Rhodocytin (Aggretin) Activates Platelets Lacking α2β1 Integrin, Glycoprotein VI, and the Ligand-binding Domain of Glycoprotein Ibα

Although α2β1integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the venom ofCalloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against α2β1 integrin. This finding suggested that clustering of α2β1 integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to α2β1 integrin. Here we show that the Cre/loxP-mediated loss of β1 integrin on mouse platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of α2β1integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibα either on normal or on β1-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets lacking both α2β1 integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbα rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin induces platelet activation by mechanisms that are fundamentally different from those induced by collagen. Although α2β1integrin (glycoprotein Ia/IIa) has been established as a platelet collagen receptor, its role in collagen-induced platelet activation has been controversial. Recently, it has been demonstrated that rhodocytin (also termed aggretin), a snake venom toxin purified from the venom ofCalloselasma rhodostoma, induces platelet activation that can be blocked by monoclonal antibodies against α2β1 integrin. This finding suggested that clustering of α2β1 integrin by rhodocytin is sufficient to induce platelet activation and led to the hypothesis that collagen may activate platelets by a similar mechanism. In contrast to these findings, we provided evidence that rhodocytin does not bind to α2β1 integrin. Here we show that the Cre/loxP-mediated loss of β1 integrin on mouse platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of α2β1integrin in this process. Furthermore, proteolytic cleavage of the 45-kDa N-terminal domain of glycoprotein (GP) Ibα either on normal or on β1-null platelets had no significant effect on rhodocytin-induced platelet activation. Moreover, mouse platelets lacking both α2β1 integrin and the activating collagen receptor GPVI responded normally to rhodocytin. Finally, even after additional proteolytic removal of the 45-kDa N-terminal domain of GPIbα rhodocytin induced aggregation of these platelets. These results demonstrate that rhodocytin induces platelet activation by mechanisms that are fundamentally different from those induced by collagen. von Willebrand factor Fc receptor fluoresceine isothiocyanate glycoprotein antibody monoclonal antibody R-phycoerythrin 4-morpholineethanesulfonic acid Collagen is one of the major components of the vessel wall and contributes to platelet activation and adhesion at sites of vascular injury. The interaction between platelets and collagen can either occur indirectly via immobilized von Willebrand factor (vWf)1 binding to platelet receptors glycoprotein (GP) Ib-V-IX and/or activated αIIbβ3 integrin (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar) or by direct recognition of collagen by specific receptors expressed on the platelet surface. Several receptors for collagen have been identified on platelets, most importantly the Ig-like receptor GPVI (2Moroi M. Jung S.M. Okuma M. Shinmyozu K. J. Clin. Invest. 1989; 84: 1440-1445Crossref PubMed Scopus (387) Google Scholar) and α2β1 integrin (3Santoro S.A. Cell. 1986; 46: 913-920Abstract Full Text PDF PubMed Scopus (331) Google Scholar). In contrast to earlier reports, we have recently shown with β1 integrin-null platelets that α2β1 integrin is not essential for platelet adhesion to fibrillar collagen. GPVI, however, was found to be indispensable for this process (4Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W.M. Zirngibl H. Fässler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (463) Google Scholar). Although GPVI has been established as the major activating platelet collagen receptor, the way α2β1 integrin modulates the activation process is still unclear. Experimental evidence suggests that collagen contains two distinct epitopes contributing to activation of (murine) platelets. One of these epitopes specifically binds to GPVI, and this interaction is blocked by the anti-GPVI mAb JAQ1 (5Schulte V. Snell D. Bergmeier W. Zirngibl H. Watson S.P. Nieswandt B. J. Biol. Chem. 2001; 276: 364-368Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In contrast, activation through the second epitope is not blocked by JAQ1 and involves GPVI, α2β1 integrin, and high concentrations of fibrillar collagen (4Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W.M. Zirngibl H. Fässler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (463) Google Scholar). The mechanisms underlying this activation pathway and the role of α2β1 integrin are unclear. In addition to α2β1 and GPVI, other receptors may be involved in this activation pathway. One candidate is GPIbα, because this receptor indirectly interacts with collagen via vWf (1Savage B. Saldivar E. Ruggeri Z.M. Cell. 1996; 84: 289-297Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar). Snake venom-derived proteins are frequently used to study mechanisms of platelet activation and aggregation because many of them specifically bind to platelet surface glycoprotein receptors and interfere with their function. Rhodocytin (also termed aggretin (6Chung C.H. Au L.C. Huang T.F. Biochem. Biophys. Res. Commun. 