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; Tivadar Orban; Paul Y. Kim; Michael E. Nesheim; Michael Kalafatis

doi:10.1160/TH07-08-0532

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2008; 99(03): 511-522
DOI: 10.1160/TH07-08-0532

Blood Coagulation, Fibrinolysis and Cellular Haemostasis

Schattauer GmbH

The interaction of fragment 1 of prothrombin with the membrane surface is a prerequisite for optimum expression of factor Va cofactor activity within prothrombinase

Authors

Michael A. Bukys

¹Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA
Tivadar Orban

¹Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA
Paul Y. Kim

²Departments of Biochemistry
Michael E. Nesheim

²Departments of Biochemistry

³Medicine, Queen’s University, Kingston, Ontario, Canada
Michael Kalafatis

¹Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA

⁴Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio, USA

Abstract

Summary

Incorporation of factor (F) Va into prothrombinase directs prothrombin activation by FXa through the meizothrombin pathway, characterized by initial cleavage at Arg³²⁰ We have shown that a pentapeptide with the sequence DYDYQ specifically inhibits this pathway. It has been also established that Hir^54–65(SO₃ -) 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 Arg³²⁰ and Arg²⁷¹ occurring with similar rates. Addition of purified fragment 1 to prethrombin 1 accelerates both the rate of cleavage at Arg³²⁰ and thrombin formation.Both reactions were inhibited by Hir^54–65(SO₃ -) 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 Hir54–65(SO3 -), but is not affected by DYDYQ.Addition of purified fragment 1•2 to prethrombin 2 accelerates the rate of cleavage at Arg³²⁰ 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(SO₃ -) on the same reaction. Finally, a membrane-bound ternary complex composed of prethrombin 2/fragment 1•2/Hir^54–65(SO₃ -) 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 Arg³²⁰.

Keywords

Prothrombin - thrombosis - factor Va - blood coagulation

Full Text

References

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