Rhodocytin (Aggretin) Activates Platelets Lacking α2β1 Integrin, Glycoprotein VI, and the Ligand-binding Domain of Glycoprotein Ibα
2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês
10.1074/jbc.m103892200
ISSN1083-351X
AutoresWolfgang Bergmeier, Daniel Bouvard, Johannes A. Eble, Rabée Mokhtari-Nejad, Valerie Schulte, Hubert Zirngibl, Cord Brakebusch, Reinhard Fässler, Bernhard Nieswandt,
Tópico(s)Blood properties and coagulation
ResumoAlthough 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 transgene (mx-cre+) (14Kuhn R. Schwenk F. Aguet M. Rajewsky K. Science. 1995; 269: 1427-1429Crossref PubMed Scopus (1559) Google Scholar). Deletion of the β1 gene was induced in 4–5-week-old (β1(fl/fl)/Mx-cre+) mice by three intraperitoneal injections of 250 μg of polyinosinic-polycytidylic acid at 2-day intervals. Control mice β1(fl/fl) received the same treatment and were derived from same litters. For experiments, mice were used at least 2 weeks after polyinosinic-polycytidylic acid injection. The absence of the α2 and β1integrin subunits on the platelets from these mice was always confirmed by flow cytometry and Western blotting 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). C57Bl/6 mice deficient in the FcRγ chain (15Takai T. Li M. Sylvestre D. Clynes R. Ravetch J.V. Cell. 1994; 76: 519-529Abstract Full Text PDF PubMed Scopus (838) Google Scholar) were obtained from Taconics (Germantown, NY). C57Bl/6 × SV129 mice deficient in GPV were kindly provided by F. Lanza (Strasbourg, France). Mice were injected with 100 μg of JAQ1 intraperitoneally, and platelets were isolated on day 7. As reported previously, GPVI was not detectable on those platelets by flow cytometry and Western blotting (16Nieswandt B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-470Crossref PubMed Scopus (308) Google Scholar). High molecular weight heparin (Sigma), α-thrombin (Roche Molecular Biochemicals), collagen (Nycomed GmbH, Munich, Germany), and O-sialoglycoprotein endopeptidase (Cedarlane, Hornby, Canada) were purchased. Apyrase was purified from potatoes as described previously (17Cazenave J.P. Hemmendinger S. Beretz A. Sutter-Bay A. Launay J. Ann. Biol. Clin. 1983; 41: 167-179PubMed Google Scholar). Acetylsalicylic acid was from Sanofi-Synthelabo (Paris, France). Botrocetin and purified human vWf were kindly provided by F. Lanza (Strasbourg, France) and G. Dickneite (Marburg, Germany), respectively. C. rhodostoma(Malayain pit viper) venom was purchased from Sigma. Dissolved in 50 mm Tris/HCl, pH 7.4, 150 mm NaCl, 1 mm EDTA, 0.02% sodium azide, the venom was separated by gel filtration chromatography on a Superose 6 column. The eluate fractions of respective size were pooled. After dilution with Mono S-buffer A (20 mm MES/NaOH, pH 6.5), the pooled fractions were applied to a Mono S HR5/5 column (Amersham Pharmacia Biotech). Not binding to Mono S resin under these conditions, the rhodocytin in the flow-through was dialyzed against Mono Q buffer A (20 mm Tris/HCl, pH 8.0, 0.02% sodium azide) and applied to a Mono Q HR5/5 column (Amersham Pharmacia Biotech). Rhodocytin was eluted from the Mono Q column in a linear NaCl gradient at sodium chloride concentrations above 300 mm. The rhodocytin-containing eluate fraction was concentrated by centrifugal ultrafiltration in a Centricon 10 tube. Finally, the concentrated solution of rhodocytin was purified by gel filtration on a tandem array of TSK G3000 SWXL and TSK G2000 SWXL (TosoHass, Stuttgart, Germany). Purity was determined by SDS-polyacrylamide gel electrophoresis in a 12–18% acrylamide separating gel. Protein concentration was assayed by the BCA method according to the manufacturer's protocol (Pierce). The rat anti-mouse P-selectin mAb RB40.34 was kindly provided by D. Vestweber (Münster, Germany) and modified in our laboratories. FITC hamster anti-β1 integrin (Ha31/8), FITC hamster anti-α2 integrin, and rat anti-β1 integrin (9EG7) were from BD Pharmingen. Horseradish peroxidase-labeled rabbit anti-FITC, polyclonal rabbit anti-fibrinogen, and polyclonal rabbit anti-vWf were purchased from DAKO. All other antibodies were generated, produced, and modified in our laboratories: JAQ1 (anti-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), JON1 (anti-GPIIb/IIIa) (18Bergmeier W. Rackebrandt K. Schroder W. Zirngibl H. Nieswandt B. Blood. 