1999; 263: 723-727Crossref PubMed Scopus (37) Google Scholar)), purified from the venom of Calloselasma rhodostoma belongs to the family of C-type lectins and induces aggregation of human as well as mouse platelets (7Suzuki-Inoue K. Ozaki Y. Kainoh M. Shin Y. Wu Y. Yatomi Y. Ohmori T. Tanaka T. Satoh K. Morita T. J. Biol. Chem. 2001; 276: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Recent studies gave rise to conflicting results on the mechanisms underlying this activation process. Several experiments suggested that rhodocytin activates platelets in a collagen-like manner. First, both processes are sensitive to inhibition of thromboxane A2 formation by treatment with acetylsalicylic acid, and, second, both can be inhibited by mAbs against α2β1 integrin (7Suzuki-Inoue K. Ozaki Y. Kainoh M. Shin Y. Wu Y. Yatomi Y. Ohmori T. Tanaka T. Satoh K. Morita T. J. Biol. Chem. 2001; 276: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Inoue K. Ozaki Y. Satoh K. Wu Y. Yatomi Y. Shin Y. Morita T. Biochem. Biophys. Res. Commun. 1999; 256: 114-120Crossref PubMed Scopus (36) Google Scholar). Based on these results, the authors concluded that rhodocytin activates platelets by interacting with α2β1 integrin (8Inoue K. Ozaki Y. Satoh K. Wu Y. Yatomi Y. Shin Y. Morita T. Biochem. Biophys. Res. Commun. 1999; 256: 114-120Crossref PubMed Scopus (36) Google Scholar). Others reported that rhodocytin activates platelets through α2β1 integrin and GPIbα (9Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), results that were also based on experiments with inhibitory antibodies against both receptors. Both hypotheses, however, were challenged by our finding that rhodocytin does not bind recombinant, soluble α2β1 (10Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), and the same result has meanwhile been obtained with wild-type α2β1integrin isolated from human platelets. 2J. A. Eble, unpublished results. Additionally, rhodocytin activates platelets from FcRγ chain-deficient mice (7Suzuki-Inoue K. Ozaki Y. Kainoh M. Shin Y. Wu Y. Yatomi Y. Ohmori T. Tanaka T. Satoh K. Morita T. J. Biol. Chem. 2001; 276: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), which lack GPVI (11Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar) and do not respond to collagen (4Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W.M. Zirngibl H. Fässler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (463) Google Scholar, 12Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (399) Google Scholar), suggesting that rhodocytin uses other mechanisms than collagen to induce aggregation. Moreover, we have recently shown that β1-null platelets fail to express α2β1 integrin but display no reduced response to fibrillar collagen, indicating that α2β1 integrin is not a major signaling collagen receptor on platelets (4Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W.M. Zirngibl H. Fässler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (463) Google Scholar). To directly test whether rhodocytin induces platelet activation by mechanisms similar to those induced by collagen, we now examined the effects of this agonist on platelets lacking α2β1 integrin, GPVI, the ligand-binding domain on GPIbα, or all three of them. The results of these studies demonstrate that none of these receptors is required for platelet activation by rhodocytin. Mice carrying the β1-null allele in megakaryocytes were generated as described previously (4Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W.M. Zirngibl H. Fässler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (463) Google Scholar). Briefly, β1(fl/fl) mice (13Potocnik A.J. Brakebusch C. Fassler R. Immunity. 2000; 12: 653-663Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar) were crossed with transgenic mice carrying the Mx-cre R. M. K. PubMed Scopus Google Scholar). of the β1 was induced in mice by three of of acid at mice β1(fl/fl) the same treatment and were from same mice were used at after acid The of the and on the platelets from these mice was by and as described (4Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W.M. Zirngibl H. Fässler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (463) Google Scholar). mice in the FcRγ T. M. D. R. Cell. Full Text PDF PubMed Scopus Google Scholar) were obtained from mice in were provided by Mice were with of JAQ1 and platelets were isolated on reported GPVI was not on those platelets by and B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. C. Zirngibl H. J. 2001; PubMed Scopus Google Scholar). collagen and were was purified from as described previously A. A. J. Biol. Clin. Google Scholar). acid was from and purified human vWf were