Hsu C C, Wu W B, Huang T F
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan.
J Thromb Haemost. 2008 Sep;6(9):1578-85. doi: 10.1111/j.1538-7836.2008.03071.x. Epub 2008 Jul 4.
Injuries to the vessel wall and subsequent exposure of the matrix of the subendothelial layer resulted in thrombus formation. Platelet glycoprotein (GP) Ib and VI play a crucial role in matrix-induced activation and aggregation of platelets.
In the present study, we reported that the GPIb-cleaving snake venom metalloproteinase (SVMP), kistomin, inhibited collagen-induced platelet aggregation. Moreover, kistomin inhibited platelet aggregation induced by convulxin (CVX, a GPVI agonist) and a GPVI-specific antibody in a concentration and time-dependent manner. Kistomin treatment decreased platelet GPVI but not integrin alpha2beta1 and alphaIIbbeta3, accompanied with the formation of GPVI cleavage fragments, as determined by flow cytometric and Western blot analyses. In addition, intact platelet GPVI and recombinant GPVI were digested by kistomin to release 25- and 35-kDa fragments, suggesting that kistomin cleaved GPVI near the mucin-like region. We designed four synthetic peptides ranging from Leu180 to Asn249 as the substrates for kistomin and found that kistomin cleaved these synthetic peptides at FSE205/A206TA and NKV218/F219TT, as analyzed by MALDI-TOF-MS. In addition, GPVI-specific antibody-induced tyrosine kinase phosphorylation in platelets was reduced after kistomin pretreatment, and platelet adhesion to collagen but not to fibrinogen was attenuated by kistomin.
We provided here the first evidence that a P-I snake venom metalloproteinase, kistomin, inhibits the interaction between collagen and platelet GPVI through its proteolytic activity on GPVI, thus providing an alternative strategy for developing new anti-thrombotic agents.
血管壁损伤及随后内皮下层基质的暴露会导致血栓形成。血小板糖蛋白（GP）Ib和VI在基质诱导的血小板激活和聚集中起关键作用。
在本研究中，我们报道了能切割GPIb的蛇毒金属蛋白酶（SVMP）——基斯托明，可抑制胶原蛋白诱导的血小板聚集。此外，基斯托明以浓度和时间依赖性方式抑制convulxin（CVX，一种GPVI激动剂）和GPVI特异性抗体诱导的血小板聚集。通过流式细胞术和蛋白质印迹分析确定，基斯托明处理可降低血小板GPVI，但不影响整合素α2β1和αIIbβ3，并伴有GPVI裂解片段的形成。此外，完整的血小板GPVI和重组GPVI被基斯托明消化，释放出25 kDa和35 kDa的片段，表明基斯托明在粘蛋白样区域附近切割GPVI。我们设计了四种从Leu180到Asn249的合成肽作为基斯托明的底物，通过基质辅助激光解吸电离飞行时间质谱分析发现，基斯托明在FSE205/A206TA和NKV218/F219TT处切割这些合成肽。此外，基斯托明预处理后，GPVI特异性抗体诱导的血小板酪氨酸激酶磷酸化降低，基斯托明减弱了血小板与胶原蛋白的黏附，但不影响与纤维蛋白原的黏附。
我们在此首次提供证据表明，一种P-I型蛇毒金属蛋白酶基斯托明通过其对GPVI的蛋白水解活性抑制胶原蛋白与血小板GPVI之间的相互作用，从而为开发新型抗血栓药物提供了一种替代策略。
