Homogeneous, single-chain human factor V was used to develop a double antibody competition radioimmunoassay to measure factor V concentrations in plasma and platelets. Standard curves were constructed that allow for the detection of as little as 20 ng factor V/ml of plasma. Normal factor V concentrations range from 4 to 14 micrograms/ml of plasma with an average value of 7.0 +/- 2.0 micrograms/ml (n = 64). No correlation was observed between antigen levels and age or sex. The radioimmunoassay data are consistent with factor V clotting assays, providing freshly drawn plasma is used in the bioassay. Radioimmunoassay of washed platelets indicate that 0.63–1.93 microgram of factor V is present per 2.5 X 10(8) platelets (4612–14128 molecules of the factor V platelet). When normalized to individual hematocrits and platelet count, the data indicated that platelets contribute approximately 18%-25% of the factor V found in whole blood. In addition, two individuals with functionally deficient factor V were examined and found to be deficient in both antigen and activity.

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In vitro evaluation of prothrombin complex concentrates in a thrombin generation assay, using DAPA and purified components of the prothrombinase complex, demonstrated significant levels of coagulant- active “phospholipid replacing” activity. Quantification of this activity showed a significant correlation (r = 0.8747, p less than 0.01) with thrombogenicity measured in vivo in a stasis model in rabbits. Extracted lipid material retained full phospholipid replacing activity in the vitro assay. Thin-layer chromatographic characterization confirmed the presence of phospholipids with known coagulant activity in vitro. In vivo, the extracted material was nonthrombogenic but augmented the thrombogenicity of purified factor Xa. Substitution of a synthetic coagulant-active phospholipid (phosphatidylcholine-phosphatidylserine lipid vesicles) for the extracted phospholipid produced a similar augmentation of a factor-Xa- induced thrombogenicity in vivo. It is concluded that the coagulant- active phospholipid content of prothrombin complex concentrates is a major determinant of thrombogenicity but requires the presence of activated clotting factors for its expression in vivo.

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Human platelet factor V has been isolated using either a monoclonal or polyclonal antibody directed against human plasma factor V. The largest peptide observed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified human platelet factor V comigrates with purified human plasma factor V. However, a significant portion of the isolated protein is represented by peptides of lower apparent molecular weight (Mr). These lower Mr species that copurify with platelet factor V have been shown to be platelet factor V components by their immunological cross-reactivity with monoclonal and polyclonal antibodies to purified human plasma factor V. Platelets isolated from whole blood drawn directly into inhibitors to prevent proteolysis and platelet activation demonstrate the pattern of fragmented platelet factor V. The components of purified platelet factor V demonstrate apparent Mr ranging between 115 K and 330 K and are detectably different from the intermediates and end products observed during the thrombin cleavage of single-chain plasma factor V. Upon treatment with thrombin the platelet factor V components are cleaved and the end products are indistinguishable from those obtained upon thrombin activation of plasma factor V to plasma factor Va. Examination of the components by immunoblotting demonstrates that some of the cleavages which have occurred in the platelet factor V molecule are within the 150-K activation peptide. Bioassay indicates that platelet factor V exists as a procofactor and cleavage by thrombin yields the active cofactor, platelet factor Va. These data suggest that human platelet factor V is stored in the platelet as a partially fragmented procofactor that can be activated by thrombin to yield human platelet factor Va, the active cofactor in the human prothrombinase complex.

Highly purified human Factor V was used for the development of a competitive double antibody radioimmunoassay (RIA) using 125I-human Factor V, burro anti-human Factor V antisera as the primary antibody and goat anti-burro antisera as the precipitating antibody. The standard curve allows the detection of as little as 20 ng of Factor V per ml of plasma. With this specific RIA for human Factor V, we have measured the level of Factor V in the plasma and platelets of normal individuals. The normal level of Factor V in plasma ranges from 4 to 14 μg per ml, with the average value equal to 7.0 ± 2.0 μg/ml (n = 64; 33 females; 31 males; 22 to 61 years of age). There appeared to be no correlation between antigen levels and age or sex. Factor V clotting assays were consistent with the RIA data for any given plasma preparation providing freshly drawn plasma was used in the bioassay. The bioassay data were quantitated based upon the specific activity of purified plasma Factor V; 1.7 units of Factor V equals 1 μg of protein. Plasma Factor V antigen levels were not affected by lyophilization of the plasma, prolonged storage of the plasma at -20°C or intentional conversion of the plasma to serum. The levels of Factor V present in washed human platelets were also determined using the RIA. Assay of washed platelets lysed in 0.2% Triton X-100 indicated that 0.6 to 0.85 μg of Factor V was present per 2.5 × 108 platelets (4400 to 6200 molecules of Factor V per platelet). This result is in marked contrast to our observations for the bovine system, where we found that bovine platelets possess approximately 400 to 800 molecules of Factor V per platelet, and plasma Factor V levels range from 30 to 50 μg per ml. In the bovine system, the platelets possess approximately 1% of the total Factor V present, while in human blood, the platelets possess as much as 10 to 15% of the total Factor V present.

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