title: Prothrombin activation by platelet-associated prothrombinase proceeds through the prethrombin-2 pathway via a concerted mechanism.
abstract: The protease α-thrombin is a key enzyme of the coagulation process as it is at the cross-roads of both the pro- and anti-coagulant pathways. The main source of α-thrombin in vivo is the activation of prothrombin by the prothrombinase complex assembled on either an activated cell membrane or cell fragment, the most relevant of which is the activated platelet surface. When prothrombinase is assembled on synthetic phospholipid vesicles, prothrombin activation proceeds with an initial cleavage at Arg-320 yielding the catalytically active, yet effectively anticoagulant intermediate meizothrombin, which is released from the enzyme complex ∼30-40% of the time. Prothrombinase assembled on the surface of activated platelets has been shown to proceed through the inactive intermediate prethrombin-2 via an initial cleavage at Arg-271 followed by cleavage at Arg-320. The current work tests whether or not platelet-associated prothrombinase proceeds via a concerted mechanism through a study of prothrombinase assembly and function on collagen-adhered, thrombin-activated, washed human platelets in a flow chamber. Prothrombinase assembly was demonstrated through visualization of bound factor Xa by confocal microscopy using a fluorophore-labeled anti-factor Xa antibody, which demonstrated the presence of distinct platelet subpopulations capable of binding factor Xa. When prothrombin activation was monitored at a typical venous shear rate over preassembled platelet-associated prothrombinase neither potential intermediate, meizothrombin or prethrombin-2, was observed in the effluent. Collectively, these findings suggest that platelet-associated prothrombinase activates prothrombin via an efficient concerted mechanism in which neither intermediate is released.
authors.affiliation: Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405, USA.
authors.forename: Laura M
authors.initials: LM
authors.lastname: Haynes
authors.forename: Beth A
authors.initials: BA
authors.lastname: Bouchard
authors.forename: Paula B
authors.initials: PB
authors.lastname: Tracy
authors.forename: Kenneth G
authors.initials: KG
authors.lastname: Mann
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology; D001777:Blood Coagulation; D001792:Blood Platelets / Q000378:metabolism; D002384:Catalysis; D003094:Collagen / Q000737:chemistry; D004792:Enzyme Precursors / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D008954:Models, Biological; D010743:Phospholipids / Q000737:chemistry; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism*; D013917:Thrombin / Q000737:chemistry; D013997:Time Factors
chemical_list: D000925:Anticoagulants; D004792:Enzyme Precursors; D008051:Lipid Bilayers; D010743:Phospholipids; D011516:Prothrombin; D003094:Collagen; C012007:prethrombins; D013917:Thrombin; C026812:meizothrombin; D015951:Factor Xa