The Interaction of Fragment 1 of Prothrombin With the Membrane Surface Is a Prerequisite for Optimum Expression of Factor VA Cofactor Activity Within Prothrombinase

Michael A. Bukys, Cleveland State University, Department of Chemistry, OH 44115, USA.
Tivadar Orban
Paul Y. Kim
Michael E. Nesheim
Michael Kalafatis

This work was supported by Predoctoral Fellowships from the Cellular and Molecular Medicine Specialization at Cleveland State University (to MAB and TO), United States Public Health Services Grant HL-46703–6 from the National Institutes of Health (to MEN), National Science Foundation grant under the following NSF programs: Partnerships for Advanced Computational Infrastructure Distributed Terascale Facility, and Terascale Extensions: Enhancement to the Extensible Terascale Facility grant MCB060021T (to MK), computational grant PFS0202 from the Ohio Supercomputer Center (to MK), and grant HL-73343 from the National Heart Lung and Blood Institutes (to MK).

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).