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

目前状况：无血小板无血液转移！假设：阻断或切割血小板粘附受体可减少血液转移？血源性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到内皮细胞或血管基质分子上。随后，它们向外扩散并在远处的器官中定居。由于它们的大尺寸，粘附的tcpa强烈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分别是与胶原结合导致血小板活化的α 2 β 1 （a2b1）整合素和gpib - ix - v复合物，以及血管性血液病因子（vWF）。Eble实验室已经鉴定出rhodocetin和其他蛇毒衍生的c型凝集素相关蛋白（clrp）。rhodocsetin γ - δ亚基选择性阻断a2b1整合素，α - β亚基抑制GPIb。另一种CLRP黄黄素也有类似的双重抑制作用，尽管这两种受体结合位点都位于同一亚基内。在这个项目中，Eble实验室与Belo Horizonte FUNED的Sanchez教授合作，他发现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。我们假设双功能CLRPs和nhSVMPs，分别阻断和切割a2b1整合素和GPIb-IX-V，损害TCPAs的形成和粘附，从而减少转移。我们的目标是(i)鉴定新的双功能clrp和nhSVMPs，它们同时靶向血小板粘附受体，以及（ii）测试它们对肿瘤细胞-血小板内聚、肿瘤细胞诱导的血小板活化和血小板支持的肿瘤细胞外溢的抑制作用。新的a2b1整合素和GPIb抑制剂将从巴西蛇毒中分离出来，并在分子和细胞水平上进行结合/抑制试验。它们对TCPA形成的影响，包括随后的血小板活化，将通过流式细胞术和聚集术监测。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的剪切力下损害TCPA附着在内皮细胞或胶原蛋白和vWF上的潜力。将通过基于阻抗的方法和活细胞成像实时测量无血小板或有血小板的肿瘤细胞的粘附和侵袭。这些结果将揭示双功能CLRPs和nhSVMPs的分子机制，并将阐明两种粘附受体a2b1整合素和GPIb在血小板支持的造血肿瘤细胞传播中的作用。这项研究将揭示这些毒液成分在血小板靶向策略发展中的潜力，以减少转移。

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.