Direct measurement of the platelet:collagen interaction by affinity chromatography on collagen/Sepharose

Abstract

A method for studying the platelet:collagen interaction is described that permits simultaneous measurement of platelet adhesion to collagen and the collagen-initiated release reaction. Plasma-free platelets are passed through short columns composed of polymeric collagen covalently linked to agarose (Sepharose 2B). EDTA (0.3 mM) is used to prevent platelet aggregation. 14C-Serotonin is used to measure the extent of the release reaction. Measurement of adhesion is based upon 51Cr. In the experiments that are described, the extents of both adhesion and serotonin release were a function of the total collagen content of the column and of the number of platelets applied. Up to 100 per cent of the applied platelets adhered to the columns. As much as 70 per cent of the platelet serotonin was released. Intracellular 51Cr, lactate dehydrogenase, and pyruvate kinase, on the other hand, were not lost from the platelets. Plasma-free platelets were prepared by two different techniques: gel filtration and differential centrifugation. Both preparations gave the same results. The influence of the column temperature was also examined. At temperatures below 37 degrees C., there was a sharp drop in serotonin release, but only a slight decline in platelet adhesion to collagen. Our results suggest that the collagen/Sepharose assay system should provide a usable and greatly needed technique for studying the molecular basis for the platelet:collagen interaction for assessing platelet function in abnormal states and for investigating the mechanism of action of potential inhibitors.