Arthur Jane F, Shen Yang, Kahn Mark L, Berndt Michael C, Andrews Robert K, Gardiner Elizabeth E
Department of Immunology, Monash University, Melbourne 3004, Australia.
J Biol Chem. 2007 Oct 19;282(42):30434-41. doi: 10.1074/jbc.M701330200. Epub 2007 Aug 9.
Thrombus formation in hemostasis or thrombotic disease is initiated by adhesion of circulating platelets to damaged blood vessel walls. Exposed subendothelial collagen interacting with platelet glycoprotein (GP) VI leads to platelet activation and integrin alpha(IIb)beta(3)-mediated aggregation. We previously showed that ligand binding to GPVI also induces metalloproteinase-dependent shedding, generating an approximately 55-kDa soluble ectodomain fragment and an approximately 10-kDa membrane-associated remnant. Here, treatment of platelets with collagen or the GPVI-targeting rattlesnake toxin convulxin also induces rapid (10-30 s) formation of a high molecular weight GPVI complex (GPVIc) under nonreducing conditions, as detected by immunoblotting with anti-GPVI antibodies. The appearance of an approximately 20-kDa remnant detectable using a polyclonal antibody against the GPVI cytoplasmic tail under nonreducing, but not reducing, conditions after ectodomain shedding and nonreduced/reduced two-dimensional SDS-polyacrylamide gel analysis of biotinylated platelets confirmed that that GPVIc was a homodimer. Formation of disulfide-linked GPVIc was prolonged in the presence of metalloproteinase inhibitor GM6001 and was independent of GPVI signaling because it was unaffected by inhibitors of Src kinases, Syk, or phosphoinositide 3-kinase. To identify the thiol involved in disulfide bond formation, wild-type or mutant GPVI, where two available sulfhydryls (Cys-274 and Cys-338) were individually mutated to serine, was expressed in rat basophilic leukemia cells. Dimerization of wild-type and C274S GPVI, but not the C338S mutant, was observed after treating cells with convulxin. We conclude that (i) a subpopulation of GPVI forms a constitutive dimer on the platelet surface, facilitating rapid disulfide cross-linking, (ii) convulxin or other GPVI agonists induce disulfide-linked GPVI dimerization independent of GPVI signaling, and (iii) the penultimate residue of the GPVI cytoplasmic tail, Cys-338, mediates disulfide-dependent dimer formation.
在止血或血栓形成性疾病中，血栓形成始于循环血小板与受损血管壁的黏附。暴露的内皮下胶原蛋白与血小板糖蛋白（GP）VI相互作用，导致血小板活化以及整合素α（IIb）β3介导的聚集。我们之前表明，配体与GPVI结合还会诱导金属蛋白酶依赖性脱落，产生一个约55 kDa的可溶性胞外域片段和一个约10 kDa的膜相关残余物。在此，用胶原蛋白或靶向GPVI的响尾蛇毒素convulxin处理血小板，在非还原条件下也会诱导快速（10 - 30秒）形成高分子量GPVI复合物（GPVIc），这通过用抗GPVI抗体进行免疫印迹检测到。在胞外域脱落且对生物素化血小板进行非还原/还原二维SDS - 聚丙烯酰胺凝胶分析后，在非还原而非还原条件下，使用针对GPVI胞质尾的多克隆抗体可检测到一个约20 kDa的残余物，这证实GPVIc是一个同二聚体。在金属蛋白酶抑制剂GM6001存在的情况下，二硫键连接的GPVIc的形成会延长，并且它独立于GPVI信号传导，因为它不受Src激酶、Syk或磷脂酰肌醇3激酶抑制剂的影响。为了确定参与二硫键形成的硫醇，将野生型或突变型GPVI（其中两个可用巯基（Cys - 274和Cys - 338）分别突变为丝氨酸）在大鼠嗜碱性白血病细胞中表达。在用convulxin处理细胞后，观察到野生型和C274S GPVI的二聚化，但未观察到C338S突变体的二聚化。我们得出结论：（i）GPVI的一个亚群在血小板表面形成组成型二聚体，促进快速二硫键交联；（ii）convulxin或其他GPVI激动剂诱导二硫键连接的GPVI二聚化，独立于GPVI信号传导；（iii）GPVI胞质尾的倒数第二个残基Cys - 338介导二硫键依赖性二聚体形成。
