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Updated: Aug 7, 2026

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay
Published on: September 9, 2012
Proteolytic activation of human factors IX and X by recombinant human factor VIIa: effects of calcium, phospholipids,
Y Komiyama1, A H Pedersen, W Kisiel
1Department of Pathology, University of New Mexico School of Medicine, Albuquerque 87131.
Abstract:
Previous studies indicated that factor VIIa, in complex with tissue factor, readily activates either factor X or factor IX in the presence of calcium ions. In order to assess the relative physiological importance of the activation of factor IX versus the activation of factor X by recombinant factor VIIa, we have obtained steady-state kinetic parameters for the factor VIIa catalyzed activation of factor IX and factor X under a variety of cofactor conditions that include calcium alone, calcium and phospholipids, calcium, phospholipids, and tissue factor apoprotein, and calcium and cell-surface tissue factor. Calcium alone stimulated the activation of factors IX and X by factor VIIa maximally at 1 and 2.5 mM, respectively. In the presence of 25 microM phospholipids, maximal rates of factor IX and factor X activation were achieved at 2.5-5 mM calcium. With calcium alone, or with phospholipid and calcium, the initial rates of factor IX activation by factor VIIa were significantly higher than that observed for factor X. Kinetic studies revealed that the Km for the factor VIIa catalyzed activation of factor IX was essentially constant in the presence of 5 mM calcium and 1-500 microM phospholipid, whereas the Km for factor X activation varied with phospholipid concentration, reaching a minimum at 7-20 microM phospholipid. At all concentrations of added phospholipid, the kcat/Km ratio for the activation of factor IX by factor VIIa appeared to be considerably greater than that observed for the activation of factor X.(ABSTRACT TRUNCATED AT 250 WORDS)
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