Prothrombin activation on the activated platelet surface optimizes expression of procoagulant activity

Abstract

Effective hemostasis relies on the timely formation of-thrombin via prothrombi-nase, a Ca 2-dependent complex of factors Va and Xa assembled on the activated platelet surface, which cleaves prothrombin at Arg271 and Arg320. Whereas initial cleavage at Arg271 generates the inactive intermediate prethrom-bin-2, initial cleavage at Arg320 generates the enzymatically active intermediate meizothrombin. To determine which of these intermediates is formed when pro-thrombin is processed on the activated platelet surface, the cleavage of prothrom-bin, and prothrombin mutants lacking either one of the cleavage sites, was monitored on the surface of either thrombin-or collagen-activated platelets. Regardless of the agonist used, prothrombin was initially cleaved at Arg271 generating pre-thrombin-2, with-thrombin formation quickly after via cleavage at Arg320. The pathway used was independent of the source of factor Va (plasma-or platelet-derived) and was unaffected by soluble components of the platelet releasate. When both cleavage sites are presented within the same substrate molecule, Arg271 effectively competes against Arg320 (with an apparent IC 50 0.3M), such that more than 90% to 95% of the initial cleavage occurs at Arg271. We hypothesize that use of the prethrombin-2 pathway serves to optimize the procoagu-lant activity expressed by activated plate-lets, by limiting the anticoagulant functions of the alternate intermediate, meizothrombin. (Blood. 2011;117(5): 1710-1718) Introduction The activation of prothrombin to-thrombin is a critical step in the response to vascular injury. The generation of-thrombin is achieved through the action of prothrombinase, which is composed of the serine protease factor Xa and its nonenzymatic cofactor, factor Va, assembled on an appropriate membrane surface in the presence of Ca 2 ions. 1 In the physiologic setting, this surface is provided by the activated platelet. 2 Relative to the activity of factor Xa alone, incorporation of factor Xa into prothrombinase accelerates the rate of prothrombin cleavage by 5 orders of magnitude, 3 and both factor Va and the membrane surface are critical in this rate amplification, as removal of either component results in a substantial decrease in the rate of prothrombin cleavage. 3 Indeed, deficiencies or disorders of any component of this complex result in severe bleeding diatheses. 4-6 Prothrombin, the substrate for prothrombinase, consists of 4 domains: fragment 1, fragment 2, and the A and B chains of-thrombin (Figure 1). 1 Prothrombin is proteolytically activated to-thrombin by cleavage on the C-terminal side of 2 specific residues: Arg271 (located between fragment 2 and the A chain) and Arg320 (located between the A and B chains). Initial cleavage at Arg271 results in the generation of prethrombin-2, an inactive intermediate, and the release of fragment 1.2 (F1.2). Subsequent cleavage at Arg320 converts prethrombin-2 to-thrombin. 7 Alternatively , cleavage may occur first at Arg320, leading to the generation of the enzymatically active interme...

Histone H4 Promotes Prothrombin Autoactivation

“…In this context, it becomes of interest to ponder what conformation of prothrombin is stabilized upon interaction with the prothrombinase complex and whether the selection depends on the environment in which prothrombin activation takes place. Based on the results reported here, the fully extended conformation of prothrombin is not prone to autoactivation and may be the one stabilized by assembly of the prothrombinase complex on the surface of platelets, thereby making cleavage at Arg-271 preferred over cleavage at Arg-320 under conditions relevant to physiology (3,4). On the other hand, conditions where factor Xa and cofactor Va induce conformational transitions in prothrombin similar to those induced by histone H4 may direct cleavage at Arg-320 and promote thrombin generation along the meizothrombin pathway, where cleavage at Arg-320 precedes cleavage at Arg-271 (1).…”

“…The alternative cleavage at Arg-320 separates the A and B chains that remain connected through a disulfide bond and yields the active intermediate meizothrombin. Under physiological conditions on the surface of platelets, activation of prothrombin proceeds via the prethrombin-2 intermediate (3,4). The recent crystal structure of prothrombin supports this preferred pathway of activation based on the different solvent accessibility of the two sites of cleavage and the distinct electrostatic properties of the epitope recognizing factor Xa in prothrombin and prethrombin-2 (5).…”

Nuevos anticoagulantes orales

“…Con esto se origina una activación plaquetaria con la subsecuente secreción granular y agregación. El tapón plaquetario inicial provee la superfi cie rica en fosfolípidos aniónicos en donde se ensamblan los complejos macro-moleculares y se efectúan las reacciones enzimáticas que concluyen en la generación de trombina 8 . La exposición del factor tisular (FT), ya sea desde el subendotelio (pared vascular o ateroma) o desde células circulantes que lo pueden expresar (ej.…”

Meizothrombin Preparation and Its Role in Fibrin Formation and Platelet Aggregation

“…Active FIIa is formed as a result of following cleavage of intermediates. Both the PL composition of platelet membranes and the conformation of prothrombinase are assumed to govern the pathway choise in vivo [1]- [5].…”