Chapter 1 Overview of is

8/8/2019 Chapter 1 Overview of is

1/35

Editors: Colman, Robert W.; Clowes, Alexander W.; Goldhaber, SamuelZ.; Marder, Victor J.; George, James N.Title: Hemostasis and Thrombosis: Basic Principles and ClinicalPractice, 5th Edition

Copyright 2006 Lippincott Williams & Wilkins

> Table of Contents > Part I - Basic Principles of Hemostasis and Thrombosis > Chapter 1 -Overview of Hemostasis

Chapter 1

Overview of Hemostasis

Robert W. Colman

Alexander W. Clowes

James N. George

Samuel Z. Goldhaber

Victor J. Marder

Humans have evolved an intricate hemostatic system that is designed tomaintain blood in a fluid state under physiologic conditions, but that is primed toreact to vascular injury in an explosive manner to stem blood loss by sealing thedefect in the vessel wall. Thrombosis may occur if the hemostatic stimulus isunregulated, either because the capacity of inhibitory pathways is impaired or,more commonly, because the capacity of the natural anticoagulant mechanism isoverwhelmed by the intensity of the stimulus. Thrombosis may be regarded as anaccident of nature that has not had time to adapt through the lengthy process ofevolution to the advances of modern medicine, which allow patients to survivethe hemostatic challenge of major surgery and trauma but leave them vulnerableto venous thrombosis.

The normal vascular endothelium maintains blood fluidity by inhibiting bloodcoagulation and platelet aggregation and promoting fibrinolysis (see Fig. 1-1). Italso provides a protective barrier that separates blood cells and plasma factorsfrom highly reactive elements in the deeper layers of the vessel wall. Thesecomponents include adhesive proteins such as collagen, fibronectin, laminin,vitronectin, and von Willebrand factor (VWF), which promote platelet adhesion,and tissue factor, a membrane protein located in smooth muscle, fibroblasts, and

macrophages, that triggers blood coagulation. After the vessel is severed, itconstricts, thereby diverting blood from the site of injury, and the shed blood isexposed to these subendothelial structures that stimulate hemostatic plugformation by promoting platelet adhesion and aggregation and by activatingblood coagulation. After platelets are stimulated by subendothelial collagen, theyexpose and assemble membrane glycoprotein (GP) IIb and GP IIIa, which canthen bind fibrinogen and VWF, cofactors for platelet recruitment andaggregation. Secretion of proteins from is mediated by thromboxane synthesis,phosphorylation of specific proteins, and intracellular calcium translocation.Protein cofactors such as factor V, secreted by platelets or derived from plasma,serve as a nidus for assembling enzymecofactor complexes on the platelet

surface, thereby accelerating factor X and prothrombin activation. The result isthrombin formation, which augments its own production manyfold by converting

Pgina 1 de 35Ovid: Hemostasis and Thrombosis: Basic Principles and Clinical Practice

08/09/2010mk:@MSITStore:C:\Users\Propietario\Documents\FreeRapid-0.83u1\Descargas\Hem...


2/35

factors V and VIII into activated cofactors and stimulating platelet secretion.

This explosive cellular and molecular reaction is modulated by endothelial cellelaboration of antithrombotic lipids (prostacyclin, or PGI2), proteins

(thrombomodulin), inorganic compounds [nitric oxide (NO)], and polysaccharides(heparan); by surface-binding enzymes such as adenosine diphosphatase(ADPase) (CD39); and by several plasma protease inhibitors, most importantlyantithrombin (AT) III for factors IXa, Xa, and thrombin; C1 inhibitor, for thecontact system enzymes factor XIIa, 1-antitrypsin for factor XIIf, and 2-macroglobulin kallikrein; for factor XIa; and 2-macroglobulin as a general

backup. A major substrate of thrombin is fibrinogen, which, after initialhydrolysis, forms fibrin monomers that then undergo spontaneous polymerizationto form the fibrin clot. Covalent cross-linking by the thrombin-activated enzymefactor XIIIa increases the resistance of the clot to fibrinolysis.

Plasminogen, a plasma zymogen, is converted to plasmin by two plasminogenactivators elaborated by endothelial cells. The process is modulated by at leastthree plasminogen activator inhibitors. Plasmin normally does not act on

fibrinogen in solution because of the presence of However, plasmin on thesurface of the fibrin clot is protected from the inhibitor, and fibrinolysis occurswith the formation of fibrin degradation products. Second, plasminogen activatorinhibitor (PAI-1) released by endothelial cells and by platelets neutralizes tissue-type plasminogen activator (tPA) and prevents early lysis of clot. Third, thrombinactivates a carboxypeptidase B proenzyme, called thrombin-activated fibrinolyticinhibitor(TAFI), that impairs fibrinolytic degradation of fibrin strands. Therefore,with more active coagulation and with more complete conversion of prothrombinto thrombin, not only is more fibrin formed from fibrinogen, but such fibrin isfurther protected from fibrinolysis by TAFI. Dissolution of the clot paves the wayfor the deposition of collagen, formation of fibrous tissue, and wound healing.

ENDOTHELIUMNormal endothelium (see Fig. 1-2) maintains blood fluidity by producinginhibitors of blood coagulation and platelet aggregation, modulating vasculartone and permeability, and providing a protective envelope, thereby separatinghemostatic blood components from reactive subendothelial structures.Endothelial cells synthesize and secrete basement membrane and extracellularmatrix, which contain adhesive proteins, collagen, fibronectin, laminin,vitronectin, and VWF. The endothelium inhibits blood coagulation by synthesizingand secreting thrombomodulin and heparan sulfate onto its surface; modulatesfibrinolysis by synthesizing and secreting tPA, urokinase plasminogen activator

(uPA), and plasminogen activator inhibitors; inhibits platelet aggregation byreleasing PGI2 and NO; and regulates vessel wall tone by synthesizingendothelins, which induce vasoconstriction, and PGI2 and NO, which produce

vasodilation.

Defective vascular function can lead to abnormal bleeding if the endotheliumbecomes more permeable to blood cells, if the vasoconstrictive response isimpaired because of structural abnormalities of the vessel wall or extravascularsupporting tissues, or if physiologic fibrinolysis is not controlled by the normalproduction of plasminogen activator inhibitor. Bleeding associated withendothelial injury may be mediated by immune complexes and viruses (1,2).Proteolytic enzymes released from leukocytes in inflammatory states perturbendothelial cells and alter connective tissue proteins and also could contribute topetechial hemorrhage in vasculitic disorders (3,4). Attenuation and fenestration




3/35

of the vascular endothelium may contribute to the hemorrhagic manifestations

of idiopathic thrombocytopenia purpura, which may respond to prednisonetherapy promptly, even before a detectable rise in platelet count (5).

Endothelial cells lose their nonthrombogenic protective properties after they arestimulated by enzymes such as thrombin, hypoxia, fluid shear stress, oxidants;cytokines such as interleukin-1, tumor necrosis factor, and -interferon;synthetic hormones such as desmopressin acetate and endotoxin. Synthesis oftissue factor and PAI-1 is induced and the concentration of surface-boundthrombomodulin is reduced by cytokines and endotoxin, whereas desmopressinacetate results in the release of high-molecular-weight VWF multimers fromWeibel-Palade bodies that may augment platelet adhesion to the injured vesselwall. Endothelial cells contain receptors, termed integrins, of the very lateantigen (VLA) type, that allow binding of fibronectin (1 1), collagen (V 1),and laminin (3 1, 6 1) and of the cytoadhesive type, notably the vitronectinreceptor (2 3) (6). The stimulated endothelial cell synthesizes chemokines,

such as monocyte chemoattractant protein, interleukin-8, regulated on activationnormal T-cell expressed and secreted (RANTES), and GRO. Endothelial cells alsocontain intercellular adhesion molecules (see Chapter 45) such as ICAM-1 andICAM-2, and VCAM-1, which act as counterreceptors for leukocyte integrins

(7,8). Before tight adhesion to the endothelium, platelets and leukocytes roll, aninteraction mediated by E selectin and P selectin (which is stored in Weibel-

P.4

FIGURE 1-1. Overview of hemostasis. ADP, adenosine diphosphate;ADPase, adenosine diphosphatase; Epi, epinephrine; TxA2, thromboxane A2;PAI, plasminogen activator inhibitor; PGI2 prostacyclin; NO, nitric oxide;

VWF, von Willebrand factor.




4/35

Palade bodies).

Endothelium is heterogeneous, both metabolically and structurally (9). Forinstance, angiotensin-converting enzyme apparently is synthesized by most

endothelial cells but is principally synthesized by aortic endothelium, not bycardiac microvessel endothelium; on the other hand, thromboxane is notsynthesized by most endothelial cells but is synthesized by pulmonary arterialendothelium. Endothelial cell turnover is low under resting conditions but varieswith location. At sites of hemodynamic stress and injury, proliferation isespecially increased. Endothelial cells contain a full array of contractile proteins,but of special importance are the stress fibers involved in cell attachment andmaintenance of endothelial junctional apposition by vascular endothelial (VE)cadherin and plateletendothelial cell adhesion molecule (PECAM). Endothelialcells contain caveolae that concentrate glycosylphosphatidyl-linked proteins suchas the urokinase receptor.

Endothelial cell permeability is influenced by the functional adaptations that jointhe cells to their neighbors. Macromolecules pass across the endothelium intothe vessel wall through

patent intercellular junctions, by endocytosis, and through the transendothelialpores. Vessel permeability is increased by vasodilation, by induction of severethrombocytopenia, and by high doses of heparin. Spontaneous bleeding observedwith a low platelet count or after heparin infusion may be induced by increasedvascular permeability.

