Purification of a platelet protein which stimulates fibrinolytic inhibition and tissue factor in human fibroblasts.
Platelets are able to stimulate an increase in two distinct activities, tissue factor (thromboplastin) and fibrinolytic inhibition, in human fibroblasts in vitro. A procedure has been developed which allows the purification of a platelet macromolecule which is able to stimulate both of these changes. Washed human platelets were homogenized, sonicated, and then centrifuged at 90,000 x g for 2 h. The resulting pellet was solubilized in 0.05 M sodium carbonate, pH 10.5, and chromatographed on Sephadex G-200, then on hydroxylapatite, resulting in a 135-fold purification and a 20% yield. When the purified material was further fractionated on sodium dodecyl sulfate-polyacrylamide gels, stimulatory activity for both tissue factor and fibrinolytic inhibition was found only in the 75,000-dalton region. The active material could be inactivated by mercaptoethanol with no change in its apparent molecular weight. It was readily inactivated by trypsin with the concomitant loss of the 75,000-dalton Coomassie-staining band. Assay of the purified material for carbohydrate was negative. After isoelectric focusing, the purified material had a major band at pH 5.8 which stimulated both tissue factor and fibrinolytic inhibition. Subcellular fractionation of platelet homogenates by sucrose density gradient centrifugation resulted in a 2-fold increase in stimulatory material in the granule/mitochondrial fraction. This platelet-derived protein may represent a physiologically important regulator for both cellular procoagulant and the net fibrinolytic activity of systemic cells.
D001792:Blood Platelets / Q000032:analysis*; D002457:Cell Extracts / Q000302:isolation & purification* / Q000494:pharmacology; D002850:Chromatography, Gel; D005342:Fibrinolysis / Q000187:drug effects*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D013925:Thromboplastin / Q000378:metabolism*; D013997:Time Factors; D014020:Tissue Extracts / Q000302:isolation & purification*
D002457:Cell Extracts; D014020:Tissue Extracts; D013925:Thromboplastin