Stroma free human platelet lysates potentiate the in vivo thrombogenicity of factor Xa by the provision of coagulant-active phospholipid

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Abstract
A stroma free platelet lysate supernatant (PLS) was prepared by repeatedly freezing and thawing a human platelet suspension separated from platelet rich plasma by gel filtration. The material was potently thrombogenic in a stasis model in rabbits, but only when combined with a purified preparation of factor Xa. The dose of factor Xa used was not thrombogenic when given alone. Initial evaluation suggested the presence of a factor V activator when PLS shortened the clotting time of normal but not a V deficient plasma. Subsequent evaluation, in a system employing purified components of the prothrombinase complex and a marker of thrombin generation, dansyl‐arginine‐N‐(3‐ethyl‐1,5‐pentanediyl)amide (DAPA), failed to confirm the presence of a factor V activator. Further evaluation in this system demonstrated that the procoagulant activity related to the content of coagulant‐active phospholipid. Thin layer chromatography confirmed the presence of phospholipids known to have coagulant activity. These components were isolated by a lipid extraction procedure and the phospholipid replacing activity in the in vitro assay with DAPA was quantitatively retained. The extracted material was non‐thrombogenic in vivo but augmented the thrombogenicity of a purified preparation of Xa.