Increased fenestration and attenuation of endothelium may account for the loss

of barrier function associated with experimental thrombocytopenia. Thethrombocytopenia-induced extravasation of erythrocytes, manifest clinically aspetechiae, occurs principally through postcapillary venular interendothelial

FIGURE 1-2. Thromboresistant properties of endothelium. The endothelialcells express adenosine diphosphatase (ADPase), and synthesizeprostacyclin (PGI2) thrombomodulin, heparan, and plasminogen activators,

all of which inhibit hemostasis (and thrombus formation) and contribute tothe maintenance of vascular patency. ADP, adenosine diphosphate; AT,antithrombin; NO, nitric oxide; tPA, tissuetype plasminogen activator; uPA,urokinase plasminogen activator.

P.5




5/35

channels. The loss of endothelial barrier function, associated with extremeplatelet depletion, may be related to a loss of serotonin and norepinephrinedelivered by platelets to the microvascular milieu, as exogenous sources ofeither amine prevent petechial formation in severely thrombocytopenic animalsor failure of platelets to plug gaps at intracellular junctions between retractedendothelial cells.

Endothelial cells are highly negatively charged, a feature that may repel thenegatively charged platelets. This anionic surface, as well as otherantithrombotic properties of endothelium, could be important in limiting theintravascular extension of the hemostatic reaction induced by vessel injury (10).Therefore, synthesized and released from endothelial cells close to the site ofhemostatic plug formation, could inhibit intravascular platelet aggregation(11,12,13 and 14). Thrombomodulin and heparan sulfate, the two endothelialsurfacebound thrombin inhibitors, could limit the intravascular spread offibrin beyond the confines of the hemostatic plug (15,16,17,18 and 19). Heparansulfate, a glycosaminoglycan, activates AT and, therefore, catalyzes theinhibition of thrombin and factor Xa. Endothelial cellassociated ADPase

(CD39) cleaves adenosine diphosphate (ADP) to adenosine monophosphate(AMP), thereby modulating this stimulatory agonist (20).

Thrombomodulin binds thrombin and inhibits the ability of the enzyme to cleavefibrinogen and activate platelets and factors Va and VIIIa. Thrombomodulin alsomarkedly enhances thrombin's ability to activate protein C. Protein C binds toendothelial cell protein C receptor, which enhances its activation (21). Protein C,in turn, inactivates factors Va and VIIIa and enhances fibrinolysis, probably bybinding an inhibitor of plasminogen activators (22). Thrombin bound tothrombomodulin also is inactivated by circulating AT, a step accelerated byheparan sulfate. Protein C activity is controlled by protein C inhibitor (PAI-3)

and inhibitor and is stimulated by protein S, a cofactor (23,24). Protein S, inturn, is controlled by C4b, which forms a complex with it, thereby preventing itsaction (25). The enhancement of fibrinolysis by protein C also may depend onprotein S (26). Therefore, the binding of thrombin with thrombomodulin resultsin the loss of the coagulant effect and in the enhancement of thrombin's abilityto activate protein C and therefore to inhibit thrombogenesis.

The synthesis of PGI2 by endothelial cells is stimulated by thrombin and other

stimuli, including epinephrine and trauma (27). Other agonists, includinghistamine, adenosine triphosphate (ATP), bradykinin, and acetylcholine,stimulate endothelial cell guanylate cyclase, raising the levels of intracellularcyclic 3,5-guanosine monophosphate (cGMP), which results in the

synthesis of NO. Therefore, endothelial cells exposed to appropriate stimulisynthesize and release two distinct mediators of vasodilation and inhibition ofplatelet function (28). Stimulated endothelial cells also synthesize a group ofpeptides known as endothelins that have counterregulatory properties, includingvasoconstriction (29). Endothelial cells also elaborate plasminogen activators,which, in the presence of fibrin, promote fibrinolysis and can aggravate ableeding tendency in susceptible patients. The bleeding tendency can becontrolled by synthetic and natural fibrinolytic inhibitors (see Chapter 79). PAI-1also is elaborated with a different time course and in response to differentstimuli (30). Deficiency of PAI-1 causes a bleeding tendency, an indication thatunopposed physiologic fibrinolysis disrupts the hemostatic balance.

Endothelial cells are stimulated by cytokines and other mediators to mount aprocoagulant response characterized by an increased synthesis and release of




6/35


7/35

in the formation of a bridge from platelets to subendothelial connective tissue,although this may be an oversimplification.

The importance of these events is illustrated by the occurrence of hemorrhage inBernard-Soulier disease, in which patients lack GP Ib/IX, or in von Willebranddisease, in which VWF is decreased or defective. At high-shear rates (i.e., > 800per second), comparable to those found in arteries in the microvasculature,plasma VWF is required for normal adhesion of platelets to subendothelium,perhaps as a bridge between platelets and the fibrillar surface (38). At low-shearrates, adhesion of platelets to subendothelium is normal in patients with thesedisorders, suggesting that other proteins can substitute for the action of VWF, atleast to some extent. Signaling through GP Ib/IX/V activates GP IIb/IIIa withoutinvolvement of other receptors (39).

Other adhesive events that are involved include interactions of collagen with theplatelet GP Ia/IIa (40) followed by activation of intracellular signaling pathwaysby platelet GP VI (41). Abnormalities in either of these platelet receptors for

collagen cause bleeding defects.Once adherent to subendothelium, platelets spread out on the surface, andadditional platelets, delivered by the flowing blood, adhere first to the basallayer of adherent platelets and eventually to one another, forming a mass ofaggregated platelets. A crucial event in platelet aggregation is induction of achange in the disposition of surface membrane GP IIb/IIIa (42), which acquiresthe capacity to bind fibrinogen, as well as VWF, fibronectin, and vitronectin (43).Fibrinogen appears to be the most important in aggregation by virtue of itsdivalent structure, possibly allowing it to form a bridge from platelet to plateletand thereby mediating aggregation.

The current paradigm for bidirectional signaling has been well summarized (44).Inside-out signaling from the cytoplasmic tail of this integrin to the ligandrecognition site results in conformational changes leading to increased ligandaffinity. An increase in the number of surface receptors is derived from fusion ofthe platelet membranes with the plasma membranes. Shuttling of GP IIb/IIIabetween these two membranes is responsible for acquisition of fibrinogen fromplasma.

Several other integrins in the platelet membrane act as receptors for adhesiveplasma proteins. These heterodimers, such as the vitronectin receptor, arepresent on the surface of both blood cells and endothelial cells (45). WhereasVWF and collagen can interact with resting platelets, fibrinogen forms a high-

affinity bond only with an integrin GP IIb/IIIa on activated platelets (46). In thecongenital disorder Glanzmann thrombasthenia, the GP IIb/IIIa complex isdeficient, and the associated defect in fibrinogen binding results in a bleedingtendency (47). Likewise, the bleeding in congenital afibrinogenemia is caused inpart by an abnormality of platelet aggregation.

Interaction with GP receptors in the platelet membrane also is a feature ofparticipation in platelet aggregation by fibronectin and thrombospondin. Theinteraction of the latter with GP IV may act to stabilize platelet aggregates.

Of the many platelet agonists whose ability to induce aggregation and secretionhas been studied in vitro, those having the greatest physiologic relevance are

the proteolytic enzyme thrombin, ADP, collagen, arachidonic acid, andepinephrine. Epinephrine is the only one of these that does not result in a

P.6




8/35

detectable change in platelet shape.

Specific receptors exist on the platelet surface for these agonists (see Fig. 1-3)(48). Many of the receptoragonist complexes interact in the plateletmembrane with coupling proteins that hydrolyze guanosine triphosphate, the Gproteins. Evidently, some interact with target proteins coupled to ion-permeablechannels in the platelet membrane, modulating ion flux, especially the inwardmovement of ionized calcium. Others are linked to protein tyrosine kinases (TK)that phosphorylate other sites on the receptor protein itself. Accompanying thesebiochemical events are visible effects, such as the disappearance of theequatorial band of microtubules that normally maintain the platelet's discoidshape, centralization of storage granules, and formation of pseudopodia.Stimulatory agonists lead to activation of phospholipase C, which cleavesphosphatidylinositol bisphosphate (PIP2) to form inositol triphosphate (IP3) anddiacylglycerol. IP3 reacts with receptors on calcium storage organelles known as

the dense tubular system, analogous to the sarcoplasmic reticulum of muscle,leading to mobilization of ionized calcium and increasing its cytoplasmicconcentration (49). Familial abnormalities in a G protein (50) and a

phospholipase C (51) have been shown to lead to mild hemorrhagic disorders.

Many processes involved in platelet activation are calcium dependent, includingphosphorylation of the light chain of myosin by a specific kinase enzyme andliberation of arachidonic acid from membrane phospholipids by the enzymephospholipase A2 (52,53). Phospholipase liberates arachidonic acid from

phosphatidylcholine and probably other phospholipids. Arachidonic acid isconverted by the enzyme cyclooxygenase to prostaglandin endoperoxides andultimately to the potent platelet agonist thromboxane as well as to stableprostaglandins such as prostaglandin The latter inhibits platelet activation and,in a negative feedback system, may serve to modulate platelet activities. A

reactive serine in cyclooxygenase is alkylated by aspirin, inactivating the enzymepermanently; this accounts for the substantial pharmacologic action on plateletsof this widely used drug. High concentrations of intracellular calcium (occurring,for example, after thrombin stimulation) lead to activation of acalciumdependent neutral cysteine protease (calpain), which may participate inremodeling of cytoskeletal proteins, cleavage of receptor proteins (54), andthrombin-induced activation of platelets.

Diacylglycerolsuch as IP3, a product of the action of phospholipase

Cactivates a ubiquitous enzyme, protein kinase C, in platelets (55). Proteinkinase C phosphorylates (among other substrates) pleckstrin, a 47-kDa proteinthat is a marker for activation of the kinase. Phosphatases provide a negative

feedback, reducing the elevation of ionized calcium by IP3 (56). Diacylglycerolmay be responsible for the alleged calcium-independent reactionsoccurring during platelet activation, or may act together with ionized calcium toactivate protein kinase C and stimulate secretion (57).

