Fox J E, Reynolds C C, Morrow J S, Phillips D R
Blood. 1987 Feb;69(2):537-45.
We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)-polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X-100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), or N-ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.
我们最近发现血小板含有与质膜上糖蛋白（GP）Ib相连的膜下肌动蛋白丝。在本研究中，进行了实验以确定血影蛋白是否与这些丝相关。通过差速离心从未刺激血小板的Triton X - 100（Sigma，圣路易斯）裂解物中分离出膜结合丝。使用针对人脑和红细胞血影蛋白的抗体通过免疫方法检测血小板血影蛋白。免疫印迹显示血小板血影蛋白由两种多肽组成（分子量240,000和235,000），其表观分子量与人脑血影蛋白的α链和β链相似，但与红细胞血影蛋白（分子量240,000和220,000）略有不同。免疫沉淀实验将血小板血影蛋白鉴定为在十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶上迁移于肌动蛋白结合蛋白（分子量250,000）和血小板多肽P235（分子量235,000）之间的两种次要多肽。对从Triton X - 100裂解的血小板中分离的组分进行免疫印迹显示，血小板血影蛋白的α链和β链几乎完全与连接到质膜的肌动蛋白丝相关。在Triton X - 100可溶部分或与未与膜结合的肌动蛋白丝中回收的血影蛋白很少。在用凝血酶或离子载体A23187激活血小板的过程中，血影蛋白的α链和β链被水解，产生分子量为160,000的主要降解产物和分子量为170,000的次要降解产物。在存在Ca2 +的情况下孵育的Triton X - 100裂解物中也产生这两种水解产物，但当裂解物用亮抑酶肽、乙二醇双（β-氨基乙醚）-N，N，N'，N'-四乙酸（EGTA）或N - 乙基马来酰胺（已知的Ca2 +依赖性蛋白酶抑制剂）处理时则不产生。这些实验表明血影蛋白是膜结合肌动蛋白丝网络中以前未被识别的成分，并且Ca2 +依赖性蛋白酶对血影蛋白的水解可能在血小板激活过程中调节丝的相互作用。
