Activation of Coagulation Factor V by a Platelet Protease

William H. Kane; Jozef S. Mruk; Philip W. Majerus

doi:10.1172/JCI110697

Published November 1, 1982 - More info

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Abstract

Factor V must be converted to Factor V_a in order to bind to a high affinity platelet surface site and participate in prothrombin activation. Osterud et al. (10) presented data that suggested that human platelets contain an activated form of Factor V and a Factor V activator. We find that the Factor V released when platelets are disrupted by freezing and thawing or sonication is activated 3- to 10-fold by thrombin as determined by coagulation assay and is therefore stored as the relatively inactive procofactor rather than in the active form Factor V_a.

We incubated purified Factor V, which had a specific activity of 140±30 U/mg, with Factor V-deficient frozen and thawed platelets (10⁹ platelets/ml) obtained from a patient with Factor V deficiency. The specific activity of the Factor V increased to a maximum of 740±240 U/mg (mean±SD of three experiments). When this partially activated Factor V was incubated with thrombin its specific activity increased further to 1,440±280 U/mg, which is similar to the activity of Factor V activated with thrombin alone (1,540±60 U/mg).

The platelet Factor V activator is not inhibited by dansyl arginine-4-ethylpiperidine amide, 93 μM, indicating that it is not thrombin. When thrombin-stimulated platelets, to which dansyl arginine-4-ethylpiperidine amide had been added to inhibit the further action of thrombin, were incubated with ¹²⁵-labeled Factor V, there was no detectable proteolysis of the Factor V molecule. Our failure to detect activation of Factor V under these conditions suggests that <4% of the platelet protease is released by thrombin. Subcellular fractionation of platelets indicates that the platelet protease that activates Factor V is in the soluble fraction.

When Factor V_a formed by the action of platelet protease is incubated with platelets, peptides with M_r = 105,000, 87,000, and 78,000 bind to the platelet surface. All three radiolabeled peptides are displaced from platelets by unlabeled Factor V_a formed by the action of thrombin. The stoichiometry of binding suggests that the 105,000-dalton peptide is associated with either an 87,000- or a 78,000-dalton peptide. The 78,000-dalton peptide binds with greater affinity and probably accounts for the bulk of the activity of Factor V_a in coagulation assays. Whether or not the platelet protease serves to activate Factor V before thrombin formation during normal hemostasis remains to be determined.

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