2000; 95: 886-893Crossref PubMed Google Scholar), p0p4 (anti-GPIbα) (18Bergmeier W. Rackebrandt K. Schroder W. Zirngibl H. Nieswandt B. Blood. 2000; 95: 886-893Crossref PubMed Google Scholar), DOM1 (anti-GPV) (19Nieswandt B. Bergmeier W. Rackebrandt K. Gessner J.E. Zirngibl H. Blood. 2000; 96: 2520-2527Crossref PubMed Google Scholar), and ULF1 (anti-CD9) (19Nieswandt B. Bergmeier W. Rackebrandt K. Gessner J.E. Zirngibl H. Blood. 2000; 96: 2520-2527Crossref PubMed Google Scholar). Mice were bled under ether anesthesia from the retroorbital plexus. The blood was collected in a tube containing 10% (v/v) 7.5 units/ml heparin, and platelet-rich plasma was obtained by centrifugation at 300 × g for 10 min at room temperature. The platelets were washed twice in Tyrode's buffer (137 mm NaCl, 2 mm KCl, 12 mm NaHCO3, 0.3 mmNaH2PO4, 1 mm MgCl2, 2 mm CaCl2, 5.5 mm glucose, 5 mm Hepes, pH 7.3) containing 0.35% bovine serum albumin and finally resuspended at a density of 2 × 105platelets/μl in the same buffer in the presence of 0.02 unit/ml of the ADP scavenger apyrase, a concentration sufficient to prevent desensitization of platelet ADP receptors during storage. Platelets were kept at 37 °C throughout all experiments. Platelets (108) were solubilized in 1 ml of lysis buffer (Tris-buffered saline containing 20 mm Tris/HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 0.5 μg/ml leupeptin, and 0.5% Nonidet P-40; all from Roche Molecular Biochemicals). After lysis, whole cell extract was run on a 9% SDS-polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. The membrane was first incubated with 5 μg/ml FITC-labeled mAb followed by rabbit anti-FITC-horseradish peroxidase (1 μg/ml). The proteins were visualized by ECL. Washed platelets (2 × 106) were incubated with the indicated amounts of agonists for 5 min followed by staining with fluorophore-conjugated Abs (5 μg/ml) for 10 min at 37 °C and immediately analyzed on a FACScalibur (Becton Dickinson). Platelets were gated by forward scatter/side scatter characteristics. The washed platelets (2 × 109/ml) were resuspended in Tyrode's buffer (1 mmMgCl2, 1 mm CaCl2) and incubated at 37 °C for 30 min with 100 μg/ml O-sialoglycoprotein endopeptidase. Aliquots of the platelet suspensions were analyzed in flow cytometry and Western blotting to estimate markers of platelet activation and alterations in platelet glycoproteins. To determine platelet aggregation, light transmission was measured using washed platelets (200 μl with 0.5 × 106 platelets/μl). Transmission was recorded on a Fibrintimer 4 channel aggregometer (LAbor, Hamburg, Germany) over 10 min and was expressed as arbitrary units with 100% transmission adjusted with Tyrode's buffer. It 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 might 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 findings were challenged by our result showing that rhodocytin does not bind to a recombinant soluble form 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 investigate whether rhodocytin activates platelets through mechanisms similar to those induced by collagen, we compared the effects of rhodocytin and fibrillar collagen on mouse platelets lacking the collagen receptors α2β1 integrin and GPVI. As reported previously, FcRγ chain-deficient platelets lack GPVI but express normal amounts of α2β1integrin (Fig. 1a) 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) (Fig. 1b). In contrast, platelets from mice with a Cre/loxP-mediated deletion of the β1 gene in megakaryocytes lack all β1integrins but express normal amounts of GPVI (Fig. 1a). β1-null platelets display delayed 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) (Fig. 1b). 