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, Inhaltsverzeichnis

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

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

› Institutsangaben

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

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