Surface-dependent reactions of the vitamin K-dependent enzyme complexes

Abstract

During the past 20 years contributions from many laboratories have led to the development of isolation procedures, delineation of primary structures, and more recently, to the expression of recombinant proteins associated with the coagulation cascade. In general, studies of coagulation proteins under defined conditions have demonstrated the prescience of Davie and Ratnoff and MacFarlane in their proposals of the coagulation cascade. The more recent discovery of thrombomodulin by Esmon et al has led to the identification and characterization of components of the vitamin K-dependent anticoagulant pathway. In this review we have attempted to analyze and compare the functional properties of each of the vitamin K-dependent enzyme complexes associated with the procoagulant and anticoagulant phases of blood clotting. Although dissimilarities exist, the vitamin K-dependent complexes have analogous requirements and appear to function with a common general mode of organization. Membrane-bound cofactors serve as anchoring sites for the appropriate membrane-binding enzymes. This process localizes the complex on the membrane surface and increases the catalytic efficiency for substrate utilization. Complex formation provides extraordinary improvements in the catalytic efficiency for the complexes as compared with their soluble enzyme components. Membrane- bound complexes provide a mechanism that can be regulated at a site by membrane presentation, zymogen activation, and cofactor activation or presentation. The kinetic constants obtained for the various coagulation reactions determined in vitro provide some insights into how these pathways may function in vivo. The catalytic efficiency (kcat/Km) for factor X activation by factor VIIIa/factor IXa is far in excess of the catalytic efficiency of activation of factor X by tissue factor/factor VIIa (Table 3). This may provide a rational interpretation for the observation that patients with hemophilia A and B bleed even though they appear to have an alternative pathway to factor X activation. In addition, tissue factor is not ordinarily presented by the vascular tissue that has direct access to blood. However, it appears that extravascular constitutive tissue factor is available once the blood vessel becomes disrupted. The efforts to identify the initiating reactions of the blood coagulation process have not been unambiguously successful. We conclude that factor VII is most likely a zymogen, just as are the other proenzymes of the blood clotting process. In addition, it is difficult to rationalize the importance of the intrinsic pathway of coagulation involving factor XII, prekallikrein, and high molecular weight kininogen since the congenital absence of any one of these factors does not result in abnormal bleeding.

Coagulation Factor VIIa

“…The activated enzyme (factor Xa) is an Arg-specific serine protease related to trypsin, but has much narrower substrate specificity. The N-terminal γ-carboxyglutamic acid (Gla) domain binds B8À10 calcium ions in a cooperative manner (average K d B 0.5 mM) and mediates the interaction of factor X/Xa with phospholipid membranes [4,5,6]. In the presence of calcium ions, factor Xa forms a phospholipid-bound complex with a cofactor, factor Va.…”

“…This 'prothrombinase' complex activates prothrombin (Chapter 643) B300 000fold more rapidly than does free factor Xa. Studies of this reaction have demonstrated that phospholipid reduces the K m for prothrombin, whereas factor Va increases the V max [5]. In vivo, the surface of activated platelets is the main site at which the prothrombinase complex is assembled.…”

“…Factor Va is derived from factor V by specific proteolytic cleavages mediated by thrombin or factor Xa. Membrane-bound factor Va binds factor Xa with high affinity (K d B 1 nM), due at least in part to interactions between the catalytic domain (residues 163À170 [7]) and residues 499À505 of factor Va [8], but has no measurable affinity for the zymogen [5]. In the absence of factor Va, factor Xa binds to phospholipid membranes with a K d of B30À100 nM.…”

“…Factor Xa activates prothrombin, a single-chain molecule, by hydrolyzing two peptide bonds and the reaction may thus proceed via two pathways [5,10]. In one pathway, cleavage of the Arg320kIle321 bond yields an intermediate, meizothrombin, which may then be cleaved at the Arg271kThr272 bond to yield fragment 1-2 and thrombin [11].…”

“…This pathway is called the 'extrinsic pathway' and is responsible for the initiation of coagulation, proceeding mainly on the surface of damaged endothelial cells and macrophages, but probably also on activated platelets [58,59]. Alternatively, factor X is activated on the platelet surface by a membrane-bound 'tenase' complex comprising factor IXa, its cofactor factor VIIIa, and calcium ions, which activates factor X B 10 6 -fold more rapidly than factor IXa alone [5]. This 'intrinsic pathway' is responsible for amplifying the coagulation process (see also Chapter 640) and its importance is illustrated by the fact that hereditary deficiency of factors IX or VIII causes hemophilia B and A, respectively.…”