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). Aggregation occurred with a dose-dependent lag phase (Fig.2a) and was sensitive to inhibitors of the thromboxane A2-producing system (not shown). Platelet activation by rhodocytin occurred independently of GPVI/FcRγ (Fig. 2a) and was accompanied by marked degranulation (as shown by P-selectin expression) and strong fibrinogen binding (Fig. 2b). Strikingly, rhodocytin also induced aggregation of β1-null platelets. The extent of degranulation and fibrinogen binding was indistinguishable between β1-null and control platelets (Fig. 2b). Furthermore, no differences were found in the dose-response characteristics between normal and β1-null platelets (not shown). These results demonstrate that β1 integrins, including α2β1, are not essential for rhodocytin-induced platelet activation. It has been shown that snake venom toxins may induce platelet activation by interacting with multiple receptors (20Dormann D. Clemetson J.M. Navdaev A. Kehrel B.E. Clemetson K.J. Blood. 2001; 97: 929-936Crossref PubMed Scopus (65) Google Scholar). Thus, blocking only one of them would not inhibit platelet activation/aggregation. Therefore, we examined the effects of rhodocytin on platelets lacking both α2β1 and GPVI. To generate such platelets, GPVI was depleted in mice with β1-null platelets by injection of JAQ1 (100 μg/mouse). This treatment induces a virtually complete internalization and proteolytic degradation of GPVI in circulating platelets but does not affect other receptors, including αIIbβ3, GPIb-V-IX, and CD9 (16Nieswandt B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-470Crossref PubMed Scopus (308) Google Scholar). GPVI-depleted platelets do not respond to collagen, whereas activation by other agonists is not affected (16Nieswandt B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-470Crossref PubMed Scopus (308) Google Scholar). As shown in Fig.3a, platelets from JAQ1-treated β1-null mice lacked both collagen receptors. However, these β1/GPVI-deficient platelets responded normally to rhodocytin as demonstrated by aggregometry and flow cytometric analysis (Fig. 3, b and c). This finding excludes an essential role of α2β1and GPVI in rhodocytin-induced platelet activation. A very recent report showed that rhodocytin (aggretin)-induced 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 known ligands, including vWf, thrombin (21Lopez J.A. Dong J.F. Curr. Opin. Hematol. 1997; 4: 323-329Crossref PubMed Scopus (94) Google Scholar), P-selectin (22Romo G.M. Dong J.F. Schade A.J. Gardiner E.E. Kansas G.S. Li C.Q. McIntire L.V. Berndt M.C. Lopez J.A. J. Exp. Med. 1999; 190: 803-814Crossref PubMed Scopus (300) Google Scholar), and MAC-1 (23Simon D.I. Chen Z. Xu H. Li C.Q. Dong J. McIntire L.V. Ballantyne C.M. Zhang L. Furman M.I. Berndt M.C. Lopez J.A. J. Exp. Med. 2000; 192: 193-204Crossref PubMed Scopus (533) Google Scholar), as well as snake venom-derived C-type lectins like jararaca GPIb-BP (24Kawasaki T. Fujimura Y. Usami Y. Suzuki M. Miura S. Sakurai Y. Makita K. Taniuchi Y. Hirano K. Titani K. J. Biol. Chem. 1996; 271: 10635-10639Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), alboaggregin A (20Dormann D. Clemetson J.M. Navdaev A. Kehrel B.E. Clemetson K.J. Blood. 2001; 97: 929-936Crossref PubMed Scopus (65) Google Scholar), and echicetin (25Peng M. Lu W. Beviglia L. Niewiarowski S. Kirby E.P. Blood. 