Nesheim, ME

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.

It has generally been assumed that the thrombogenicity of PCC relates to their invariable contamination by activated products of their component clotting factors. In earlier studies we demonstrated a lack of correlation between the activated clotting factor content ᶓ in vivo thrombogenicity of some PCC. In contrast, in vitro measurement of coagulant active PL content appeared to be a more reliable determinant of subsequent in vivo activity. In recent studies we have confirmed the presence of PL of known coagulant activity in PCC ᶓ studied its activity both in vitro ᶓ in vivo after its extraction from the parent PCC.PCCs were obtained from various agencies or prepared in our laboratory to a standard protocol either from fresh frozen plasma or plasma obtained from outdated platelet concentrates. Each was assayed for Xa (S-2222) ᶓ PL (thrombin generation assay with DAPA) ᶓ an index of thrombogenicity in vivo determined in a stasis model in rabbits. There was a significant correlation between the latter ᶓ PL (r=0.8727 p<0.01) but not Xa. Thin layer chromatography of an ethanol ether extract of the most thrombogenic PCC showed the presence of PL known to be coagulant active including Phospho- tidylserine ᶓ -choline. This extract retained full PL replacing activity in the DAPA assay in vitro but was non- thrombogenic in vivo. When, however, a highly purified preparation of Xa was added, in the same relative proportions as in the parent PCC, thrombogenicity in vivo was restored.It is concluded that the content of coagulant active PL is a critical determinant of the potential thrombogenicity of PCC in vivo. It appears to act in concert with Xa ᶓ conceivably, other serine proteases perhaps by protecting them from inactivation by AT III. This may explain why relatively “non-activated” PCCs may still be associated with thromboembolic complications.