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

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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.