1993; 81: 2321-2328Crossref PubMed Google Scholar). Based on their results, Navdaev et al. (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 plays an essential role in rhodocytin-induced platelet activation. To directly test this hypothesis, we treated platelets withO-sialoglycoprotein endopeptidase (26Liu L. Freedman J. Hornstein A. Fenton J.W. Song Y. Ofosu F.A. J. Biol. Chem. 1997; 272: 1997-2004Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). This treatment resulted in complete proteolytic removal of the 45-kDa N-terminal domain of GPIbα as demonstrated by flow cytometric analysis (Fig.4a). Interestingly, Western blot analysis revealed that, in addition to cleavage of the 45-kDa N-terminal domain, the truncated remainder of GPIbα (105 kDa) was further cleaved in close vicinity to the transmembrane region of GPIbα, resulting in the release of glycocalicin lacking the 45-kDa N-terminal region (∼85 kDa) (Fig. 4b). Because of the complete lack of the 45-kDa N-terminal domain of GPIbα, botrocetin-induced vWf binding was abolished inO-sialoglycoprotein endopeptidase-treated platelets (Fig.4c). However, these platelets responded normally to rhodocytin (Fig. 4, d and e), demonstrating that the ligand-binding domain on GPIbα is not essential for this activation process. Although our results demonstrated that neither the collagen receptors α2β1 integrin and GPVI nor GPIbα are essential for rhodocytin-induced platelet activation, it could not be excluded that rhodocytin binds to all three receptors that independently elicit aggregation. Therefore, we removed the 45-kDa N-terminal region of GPIbα from β1- and β1/GPVI-deficient platelets and examined their response to rhodocytin. As shown in Fig. 5, even the absences of α2β1, GPVI, and the 45-kDa N-terminal domain of GPIbα had no significant effect on rhodocytin-induced platelet activation and aggregation. The mechanism underlying platelet activation by rhodocytin (aggretin) has been debated. Several investigators suggested that rhodocytin activates platelets in a collagen-like manner 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, 20Dormann D. Clemetson J.M. Navdaev A. Kehrel B.E. Clemetson K.J. Blood. 2001; 97: 929-936Crossref PubMed Scopus (65) 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 present study we demonstrate that rhodocytin induces activation of murine platelets in the absence of α2β1integrin, GPVI, and the ligand-binding domain of GPIbα. These are the three major receptors that directly or indirectly interact with collagen. Our findings exclude an essential role for these receptors in rhodocytin-induced activation and demonstrate that the activation process follows mechanisms that are fundamentally different from those induced by collagen. Our present findings are in contrast to those reported by others (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), who showed that mAbs against α2β1 blocked rhodocytin-induced platelet aggregation and concluded that the integrin plays an essential role in the activation process. An explanation for this discrepancy could be that treatment of platelets with mAbs against α2β1 may have different effects than the absence of the receptor. Antibodies may exert steric effects on other cell surface proteins or may elicit inhibitory signals. Previously, results from inhibition studies with some antibodies against α2β1 could not be confirmed in a genetic model in which the β1 gene is deleted in all hematopoietic cells, including megakaryocytes. It was shown that antibodies against α2β1 integrin markedly reduced or abolished platelet adhesion and aggregate formation on collagen in stasis and flow (27Saelman E.U. Nieuwenhuis H.K. Hese K.M. de Groot P.G. Heijnen H.F. Sage E.H. Williams S. McKeown L. Gralnick H.R. Sixma J.J. Blood. 