein S

Thrombin ProthrombinThrombomodulin

Protein S

Protein C

Va

Figure 5 Negative feedback controls (red arrows). (1) Inhibition of tissuefactor (TF)–VIIa by tissue factor pathway inhibitor (TFPI) first requiresreaction with factor Xa, the TFPI–Xa complex formed being the inhibitor ofTF–VIIa. (2) The protein C pathway, initiated by thrombin in the presenceof thrombomodulin, entails the proteolytic inactivation of factors VIIIa andVa by activated protein C (APC). Protein S plays a cofactor role.

Blood Coagulation

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sians. But in this group it is a major risk factor, theheterozygous state occurring in 5–15% of the population,and being found in 20–60% of cases of venous thrombosis.

Inhibitors

Antithrombin III

ATIII is a member of the serpin family of proteaseinhibitors and forms a 1:1 complex linked between aspecific Arg residue of the inhibitor and the active site Serof the target protease. Although all the clotting proteasesare inhibited at measurable rates by ATIII in thelaboratory, the enzymes most rapidly inhibited (i.e. themajor physiological targets) are thrombin and factor Xa.ATIII is present in the plasma at levels in large excess overits targets, at a concentration of about 4 mmol L2 1. Even ifthe clotting zymogens were completely converted toenzymes – which never occurs, even in the most severeDIC – their total concentration would amount to onlyabout one-third of the ATIII available. However, despitethere being no scarcity of ATIII, heterozygous geneticdefects that cause 40–60% reductions in ATIII concentra-tion none the less greatly increase the risk of thrombosis.The reason is a kinetic one, the rates of inactivation of thetarget enzymes being proportional to the inhibitorconcentration. It is likely that as well as effecting a‘mopping up’ function in inactivating enzymes generatedduring clotting, ATIII provides a permanent inhibitorycapacity that regulates the proposed system thresholddescribed under Positive Feedback Controls.

The incidence of heterozygous ATIII deficiency in thegeneral population has been variously estimated atbetween 0.02 and 0.05%. However, although this is lessthan the 1 in 100 incidence of factor V Leiden, the risk ofthrombosis is much greater. Approximately two-thirds ofpeople with heterozygous ATIII deficiency will suffer athrombosis during their lifetime, accounting for 2–5% ofcases of venous thrombosis (Hirsh et al., 1989).

Heparin

Heparin is a highly sulfated polysaccharide that isfrequently used for anticoagulant therapy in the hospitalsetting. Unlike coumarin therapy (see Vitamin K, above),heparin therapy is relatively easily controlled, and has thebenefit that its anticoagulant effect is immediate, althoughit does have to be infused. Heparin is the anticoagulant ofchoice in many hospital settings, including the immediatetreatment of acute thrombosis (heart attack, thromboem-bolic stroke, pulmonary embolism, etc.), and is also usedfor anticoagulation in surgical procedures that entail asignificant risk of postoperative thrombosis. It exerts itsanticoagulant action by greatly increasing the rate of

inhibition of clotting proteases by ATIII, the extent ofacceleration (and hence the degree of anticoagulation)being controlled by the heparin dose. Both natural andsynthetic variants of heparin exist. Normal heparin,usually isolated from pig mucosa, is a mixture ofpeptidoglycans of varying sulfation and length, and offairly high molecular weight (approximately 8–12 kDa). Ithas a fairly broad spectrum of acceleratory effects on theinhibition of both thrombin and factor Xa by ATIII. Morestructurally specific heparin formulations are now avail-able of lower molecular weight, more specific properties,and considerably higher cost. Most of these preparationsaccelerate the inhibition of factor Xa by ATIII more thanthat of thrombin, but the differences are variable and thereis little evidence that selective targeting of factor Xa isbeneficial per se in regard to anticoagulant function.However, they do have other major advantages: they areroutinely and effectively administered intramuscularly;they have increased and more reliable bioavailability, atleast in part because they are less subject to inactivation byantiheparin (chiefly platelet-derived) agents in the blood;they have substantially longer half-lives; and they aresufficiently predictable (particularly with regard to bioa-vailability) to be used without any monitoring of plasmalevels (Hirsh, 1998).

Tissue factor pathway inhibitor

A human deficiency of TFPI has not been described, butstudies in transgenic mice suggest that it is of keyimportance in regulating system initiation (see NegativeFeedback Controls). It is an unusual three-domaininhibitor with two target enzymes. In order to inhibit theTF–VIIa complex, TFPI must first combine with andinhibit factor Xa, in a reaction involving the middledomain. The TFPI–Xa complex formed then inactivatesthe TF–VIIa complex in a reaction involving the N-terminal domain. The final product is a TF–VIIa–TFPI–Xa complex (Broze, 1995). The third (C-terminal) domain,although homologous to the other two, apparently has noantiprotease activity, but is none the less required for fullbiological TFPI activity.

The plasma concentration of TFPI is very low, at 1–3 nmol L2 1, but rather more exists bound to the endothe-lium, amounting to about three to four times the plasmacontent. Additionally TFPI is released from the a granulesof platelets upon their activation. It seems likely that theplasma TFPI may be of minor importance, with these localsources playing the prominent role in TF–VIIa regulation.

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