Specific and quantitative method for estimation of platelet adhesion to fibrillar collagen

A quantitative method (Sepharose test) was devised to measure the adhesion of blood platelets to fibrillar collagen. [14C]5HT-labeled platelets were isolated from plasma, resuspended in EDTA buffer, and incubated with buffer (control) or with fibrillar collagen for 150 sec at 33 degrees C. The mixtures were then filtered through Sepharose 2B columns. In controls the platelets were rapidly eluted, and this was confirmed after 51Cr labeling. [C]5HT was recovered in two stages: 60% with the platelets and 40% retarded, as free 5HT. After incubation with fibrillar collagen (50 micrograms), platelets were retained with the fibrils on the top of the column, and only free [14C]5HT (released from the platelets) was eluted. The percentage of adhesion depended on the number of platelets, the amount of collagen, its degree of polymerization, and the time of incubation at 33 degrees C. [14C]5HT release was markedly diminished when both incubation and filtration were performed at low temperature. ASA, used either in vitro or in vivo in rabbits, did not change the percentage of adhesion but significantly diminished the total amount of [14C]5HT eluted. This method offers a quantitative and reproducible system for the differentiation of adhesion and release, independent of platelet aggregation.