Lyman B, Rosenberg L, Karpatkin S
J Clin Invest. 1971 Sep;50(9):1854-63. doi: 10.1172/JCI106677.
A method has been developed for measuring the adhesion of platelets to purified collagen fibers obtained from bovine tendon. This method differs from others in that: (a) platelet adhesion is measured in the absence of platelet aggregation; (b) platelet-rich plasma collected in ACD (acid citrate dextrose) or EDTA, or washed platelets can be employed; (c) adherent platelets are enumerated directly; (d) erythrocytes and leukocytes do not adhere. Washed platelets suspended in human Ringer solution exhibit negligible adhesion (at the platelet concentrations employed) in contrast to washed platelets suspended in plasma. Addition of purified human fibrinogen (95% clottable, 2-4 mg/ml) to human Ringer solution completely restores the ability of washed platelets to adhere to collagen fibers. Albumin (fatty acid free, 50 mg/ml) is also capable of restoring adhesion. Albumin and seven other proteins at concentrations of 5-10 mg/ml, with varying molecular weights, isoelectric points, and frictional coefficients are incapable of supporting the adhesion of washed platelets. The proteins tested were human globulin, hexokinase, hemoglobin, cytochrome-C, insulin, thyroglobulin, and muramidase. Platelet adhesion is proportional to both platelet concentration and fibrinogen concentration, but is independent of temperature or glycogen stores. Modification of fibrinogen by acylation of amino groups or removal of sialic acid has no effect on its ability to support platelet adhesion. Degradation of fibrinogen with purified plasmin results in decreased support of platelet adhesion. This accompanied formation of early breakdown products with clottability ranging from 84-0%. Formation of fibrinogen degradation products was monitored by SDS-polyacrylamide gel electrophoresis of the corresponding fibrins after reduction of disulfide bonds (a method capable of distinguishing alpha-, beta- and gamma-chains). Decreased support of platelet adhesion is associated with the disappearance of intact alpha- chains and early modification of the beta-chains. Purified proteinpolysaccharide macromolecules obtained from bovine nasal and humeral cartilage, and from nucleosus pulposus are as effective as fibrinogen on a weight basis and ten to thirty times more effective on a molar basis in supporting platelet adhesion. The purified mucopolysaccharide side chains: chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan-sulfate are incapable of supporting platelet adhesion.
已开发出一种用于测量血小板与从牛腱中获得的纯化胶原纤维的黏附力的方法。该方法与其他方法的不同之处在于：(a) 在无血小板聚集的情况下测量血小板黏附力；(b) 可以使用收集在ACD（酸性枸橼酸葡萄糖）或EDTA中的富含血小板的血浆，或洗涤过的血小板；(c) 直接计数黏附的血小板；(d) 红细胞和白细胞不黏附。与悬浮在血浆中的洗涤过的血小板相比，悬浮在人林格溶液中的洗涤过的血小板（在所使用的血小板浓度下）表现出可忽略不计的黏附力。向人林格溶液中添加纯化的人纤维蛋白原（95%可凝，2 - 4mg/ml）可完全恢复洗涤过的血小板黏附胶原纤维的能力。白蛋白（无脂肪酸，50mg/ml）也能够恢复黏附力。浓度为5 - 10mg/ml的白蛋白和其他七种蛋白质，具有不同的分子量、等电点和摩擦系数，均不能支持洗涤过的血小板的黏附。所测试的蛋白质为人球蛋白、己糖激酶、血红蛋白、细胞色素C、胰岛素、甲状腺球蛋白和溶菌酶。血小板黏附力与血小板浓度和纤维蛋白原浓度均成正比，但与温度或糖原储备无关。通过氨基酰化或去除唾液酸对纤维蛋白原进行修饰对其支持血小板黏附的能力没有影响。用纯化的纤溶酶降解纤维蛋白原会导致对血小板黏附的支持作用降低。这伴随着早期降解产物的形成，其可凝性范围为84% - 0%。通过在二硫键还原后对相应纤维蛋白进行SDS - 聚丙烯酰胺凝胶电泳（一种能够区分α - 、β - 和γ - 链的方法）来监测纤维蛋白原降解产物的形成。对血小板黏附支持作用的降低与完整α - 链的消失和β - 链的早期修饰有关。从牛鼻软骨、肱骨软骨和髓核中获得的纯化蛋白多糖大分子在重量基础上与纤维蛋白原一样有效，在摩尔基础上支持血小板黏附的效果要高10到30倍。纯化的黏多糖侧链：硫酸软骨素 - 4、硫酸软骨素 - 6和硫酸角质素均不能支持血小板黏附。
