Fate of Membrane-bound Reactants and Products during the Activation of Human Prothrombin by Prothrombinase

Abstract

Membrane binding by prothrombin, mediated by its N-terminal fragment 1 (F1) domain, plays an essential role in its proteolytic activation by prothrombinase. Thrombin is produced in two cleavage reactions. One at Arg 320 yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg 271 , yields thrombin and severs covalent linkage with the N-terminal fragment 1.2 (F12) region. Covalent linkage with the membrane binding domain is also lost when prethrombin 2 (P2) and F12 are produced following initial cleavage at Arg 271 . We show that at the physiological concentration of prothrombin, thrombin formation results in rapid release of the proteinase into solution. Product release from the surface can be explained by the weak interaction between the proteinase and F12 domains. In contrast, the zymogen intermediate P2, formed following cleavage at Arg 271 , accumulates on the surface because of a ϳ20-fold higher affinity for F12. By kinetic studies, we show that this enhanced binding adequately explains the ability of unexpectedly low concentrations of F12 to greatly enhance the conversion of P2 to thrombin. Thus, the utilization of all three possible substrate species by prothrombinase is regulated by their ability to bind membranes regardless of whether covalent linkage to the F12 region is maintained. The product, thrombin, interacts with sufficiently poor affinity with F12 so that it is rapidly released from its site of production to participate in its numerous hemostatic functions.Thrombin, the key effector serine proteinase of the blood coagulation cascade, is produced by specific and limited proteolysis of the zymogen, prothrombin. The physiologically relevant catalyst for this reaction is the prothrombinase complex consisting of the serine proteinase, factor Xa, and the cofactor, factor Va, assembled on membranes in the presence of Ca 2ϩ(1). In addition to facilitating the assembly of the enzyme complex, membranes containing acidic or amino phospholipids play an important role in mediating the delivery of prothrombin to the membrane-bound enzyme (1, 2). This arises from the ability of prothrombin to bind to these membranes through the fragment 1 (F1) 2 domain present at its N terminus (1, 3, 4). Thrombin, derived from the C-terminal half of prothrombin, is produced as a result of cleavages 3 following Arg 271 and Arg 320(1, 3, 5). Cleavage at Arg 320 converts the zymogen to a proteinase, whereas cleavage at Arg 271 severs covalent linkage with the N-terminal fragment 1.2 (F12) domain harboring the membrane binding site (Scheme 1). Covalent linkage of the C-terminal domain with F12 is also lost in the zymogen intermediate, prethrombin 2 (P2), produced following cleavage only at Arg 271 (Scheme 1). In contrast, meizothrombin (mIIa), produced following cleavage only at Arg 320 is covalently linked to the membrane binding domain through a disulfide bond (Scheme 1). Accordingly, both prothrombin and mIIa are established to bind to membranes, and this binding interaction impacts the...

Ligand Binding Shuttles Thrombin along a Continuum of Zymogen- and Proteinase-like States

“…Isothermal titration calorimetry of F12 binding to IIa TAT yielded heat traces and an isotherm (Fig. 1) intermediate to those expected from previous measurements of F12 binding to P2 or thrombin (18). Thus, the thermodynamic constants for F12 binding are apparently sensitive to subtle aspects of proteinase formation beyond simply reporting on the cleavage, or lack thereof, at the Arg 15 -Ile 16 bond.…”

“…The large thermodynamic differences in the binding of F12 to zymogen relative to proteinase are surprisingly not observed with fragment 2 produced by proteolysis of F12 (18). Studies with EDTA did not reduce F12 binding to that observed with fragment 2 (data not shown).…”

“…Thrombin-Thermodynamic studies have previously established that F12 binds with ϳ20-fold higher affinity to the zymogen P2 than it does to thrombin (18). We therefore investigated the binding of F12 to a thrombin variant (IIa TAT ) bearing ThrAla-Thr in place of the N-terminal Ile 16 -Val 17 -Glu 18 sequence, intended to impair salt bridge formation and the conformational transition to proteinase.…”

“…F12 represents the authentic protein ligand for ABE2, with particular relevance to the process of thrombin formation. The recent observation that F12 binds to a zymogen precursor with higher affinity than to thrombin unexpectedly suggests that the conversion of zymogen to proteinase is accompanied by reciprocal changes in ligand affinity for the two exosites in thrombin (18). Whereas ligand binding to ABE1 is expected to favor the proteinase, ligation at ABE2 would instead favor the zymogen.…”

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

“…Researchers have used synthetic lipids (16,20), as well as lipids derived from natural sources (21,22). In our work, we use synthetic lipids because they are chemically more stable.…”