provided by and C. venom was from in the venom was by on a The of were with the were to a binding to these the rhodocytin in the was against and to a Rhodocytin was from the in a at concentrations The was by in a Finally, the of rhodocytin was purified by on a of and was by in a was by the to the The mAb was provided by D. and in our integrin integrin, and integrin were from and were from other antibodies were and in our JAQ1 (11Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), W. Rackebrandt K. W. Zirngibl H. Nieswandt B. 2000; PubMed Google Scholar), W. Rackebrandt K. W. Zirngibl H. Nieswandt B. 2000; PubMed Google Scholar), B. Bergmeier W. Rackebrandt K. Gessner J.E. Zirngibl H. 2000; PubMed Google Scholar), and B. Bergmeier W. Rackebrandt K. Gessner J.E. Zirngibl H. 2000; PubMed Google Scholar). Mice were from the The was in a and was obtained by at for at The platelets were in and at a of in the same in the of of the a sufficient to of platelet receptors were at all were in of and all from was on a and a The was with mAb by The proteins were by platelets were with the of for by with for at and on a were by The platelets were in and at for with of the platelet were in and to of platelet activation and in platelet To platelet was platelets with was on a and was expressed as with with has been reported that rhodocytin induces platelet aggregation by interacting with the collagen receptor α2β1 integrin. Based on these results, it was suggested that collagen also activate platelets via α2β1 integrin (7Suzuki-Inoue K. Ozaki Y. Kainoh M. Shin Y. Wu Y. Yatomi Y. Ohmori T. Tanaka T. Satoh K. Morita T. J. Biol. Chem. 2001; 276: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). These were challenged by our result that rhodocytin does not bind to a soluble of α2β1 integrin (10Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). To directly whether rhodocytin activates platelets through mechanisms similar to those induced by collagen, we the effects of rhodocytin and fibrillar collagen on mouse platelets lacking the collagen receptors α2β1 integrin and reported FcRγ chain-deficient platelets lack GPVI but express normal of α2β1integrin and do not respond to collagen (11Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 12Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (399) Google Scholar) In contrast, platelets from mice with a Cre/loxP-mediated of the β1 in megakaryocytes lack all but express normal of GPVI β1-null platelets display but not reduced aggregation to fibrillar collagen (4Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W.M. Zirngibl H. Fässler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (463) Google Scholar) The effects of rhodocytin on mouse platelets were similar to those reported for human platelets (7Suzuki-Inoue K. Ozaki Y. Kainoh M. Shin Y. Wu Y. Yatomi Y. Ohmori T. Tanaka T. Satoh K. Morita T. J. Biol. Chem. 2001; 276: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 8Inoue K. Ozaki Y. Satoh K. Wu Y. Yatomi Y. Shin Y. Morita T. Biochem. Biophys. Res. Commun. 1999; 256: 114-120Crossref PubMed Scopus (36) Google Scholar, 9Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). with a and was sensitive to of the thromboxane activation by rhodocytin of and was by shown by and binding rhodocytin also induced aggregation of β1-null platelets. The of and binding was between β1-null and platelets Furthermore, no were found in the between normal and β1-null platelets These results demonstrate that β1 are not essential for rhodocytin-induced platelet activation. has been shown that snake venom may induce platelet activation by interacting with receptors D. Clemetson J.M. A. Kehrel B.E. Clemetson K.J. 2001; PubMed Scopus Google Scholar). one of them not platelet we examined the effects of rhodocytin on platelets lacking both α2β1 and To platelets, GPVI was in mice with β1-null platelets by of JAQ1 This treatment induces a and proteolytic of GPVI in platelets but does not other and B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. C. Zirngibl H. J. 2001; PubMed Scopus Google Scholar). platelets do not respond to collagen, activation by other is not B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. C. Zirngibl H. J. 2001; PubMed Scopus Google Scholar). shown in platelets from β1-null mice both collagen receptors. these platelets responded normally to rhodocytin as demonstrated by and and This finding an essential role of GPVI in rhodocytin-induced platelet activation. that rhodocytin platelet activation was inhibited by a mAb against the 45-kDa N-terminal domain on GPIbα (9Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). This domain contains the binding sites for all J.A. 1997; PubMed Scopus Google Scholar), A.J. J.A. J. 1999; PubMed Scopus (300) Google Scholar), and H. J. J.A. J. 2000; PubMed Scopus Google Scholar), as well as snake venom-derived C-type lectins T. Y. Y. M. Y. K. Y. K. K. