ultimately in the formation of the stable, insoluble fibrin clot. Many of the activated zymogens are serine proteases, descended from the same evolutionary tree as the digestive enzymes, trypsin, chymotrypsin and elastase. They contain the catalytic triad consisting of histidine, aspartic acid and serine. These include kallikrein, thrombin, factors Vila, IXa, Xa, XIa and Xlla from the coagulation cascade and plas-min, t-PA, and urinary-plasminogen activator (u-PA) from the fibrinolytic enzyme system. This hemostatic process was historically conceived to meld an intrinsic pathway with an extrinsic pathway at a juncture resulting in the activation of factor X to factor Xa, whereupon a common pathway concluded with the formation of the fibrin clot. The clotting cascade including the extrinsic and the 'historic* intrinsic pathways, together with the fibrinolytic system for dissolution of the clot, is depicted in Figure 1. Activation of the historic intrinsic pathway could be accomplished upon contact of factor XII with a negatively charged surface to yield the activated factor Xlla. It was observed that factor Xlla could activate two zymogens, prekallikrein (PK) and factor XI, each in the presence of HMWK. Activation of PK yields the protease kallikrein (K). K, in turn, activates factor XII in the presence of HMWK to produce additional factor Xlla, a positive feedback step as the intrinsic cascade is initiated. (K activity is inhibited by a,-AT, arMG, CI-inhibitor and APCI.) The activation of factor XI by factor Xlla yields factor XIa. Factor XI is also activated by factors XIa and Ha (thrombin), the latter activation requiring heparin as a cofactor. The activation of factor XIa by thrombin is inhibited by fibrinogen. (Antithrombin III (AT III) and APCI inhibit the subsequent proteolytic activity of factor XIa).

All subsequent steps involving the intrinsic system factors occur on membrane surfaces such as the platelet membrane. Endothelial cell membranes also bind coagulation factors. PL is required, serving as the negatively charged surface. The localization of PL in the membrane permits the formation of ternary and quaternary complexes as necessary, for the accomplishment of the clotting steps. Factor XIa activates factor IX in the presence of Ca2+. (AT III inhibits the proteolytic activity of factor IXa.) Factor IXa, in the presence of factor Villa and Ca2+, activates factor X to factor Xa. The activation of factor Villa will be dealt with below. Factor Xa then activates prothrombin (factor II) to yield thrombin (factor Ila). This occurs with the mediation of factor Va in the presence of Ca2+. Thrombin subsequently acts upon circulating or locally entrapped fibrinogen to generate the initial fibrin gel which is then cross-linked with the assistance of a transglutaminase, fac tor XHIa, in the presence of Ca'+. Fibrinogen is a protein containing six polypeptide chains, two Act, two B|3 and two y chains, appropriately linked via disulfide bridges. Initial activation of fibrinogen by thrombin causes the release of fibrinopeptide A from the a chain in a relatively rapid reaction, permitting polymerization of the remaining protein. A rate-lim-iting cleavage of fibrinopeptide B from the Bp chain subsequently occurs. The resultant fibrin molecules may then be cross-linked longitudinally and transversely by factor XHIa. Factor XHIa is activated by thrombin from its inactive precursor, factor XIII, in the presence of Ca2+. Factor XHIa also functions in the presence of Ca2+. The transglutaminase excises the amide groups of select glutamines in the carboxyl terminal region of y chains and links the resultant glutamic acids with select e-amino groups of lysines in the carboxyl terminal region of the y chains. It also acts similarly with the a chains. Longitudinal cross-links are established with y-y as well as a-a chains, whereas transverse links are formed only with a-a chains. The resultant polymeric product is increasingly insoluble and more resistant to proteolysis. Factor XHIa cross-links other localized proteins in addition to fibrin, such as fibronectin, thrombo-spondin, collagen, actin, vWF and a,-AP, in forming a tight mesh. Hence, components related to coagulation and fibronolytic mechanisms may be effectively entrapped at the coagulation site. As will be discussed below in the fibrinolytic section, plasminogen is incorporated in the fibrin clot. Furthermore, t-PA binds to fibrin.

Factor Xa and thrombin have multiple roles in the clotting system. Thrombin activates the inactive factor XIII to the active factor XHIa in the presence of Ca2+. The initial fibrin gel appears to promote this step. In addition to activating factors XI and XIII, thrombin activates factor V to factor Va, factor VIII to factor Villa in the presence of Ca2% and the tissue factor-VII (TF-VII) complex to its active form, TF-VIIa.

TF is considered as part of the extrinsic blood clotting pathway (to be described shortly, after further discussions of factors Va, Villa, Ila and Xa). Factor Xa also catalyzes the activation of factor V, as does thrombin. The activation of factor VIII by thrombin is more complex. Factor VIII circulates in blood as a complex with vWF. In the process of activation of factor VIII by thrombin, vWF dissociates from the complex, thereby availing itself of the opportunity to complex with more circulating factor VIII. vWF stabilizes factor VIII, thereby prolonging its circulating half-life. Factor Villa can apparently be degraded to degradation products spontaneously. This degradation occurs at a reduced rate in the presence of high concentrations of factor IXa. Factor Xa also activates factor VIII to factor Villa. The activation of factors Va and Villa by thrombin and factor Xa serve as additional examples of positive feedback.

