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Meizothrombin-Thrombin-Prothrombin activation lntroduction The factor Xu-catalyzed conversion of the zymogen prothrombin into the active serine protease thrombin is one of the crucial steps of blood coagulation (1). Thrombin is the enzyme responsible for the conversion of fibrinogen into fibrin , a reaction that is followed by fibrin polymerization and clot formation. Thrombin also activates the transglutaminase factor XIII to factor XIII., an enzyme which stabilizes the fibrin clot. Apart from its role in the formation and stabilization of the fibrin clot, thrombin also has an important function in the regulation of the overall hemostatic process. Thus, thrombin accelerates its own rate of formation by activating the blood coagulation factors V and VIII and by stimulating blood platelets. Potential negative feedback control is exerted through the thrombin-dependent activation of protein C, producing an enzyme with anticoagulant properties that inactivates factors % and VIII. and that may also stimulate the fibrinolytic pathway. Considering the key role of thrombin in hemostatic plug formation, it is not surprising that the activation of prothrombin is one of the most intensively studied coagulation reactions (2) and that the product of this reaction, thrombin, is one of the best charact erized coagulation enzymes (3). Thrombin is not the only catalytically active product that is formed during prothrombin activation. In recent years it has been shown that during factor Xu-catalyzed prothrombin activation in addition to thrombin substantial amounts of another enzymatically active product i.e. meizothrombin can be formed (4,5). Meizothrombin differs significantly from thrombin in many of its properties and therefore, it may have different functions during the hemostatic process. In view of the pivotal role of thrombin in blood coagulation it will not be surprising that further studies of another enzymatically active prothrombin derivative with different catalytic properties may add to our understanding of the mechanisms of the reactions leading to thrombus formation. This The nomenclature used for proteolytically derived products of prothrombin is that recommended by the International Committee on Thrombosis and Haemostasis (Jackson C M. Thromb Haemostas 1977;

In this article we have reviewed the current knowledge regarding the involvement of Factor XII in contact activation. Clearly in the past decade an overwhelming amount of data and hypotheses have been published regarding the central role of this zymogen in the initiation and further propagation of contact activation reactions. Therefore we feel that it will be helpful to conclude this article with a figure that summarizes those interactions and reactions that are generally believed to reflect the major molecular events occurring during surface-dependent contact activation. The contact factors are capable of very efficient interation with each other, provided a suitable negatively charged surface is present. Such surfaces are thought to stimulate the interactions between the contact factors through binding of the proteins and thus bringing the proteins together. Factor XII readily binds to the negatively charged surface, but for the binding of prekallikrein and Factor XI, the cofactor HMW kininogen is likely to be necessary. Bound at the surface, the zymogens Factor XII and prekallikrein are thought to be involved in a so-called reciprocal activation mechanism in which Factor XIIa activates prekallikrein to kallikrein, which in turn converts Factor XII to Factor XIIa. The formation of Factor XIIa is further promoted by the fact that surface-bound Factor XII is likely more susceptible to proteolytic cleavage and by the fact that the activated Factor XIIa is capable of auto-activating its own zymogen Factor XII. However, the latter effect, although undoubtedly contributing to the formation of Factor XIIa at the surface, seems to be of less importance than the reciprocal activation mechanism. This is underscored by the fact that Factor XII activation is rather slow in prekallikrein-deficient plasma. Surface-bound Factor XIIa is then responsible for the activation of Factor XI to Factor XIa, thereby propagating the initial trigger. Presumably, Factor XIa must leave the surface in order to be able to become involved in the activation of blood coagulation Factor IX.

Factor Xa catalyzed prothrombin activation is strongly stimulated by the presence of negatively charged membranes plus calcium ions. Here we report experiments in which we determined the prothrombin-converting activity of phosphatidylcholine (PC) membranes that contain varying amounts of different anionic lipids, viz., phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylmethanol (MePA), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidyl-beta-lactate (PLac), sulfatides (SF), sodium dodecyl sulfate (SDS), and oleic acid. All anionic lipids tested were able to accelerate factor Xa catalyzed prothrombin activation, in both the absence and presence of the protein cofactor Va. This shows that the prothrombin-converting activity of negatively charged membranes is not strictly dependent on the presence of a phosphate group but that lipids which contain a carboxyl or sulfate moiety are also able to promote the formation of a functionally active prothrombinase complex. In the absence of factor Va, the prothrombin-converting activity of membranes with MePA, PG, PE, PLac, SF, or SDS was strongly inhibited at high ionic strength, while the activity of PS- and PA-containing membranes was hardly affected by ionic strength variation. This suggests that in the case of the ionic strength sensitive lipids electrostatic forces play an important role in the formation of the membrane-bound prothrombinase complex. For PS and to a lesser extent for PA we propose that the formation of a coordinated complex (chelate complex) with Ca2+ as central ion and ligands provided by the gamma-carboxyglutamic acid residues of prothrombin and factor Xa and the polar head group of phospholipids is the major driving force in protein-membrane association. Our data indicate that the anionic lipids used in this study can be useful tools for further investigation of the molecular interactions that play a role in the assembly of a membrane-bound prothrombinase complex. Membranes that were solely composed of PC can also considerably enhance prothrombin activation in the presence of factor Va. This activity of PC is only observed on membranes which are composed of PC that contains unsaturated hydrocarbon side chains. Membranes prepared from phosphocholine-containing lipids with saturated hydrocarbon side chains such as dimyristoyl-PC, dipalmitoyl-PC, distearoyl-PC, and dioctadecylglycerophosphocholine hardly accelerated prothrombin activation.(ABSTRACT TRUNCATED AT 250 WORDS)

Incubation of purified human plasma prekallikrein with sulfatides or dextran sulfate resulted in spontaneous activation of prekallikrein as judged by the appearance of amidolytic activity towards the chromogenic substrate H-D-pro-phe-arg-p-nitroanilide (S 2302). The time course of generation of amidolytic activity was sigmoidal with an apparent lag phase followed by a rapid activation until finally a plateau was reached. Soybean trypsin inhibitor completely blocked prekallikrein activation whereas corn, limabean and ovomucoid trypsin inhibitor did not. The Ki of the reversible inhibitor, benzamidine, for autoactivation (240 uM) was identical to the Ki of benzamidine for kallikrein. Thus, spontaneous prekallikrein activation and kallikrein showed the same specificity for a number of serine protease inhibitors, indicating that prekallikrein is activated by its own enzymatically active form, kallikrein. Immunoblotting analysis showed that, concomitant with the appearance of amidolytic activity, prekallikrein was cleaved. However, prekallikrein was not quantitatively converted into two-chain kallikrein since other polypeptide products were visible on the gels. This accounts for the observation that in amidolytic assays not all prekallikrein present in the reaction mixture was measured as active kallikrein. Kinetic analysis showed that prekallikrein activation can be described by a second-order reaction mechanism in which prekallikrein is activated kallikrein. The apparent second order rate constant was 27000 M-ls-1 (pH 7.2, 50 uM sulfatides, ionic strength 1=0.06, at 37°C). Autocatalytic prekallikrein activation was strongly dependent on the ionic strength, since there was a considerable decrease in the rate of the reaction at high salt concentrations. Our data support a prekallikrein autoactivation mechanism in which surface-bound kallikrein activates surface-bound prekallikrein. The rate constant of autoactivation is considerably lower than the rate constants reported for Factor Xlla dependent prekallikrein formation. Autocatalytic prekallikrein activation may, however, contribute to kallikrein formation during the initiating phase of contact activation.