Platelets contain several classes of granules in which intracellular constituentsare sequestered, including dense bodies (containing serotonin, ATP, ADP,pyrophosphate, and calcium), -granules [containing fibrinogen, VWF, factor V,highmolecular-weight kininogen (HK), fibronectin, 1-antitrypsin, (A5)b-

thromboglobulin, platelet factor 4, and platelet-derived growth factor], andlysosomes (containing a variety of acid hydrolases) (58). Centralization of thesegranules after stimulation of platelets results from activation of the plateletcytoskeletal contractile apparatus; polymerization of filamentous actin andphosphorylation of myosin are prominent reactions in platelets responding to




9/35

receptor-mediated stimulation. In the presence of elevated cytoplasmic calcium,this leads to fusion of the granular envelope with the lining membranes ofintracellular canaliculi and to external secretion of the granule contents.

ADP is thought to react with three receptors. The first, is an ADP-operatedcalcium channel with little functional effect. P2Y1 mediates shape change byactivating phospholipase C, whereas P2Y12 decreases stimulated adenylate

cyclase activity and reduces platelet cyclic 3,5-adenosine monophosphate(cAMP) (59). Both P2Y1 and P2Y12 are required for aggregation (see Chapter 32)(60). A molecular defect in P2Y12 results in hemorrhagic defect (61). The 2-

adrenergic receptor responsible for epinephrine interaction with platelets hasbeen cloned and sequenced, and a thromboxane A 2 and PGH2 receptor has been

demonstrated in binding studies (62,63). All

these receptoragonist interactions result in unmasking of functionalfibrinogen-binding sites by outside-in signaling through G proteins.

During activation, platelets expose receptors for specific plasma clotting factors,

particularly activated factor V (Va), which may be either secreted and expressedby the platelet or bound from plasma. This acquired receptor, inconjunction with anionic phospholipids exposed on activated platelets, alsofunctions as a binding site for factor Xa and thereby provides an efficientcatalytic environment for the conversion of prothrombin to thrombin by factor Xa(64). An analogous system appears to exist for the binding of factor IXa andconversion of factor X to Xa on platelets.

Platelet activation and its effects are modulated by several regulatorysubstances, of which the most important is cAMP (65). Like virtually all otheranimal cells except human red cells, platelets contain adenylate cyclase, theenzyme that converts ATP to cAMP. Its action is powerfully stimulated by the

arachidonic acid products prostaglandin in platelets and (prostacyclin) inendothelial cells. Platelets also contain cyclic nucleotide phosphodiesterases thatcleave cAMP to AMP, modulating intracellular cAMP concentration (66). The majorcAMP phosphodiesterase in platelets, PDE3A, is inhibited by cGMP. Therefore,compounds that increase cGMP also inhibit platelet activation. cAMP stimulates aprotein kinase that mediates phosphorylation of an ATP-dependent calcium-pumping system that removes calcium from the cytosol. In sufficientconcentration, cAMP inhibits not only platelet aggregation, secretion, and shapechange but adhesion to surfaces as well.

P.7




10/35

FIGURE 1-3. Platelet function. Adhesion to endothelial cells is mediated byglycoprotein (GP) Ib, which binds von Willebrand factor (VWF) on theendothelial cells. Aggregation is mediated by GP IIb/IIIa bridged to GPIIb/IIIa on another platelet by fibrinogen. Various agonists such asadenosine diphosphate (ADP) and platelet activating factor (PAF) are

pictured as interacting with specific receptors and activating phospholipaseC, probably through G proteins. This enzyme catalyzes the cleavage ofphosphatidyl inositol bisphosphate (PIP2) to IP3, which mobilizes Ca

2+ from

the dense tubular system to activate myosin light chain kinase (MLCK),which phosphorylates myosin light chain (MLC). Ca2+ also activatesphospholipase A2 (PLA2) to release arachidonic acid from phospholipids,which is in turn converted by cyclooxygenase (CO) to PGG2 and PGH2, andthen by thromboxane synthetase (TS) to thromboxane A2.The other productof the cleavage of PIP2 is diacylglycerol (DAG), which stimulates proteinkinase C (PKC) to phosphorylate the intracellular protein P47 to P47-PO4.The latter, thromboxane and MLC-PO4, together stimulate secretion of

products of the dense - and lysosomal granules. Platelet coagulantactivity is generated by coagulation factors, shown in roman numerals form




11/35

Other checks on unbridled platelet activation exist on the surface of endothelialcells, including an ADP-destroying ectoenzyme (ADPase), and thrombomodulin, apowerful thrombin inhibitor. Endothelial cells, when stimulated by agonists suchas ATP, produce NO, a potent vasodilator that inhibits platelet function by raisingplatelet cGMP (67). There is evidence to indicate that platelets themselves havethe capacity to form NO from L-arginine and that this results in a rise in theconcentration of cGMP, which is a powerful intracellular regulator of plateletactivity (68).

COAGULATIONAlthough it has been traditional (and useful for in vitro laboratory testing) todivide the coagulation system into intrinsic and extrinsic pathways, such adivision does not occur in vivo because tissue factorfactor VIIa complex is apotent activator of both factor IX and factor X.

Extrinsic SystemThe principal initiating pathway ofin vivo blood coagulation is the extrinsicsystem, which involves components from both the

blood and vascular elements (see Chapter 5). The crucial component is tissuefactor, an intrinsic membrane protein composed of a single polypeptide chainthat functions as a cofactor to factor VIII in the intrinsic system, and to factor Vin the final common pathway (see Fig. 1-4). Tissue factor pathwayinhibitor (TFPI) is a protein that in association with factor Xa inhibits the tissuefactorfactor VII complex (69,70). Tissue factor synthesis in macrophages andendothelial cells is induced by endotoxin and by such cytokines as interleukin-1and tumor necrosis factor (71,72).

The major plasma component of the extrinsic pathway is factor VII, one of agroup of vitamin Kdependent proteins (including factors IX and X,prothrombin, and protein C) synthesized as prozymogens and converted(activated) to serine proteases by a limited number of proteolytic cleavages (see

Fig. 1-5). Protein S, also a vitamin Kdependent protein, is a cofactor ratherthan a zymogen. Common to these proteins are unique -glutamyl carboxyl acid(Gla) residues at the N-terminal end of the molecule that require vitamin K forproper synthesis by hepatocytes. This postribosomal modification of the proteinis required for calcium binding, one calcium with the two carboxyl groups of aGla residue, thereby serving as a bridge for protein binding to the phospholipidsurface.

Both factor IX and factor X are activated by the TFFVIIa complex and byfactor Xa itself. The active form is designated g-glutamyl factor VIIa andrepresents approximately 1% of total factor VII. Interaction between theintrinsic and extrinsic pathways occurs at several levels of the clotting cascade.

The zymogen factor VII itself has minimal but definite protease activity and iscapable of autoactivation. It converts factor VII to VIIa and then activates it,

tenase (VIII, IXa, Ca2+) and prothrombinase (Va, Xa, Ca2+ ),on the platelet external membrane phospholipid to convert prothrombin (II)to thrombin (IIa). (Courtesy of Dr. A. Koneti Rao.)

P.8




12/35

thereby displaying both positive and negative feedback effects.

The factor VIIatissue factor enzyme complex, which assembles on theactivated monocyte or perturbed endothelial cell, has two principal substrates,

FIGURE 1-4. The clotting cascade. The central precipitating event isconsidered to involve tissue factor (TF), which, under physiologic conditions,is not exposed to the blood. With vascular or endothelial cell injury, TF actsin concert with activated factor VIIa and phospholipid (PL) to convert factor

IX to IXa and factor X to Xa. The intrinsic pathway includescontact activation of factor XI by the XIIaactivated high-molecular-weight kininogen (HKa) complex. It should be noted that thecontact system contributes to fibrinolysis and bradykinin formation in vivo.Factor XIa also converts factor IX to IXa, and factor IXa, in turn, convertsfactor X to Xa, in concert with factor VIIIa and PL (the tenase complex). However factor Xa is formed, it is the active catalytic ingredientof the prothrombinase complex, which includes factor Va and PL andconverts prothrombin to thrombin. Thrombin cleaves fibrinopeptides (FPA,FPB) from fibrinogen, allowing the resultant fibrin monomers to polymerize,and converts factor XIII to XIIIa, which cross-links (XL) the fibrin clot.

Thrombin accelerates the process (interruptedlines) by its potential toactivate factors V and VIII, but continued proteolytic action also dampensthe process by activating protein C, which degrades factor Va and VIIIa.Thrombin activation of factor XI to XIa is a proposed pathway. Naturalplasma inhibitors retard clotting: C1-inhibitor (C1 INH) neutralizes factorXIIa, tissue factor pathway inhibitor (TFPI) blocks factor VIIa/TF, andantithrombin III (ATIII) blocks factors IXa and Xa and thrombin. Arrows,active enzymes; filled rectangles, sites of inhibitor action; dashed lines,feedback reactions.




13/35

factor IX (see Chapter 7) and factor X (see Chapter 10), both vitaminKdependent proteins. Cleavage of either protein results in a serine protease,factor IXa or Xa, that remains membrane-bound. Its Gla residues facilitatefurther reactions if appropriate cofactors are present. The required cofactor forfactor IXa to catalyze the conversion of factor X to factor Xa is factor VIII (seeChapter 8), whereas that for Xa conversion of prothrombin to thrombin is factor

V (see Chapter 9).

