Effect of bifunctional venom-derived antagonists of platelet adhesion receptors (a2b1 integrin and GPIb) on platelet-supported hematogenous metastasis

双功能毒液衍生血小板粘附受体拮抗剂（a2b1整合素和GPIb）对血小板支持的血行转移的影响

• 批准号: 289218518
• 负责人: Professor Dr. Johannes Andreas Eble
• 金额: --
• 依托单位: Fundação de Amparo à Pesquisa do Estado de Minas Gerais
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/289218518?language=en
• 关键词: Effect; bifunctional; venom; derived; antagonists

State of art: No hematogenous metastasis without platelets! Hypothesis: Less hematogenous metastases by blocking or cleaving adhesion receptors of platelets?Blood-borne tumor cells associate with platelets into tumor cell-platelet-aggregates (TCPAs). They adhere via platelet adhesion receptors to endothelial cells or vascular matrix molecules. Subsequently, they extravasate and colonize distant organs. Because of their large size, adherent TCPAs are strongly exposed to shear forces of the blood stream and hence have to adhere firmly. Important platelet adhesion receptors are alpha2beta1 (a2b1) integrin and GPIb-IX-V-complex which bind to collagens, leading to platelet activation, and von Willebrand factor (vWF), respectively.The Eble lab has identified rhodocetin and other snake venom-derived C-type lectin-related proteins (CLRPs). The rhodocetin gamma-delta subunit blocks a2b1 integrin selectively, and its alpha-beta subunit inhibits GPIb. A similar dual inhibition was unraveled for another CLRP, flavocetin, although both receptor binding sites are localized within the same subunit. In this project, the Eble lab cooperates with Prof. Sanchez at FUNED, Belo Horizonte, who identified a non-hemorrhagic snake venom metallo-proteinase (nhSVMP) which cuts platelet adhesion receptors. We hypothesize that bifunctional CLRPs and nhSVMPs, which block and cleave, respectively, a2b1 integrin and GPIb-IX-V, impair formation and adhesion of TCPAs and thus reduce metastasis.Our aims are (i) to identify novel bifunctional CLRPs and nhSVMPs which target both platelet adhesion receptors, and (ii) to test their inhibitory effects on tumor cell-platelet cohesion, tumor cell-induced platelet activation, and platelet-supported tumor cell extravasation. Novel inhibitors of a2b1 integrin and GPIb will be isolated from Brazilian snake venoms and characterized by binding/inhibition assays at the molecular and cellular level. Their effect on TCPA formation, including subsequent platelet activation, will be monitored by flow cytometry and aggregometry. Various adhesion and migration assays will show the potential of these adhesion receptor-interfering components to impair TCPA attachment to endothelial cells or to collagen and vWF at defined shear forces. Adhesion and invasion of tumor cells without or with platelets treated with isolated venom components will be measured biometrically and in real time by impedance-based methods and by live cell imaging.The results will unravel the molecular mechanisms of bifunctional CLRPs and nhSVMPs and will shed light on the role of the two adhesion receptors, a2b1 integrin and GPIb, in platelet-supported hematogenic tumor cell dissemination. This study will reveal the potential of these venom components for the development of platelet-targeting strategies to reduce metastasis.

最新技术水平：无血小板情况下无血行转移！假说：阻断或裂解血小板粘附受体减少血行转移？血液传播的肿瘤细胞与血小板结合形成肿瘤细胞-血小板-聚集体（TCPAs）。它们通过血小板粘附受体粘附于内皮细胞或血管基质分子。随后，它们外渗并定殖于远处器官。由于它们的大尺寸，粘附的TCPA强烈地暴露于血流的剪切力，因此必须牢固地粘附。重要的血小板粘附受体是α 2 β 1（a2 b1）整联蛋白和GPIb-IX-V复合物，它们分别与胶原蛋白结合，导致血小板活化，以及血管性血友病因子（vWF）。rhoorectin γ-δ亚基选择性阻断α 2b 1整联蛋白，其α-β亚基抑制GPIb。一个类似的双重抑制被解开另一个CLRP，flavocetin，虽然两个受体结合位点位于同一个亚基。在这个项目中，Eble实验室与贝洛奥里藏特FUNED的Sanchez教授合作，后者鉴定了一种非出血性蛇毒金属蛋白酶（nhSVMP），可以切割血小板粘附受体。我们假设，分别阻断和切割α 2 β 1整合素和GPIb-IX-V的双功能CLRP和nhSVMP损害了TCPA的形成和粘附，从而减少了转移。我们的目的是（i）鉴定靶向血小板粘附受体的新型双功能CLRP和nhSVMP，和（ii）测试它们对肿瘤细胞-血小板粘附、肿瘤细胞诱导的血小板活化、和血小板支持的肿瘤细胞外渗。新的α 2 β 1整联蛋白和GPIb的抑制剂将从巴西蛇毒中分离出来，并通过分子和细胞水平的结合/抑制试验进行表征。将通过流式细胞术和聚集测定法监测其对TCPA形成的影响，包括随后的血小板活化。各种粘附和迁移试验将显示这些粘附受体干扰成分在规定剪切力下损害TCPA与内皮细胞或胶原蛋白和vWF的粘附的潜力。肿瘤细胞的粘附和侵袭，没有或与血小板处理与分离的毒液成分将生物计量学和真实的时间通过阻抗为基础的方法和活细胞imaging.The结果将解开双功能CLRP和nhSVMPs的分子机制，并将阐明两个粘附受体，a2 b1整合素和GPIb，在血小板支持的造血肿瘤细胞传播的作用。这项研究将揭示这些毒液成分用于开发血小板靶向策略以减少转移的潜力。