Role of Procoagulant Lipids in Human Prothrombin Activation. 2. Soluble Phosphatidylserine Upregulates and Directs Factor X_a to Appropriate Peptide Bonds in Prothrombin

Abstract

Activation of human prothrombin to thrombin (II(a)) by factor X(a) during blood coagulation requires proteolysis of two bonds and thus involves two possible activation pathways (parallel-sequential activation model). Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). A soluble lipid, dicaproylphosphatidylserine (C6PS), enhances activation by 60-fold [Koppaka et al. (1996) Biochemistry 35, 7482]. We report here that C6PS binding to factor X(a) not only enhances the rate of activation but also alters the pathway. Activation was monitored using a chromogenic substrate (S-2238) to detect both II(a) and MzII(a) active site formation and SDS-PAGE to detect Pre2-F1.2 as well as II(a) and MzII(a). Of the four kinetic constants needed to describe activation, two (MzII(a) and Pre2-F1.2 consumption) were measured directly, and two (MzII(a) and Pre2-F1.2 formation) were obtained by fitting the three time courses simultaneously to the parallel-sequential reaction model. The time courses of II(a), MzII(a), and Pre2-F1.2 formations were all well described below the C6PS critical micelle concentration (CMC) by this activation model. The rate of Arg(322)-Ile cleavage leading to MzII(a) formation increased by 150-fold, while the rate of Arg(273)-Thr cleavage leading to Pre2-F1.2 formation was inhibited slightly. At concentrations of water-soluble C6PS above its CMC, all four proteolytic reactions increased in rate by 2-5-fold at the C6PS CMC. We conclude that soluble C6PS differentially affects the rate of individual bond cleavages during prothrombin activation in solution such that activation occurs almost exclusively via MzII(a) formation. Finally, C6PS enhanced the rates of all proteolytic reactions to within a factor of 3 of the enhancement seen with PS-containing membranes. We conclude that PS-containing membranes regulate prothrombin activation by factor X(a) mainly via interaction of individual PS molecules with factor X(a).

A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex

“…These observations and other previously published observations on the C2 domain 16,[20][21][22][23]25,33,39 suggest a model for FVa membrane binding and activation by PS, as summarized in Figure 6. In this model, binding of wild-type rFVa 2 to a PS-containing membrane (top panel) occurs primarily (but not exclusively 16,20 ) via the C2 domain.…”

Soluble Phosphatidylserine Triggers Assembly in Solution of a Prothrombin-activating Complex in the Absence of a Membrane Surface

“…This means that activation proceeds via two possible intermediates and that four distinct proteolytic reactions must be characterized to define the process (22). We showed that, even in the absence of FV a , PS-containing membranes as well as C6PS 1) alter the rates of all four reactions, 2) alter the preferred pathway of activation, and 3) cause some intermediate to be converted processively to thrombin without release from the prothrombinase complex (22,31). In order to avoid these complications in characterizing the activity of the soluble FX a ⅐FV a2 complex, we focused here on only one of the four possible reactions, conversion of the intermediate MzII a to thrombin.…”

“…This implies that it is the exposure of PS on the surface of activated platelet membranes, not the membrane surface itself, that is crucial to assembly of the prothrombinase complex. It also suggests that PS exposure acts not only to locate factor X a to the membrane surface but also to activate this enzyme (8,31). 2 Similarly, PS exposure both locates factor V a to the membrane and activates it (10) to bind factor X a (Fig.…”

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

“…4), even a small amount of PS in the 30% DOPE membrane will occupy the regulatory sites on Xa and Va to increase in V sat and decrease in K d app (Table 1). Even though prothrombin does not bind tightly to a 30% PE membrane, the well known reduction of K m upon binding to PS-containing membranes is due to binding of lipids to the regulatory site on Xa and not to the availability of membrane-associated prothrombin (40). In this picture, the effect of PE on membrane interfacial packing contributes only to bringing factor Va to the membrane, but not for the reduction in K m , the increase in k cat , and the decrease in K d app , which result from PE regulation of both factors Xa and Va.…”