Factor VIII exists in plasma mostly as a noncovalent complex with VWF, and itscoagulant function is to accelerate factor IXa conversion of factor X to Xa. Theabsence of factor VIII or IX underlies the hemophilia syndromes, classichemophilia A and hemophilia B, which produce identical hemorrhagic states.Perhaps the similarity of the hemorrhagic condition with either factor VIII or IXdeficiency results from a lack in each case of a

proper tenase complex that is crucial for factor X activation (Fig. 1-4).The severity of the clinical disorder reflects the concentration of factor VIII orIX. The most severe clinical disease, manifest by spontaneous joint hemorrhage

(hemarthroses), occurs with factor VIII or factor IX levels of 0% to 1% (seeChapter 50). At factor levels of 5% to 30%, symptoms may be mild or evennonexistent, except in serious trauma such as surgery, and activity above 30%usually suffices for normal hemostasis. The presence of more than twice as muchfactor VIII or IX in healthy persons as is present in carriers (mean 50%)indicates that clotting proteins usually are present in excess and thatdeficiencies must be relatively severe to produce clinically significant effects.

P.9




14/35

The direct conversion of factor X to factor Xa by factor VIIatissue factorcomplex bypasses the need for factor VIII or IX. Nonetheless, a congenitaldeficiency of factor VII or X produces a similar hemorrhagic condition, anddistinguishing one from the other requires the determination of specificcoagulation factor activities (see Chapter 58). A clinically definable decrease intissue factor has not been described.

Intrinsic SystemParallel with the extrinsic system is the intrinsic system, which could be definedas coagulation initiated by components entirely contained within the vascularsystem. This pathway results in the activation of factor IX by a novel dimericserine protease, factor XIa (Fig. 1-4), providing a pathway independent of factor

FIGURE 1-5. Tentative structures of human prothrombin, factor IX, factorX, and protein C. The Ys refer to the -carboxyglutamic acid residuespresent in the Gla domains. The open diamonds refer to potential N-l inkedcarbohydrate chains. Cleavage sites and the enzymes involved during theactivation of each protein are identified by solid arrows. Amino acidsinvolved in catalysis (H, D, S) also are identified. Proposed disulfide bondshave been placed by analogy to those in bovine prothrombin and epidermalgrowth factor.




15/35

VII for blood coagulation. However, an important difference

exists between these two pathways in the clotting cascade. Whereas theactivation of factor IX by XIa requires only the presence of ionized calcium, theactivation of factor IX by VIIa requires calcium and the protein cofactor, tissuefactor, embedded in a cell membrane (lipid bilayer).

The role of the contact system proteins (see Chapter 6) in initiation of theintrinsic pathway of coagulation in hemostasis is questionable, because only adeficiency of factor XI is associated with a hemorrhagic tendency. These proteinsparticipate instead in the initiation of the inflammatory response, complementactivation, fibrinolysis, angiogenesis (73), and kinin formation (74), and studiesshow that kininogen is an anticoagulant protein in vivo (75). The mechanismmay be the inhibition of binding of low concentrations of thrombin to platelet GPIb/IX (76). The contact system is involved when blood interacts with a foreignsurface, as in cardiopulmonary bypass. The zymogen factor XII (Hageman factor)is the first protein in the series of tightly regulated reactions (Fig. 1-4) andbinds to negatively charged surfaces such as kaolin, dextran sulfate, and

sulfatides. The heavy chain of factor XII binds to the surface, allowing a largeincrease in local concentration of the enzyme, autoactivation, and action on itssubstrates, prekallikrein and factor XI, to form kallikrein and factor XIa (77). Inmost coagulation enzymes, the light chain contains the active site residuesserine, histidine, and aspartic acid, homologous to the archetypal serineprotease chymotrypsin, whereas the heavy chain contains binding regions tosurfaces, phospholipids, cell membrane, and connective tissue, which define theunique role of each coagulation proteolytic enzyme.

The assembly of cofactor, enzyme, and substrate is a recurrent theme in bloodcoagulation, resulting in maximal efficiency and speed of the molecular

reactions, especially as a phospholipid or cell membrane provides the surface forefficient positioning of interacting enzyme complexes with proenzyme substrates.

Kallikrein cleaves HK to liberate the nonapeptide bradykinin, and the remainingkinin-free kininogen (activated HK) binds at least 10-fold better to surface thanto the intact procofactor, thereby allowing more prekallikrein to associate withthe urokinase receptor on the endothelial cell (78), enhance fibrinolysis (79),and inhibit angiogenesis (73).

Negative feedback regulation is characteristic of the coagulation system. Onesuch reaction is factor XIa cleavage of the light chain of HK, which contains thecoagulant activity, thereby destroying its cofactor activity and allowing factorXIa to dissociate from the activating surface (80). Similarly, thrombin activatesfactors V and VIII, but conversion of protein C to activated protein C leads to thedestruction of factors Va and VIIIa. Although deficiency of any of the threeproteins involved in the contact system pathway results in slow generation ofthrombin and a prolonged in vitro partial thromboplastin time, their effect in vivoappears to be unrelated or the opposite. HK is an antithrombotic protein afterendothelial injury (75), and a deficiency may predispose to thrombosis. FactorXII deficiency has been implicated as a risk factor in venous and perhaps arterialthrombosis (81), so it may be a natural anticoagulant. Only a deficiency of factorXI may result in a hemorrhagic disorder. Even factor XI deficiency results only ina mild disorder of hemostasis in half of the affected individuals, and it is likelythat blood coagulation in vivo is initiated by factor IX or X through TFVIIamechanisms.

P.10




16/35

Coagulation Common PathwayOnce factor Xa is formed by either the extrinsic or the intrinsic pathway, itconverts prothrombin to thrombin (see Chapter 10). As with the other vitaminKdependent factors (Fig. 1-5), prothrombin has distinct functional domainsdevoted to calcium binding to phospholipids (10 Gla residues at the N-terminal

portion). This region resembles epidermal growth factor containing -hydroxyaspartic acid or asparagine, which can bind Ca2+ a region for cofactor(factor V) interaction, an activation peptide region, and a portion containing thecatalytic center. Elevated prothrombin levels due to a mutation in theuntranslated region, G20120A, result in a common genetic cause of thehypercoagulable state (82). After appropriate cleavage of prothrombin by factorXa, the N-terminal Gla portion is removed, and the resultant two-chain thrombinmolecule detaches from the phospholipid surface. The interaction of the fourcomponents of the prothrombinase complex (factor Xa, factor V, phospholipid,and calcium) provides a markedly increased rate of prothrombinactivationmore than 300,000-fold more than that achievable with only the

enzyme (factor Xa) and substrate (prothrombin). Factor V that participates inthis prothrombinase complex on the platelet membrane probably issupplied as the result of its secretion from platelet or fusion with the plasmamembrane, and it serves as a receptor for factor Xa binding to the activatedplatelet (83). Because of this involvement of platelets, the bleedingmanifestations of factor V deficiency may resemble those of qualitative plateletdisorders. Alternative pathways for prothrombin activation by factor Xaindependent of factor V have been described in malignant cells, hypoxicendothelial cells, and macrophages (84,85 and 86).

Blood coagulation proteins can be grouped according to shared properties,activities, or localization. For instance, phospholipid-oriented enzymes require

vitamin Kdependent carboxylation of glutamic acid residues at their N-terminal domains, and procofactors with no enzymatic activity share an ability tofacilitate attachment and interaction of clotting factors on biological surfaces.Another grouping of factors includes those that serve as a substrate forthrombin: for instance, cofactors V and VIII (activated, then inactivated),protein C (activated), prothrombin (cleaved to prethrombin), protein S(inactivated), factor XIII (to form active fibrin-stabilizing factor), and fibrinogen(liberating the fibrinopeptides). Further, deficiency of factor VIII or IX producesthe same clinical disorder by virtue of their cooperation in the tenase complex. In addition, factor V, fibrinogen, and the adhesive proteins fibronectin,VWF, and thrombospondin are all stored in platelet -granules.

The mapping of the entire human genome has uncovered new relationships of oldpairings of coagulation proteins. Mutations in the LMANIgene (87) leads todefects in the processing of both factor V and VIII in the ERGogli subcellularsystem, explaining the combined deficiency of these coagulation cofactors.Deficiency of vitamin K epoxide reductase (88) leads to warfarin resistance offactor II, VII, IX, and X. Another important new hemostatic gene recentlydiscovered (89) is ADAMTS, which controls the proteolytic breakdown of VWFmutimers and ADAMTS deficiency, as associated with thromboticthrombocytopenic purpura (TTP).

Plasma proteolytic inhibitors (see Chapters 11, 13, 19, and 21) serve to limit

and control the extent and speed of both blood coagulation and fibrinolyticreactions (see Table 1-1).




17/35

Inhibition of CoagulationThe major inhibitor of the contact system is C1 inhibitor, which accounts for 95%of the plasma inhibitory capacity for factor XIIa and more than 50% towardkallikrein; however, hereditary deficiency of C1 inhibitor results in angioedemarather than bleeding (90). 1-Antitrypsin is the major inhibitor of factor XIa,

but a more critical role is its inhibition of neutrophil elastase; inhibitor deficiencyresults in emphysema due to the unopposed effects of elastase in the lungalveoli (91).

