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

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.