Factors Va and Villa, which participate in the coagulation system as cofactors of factor Xa and IXa, respectively, are also subject to enzymic degradation. Both factors Va and Villa are degraded by a complex of activated protein C (APC) with protein S (PS), APC-PS. The formation of the APC-PS complex occurs on the membrane surface, as do all the reactions in the clotting cascade from the functioning of factor XIa to the activation of prothrombin. The series of reactions begins with the initial formation of a complex comprising thrombin, thrombomodulin (an EC membrane protein) and Ca2+ on the membrane. This complex catalyzes the transformation of protein C (PC) to APC. The APC interacts with PS to yield the APC-PS complex in which the APC contains the catalytic degradative potential to fragment factors Va and Villa. Under such circumstances, the catalytic functions of factors IXa and Xa are depressed, thereby reducing the generation of thrombin from prothrombin. Hence, thrombin can act not only as a positive modulator (positive feedback) in activating factors Va and Villa, but also as a negative modulator by initiating the formation of APC-PS which degrades factors Va and Villa, limiting thrombin production. An inhibitor of APC (APCI) has been identified which inhibits the formation of the APC-PS complex from its APC and PS components. It further inhibits the functioning of factor XIa. It therefore functions overall to promote clotting at the Va and Villa steps by 'lowering' their 'inactivation' rate. Its effect on factor XIa may be of questionable impact, as discussed below. Another protein, a plasma component, C4b-binding protein (C4b-BP), also negatively modulates the degradation of factors Va and Villa by forming a complex with PS, the PS-C4b-BP complex which competitively blocks the formation of the APC-PS complex.

Thrombin activity is inhibited by four naturally occurring proteins: antithrombin III (ATIII), a2-MG, HCII, and APCI. The APCI also inhibits factor Xa. ATIII, HCII and APCI are serpins (serine protease inhibitors). ATIII is a glycoprotein which is capable of forming a stable tetrahedral complex with thrombin thereby inactivating it. ATIII also inhibits factors IXa, Xa, XIa and Xlla. ATIII contains two domains for binding heparin. Both domains are rich in basic amino acids. Hence they readily bind to the negatively charged sulfate esters in heparin. They also bind to heparan sulfate, a proteoglycan which is synthesized by ECs and expressed to their surface. Hep arin is synthesized by mast cells and is detected in granules of these cells beneath endothelial tissue. When ATIII binds to heparin to form an ATIII—heparin complex, the conformation of the ATIII is presumably altered, significantly enhancing reaction with thrombin. Following formation of the ATIII-thrombin complex, heparin is released, thereby permitting it to react with free ATIII molecules. The heparan sulfate-ATIII complex is also a powerful inhibitor of thrombin. HCII inactivates thrombin. Its inactivation of thrombin can be accelerated 1000-fold by complexing with dermatan sulfate, a muco-polysaacharide component of the vascular wall.

As may be seen in Figure 1, factor Xa exerts multiple roles in coagulation, as does thrombin. It activates factors II, V and VIII, thereby promoting the clotting process. It also activates factors IX on a membrane surface, a positive feedback step, thereby stimulating its own activation from factor X, and is further involved in the activation of factor IX by activating the TF-VII complex of the extrinsic clotting system. Lastly, it is a component of a negative feedback system whereby it complexes with tissue factor pathway inhibitor (TFPI) to form the Xa-TFPI complex which inhibits factor Xa function as well as TF-Vlla function (Factor Xa is also inhibited by ATIII and by APCI.)

The significance of the contact system for the initial activation of blood clotting in the intrinsic clotting pathway has been questioned as a functional pathway in vivo as a deficiency in factor Xfl does not lead to a clotting abnormality. In addition, a deficiency in factor XI leads to a mild clotting abnormality, if at all. Hence, current views accept the proposal that the exposure of damaged vascular tissue initiates the clotting cascade in what historically was considered as the extrinsic clotting system. Damaged vascular tissue causes an immediate response with the induction of mRNA for TF. TF is a glycoprotein which is associated with the membrane of medial smooth muscle cells and adventitia. It has not been observed in the EC. However, it is inducible in the EC upon vascular injury, and exposure to thrombin. Other factors inducing enhancement of TF are platelets, granulocytes, macrophages, IL-1, and tumor necrosis factor a. Exposed TF binds to factor VII to form the TF-VII complex, a mostly inactive complex. It is activated by thrombin, which may be present in small amounts at the initial site of damage, as well as factor Xa. The TF-VII complex can also be activated by factors IXa and Xlla, and by its product, TF-VIIa, the latter being a positive feedback step. The TF-VIIa complex reacts with factor X on the membrane surface to transform factor X to the active factor Xa. Subsequently factor Xa activates

Table 1 Inhibitors of thrombotic and fibrinolytic enzymes

Coagulation/ ATI 11 ATIII-H a2-MG HCII TFPI TFPI Xa fibrinolytic enzyme a^-AT Fg a2-AP PAI-1 PAI-2 C1 inhibitor Heparan Protein C

sulfate-ATIII inhibitor

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