TABLE 1-1 PLASMA PROTEASE INHIBITORS OFCONTACT SYSTEM COAGULATION AND

FIBRINOLYSIS

Inhibitor

Plasma

concentration Molecularweight

(daltons)Major target

enzymesg/mL nM

1-Protease

inhibitor2,500 45,000 55,000 Factor XI,

elastase

AntithrombinII I

290 4,700 62,000 Factor Xa,thrombin

2-

Macroglobulin2,500 3,400 725,000 Kall ikrein,

plasmin,thrombin

C1 inhibitor 240 2,300 105,000 Activatedfactor XII,kall ikrein

2-Antiplasmin 70 1,050 67,000 Plasmin

Heparincofactor II

40 600 65,000 Thrombin

Plasminogenactivatorinhibitor-1(PAI-1)

10 200 50,000 Tissueplasminogenactivator,urokinase

Protein C

inhibitor (PAI-

5 10 53,000 Protein C,

kall ikrein




18/35

ATIII is the major inhibitor of factors IXa, Xa, and thrombin. Although enough

ATIII is present to neutralize three times the total amount of thrombin that couldform in the blood, a decrease to 40% to 50% predisposes to thromboticdisorders. That congenital ATIII deficiency is associated with a strikinglyincreased risk of venous thromboembolism indicates that inhibitors play a majorregulatory role and that a delicate balance exists between the procoagulant andanticoagulant forces. The catalytic-site serine of thrombin reacts with anarginine in the active center of ATIII to form a covalent inactive complex. Theinhibition produced by ATIII is potentiated by heparin, a sulfated polysaccharidewith the highest negative charge of any naturally occurring polymer and a closerelative to the heparan sulfate that exists on the endothelial surface (Fig. 1-2).Heparin binds to a basic group in ATIII to increase its rate of inactivation of

thrombin. Once thrombin is bound to fibrin, it is resistant to ATIII and evenmore so to ATIIIheparin complex (92,93). Heparin cofactor II is a serpin(serine protease inhibitor) that selectively inactivates thrombin (not factor Xa) inthe presence of heparin or dermatan sulfate (94).

Factor Xa also has a specific inhibitor, Z protease inhibitor, the action of which ismarkedly enhanced by protein Z, a vitamin Kdependent protein, in aphospholipid and manner Ca2+-dependent (95).

2-Macroglobulin is a secondary or backup inhibitor for many plasma coagulant

and fibrinolytic enzymes, including kallikrein, thrombin, and plasmin. Becauseenzymes trapped in the cage structure of this inhibitor exhibit some activity,

complexes may serve as a repository of enzymatic activity that is protectedagainst other inhibitors. No clinical disorder of severe deficiency has beendescribed. is the primary inhibitor of plasmin acting to prevent a systemicfibrinogenolytic response to noxious stimuli, limiting the fibrinolytic response tothrombi in the affected region and allowing hemostatic plugs to remain intactuntil healing is complete (96). In the absence of hemostatic plugs dissolvebefore healing has occurred and a hemorrhagic state results (97). A deficiency inPAI-1 also results in a hemorrhagic tendency (98). Protein C inhibitor also is aserpin that can inactivate protein C and thereby function as a potentialprocoagulant molecule (99).

FIBRIN FORMATION AND FIBRINOLYSISThrombin acts on multiple substrates, including fibrinogen; factors XIII, V, and

3)

Tissue factor

pathwayinhibitor

0.1 0.25 40,000 Factor VIIa

(tissue factor),factor Xa(tissue factor)

P.11




19/35

VIII; platelet membrane GP V; protein S; and protein C (see Fig. 1-6). In sodoing, thrombin occupies a central role in the process of hemostatic plugformation, influencing its form, rate of formation, and limitation. Its potentiatingeffect on factors VIII and V produces an increase in the tenase andprothrombinase complexes (Fig. 1-4), resulting in a burst of thrombin activityand fibrin strand formation. When thrombin hydrolyzes factor V too slowly

because of a point mutation at a cleavage site, the result is the most commongenetic cause of thrombosis factor VLe iden (100). Thrombin also helps to recruit

platelets into the hemostatic plug, depending on the relative influences ofintrinsic or extrinsic clotting systems that are operative. When coagulation startsprincipally on the altered platelet surface (intrinsic system), thrombin formationis slower than when the extrinsic coagulation system is initiated by exposure totissue factor, a membrane protein found in macrophages, activated endothelialcells, and tumor cells. In the latter situation, thrombin might have a greaterinfluence on platelet aggregation.

The formation of fibrin strands represents the second phase in hemostasis (thefirst being the primary platelet aggregate). The precursor of fibrin is fibrinogen,

a large glycoprotein ofMr 340,000 present in high concentration in both plasmaand platelet granules that interacts with other proteins, including factor XIII, 2-plasmin fibronectin, inhibitor, plasminogen, and plasminogen activator

(101). The location and surface concentration of these modifying proteinsinfluence the orderly process of fibrin formation, cross-linking, and fibrin lysis.Thrombin binds to the fibrinogen central domain and liberates fibrinopeptides Aand B, resulting in fibrin monomer and polymer formation (102). Progressivelengthening of the polymer chain occurs by a half-overlap, side-to-sideapproximation of fibrin monomer molecules (see Fig. 1-7), and the two-strandedprotofibrils interact laterally to form long, thin fibrin strands or short, broadsheets of fibrin (103,104). Although the degree of lateral strand association

probably contributes to the tensile strength of the

clot, its resistance to plasmin degradation is influenced mainly by cross-linkingmediated by factor XIIIa (105). In addition, factor XIIIa, by linking 2-plasmin

inhibitor to fibrin, may protect the clot against fibrinolysis. Factor XIII exists inplasma as a four-chain precursor molecule ofMr 320,000, and after thrombinactivation, the enzyme (with calcium) induces cross-linking of the fibrin polymer(106). Covalent isopeptide bonds form between lysine donors and glutaminereceptors, with two cross-linked rapidly to form - dimers; chains arecrosslinked more slowly, each with two other such chains, to form a polymernetwork (107,108). In mature forms, the fibrin fiber contains approximately 100

protofibrils, with a somewhat random pattern of branching that links the fiberstogether.

P.12




20/35

The fibrin mesh binds the platelets together and contributes to their attachmentto the vessel wall, mediated by binding to platelet receptor glycoproteins and byinteractions with other adhesive proteins such as thrombospondin, fibronectin,and platelet fibrinogen (released from platelet granules but probably otherwiseequivalent to plasma fibrinogen) (109). After attachment to platelet-bindingsites, these proteins may serve as molecular bridges between plasma proteinsand the platelet interior, between platelets and the vessel wall, and betweenplasma

fibrin fibers and the subendothelial matrix. For instance, fibronectin is cross-linked by factor XIIIa to fibrin, and its separate binding site for collagen couldserve to bridge fibrin to the vessel wall (110,111). VWF also could serve as abridge between platelet membrane GP Ib (or IIb/IIIa) and a subendothelialmatrix component (112). Additionally, the platelet membrane GP IIb/IIIa could

join plasma fibrinogen (or -granule fibrinogen) to intracellular actin, therebymediating clot retraction and vessel wall constriction (113).

FIGURE 1-6. The multitude of actions of thrombin, resulting inprocoagulant tendencies, potentiation of ongoing reactions, feedbackinhibition, and limitation of clotting.

P.13




21/35

The potential for hemorrhagic or thrombotic disease that results fromderangements in fibrinogen structure, concentration, or interaction withthrombin or factor XIII, is great and varied. It could, for instance, manifest as apoorly polymerizing protein, by slow or absent liberation of a fibrinopeptide, oras an inadequately cross-linked fibrin (114). The latter situation similarly couldbe produced by an absent or faulty factor XIII molecule, which could contribute

FIGURE 1-7. Fibrinogen and thrombin-induced fibrin monomer and polymerformation, factor XIIIainduced fibrin cross-linking, and plasmininducedcross-linked fibrin degradation. The curly lines represent coiled coilsbetween central and terminal domains, and the double horizontal linesrepresent cross-link sites induced by factor XIIIa between chains of twocontiguous fragment D-domains. The central and two terminal domains offibrinogen are included in the fragment E- and D-domains, respectively. Thefibrinopeptides are indicated as small vertical lines connected to the central(E) domain of fibrinogen and are absent from the fibrin monomer molecules

after thrombin action. Plasmin action is depicted here as limited to cleavageof the coiled coils between center (E) and terminal (D) domains, to yield thecomplexes noted at the bottom. These complexes consist of twononcovalently bound fragments (e.g., fragments DD and E in the DDEcomplex).




22/35

to both a hemorrhagic condition and inadequate wound healing. The mostcommon acquired disorders of fibrinogen are those of consumption, thedisseminated intravascular coagulation (DIC) syndromes, which may reflectexcessive or inappropriate coagulation or proteolytic degradation of plasmafibrinogen and can result in a variety of hemorrhagic and thromboticmanifestations, depending to a great extent on the underlying pathologic process

(115).

Several distinct mechanisms for controlling and localizing hemostasis exist,including the effects of vascular flow and hemodilution, proteolytic feedback bythrombin, inhibition by plasma proteins and endothelial celllocalizedactivation of an inhibitory enzyme (protein C), and fibrinolysis. First, thehemostatic plug is exposed to the disruptive pull of blood flow, and small clumpsof platelets that are inadequately attached to the main body of platelets or tothe vessel wall can be washed free into the blood. Second, thrombin that ispresent in the hemostatic plug and that has already contributed to its formationby potentiating the activation of factors V and VIII ultimately inactivates thesesame cofactors (Fig. 1-6) in the presence of thrombomodulin, a membrane

protein of endothelial cells. This complex effect of thrombin is an exquisite yetsimple example of self-dampening that effectively limits the growth of the fibrin-platelet plug. Third, soluble activated coagulant proteins such as factor Xa orthrombin may diffuse away from the clot, to be bound to inhibitory plasmaproteins that destroy or at least markedly decrease their coagulant potential.Principal among these inhibitors is ATIII, which forms a tight complex not onlywith thrombin but also with other serine protease coagulant proteins and withthe fibrinolytic enzyme plasmin. However, although thrombin can be readilyinactivated by ATIII in solution, thrombin's catalytic site is inaccessible to theinhibitor while the enzyme is bound to fibrin, and it may retain the ability tocleave fibrinopeptides even in the presence of heparin. Fourth, thrombin that

diffuses into the endothelial cell surface may bind to a specific receptor,thrombomodulin, thereby setting into motion another restraint on localcoagulation. As previously stated, the thrombinthrombomodulin complexserves as a receptor for the vitamin Kdependent protein C (Figs. 1-2 and 1-5), which is activated and released from the endothelial cell surface. Activatedprotein C reacts with factors V and VIII to destroy their coagulant properties,thereby limiting the effect of thrombin. Patients with deficiencies of protein C,protein S (a cofactor of protein C), and ATIII have been described in whom thehemostatic process is not effectively limited and in whom there is a lifelongtendency for pathologic thromboembolic disease.

