A Control Switch for Prothrombinase: CHARACTERIZATION OF A HIRUDIN-LIKE PENTAPEPTIDE FROM THE COOH TERMINUS OF FACTOR Va HEAVY CHAIN THAT REGULATES THE RATE AND PATHWAY FOR PROTHROMBIN ACTIVATION

Michael A Bukys,Paul Y Kim,Michael E Nesheim,Michael Kalafatis

doi:10.1074/jbc.m604482200

Abstract

Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg(271) and prethrombin 2 formation (pre2 pathway). Factor Va directs prothrombin activation by factor Xa through the meizothrombin pathway, characterized by initial cleavage at Arg(320) (meizo pathway). We have shown previously that a pentapeptide encompassing amino acid sequence 695-699 from the COOH terminus of the heavy chain of factor Va (Asp-Tyr-Asp-Tyr-Gln, DYDYQ) inhibits prothrombin activation by prothrombinase in a competitive manner with respect to substrate. To understand the mechanism of inhibition of thrombin formation by DYDYQ, we have studied prothrombin activation by gel electrophoresis. Titration of plasma-derived prothrombin activation by prothrombinase, with increasing concentrations of peptide, resulted in complete inhibition of the meizo pathway. However, thrombin formation still occurred through the pre2 pathway. These data demonstrate that the peptide preferentially inhibits initial cleavage of prothrombin by prothrombinase at Arg(320). These findings were corroborated by studying the activation of recombinant mutant prothrombin molecules rMZ-II (R155A/R284A/R271A) and rP2-II (R155A/R284A/R320A) which can be only cleaved at Arg(320) and Arg(271), respectively. Cleavage of rMZ-II by prothrombinase was completely inhibited by low concentrations of DYDYQ, whereas high concentrations of pentapeptide were required to inhibit cleavage of rP2-II. The pentapeptide also interfered with prothrombin cleavage by membrane-bound factor Xa alone in the absence of factor Va increasing the rate for cleavage at Arg(271) of plasma-derived prothrombin or rP2-II. Our data demonstrate that pentapeptide DYDYQ has opposing effects on membrane-bound factor Xa for prothrombin cleavage, depending on the incorporation of factor Va in prothrombinase.

Highlights

Membrane-bound factor Xa alone catalyzes prothrombin activation following initial cleavage at Arg271 and prethrombin 2 formation
Whereas factor Xa alone can activate prothrombin following sequential cleavages at Arg271 and Arg320 to produce thrombin (Fig. 1, pathway I, pre2 pathway), its catalytic efficiency is poor in the absence of factor Va, and the overall reaction is incompatible with survival and the rapid arrest of bleeding
The prothrombinase complex, which is formed following the interaction of factor Va with membranebound factor Xa in the presence of divalent metal ions, catalyzes the activation of prothrombin following the opposite pathway (Arg320 followed by Arg271; see Fig. 1, pathway II, meizo pathway), resulting in a substantial increase in the catalytic efficiency of factor Xa required for normal hemostasis [15]

Summary

A Control Switch for Prothrombinase

CHARACTERIZATION OF A HIRUDIN-LIKE PENTAPEPTIDE FROM THE COOH TERMINUS OF FACTOR Va HEAVY CHAIN THAT REGULATES THE RATE AND PATHWAY FOR PROTHROMBIN ACTIVATION*. The prothrombinase complex, which is formed following the interaction of factor Va with membranebound factor Xa in the presence of divalent metal ions, catalyzes the activation of prothrombin following the opposite pathway (Arg320 followed by Arg271; see Fig. 1, pathway II, meizo pathway), resulting in a substantial increase in the catalytic efficiency of factor Xa required for normal hemostasis [15] Both cleavages are phospholipid-dependent, only initial cleavage of prothrombin at Arg320 is strictly dependent on factor Va. The increase in the rate of the overall enzymatic reaction is attributed to an increase in the kcat, which in turn is solely credited to the interaction of the cofactor molecule with both the membrane-bound enzyme and the membrane-bound substrate [11,12,13,14,15,16,17,18].

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

Methods