Andrews R K, Kroll M H, Ward C M, Rose J W, Scarborough R M, Smith A I, López J A, Berndt M C
Hazel and Pip Appel Vascular Biology Laboratory, Baker Medical Research Institute, Victoria, Australia.
Biochemistry. 1996 Sep 24;35(38):12629-39. doi: 10.1021/bi960704e.
Binding of the multimeric adhesive glycoprotein, von Willebrand Factor (vWF), to the platelet membrane glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion and initiates signal transduction leading to platelet activation. Recently described viper venom proteins that bind to the GP Ib alpha-chain and inhibit vWF binding provide novel probes for studying receptor function. We have purified a 50-kDa form of alboaggregin from the white-lipped tree viper (Trimeresurus albolabris) and two 25-kDa proteins, CHH-A and CHH-B, from the timber rattlesnake (Crotalus horridus horridus) in addition to a previously described 25-kDa alboaggregin and echicetin. Complete or partial amino acid sequencing of CHH-A, CHH-B, and 50-kDa alboaggregin and cross-reactivity of these proteins with an anti-botrocetin antiserum confirmed that they were disulfide-linked heterodimers or higher multimers of the C-type lectin protein family. These proteins, together with 25-kDa alboaggregin and echicetin, specifically bound to GP Ib alpha within the N-terminal peptide domain, His-1-Glu-282, and inhibited vWF binding with comparable IC50 values (approximately 0.2 microgram/mL). However, cross-blocking studies between these structurally related proteins and anti-GP Ib alpha monoclonal antibodies demonstrated that the venom protein binding sites were not congruent. Further, the 50-kDa alboaggregin, but not the other venom proteins, potently induced platelet activation as assessed by dense granule serotonin release or elevation of cytosolic ionized calcium. Treatment of platelets with the 50-kDa alboaggregin was associated with activation of protein kinase C and tyrosine kinase(s), resulting in a platelet protein phosphorylation profile similar to that seen on shear-stress-induced vWF binding to platelets. These results suggest that the 50-kDa alboaggregin induces cytoplasmic signaling coincident with its binding to the GP Ib-IX-V complex and provides a potentially useful probe for studying the mechanism of vWF-dependent platelet activation.
多聚体黏附糖蛋白血管性血友病因子（vWF）与血小板膜糖蛋白（GP）Ib-IX-V复合物的结合介导血小板黏附，并启动导致血小板活化的信号转导。最近描述的与GP Ibα链结合并抑制vWF结合的蝰蛇毒蛋白为研究受体功能提供了新的探针。我们从白唇竹叶青（Trimeresurus albolabris）中纯化了一种50 kDa的白唇竹叶青凝集素，从东部菱斑响尾蛇（Crotalus horridus horridus）中纯化了两种25 kDa的蛋白CHH-A和CHH-B，此外还有之前描述的25 kDa白唇竹叶青凝集素和echicetin。对CHH-A、CHH-B和50 kDa白唇竹叶青凝集素进行完整或部分氨基酸测序，以及这些蛋白与抗博曲毒素抗血清的交叉反应证实它们是C型凝集素蛋白家族的二硫键连接的异二聚体或更高聚体。这些蛋白与25 kDa白唇竹叶青凝集素和echicetin一起，特异性结合于N端肽段His-1-Glu-282内的GP Ibα，并以相当的IC50值（约0.2 μg/mL）抑制vWF结合。然而，这些结构相关蛋白与抗GP Ibα单克隆抗体之间的交叉阻断研究表明，蛇毒蛋白结合位点并不一致。此外，50 kDa白唇竹叶青凝集素，而非其他蛇毒蛋白，通过致密颗粒5-羟色胺释放或胞质游离钙升高评估，能有效诱导血小板活化。用50 kDa白唇竹叶青凝集素处理血小板与蛋白激酶C和酪氨酸激酶的活化有关，导致血小板蛋白磷酸化谱类似于剪切应力诱导vWF与血小板结合时所见。这些结果表明，50 kDa白唇竹叶青凝集素在与GP Ib-IX-V复合物结合的同时诱导细胞质信号传导，并为研究vWF依赖性血小板活化机制提供了一个潜在有用的探针。