The last mechanism for limiting clot formation is fibrinolysis, which alsoconstitutes a repair mechanism, along with endothelial cell regrowth and vesselrecanalization. Fibrinolysis resembles the cascade mechanism of clotting factoractivation in that it involves zymogen-to-enzyme conversions, feedbackpotentiation and inhibition, and a finely tuned balance with inhibitors. Theinactive precursor protein is plasminogen, present in plasma at twice the molarconcentration of inhibitor. During the initial period of hemostatic plug formation,platelets and endothelial cells release plasminogen activator inhibitors thatfacilitate fibrin formation (116). However, in response to a poorly understood butprecisely timed and orchestrated sequence of stimuli, endothelial cells liberatetissue plasminogen activator (117). Both tissue plasminogen activators andprourokinase have the capacity to convert plasminogen (especially aplasminogen molecule bound to fibrin) to the serine protease-active form,




23/35

plasmin (118).

As with thrombin feedback that leads to accelerated factor Xa formation, plasminalso exerts positive feedback by cleavage of an activation peptide fromplasminogen (connecting Gluplasminogen to Lys-plasminogen), rendering it moresusceptible to surface binding and subsequent activation by plasminogenactivators. Perhaps more critical is the markedly heightened reactivity ofplasminogen after it is bound to fibrin by lysinebinding sites located on itskringle structures. Lipoprotein A with multiple kringles and histidine-richglycoprotein also modulates fibrinplasminogen interactions by inhibitingplasminogen binding to fibrin (119,120).

Although only a small proportion of plasma plasminogen is bound to fibrin duringparticulate clot formation, this is sufficient to influence subsequent physiologicfibrinolysis (121). The process is a balanced one, however, because 2-plasmin

inhibitor also is bound to the fibrin, covalently attached by factor XIIIa action(122). The relative proportions and positions of profibrinolytic plasminogen andplasminogen activator molecules and antifibrinolytic 2-plasmin inhibitor

molecules on the fibrin strand influence the timing and degree of clotdissolution. Clinical derangements related to the molecular disorders include ahemorrhagic disorder due to deficient or defective 2-plasmin inhibitor and PAI-

1 (123).

Studies have elucidated an important connection between the coagulation andfibrinolytic pathways by virtue of thrombinthrombomodulin mediation of bothprotein C and TAFI activation. Whereas activation of protein C leads toinactivation of factors Va and VIIIa and curtailment of further clot formation,TAFI activation promotes stabilization of fibrin and therefore persistence offormed fibrin clots. The mode of action of TAFI is to cleave C-terminal lysineresidues from fibrin, thereby preventing plasminogen, plasmin, or tissue-type

plasminogen activators (tPA) from binding to fibrin, and secondarily, inhibition offibrinolysis. Clinical conditions of decreased coagulation, such as classichemophilia, not only are deficient in thrombin and fibrin formation but, by virtueof low TAFI formation, allow fibrinolysis to proceed relatively unimpeded. Thecombination of less fibrin and more lysis contributes to the bleeding seen inpatients with factor VIII deficiency. Similarly, patients with deficiency ofcontact-induced coagulation also appear to have decreased TAFI activation,perhaps by inadequate completion of clotting after initial fibrin formation. On theother hand, patients with deficiency of protein C manifest a thrombotic tendencyby virtue of a failure of feedback inhibition of factors Va and VIIIa bythrombin. The predilection to thrombosis also may have a contribution by

excessive TAFI formation due to continuously high production of thrombin. Inthis case, not only are thrombin and fibrin formed but such fibrin is renderedresistant to plasmin lysis by TAFI.

The enhanced fibrinolysis seen with activation of the plasma kallikreinkininsystem has now been explained. When HK is cleaved by kallikrein, bradykinin isreleased, which stimulates release of tPA. The cleaved HK (HKa) binds toendothelial urokinase plasminogen [urokinase plasminogen activator receptor(uPAR)] (78). The event places HKa [in complex with prekallikrein (77)] inposition to bind to domains 2 and 3 of uPAR in close proximity to prourokinasebound to domain 1

of uPAR, thereby enhancing the possibility of activation of prourokinase tourokinase. PK is converted to kallikrein by the endothelial cell membrane

P.14




24/35

protease prolylcarboxypeptidase (124). This localization allows kallikrein toefficiently activate prourokinase to urokinase (125) without inhibition by plasmaserpins such as C1 inhibitor. Direct evidence for this is provided by a studyshowing that HKPK interaction is required (79) for plasmin formation byprourokinase on the endothelial cell membrane.

Once plasmin is produced locally on the hemostatic plug, the potential for fibrindegradation exists. By an intricate balance of the simultaneous forces ofcoagulation and platelet aggregation, inhibition of coagulation, profibrinolyticand antifibrinolytic reactions, and cellular mechanisms for both coagulation andlysis (in leukocytes as well as in platelets and endothelial cells), the clot isgradually reduced. The neutral serine protease (elastase) released from theprimary granules of neutrophils also contributes to the local fibrinolytic potential(126). The surface of the clot may be removed first, revealing fresh surfacesthat are progressively attacked until the process is completed (127).

During hemostatic plug or thrombus dissolution, solubilized fibrin degradationproducts are liberated into the circulation, some of which represent unique

cross-linked derivatives such as D-dimer that can be distinguished fromfibrinogen degradation products (128). The circulating degradation productsserve as diagnostic markers of thrombin or factor XIIIa, or both, plus plasminaction that reflects prior clot formation and ongoing fibrinolysis. The surface of aclot and circulating fibrin derivatives may possess a small but significant amountof active thrombin that could serve to propagate the coagulant processelsewhere in the circulation (129). Active plasmin molecules also may bereleased into the circulation during fibrinolysis, but just as free thrombin isneutralized by ATIII, plasmin is extremely susceptible in solution to inhibitorneutralization by inhibitor (130). This latter reaction serves to limitfibrinogenolysis to the region of the clot, just as ATIII serves to prevent

disseminated coagulation by the spread of a regional hemostatic process.When hemostatic plug formation is defective (e.g., in hemophilia), naturallyoccurring fibrinolysis may aggravate bleeding; conversely, the use of epsilonaminocaproic acid aids in hemostasis. This mechanism also may apply inbleeding after dextran infusion, 2-antiplasmin deficiency, and factor XIII

deficiency. In the latter case, the lack of cross-links leads to increasedsusceptibility to plasmin and to failure to cross-link antiplasmin to the fibrin clot,which also may lead to increased fibrinolysis and hemorrhage.

The entire process involving endothelial cells and platelets, clotting factors andadhesive proteins, and inhibitory mechanisms of clotting, fibrinolysis, andplatelet aggregation serves to promote the right balance and location ofhemostasis and recovery. This highly developed system of checks and balancesallows a rapid and efficient hemostatic response to bleeding but avoids athrombogenic response away from the site of injury or persisting beyond thetime of its physiologic need. Derangement of any portion of the intricate processcan produce an imbalance, sometimes only slight, with a resultant hemorrhagicor thrombotic clinical disorder. A further complicating feature of this delicatebalance is therapeutic intervention, which must be carefully regulated to correcta hemostatic defect without upsetting the balance too far and thereby leading tothrombosis.

References1. Cines DB, Tomaski A, Tannenbaum S. Immune endothelial-cell injury in




25/35

heparin-associated thrombocytopenia. N Engl J Med1987;316(10):581589.

2. MacGregor RR, Friedman HM, Macarak EJ, et al. Virus infection ofendothelial cells increases granulocyte adherence.J Clin Invest1980;65(6):14691477.

3. Janoff A, Sloan B, Weinbaum G, et al. Experimental emphysema inducedwith purified human neutrophil elastase: tissue localization of the instilledprotease. Am Rev Respir Dis 1977;115(3):461478.

4. LeRoy EC, Ager A, Gordon JL. Effects of neutrophil elastase and otherproteases on porcine aortic endothelial prostaglandin I2 production, adeninenucleotide release, and responses to vasoactive agents. J Clin Invest1984;74:10031010.

5. Kitchens CS. The anatomical basis of purpura. Prog Hemost Thromb1982;5:211210.

6. Hynes RO. Integrins: a family of cell surface receptors. Cell1987;48(4):549554.

7. Dustin ML, Garcia Aguilar J, Hibbs ML, et al. Structure and regulation ofthe leukocyte adhesion receptor LFA-1 and its counterreceptors, ICAM-1 andICAM-2. Cold Spring Harb Symp Quant Biol1989;54(Pt 2):753765.

8. Hession C, Osborn L, Goff D, et al. Endothelial leukocyte adhesionmolecule 1: direct expression cloning and functional interactions. Proc Natl

Acad Sci U S A 1990;87(5):16731677.

9. Rosenberg RD, Aird WC. Vascular-bedspecific hemostasis andhypercoagulable states. N Engl J Med1999;340(20):15551564.

10. Ofosu FA, Buchanan MR, Anvari N, et al. Heparin, heparan sulfate anddermatan sulfate. Ann N Y Acad Sci1989;556:123.

11. Marcus AJ, Weksler BB, Jaffe EA. Enzymatic conversion of prostaglandinendoperoxide H2 and arachidonic acid to prostacyclin by cultured humanendothelial cells.J Biol Chem 1978;253(20):71387141.

