Effekt bifunktioneller Schlangengiftantagonisten gegen Plättchenadhäsionsrezeptoren (a2b1 Integrin, GPIb) auf die Plättchen-unterstützte hämatogene Metastasierung
Zellbiologie
Zusammenfassung der Projektergebnisse
This project has identified novel venom derived proteins, likely from the CLRP family, that interact with integrin α2β1 and/or GPIb. Whether or not the venom protein from Bothrops neuwiedi, termed Neuwiedicetin and Bn-GPIb-bp, are the same proteins has not yet been demonstrated. The isolated venom components directed against integrin α2A-domain inhibit its interaction with collagen. The role of the GPIb-binding venom proteins have remained elusive due to the high background problem in the binding-ELISA of soluble vWF-A1-domain to immobilized recombinant GPIb(290) or platelet-extracted glycocalicin. We established a new “on-line” workflow, which includes aggregometric monitoring of the TCPA formation and their flow cytometric separation and imaging to characterize the different cellular clusters. The aggregometric experiments revealed that serum, most likely the containing fibrinogen and its conversion product, fibrin, are essential for TCPA formation. We showed that fibrinogen conversion is likely mediated by the extrinsic coagulation pathway and triggered by tissue factor (TF). The more TF the cancer cell express, the shorter the lag period after which cancer cell-induced formation of TCPAs occurred. The aggregation curve can be approximated by a two phase-exponential curve, indicating that platelets are activitated during TCPA formation in two subsequent activation steps. Inhibitory studies using venom CLRPs, such as rhodocetin, suggested that GPIb and integrin α2β1 are involved in platelet activation during TCPA formation. Moreover, other candidates, such as P-selectin (potentially as the second phase activator) and CLEC-2 (due to its interaction with cancer cell podoplanin) are still highly relevant in this process, as they could not be ruled out in our study. In contrast, inhibiting Fab-fragments against CLEC2 indicated an important, yet unclear role of of CLEC2 in tumor cell-induced platelet aggregation. Signaling within platelets via α2β1 integrin and CLEC-2 was further analyzed in this study by using minicollagen-coated nanobeads and rhodocytin. Integrin α2β1 activates a special set of kinases, such as BTK, in platelets. This information about the α2β1 integrin signaling pathway and the insights, that cancer cell activate platelets while forming TCPAs and that GPIb, CLEC-2 and integrin α2β1 are involved in the platelet activation by tumor cells, spurs the further search for CLRPs that inhibit any of these receptors. The test system for GPIb-targeting venom components would need improvement. Future work will also aim to fully elaborate the novel workflow of aggregometry and flow cytometric imaging as well as the tube-flow system to obtain TCPAs for attachment studies.
Projektbezogene Publikationen (Auswahl)
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Direct Fibrinolytic Snake Venom Metalloproteinases Affecting Hemostasis: Structural, Biochemical Features and Therapeutic Potential. Toxins (Basel) (2017) 9(12)
Sanchez EF, Flores-Ortiz RJ, Alvarenga VG, Eble JA
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Dramatic and concerted conformational changes enable rhodocetin to block α2β1 integrin selectively. PLoS Biol (2017) 15(7): e2001492
Eble JA, McDougall M, Orriss GL, Niland S, Johanningmeier B, Pohlentz G, Meier M, Karrasch S, Estevao-Costa MI, Martins Lima A, Stetefeld J
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Snake venom components in medicine: From the symbolic rod of Asclepius to tangible medical research and application. Int J Biochem Cell Biol (2018) 104: 94-113
Estevao-Costa MI, Sanz-Soler R, Johanningmeier B, Eble JA
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The spatial molecular pattern of integrin recognition sites and their immobilization to colloidal nanobeads determine α2β1 integrin-dependent platelet activation. Biomaterials (2018) 167: 107-120
Lima AM, Wegner SV, Martins Cavaco AC, Estevao-Costa MI, Sanz-Soler R, Niland S, Nosov G, Klingauf J, Spatz JP, Eble JA
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Atroxlysin-III, A Metalloproteinase from the Venom of the Peruvian Pit Viper Snake Bothrops atrox (Jergon) Induces Glycoprotein VI Shedding and Impairs Platelet Function. Molecules (2019) 24(19)
Oliveira LS, Estevao-Costa MI, Alvarenga VG, Vivas-Ruiz DE, Yarleque A, Lima AM, Cavaco A, Eble JA, Sanchez EF
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Structurally Robust and Functionally Highly Versatile-C-Type Lectin (-Related) Proteins in Snake Venoms. Toxins (Basel) (2019) 11(3)
Eble JA
