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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
1   Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA
,
Tivadar Orban
1   Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA
,
Paul Y. Kim
2   Departments of Biochemistry
,
Michael E. Nesheim
2   Departments of Biochemistry
3   Medicine, Queen’s University, Kingston, Ontario, Canada
,
Michael Kalafatis
1   Department of Chemistry, Cleveland State University, Cleveland, Ohio, USA
4   Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic, Cleveland, Ohio, USA
› Author Affiliations Financial support: 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).
Further Information

Publication History

Received: 31 August 2007

Accepted after major revision: 04 January 2008

Publication Date:
07 December 2017 (online)

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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 Hir54–65(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 Hir54–65(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 Hir54–65(SO3 -) on the same reaction. Finally, a membrane-bound ternary complex composed of prethrombin 2/fragment 1•2/Hir54–65(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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