12. Moncada S, Vane JR. The role of prostacyclin in vascular tissue. Fed Proc1979;38(1):6671.

13. Weiss HJ, Turitto VT. Prostacyclin (prostaglandin I2, PGI2) inhibits

platelet adhesion and thrombus formation on subendothelium. Blood1979;53(2):244250.




26/35

14. Weksler BB, Marcus AJ, Jaffe EA. Synthesis of prostaglandin I2(prostacyclin) by cultured human and bovine endothelial cells. Proc Natl AcadSci U S A 1977;74(9):39223926.

15. Busch C, Owen WG. Identification in vitro of an endothelial cell surface

cofactor for antithrombin III. Parallel studies with isolated perfused rathearts and microcarrier cultures of bovine endothelium. J Clin Invest1982;69(3):726729.

16. Esmon CT, Owen WG. Identification of an endothelial cell cofactor forthrombin-catalyzed activation of protein C. Proc Natl Acad Sci U S A 1981;78(4):22492252.

17. Hatton MW, Berry LR, Regoeczi E. Inhibition of thrombin by antithrombinIII in the presence of certain glycosaminoglycans found in the mammalian

aorta. Thromb Res 1978;13(4):655670.

18. Lollar P, Owen WG. Clearance of thrombin from circulation in rabbits byhigh-affinity binding sites on endothelium. Possible role in the inactivation ofthrombin by antithrombin III. J Clin Invest1980;66(6):12221230.

19. Teien AN, Abildgaard U, Hook M. The anticoagulant effect of heparansulfate and dermatan sulfate. Thromb Res 1976;8(6):859867.

20. Marcus AJ, Safier LB, Hajjar KA, et al. Inhibition of platelet function byan aspirin-insensitive endothelial cell ADPase. Thromboregulation byendothelial cells.J Clin Invest1991;88(5):16901696.

21. Fukudome K, Esmon CT. Identification, cloning, and regulation of a novelendothelial cell protein C/activated protein C receptor.J Biol Chem1994;269:2648626491.

22. Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound cofactorfor thrombin-catalyzed activation of protein C.J Biol Chem 1982;257

(2):859864.

23. Heeb MJ, Espana F, Geiger M, et al. Immunological identity ofheparindependent plasma and urinary protein C inhibitor and plasminogenactivator inhibitor-3.J Biol Chem 1987;262(33):1581315816.

24. Walker FJ. Regulation of activated protein C by protein S. The role ofphospholipid in factor Va inactivation.J Biol Chem 1981;256(21):1112811131.

25. Dahlback B. Inhibition of protein Ca cofactor function of human andbovine protein S by C4b-binding protein. J Biol Chem 1986;261(26):




27/35

1202212027.

26. de Fouw NJ, Haverkate F, Bertina RM, et al. The cofactor role of proteinS in the acceleration of whole blood clot lysis by activated protein C in vitro.Blood1986;67(4):11891192.

27. MacIntyre DE, Pearson JD, Gordon JL. Localisation and stimulation ofprostacyclin production in vascular cells. Nature 1978;271(5645):549551.

28. Warner TD, Mitchell JA, de Nucci G, et al. Endothelin-1 and endothelin-3release EDRF from isolated perfused arterial vessels of the rat and rabbit.JCardiovasc Pharmacol1989;13(Suppl. 5):S85S88; discussion S102.

29. MacCumber MW, Ross CA, Glaser BM, et al. Endothelin: visualization ofmRNAs by in situ hybridization provides evidence for local action. Proc Natl

Acad Sci U S A 1989;86(18):72857289.

30. Wiman B, Chmielewska J, Ranby M. Inactivation of tissue plasminogenactivator in plasma. Demonstration of a complex with a new rapid inhibitor. JBiol Chem 1984;259(6):36443647.

31. Nesheim ME, Wang W, Boffa M, et al. Thrombin, thrombomodulin andTAFI in the molecular link between coagulation and fibrinolysis. ThrombHaemost1997;78:386.

32. George JN. Platelets. Lancet2000;355:15311539.

33. De Botton S, Sabri S, Daugas E, et al. Platelet formation is theconsequence of caspase activation within megakaryocytes. Blood2002;100(4):13101317.

34. Kaushansky K. Thrombopoietin. N Engl J Med1998;339(11):746754.

35. Mikkola HK, Klintman J, Yang H, et al. Haematopoietic stem cells retainlong-term repopulating activity and multipotency in the absence of stemcellleukaemia SCL/tal-1 gene. Nature 2003;421(6922):547551.

36. Ruggeri ZM, Dent JA, Saldivar E. Contribution of distinct adhesiveinteractions to platelet aggregation in flowing blood. Blood1999;94(1):172178.

37. Pytela R, Pierschbacher MD, Ginsberg MH, et al. Platelet membraneglycoprotein IIb/IIIa: member of a family of Arg-Gly-Aspspecific adhesion

receptors. Science 1986;231(4745):15591562.

P.15




28/35

38. Weiss HJ, Turitto VT, Baumgartner HR. Effect of shear rate on plateletinteraction with subendothelium in citrated and native blood. I. Shearratedependent decrease of adhesion in von Willebrand's disease and theBernard-Soulier syndrome. J Lab Clin Med1978;92(5):750764.

39. Kasirer-Friede A, Cozzi MR, Mazzucato M, et al. Signaling through GP Ib-IX-V activates alpha IIb beta 3 independently of other receptors. Blood2004;103(9):34033411.

40. Staatz WD, Rajpara SM, Wayner EA, et al. The membrane glycoproteinIa-IIa (VLA-2) complex mediates the dependent adhesion of platelets tocollagen. J Cell Biol1989;108(5):19171924.

41. Howard JB. Methylamine reaction and denaturation-dependentfragmentation of complement component 3: comparison with alpha-2-

macroglobulin. J Biol Chem 1980;255:70827084.

42. Shattil SJ, Kashiwagi H, Pampori N. Integrin signaling: the plateletparadigm. Blood1998;91(8):26452657.

43. Shattil SJ, Hoxie JA, Cunningham M, et al. Changes in the plateletmembrane glycoprotein IIb.IIIa complex during platelet activation. J BiolChem 1985;260(20):1110711114.

44. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell2002;110(6):673687.

45. Lam SC, Plow EF, D'Souza SE, et al. Isolation and characterization of aplatelet membrane protein related to the vitronectin receptor. J Biol Chem1989;264(7):37423749.

46. Bennett JS, Vilaire G. Exposure of platelet fibrinogen receptors by ADPand epinephrine. J Clin Invest1979;64(5):13931401.

47. Nurden AT, Caen JP. The different glycoprotein abnormalities inthrombasthenic and Bernard- Soulier platelets. Semin Hematol1979;16(3):234250.

48. Colman RW. Platelet receptors. Hematol Oncol Clin North Am1990;4:2742.

49. Feinstein MB. The role of calcium in blood platelet function. In: WeissGB, ed. Calcium in drug action. New York: Plenum Publishing,1978:197239.

50. Gabbeta J, Yang X, Kowalska MA, et al. Platelet signal transduction




29/35

defect with Galpha subunit dysfunction and diminished Galphaq in a patientwith abnormal platelet responses. Proc Natl Acad Sci U S A 1997;94(16):87508755.

51. Lee SB, Rao AK, Lee KH, et al. Decreased expression of phospholipase C-beta 2 isozyme in human platelets with impaired function. Blood1996;88(5):16841691.

52. Adelstein RS, Conti MA. Phosphorylation of platelet myosin increasesactin-activated myosin ATPase activity. Nature 1975;256(5518):597598.

53. Pickett WC, Jesse RL, Cohen P. Initiation of phospholipase A2 activity inhuman platelets by the calcium ion ionophore A23187. Biochim Biophys Acta1976;486(1):209213.

54. Colman RW, Hoffman I. Calpains and hemostasis. In: Mellgren R, MurachiT, eds. Intracellular calcium-dependent proteolysis. CRC Press, 1990:211224.

55. Nishizuka Y. The role of protein kinase C in cell surface signaltransduction and tumour promotion. Nature 1984;308(5961):693698.

56. Berridge NJ. Inositol trisphosphate and diacylglycerol as secondmessengers. Biochem J1984;220:345340.

57. Rink TJ, Sanchez A, Hallam TJ. Diacylglycerol and phorbol ester stimulatesecretion without raising cytoplasmic free calcium in human platelets. Nature1983;305(5932):317319.

58. Holmsen H. Platelet secretion and energy metabolism. In: Hirsh J,Marder V, Salzman E, eds. Hemostasis and thrombosis: basic principles andclinical practice. Philadelphia, PA: JB Lippincott Co, 1993.

59. Mills DC, Figures WR, Scearce LM, et al. Two mechanisms for inhibition ofADP-induced platelet shape change by 5-p-Conversion to adenosine, andcovalent modification at an ADP binding site distinct from that which inhibitsadenylate cyclase. J Biol Chem 1985; 260(13):80788083.

60. Jin J, Kunapuli SP. Coactivation of two different G protein-coupledreceptors is essential for ADP-induced platelet aggregation. Proc Natl AcadSci U S A 1998;95(14):80708074.

61. Cattaneo M, Zighetti ML, Lombardi R, et al. Molecular bases of defectivesignal transduction in the platelet P2Y12 receptor of a patient with congenitalbleeding. Proc Natl Acad Sci U S A 2003;100(4):19781983.




30/35

62. Kobilka BK, Matsui H, Kobilka TS, et al. Cloning, sequencing, andexpression of the gene coding for the human platelet alpha 2-adrenergicreceptor. Science 1987;238(4827):650656.

63. Saussy DL Jr, Mais DE, Burch RM, et al. Identification of a putative

thromboxane A2/prostaglandin H2 receptor in human platelet membranes. JBiol Chem 1986;261(7):30253029.

