Inhibition of Factor X Activation at Extracellular Matrix of Fibroblasts During Flow Conditions: A Comparison Between Tissue Factor Pathway Inhibitor and Inactive Factor VIIa

Sanne Valentin; Chris P M Reutlingsperger; Ole Nordfang; Theo Lindhout

doi:10.1055/s-0038-1649969

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 1995; 74(06): 1478-1485
DOI: 10.1055/s-0038-1649969

Original Articles

Coagulation

Schattauer GmbH Stuttgart

Inhibition of Factor X Activation at Extracellular Matrix of Fibroblasts During Flow Conditions: A Comparison Between Tissue Factor Pathway Inhibitor and Inactive Factor VIIa

Authors

Sanne Valentin

¹The Biopharmaceuticals Research, Novo Nordisk A/S, Gentofte, Denmark

²The Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, The Netherlands
Chris P M Reutlingsperger

²The Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, The Netherlands
Ole Nordfang

¹The Biopharmaceuticals Research, Novo Nordisk A/S, Gentofte, Denmark
Theo Lindhout

²The Department of Biochemistry, Cardiovascular Research Institute Maastricht, University of Limburg, Maastricht, The Netherlands

Abstract

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Summary

Tissue factor pathway inhibitor (TFPI) is a naturally occurring factor Xa-dependent inhibitor of factor VIIa/tissue factor activity. In the present study, we examined the importance of the TFPI C-terminus and 3rd Kunitz-like domain for the inhibitory capacity of TFPI towards factor VIIa/tissue factor-catalyzed factor X activation and compared the inhibition with that of inactivated factor VIIa (factor VIIai). The extracellular matrix of fibroblasts, mounted in a parallel-plate flow chamber, were perfused with reaction mixtures that contained factors X, VIIa, and varying amounts of TFPI or factor VIIai. Inhibition was evaluated from the time course of factor Xa production at the outlet of the flow chamber. The factor VIIa/tissue factor-catalyzed factor Xa production was inhibited by factor VIIai and compatible with a direct competition between factor VIIai and factor VIIa for tissue factor. In contrast, TFPI showed a progressive inhibition of factor Xa production; the initial rate of factor X activation, however, was not inhibited by TFPI. Inhibition of factor Xa generation already in progress was seen for TFPI but not factor VIIai. In both cases we found that the truncated TFPI variants were as potent as full length TFPI. As to the stability of the enzyme- inhibitor complexes, TFPI/Xa/VIIa/tissue factor and factor VIIai/tissue factor, marked differences were observed. About 60% of the factor VIIa/tissue factor activity was recovered from the truncated TFPI/Xa/VIIa/tissue factor complex after 150 min of perfusion with reaction mixtures that contained factors X and VIIa. In contrast, full length TFPI did not dissociate from the complex, nor could factor VIIai be displaced by a large excess of factor VIIa.

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References

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