Brass L F, Faile D, Bensusan H B
J Lab Clin Med. 1976 Mar;87(3):525-34.
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.
本文描述了一种研究血小板与胶原蛋白相互作用的方法，该方法可同时测量血小板对胶原蛋白的黏附以及胶原蛋白引发的释放反应。将无血浆的血小板通过由共价连接到琼脂糖（琼脂糖2B）上的聚合胶原蛋白组成的短柱。使用乙二胺四乙酸（0.3 mM）来防止血小板聚集。用14C-5-羟色胺来测量释放反应的程度。黏附的测量基于51Cr。在所描述的实验中，黏附和5-羟色胺释放的程度均是柱中胶原蛋白总含量和所施加血小板数量的函数。高达100%的所施加血小板黏附到柱上。多达70%的血小板5-羟色胺被释放。另一方面，细胞内的51Cr、乳酸脱氢酶和丙酮酸激酶并未从血小板中流失。通过两种不同技术制备无血浆血小板：凝胶过滤和差速离心。两种制备方法得到相同结果。还研究了柱温的影响。在低于37摄氏度的温度下，5-羟色胺释放急剧下降，但血小板对胶原蛋白的黏附仅略有下降。我们的结果表明，胶原蛋白/琼脂糖测定系统应为研究血小板与胶原蛋白相互作用的分子基础、评估异常状态下的血小板功能以及研究潜在抑制剂的作用机制提供一种有用且急需的技术。