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus (71) Google Scholar), D. Clemetson J.M. A. Kehrel B.E. Clemetson K.J. 2001; PubMed Scopus Google Scholar), and M. W. PubMed Google Scholar). Based on their results, (9Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) concluded that GPIbα an essential role in rhodocytin-induced platelet activation. To directly test this we platelets J. A. Y. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). This treatment in proteolytic removal of the 45-kDa N-terminal domain of GPIbα as demonstrated by in addition to cleavage of the 45-kDa N-terminal the of GPIbα was in to the of GPIbα, in the of lacking the 45-kDa N-terminal of the lack of the 45-kDa N-terminal domain of GPIbα, vWf binding was platelets these platelets responded normally to rhodocytin and that the ligand-binding domain on GPIbα is not essential for this activation process. Although our results demonstrated that the collagen receptors α2β1 integrin and GPVI GPIbα are essential for rhodocytin-induced platelet activation, it not be that rhodocytin binds to all three receptors that aggregation. we the 45-kDa N-terminal of GPIbα from and platelets and examined their response to rhodocytin. shown in even the of GPVI, and the 45-kDa N-terminal domain of GPIbα had no significant effect on rhodocytin-induced platelet activation and aggregation. The underlying platelet activation by rhodocytin has been Several suggested that rhodocytin activates platelets in a collagen-like by interacting with α2β1 integrin or α2β1 integrin and GPIbα (7Suzuki-Inoue K. Ozaki Y. Kainoh M. Shin Y. Wu Y. Yatomi Y. Ohmori T. Tanaka T. Satoh K. Morita T. J. Biol. Chem. 2001; 276: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, D. Clemetson J.M. A. Kehrel B.E. Clemetson K.J. 2001; PubMed Scopus Google Scholar). In contrast to these hypotheses, we reported that rhodocytin does not bind to α2β1 integrin (10Eble J.A. Beermann B. Hinz H.J. Schmidt-Hederich A. J. Biol. Chem. 2001; 276: 12274-12284Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). In the study we demonstrate that rhodocytin induces activation of platelets in the of GPVI, and the ligand-binding domain of These are the three major receptors that directly or indirectly with collagen. an essential role for these receptors in rhodocytin-induced activation and demonstrate that the activation process mechanisms that are fundamentally different from those induced by collagen. are in contrast to those reported by (7Suzuki-Inoue K. Ozaki Y. Kainoh M. Shin Y. Wu Y. Yatomi Y. Ohmori T. Tanaka T. Satoh K. Morita T. J. Biol. Chem. 2001; 276: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), that mAbs against α2β1 blocked rhodocytin-induced platelet aggregation and concluded that the integrin an essential role in the activation process. for this be that treatment of platelets with mAbs against α2β1 may have different effects than the of the may effects on other surface proteins or may inhibitory results from inhibition studies with antibodies against α2β1 not be in a in which the β1 is in all was shown that antibodies against α2β1 integrin reduced or platelet adhesion and formation on collagen in and de PubMed Google Scholar) as well as collagen-induced platelet aggregation (7Suzuki-Inoue K. Ozaki Y. Kainoh M. Shin Y. Wu Y. Yatomi Y. Ohmori T. Tanaka T. Satoh K. Morita T. J. Biol. Chem. 2001; 276: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). the we the β1 in megakaryocytes and that α2β1 integrin is not required for platelet adhesion and formation on fibrillar collagen as well as and high β1-null platelets display a but not reduced aggregation in response to collagen, that α2β1 integrin is not a major signaling collagen receptor on platelets (4Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W.M. Zirngibl H. Fässler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (463) Google Scholar). The with in which antibody inhibition and studies conflicting results. the of can be inhibited with antibodies against α2β1 M. Okuma M. C. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), Cre/loxP-mediated of β1 integrin on platelets no role of α2β1 for platelet activation by this agonist (4Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W.M. Zirngibl H. Fässler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (463) Google Scholar). these that antibodies against α2β1 integrin induce inhibitory effects that are not based on of the integrin. treatment of platelets with antibodies may not be for on α2β1 integrin. between and platelet aggregation is the role of GPVI for collagen but not for rhodocytin. recently that GPVI is the major collagen receptor for platelet activation and that GPVI is essential for collagen-induced platelet aggregation B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. C. Zirngibl H. J. 2001; PubMed Scopus Google Scholar). aggregation processes are different from collagen-induced aggregation. Both platelets, which lack GPVI (11Nieswandt B. Bergmeier W. Schulte V. Rackebrandt K. Gessner J.E. Zirngibl H. J. Biol. Chem. 2000; 275: 23998-24002Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), and