The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase.
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).
Bukys
Michael A
MA
Cleveland State University, Department of Chemistry, OH 44115, USA.
Orban
Tivadar
T
Kim
Paul Y
PY
Nesheim
Michael E
ME
Kalafatis
Michael
M
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*
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