64. Hoyer LW, Wyshock EG, Colman RW. Coagulation cofactors: factors V andVIII. In: Colman RW, Hirsh J, Marder VJ, eds. Hemostasis and thrombosis:basic principles and clinical practice , 3rd ed. Philadelphia, PA: JB LippincottCo, 1994:109133.

65. Haslam RJ, Davidson MM, Fox JE, et al. Cyclic nucleotides in plateletfunction. Thromb Haemost1978;40(2):232240.

66. Colman RW. Platelet cyclic nucleotide phosphodiesterases. In: Rao GHR,ed. Handbook of platelet physiology and pharmacology. Boston, MA: KluwerAcademic Publishers, 1999:251267.

67. Rapoport RM, Murad F. Endothelium-dependent andnitrovasodilatorinduced relaxation of vascular smooth muscle: role of cyclicGMP. J Cyclic Nucleotide Protein Phosphor Res 1983;9(4-5):281296.

68. Radomski MW, Palmer RM, Moncada S. An L-arginine/nitric oxide pathwaypresent in human platelets regulates aggregation. Proc Natl Acad Sci U S A 1990;87(13):51935197.

69. Broze GJ Jr, Warren LA, Novotny WF, et al. The lipoprotein-associatedcoagulation inhibitor that inhibits the factor VII-tissue factor complex alsoinhibits factor Xa: insight into its possible mechanism of action. Blood1988;71(2):335343.

70. Rao LV, Rapaport SI. Studies of a mechanism inhibiting the initiation of

the extrinsic pathway of coagulation. Blood1987;69:645651.

71. Colucci M, Balconi G, Lorenzet R, et al. Cultured human endothelial cellsgenerate tissue factor in response to endotoxin. J Clin Invest1983;71(6):18931896.

72. Edwards RL, Rickles FR. Macrophage procoagulants. Prog Hemost Thromb1984;7:183209.

73. Colman RW, Jameson BA, Lin Y, et al. Domain 5 of high molecular weight

kininogen (kininostatin) down-regulates endothelial cell proliferation and




31/35

migration and inhibits angiogenesis. Blood2000;95(2):543550.

74. Colman RW. Biologic activities of the contact factors in vivo: potentiationof hypotension, inflammation, and fibrinolysis, and inhibition of celladhesion, angiogenesis, and thrombosis. Thromb Haemost1999;82:15681577.

75. Colman RW, White JV, Scovell S, et al. Kininogens are antithromboticproteins in vivo. Arterioscler Thromb Vasc Biol1999;19:22452250.

76. Bradford HN, DeLa Cadena RA, Kunapuli SP, et al. Human kininogensregulate thrombin binding to platelets through the GP Ib-IX-V complex. Blood1997;90:15081515.

77. Mandle R Jr, Colman RW, Kaplan AP, Identification of prekallikrein and

high molecular weight kininogen as a complex in human plasma. Proc NatlAcad Sci U S A 1976;73:41794183.

78. Colman RW, Pixley RA, Najamunnisa S, et al. Binding of high molecularweight kininogen to human endothelial cells is mediated via a site withindomains of the urokinase receptor. J Clin Invest1997;100:14811487.

79. Lin Y, Harris RB, Yan W, et al. High molecular weight kininogen peptidesinhibit the formation of kallikrein on endothelial cell surfaces and subsequenturokinase-dependent plasmin formation. Blood1997;90:690697.

80. Scott CF, Silver LD, Purdon AD, et al. Cleavage of human high molecularweight kininogen by factor XIa in vitro. Effect on structure and function. JBiol Chem 1985;260:1085610863.

81. Halbmayer WM, Mannhalter C, Feichtinger C, et al. The prevalence offactor XII deficiency in 103 orally anticoagulated outpatients suffering fromrecurrent venous and/or arterial thromboembolism. Thromb Haemost1992;68:285290.

82. Poort SR, Rosendaal FR, Reitsma PH, et al. A common genetic variationin the region of the prothrombin gene is associated with elevated plasmaprothrombin levels and an increase in venous thrombosis. Blood1996;88:36983703.

83. Miletich JP, Jackson CM, Majerus PW. Properties of the factor Xa bindingsite on human platelets. J Biol Chem 1978;253(19):69086916.

84. Gordon SG, Cross BA. A factor X-activating cysteine protease frommalignant tissue. J Clin Invest1981;67(6):16651671.




32/35

85. Maier RV, Hahnel GB. Microthrombosis during endotoxemia: potential roleof hepatic versus alveolar macrophages. J Surg Res 1984;36(4):362370.

86. Ogawa S, Gerlach H, Esposito C, et al. Hypoxia modulates the barrier andcoagulant function of cultured bovine endothelium. Increased monolayer

permeability and induction of procoagulant properties.J Clin Invest1990;85(4):10901098.

87. Nichols WC, Seligsohn U, Zivelin A, et al. Mutations in the ER-Golgiintermediate compartment protein ERGIC-53 cause combined deficiency ofcoagulation factors V and VIII. Cell1998;93(1):6170.

88. Li T, Chang CY, Jin DY, et al. Identification of the gene for vitamin Kepoxide reductase. Nature 2004;427(6974):541544.

89. Levy GG, Nichols WC, Lian EC, et al. Mutations in a member of theADAMTS gene family cause thrombotic thrombocytopenic purpura. Nature2001;413(6855):488494.

90. Schapira M, Scott CF, Colman RW. Protection of human plasma kallikreinfrom inactivation by C1 inhibitor and other protease inhibitors. The role ofhigh molecular weight kininogen. Biochemistry1981;20:27382743.

91. Scott CF, Schapira M, James HL, et al. Inactivation of factor XIa by

plasma protease inhibitors: predominant role of alpha 1-protease inhibitorand protective effect of high molecular weight kininogen. J Clin Invest1982;69:844852.

92. Hogg PJ, Jackson CM. Fibrin monomer protects thrombin frominactivation by heparin-antithrombin III: implications for heparin efficacy.Proc Natl Acad Sci U S A 1989;86:36193623.

93. Weitz JI, Hudoba M, Massel D, et al. Clot-bound thrombin is protectedfrom inhibition by heparin-antithrombin III but is susceptible to inactivation

by antithrombin III-independent inhibitors.J Clin Invest1990;86:385391.

94. Tollefsen DM, Majerus DW, Blank MK. Heparin cofactor II. Purificationand properties of a heparin-dependent inhibitor of thrombin in humanplasma. J Biol Chem 1982;257(5):21622169.

95. Broze GJ. Jr Protein Z-dependent regulation of coagulation. ThrombHaemost2001;86(1):813.

96. Moroi M, Aoki N. Isolation and characterization of alpha2-plasmininhibitor from human plasma. A novel proteinase inhibitor which inhibits

P.16




33/35

activator-induced clot lysis.J Biol Chem 1976;251(19):59565965.

97. Koie K, Kamiya T, Ogata K, et al. Alpha2-plasmin-inhibitor deficiency(Miyasato disease). Lancet1978;2(8104-5):13341336.

98. Schleef RR, Higgins DL, Pillemer E, et al. Bleeding diathesis due todecreased functional activity of type 1 plasminogen activator inhibitor.J ClinInvest1989;83(5):17471752.

99. Suzuki K, Deyashiki Y, Nishioka J, et al. Characterization of a cDNA forhuman protein C inhibitor. A new member of the plasma serine proteaseinhibitor superfamily.J Biol Chem 1987;262(2):611616.

100. Bertina RM, Koeleman BP, Koster T, et al. Mutation in blood coagulationfactor V associated with resistance to activated protein C. Nature

1994;369:6467.

101. Doolittle RF, Goldbaum DM, Doolittle LR. Designation of sequencesinvolved in the coiled-coil interdomainal connections in fibrinogen:constructions of an atomic scale model. J Mol Biol1978;120:311325.

102. Blomback B, Blomback M. The molecular structure of fibrinogen.Ann NY Acad Sci1972;202:7797.

103. Ferry JD. The mechanism of polymerization of fibrin. Proc Natl Acad SciU S A 1952;38:566.

104. Hermans J, McDonagh J. Fibrin: structure and interactions. SeminThromb Hemost1982;8(1):1124.

105. Robbins KC. A study on the conversion of fibrinogen to fibrin. Am JPhysiol1944;142:581580.

106. Schwartz ML, Pizzo SV, Hill RL, et al. Human factor XIII from plasma

and platelets. Molecular weights, subunit structures, proteolytic activation,and cross-linking of fibrinogen and fibrin. J Biol Chem1973;248:13951407.

107. Folk JE, Finlayson JS. The epsilon-(gamma-glutamyl)lysine crosslink andthe catalytic role of transglutaminases. Adv Protein Chem 1977;31:1133.

108. McKee PA, Mattock P, Hill RL. Subunit structure of human fibrinogen,soluble fibrin, and cross-linked insoluble fibrin. Proc Natl Acad Sci U S A 1970;66(3):738744.

109. Kaplan KL, Broekman MJ, Chernoff A, et al. Platelet alpha-granule




34/35

proteins:studies on release and subcellular localization. Blood1979;53(4):604618.

110. Mosher DF. Action of fibrin-stabilizing factor on cold-insoluble globulinand alpha2-macroglobulin in clotting plasma.J Biol Chem 1976;251(6):16391645.

111. Ruoslahti E, Pekkala A, Engvall E. Effect of dextran sulfate onfibronectincollagen interaction. FEBS Lett1979;107(1):5154.

112. Wagner DD, Urban-Pickering M, Marder VJ. Von Willebrand protein bindsto extracellular matrices independently of collagen. Proc Natl Acad Sci U S A 1984;81(2):471475.

113. Nachmias V, Sullender J, Asch A. Shape and cytoplasmic filaments in

control and lidocaine-treated human platelets. Blood1977;50(1):3953.

114. McDonagh J, Carrell N. Dysfibrinogens and other disorders of fibrinogenstructure a