platelets B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. C. Zirngibl H. J. 2001; PubMed Scopus Google Scholar) fail to activate β1 and in response to collagen. these platelets to collagen do bind or in response to this agonist (4Nieswandt B. Brakebusch C. Bergmeier W. Schulte V. Bouvard D. Mokhtari-Nejad R. Lindhout T. Heemskerk J.W.M. Zirngibl H. Fässler R. EMBO J. 2001; 20: 2120-2130Crossref PubMed Scopus (463) Google Scholar, 12Poole A. Gibbins J.M. Turner M. van Vugt M.J. van de Winkel J.G. Saito T. Tybulewicz V.L. Watson S.P. EMBO J. 1997; 16: 2333-2341Crossref PubMed Scopus (399) Google Scholar, B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. C. Zirngibl H. J. 2001; PubMed Scopus Google Scholar). these we that rhodocytin activates platelets by mechanisms that are different from those induced by collagen. and (9Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) a for rhodocytin platelet activation that involves two platelet α2β1 integrin and The of GPIbα for rhodocytin-induced aggregation was concluded from a inhibitory effect of a mAb against the on GPIbα, which is in the 45-kDa N-terminal of the receptor M. 1996; PubMed Scopus Google Scholar). This finding was in contrast to by other found no role of in rhodocytin-induced activation Y. Morita T. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, T.F. Biochem. J. PubMed Scopus Google Scholar) or no binding of rhodocytin to (7Suzuki-Inoue K. Ozaki Y. Kainoh M. Shin Y. Wu Y. Yatomi Y. Ohmori T. Tanaka T. Satoh K. Morita T. J. Biol. Chem. 2001; 276: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The in the binding studies are to may be to different used in these In our study we show that platelets lacking the 45-kDa N-terminal domain on GPIbα respond normally to rhodocytin This finding an essential role for the ligand-binding of GPIbα in the rhodocytin-induced activation process and is in contrast to the finding by and (9Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). or of inhibitory by the antibody be an for the conflicting results. is that of GPIbα induces of different signaling in J. Clin. Invest. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar, Y. J.A. 1999; PubMed Scopus Google Scholar) and that of GPIbα by mAbs platelet by mechanisms in and in W. Rackebrandt K. W. Zirngibl H. Nieswandt B. 2000; PubMed Google Scholar, Y. PubMed Google Scholar, M. van J. H. Biol. 2000; 20: PubMed Scopus Google Scholar). results do not the that rhodocytin interacts with an epitope on the that is distinct from the 45-kDa N-terminal of in studies mAbs against different epitopes on either or we were to rhodocytin-induced In mouse platelets respond normally to rhodocytin These results that the has no essential role in rhodocytin-induced platelet activation. of of GPVI, and proteolytic of we show that platelets lacking all three major receptors that directly or indirectly with collagen GPVI, and respond normally to rhodocytin. rhodocytin-induced aggregation of human and mouse platelets with a and of the FcRγ and both processes are sensitive to acetylsalicylic acid, it is that the results and in other studies (7Suzuki-Inoue K. Ozaki Y. Kainoh M. Shin Y. Wu Y. Yatomi Y. Ohmori T. Tanaka T. Satoh K. Morita T. J. Biol. Chem. 2001; 276: 1643-1652Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9Navdaev A. Clemetson J.M. Polgar J. Kehrel B.E. Glauner M. Magnenat E. Wells T.N. Clemetson K.J. J. Biol. Chem. 2001; 276: 20882-20889Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, D. Clemetson J.M. A. Kehrel B.E. Clemetson K.J. 2001; PubMed Scopus Google Scholar, T.F. Biochem. J. PubMed Scopus Google Scholar). Furthermore, other snake venom-derived B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. C. Zirngibl H. J. 2001; PubMed Scopus Google Scholar, M. T. A. B. C. W. 2000; PubMed Google Scholar), J. Ruggeri Z.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and D. Clemetson J.M. A. Kehrel B.E. Clemetson K.J. 2001; PubMed Scopus Google Scholar) unpublished results. also show similar on human and mouse platelets. it that other than GPVI, and rhodocytin-induced platelet activation. Rhodocytin induced suggesting that it signaling normally used by platelet the effects of rhodocytin be by the between two or platelet receptors. effects have previously been shown for a of that different receptors J. S.P. A. PubMed Scopus Google Scholar, J. S.P. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, M. H. K. M. Biochem. Biophys. Res. Commun. 1999; 263: PubMed Scopus Google Scholar) and Ig-like receptors and receptors B. Bergmeier W. A. Schulte V. Zirngibl H. C. 2001; PubMed Scopus Google Scholar, A. M. C. 2000; PubMed Google Scholar, C. A. J.M. H. M. B. 2000; PubMed Google Scholar). receptors for rhodocytin on the platelet Ig-like receptors the recently receptor T. C. A.J. J. 2000; PubMed Google Scholar), as well as or αIIbβ3 integrin, all of which may activation studies be to rhodocytin induces platelet aggregation. Rackebrandt and for and for the also for