Immunochemical Studies of Factor V of Bovine Platelets

Abstract

lmmunochemical techniques were employed to investigate the molecular properties of bovine platelet factor V. An antiserum prepared against purified factor V rapidly inactivated platelet factor V, indicating that platelet factor V is antigenically related to plasma factor V. A reaction ofidentity between factor V in plasma and purified factor V was documented by immunodiffusion against an antibody to platelet factor V. When platelet factor V was extracted with Triton X-100 in the presence of protease inhibitors and subjected to immunoelectrophoresis against the antiserum to plasma factor V, a single antigenic component migrating toward the anode was observed. In the absence of protease inhibitors, following release by collagen or solubilization, platelet factor V appeared close to the origin suggesting proteolytic alteration. Platelet factor V released by collagen or extracted with Triton X-100 was activated by thrombin 7.4-fold and 4.5-fold respectively, compared to a 17-fold activation of factor V in plasma under identical conditions. The ability of thrombin to activate platelet factor V as well as the close correspondence of factor V activity and antigen released by collagen indicates that the molecule is largely in the unactivated form after release. A single component of molecular weight 270000 was seen when platelet factor V released by collagen was immunoprecipitated and subjected to dodecylsulphate gel electrophoresis. Factor V coagulant assays and immunoelectrophoresis of subcellular fractions showed that platelet factor V is localized primarily in the a-granules.Platelet factor V appears to be similar to, if not identical with, plasma factor V by the criteria of immunologic identity, similar electrophoretic mobility and virtually identical molecular weights.Bovine platelets contain factor V, an accelerator of the conversion of prothrombin [I]. Human platelets freed o f plasma by high speed centrifugation also possess factor-V activity [2-41. The factor-V activity of platelets was initially assumed to be due to adsorption of plasma factor V to the platelet surface [2]. However, Breederveld et al. [5] and Osterud et al. [6] demonstrated that gel-filtered platelets possess only minimal factor-\/ activity, and that freezing and thawing increase the activity significantly. Studies of Miletich et al. [7] demonstrated that factor X a binds to the platelet surface at sites which are identical to or require the presence of factor V. Their studies suggest that specific receptors for factor X a appear on the platelet surface only after the release reaction occurs [8]. Work from our laboratory has shown that human platelet factor V is localized in the a-granules and that optimal release requires the presence of ADP and the synthesis of prostaglandin intermediates 19, 101. All of the above studies were limited in that platelet factor V was measured functionally, and increased activity could be due to either an actual increase in factor V protein of the formation of a more active m ol ecule.Bovine plasma factor V...

Biosynthesis of factor V in isolated guinea pig megakaryocytes.

“…However, Factor V may also arise from an intracellular location in platelets since human platelets separated by gel filtration do not have significant Factor V activity but develop coagulant activity upon repeated freezing and thawing (6). Studies from this laboratory show that both human (7) and bovine (8) platelets contain Factor V in the a-granules where it can be released by collagen. Thrombin can also release Factor V from human platelets (9), where it would be in the form of Factor Va and thus would exhibit high affinity binding to the surface membrane.…”