A snake venom metalloproteinase, kistomin, cleaves platelet glycoprotein VI and impairs platelet functions
A snake venom metalloproteinase, kistomin, cleaves platelet glycoprotein VI and impairs platelet functions
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DOI：
10.1111/j.1538-7836.2008.03071.x
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发表时间：
2008-09-01
影响因子：
10.4
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中科院分区：
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背景和目的：血管壁损伤和随后内皮下层基质暴露导致血栓形成。血小板膜糖蛋白(GP)Ib和Vi在基质诱导的血小板活化和聚集中起重要作用。方法和结果：在本研究中，我们报道了裂解蛇毒金属蛋白酶(SVMP)的Kistomin抑制胶原诱导的血小板聚集。此外，Kistomin以浓度和时间依赖的方式抑制由惊厥毒素(GPVI激动剂CVX)和GPVI特异性抗体诱导的血小板聚集。Kistomin治疗降低了血小板GPVI，但不减少整合素α2β1和αIIbβ3，并伴有GPVI裂解片段的形成，如流式细胞仪和Western印迹分析所确定的那样。此外，完整的血小板GPVI和重组GPVI被Kistomin消化，释放出25 kDa和35 kDa的片段，表明Kistomin在粘蛋白样区附近切割GPVI。我们设计了从Leu(180)到Asn(249)的四个合成肽作为Kistomin的底物，MALDI-TOF-MS分析发现Kistomin在FSE205/A(206)TA和NKV218/(FTT)-T-219处切割这些合成肽。此外，血吸虫素预处理后，GPVI特异性抗体诱导的血小板酪氨酸激酶磷酸化水平降低，血小板与胶原的黏附减弱，但与纤维蛋白原的黏附不减弱。结论：本研究首次证明P-I蛇毒金属蛋白酶Kistomin通过对GPVI的蛋白分解作用抑制胶原与血小板GPVI的相互作用，从而为开发新的抗血栓药物提供了新的策略。
Background and objectives: 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. Methods and results: 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 alpha 2 beta 1 and alpha IIb beta 3, 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 Leu(180) to Asn(249) as the substrates for kistomin and found that kistomin cleaved these synthetic peptides at FSE205/A(206)TA and NKV218/(FTT)-T-219, 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. Conclusions: 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.