Alteration of the Substrate and Inhibitor Specificities of Blood Coagulation Factor VIIa: Importance of Amino Acid Residue K192

Pierre F. Neuenschwander, James H. Morrissey

Research output: Contribution to journalArticle

Abstract

Initiation of blood coagulation occurs when the plasma serine protease factor VIIa (fVIIa) binds to its cell-surface receptor/cofactor, tissue factor (TF). This binding interaction mediates a large enhancement in both the proteolytic activity and the amidolytic activity (hydrolysis of small peptidylamide substrates) of fVIIa. This necessitates local changes in the catalytic center of fVIIa of which little is understood. Studies with thrombin and activated protein C have demonstrated that residue E192 (chymotrypsinogen numbering system) near the active site of these proteases is an important determinant for substrate and inhibitor specificity. By homology, residue 192 in fVIIa is K, bringing into question the potential role of this residue in fVIIa. We have prepared two mutants of fVIIa in which K192 has been replaced by either Q (as in factors IX and X) or E (as in thrombin). Both mutants were found to be defective in clotting: fVIIK192Q was 44% active, while fVIIK192E was completely ineffective. This defect was attributable to proportional decreases in specificity constants for activation of factor X. Although both mutant enzymes were catalytically competent with respect to amidolytic activity, the selectivity of fVIIaK192E was greatly altered. Inhibition of both mutants by the TF pathway inhibitor (TFPI) and bovine pancreatic trypsin inhibitor (BPTI) was also drastically altered. Neither mutant was inhibited by TFPI, while fVIIaK192Q was inhibited by BPTI better than wild-type fVIIa. In contrast, fVIIaK192E was poorly inhibited by BPTI and made more refractory to inhibition when bound to TF. These results suggest a potential role for K192 in governing the substrate and inhibitor specificities of fVIIa.

Original languageEnglish (US)
Pages (from-to)8701-8707
Number of pages7
JournalBiochemistry
Volume34
Issue number27
DOIs
StatePublished - Jul 1995

ASJC Scopus subject areas

  • Biochemistry

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