title: The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase.
abstract: Incorporation of factor (F) Va into prothrombinase directs prothrombin activation by FXa through the meizothrombin pathway, characterized by initial cleavage at Arg(320). We have shown that a pentapeptide with the sequence DYDYQ specifically inhibits this pathway. It has been also established that Hir(54-65)(SO(3)(-)) is a specific inhibitor of prothrombinase. To understand the role of FVa within prothrombinase at the molecular level, we have studied thrombin formation by prothrombinase in the presence of various prothrombin-derived fragments alone or in combination. Activation of prethrombin 1 is slow with cleavages at Arg(320) and Arg(271) occurring with similar rates. Addition of purified fragment 1 to prethrombin 1 accelerates both the rate of cleavage at Arg(320) and thrombin formation. Both reactions were inhibited by Hir(54-65)(SO(3)(-)) while DYDYQ had no significant inhibitory effect on prethrombin 1 cleavage in the absence or presence of fragment 1. Similarly, activation of prethrombin 2 by prothrombinase, is inhibited by Hir(54-65)(SO(3)(-)), but is not affected by DYDYQ. Addition of purified fragment 1*2 to prethrombin 2 accelerates the rate of cleavage at Arg(320) by prothrombinase. This addition also results in a significant inhibition of thrombin formation by DYDYQ and is concurrent with the elimination of the inhibitory effect of Hir(54-65)(SO(3)(-)) on the same reaction. Finally, a membrane-bound ternary complex composed of prethrombin 2/fragment 1*2/Hir(54-65)(SO(3)(-)) is inhibited by DYDYQ. Altogether, the data demonstrate that membrane-bound fragment 1 is required to promote optimum Fva cofactor activity which in turn is translated by efficient initial cleavage of prothrombin by prothrombinase at Arg(320).
authors.affiliation: Cleveland State University, Department of Chemistry, OH 44115, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Bukys
authors.forename: Tivadar
authors.initials: T
authors.lastname: Orban
authors.forename: Paul Y
authors.initials: PY
authors.lastname: Kim
authors.forename: Michael E
authors.initials: ME
authors.lastname: Nesheim
authors.forename: Michael
authors.initials: M
authors.lastname: Kalafatis
mesh_terms: D001120:Arginine / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism; D005165:Factor V / Q000378:metabolism*; D015943:Factor Va / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011498:Protein Precursors / Q000378:metabolism*; D011516:Prothrombin / Q000737:chemistry / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism*; D013925:Thromboplastin / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D006629:Hirudins; D010446:Peptide Fragments; D011498:Protein Precursors; C045879:prothrombin fragment 1.2; C022475:prothrombinase complex; D015943:Factor Va; C029733:prothrombin fragment 1; D005165:Factor V; D011516:Prothrombin; D013925:Thromboplastin; C012007:prethrombins; D001120:Arginine; D013917:Thrombin; C026812:meizothrombin; D015951:Factor Xa