1994; 83: 1244-1250Crossref 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). Using the Cre/loxP technology, we ablated the β1 gene in megakaryocytes and showed that α2β1 integrin is not required for platelet adhesion and thrombus formation on fibrillar collagen under static as well as low and high shear flow conditions. β1-null platelets display a delayed but not reduced aggregation in response to collagen, demonstrating 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 investigations with convulxin provide another example in which antibody inhibition and gene deletion studies showed conflicting results. Whereas the action of convulxin can be inhibited with some antibodies against α2β1 (28Jandrot-Perrus M. Lagrue A.H. Okuma M. Bon C. J. Biol. Chem. 1997; 272: 27035-27041Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), Cre/loxP-mediated ablation of β1 integrin on platelets revealed no detectable 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). Together, these findings strongly suggest that certain antibodies against α2β1 integrin induce inhibitory effects that are not based on blockage of the integrin. Therefore, treatment of platelets with anti-α2β1 antibodies may not be suitable for determining dependence on α2β1 integrin. Another striking difference between rhodocytin- and collagen-mediated platelet aggregation is the central role of GPVI for collagen but not for rhodocytin. We showed recently that GPVI is the major collagen receptor for platelet activation and that GPVI is essential for collagen-induced platelet aggregation (16Nieswandt B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-470Crossref PubMed Scopus (308) Google Scholar). Therefore, GPVI-independent aggregation processes are different from collagen-induced aggregation. Both FcRγ-null 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 GPVI-depleted platelets (16Nieswandt B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-470Crossref PubMed Scopus (308) Google Scholar) fail to activate β1 and β3integrins in response to collagen. Consequently, these platelets neither adhere to collagen nor do they bind adhesive ligands or aggregate 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, 16Nieswandt B. Schulte V. Bergmeier W. Mokhtari-Nejad R. Rackebrandt K. Cazenave J.P. Ohlmann P. Gachet C. Zirngibl H. J. Exp. Med. 2001; 193: 459-470Crossref PubMed Scopus (308) Google Scholar). From these data, we conclude that rhodocytin activates platelets by mechanisms that are different from those induced by collagen. Navdaev and co-workers (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) proposed a mechanism for rhodocytin (aggretin)-induced platelet activation that involves two platelet receptors, α2β1 integrin and GPIbα. The importance of GPIbα for rhodocytin-induced aggregation was concluded from a dose-dependent inhibitory effect of a mAb directed against the thrombin-binding site on GPIbα, which is located in the 45-kDa N-terminal region of the receptor (29Jandrot-Perrus M. Bouton M.C. Lanza F. Guillin M.C. Semin. Thromb. Hemost. 1996; 22: 151-156Crossref PubMed Scopus (23) Google Scholar). This finding was in contrast to observations made by other investigators who found no role of GPIb in rhodocytin-induced activation (30Shin Y. Morita T. Biochem. Biophys. Res. Commun. 1998; 245: 741-745Crossref PubMed Scopus (79) Google Scholar, 31Huang T.F. Liu C.Z. Yang S.H. Biochem. J. 1995; 309: 1021-1027Crossref PubMed Scopus (94) Google Scholar) or no binding of rhodocytin to GPIb (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 discrepancies in the binding studies are difficult to explain. They may be related to different experimental conditions used in these studies. In our present study we show that platelets lacking the 45-kDa N-terminal domain on GPIbα respond normally to rhodocytin (Fig. 5). This finding excludes an essential role for the ligand-binding region of GPIbα in the rhodocytin-induced activation process and is in clear contrast to the finding by Navdaev and co-workers (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). Steric hindrance or elicitation of inhibitory signals by the GPIbα-specific antibody could be an explanation for the conflicting results. It is known that occupancy of GPIbα induces tyrosine phosphorylation of different signaling molecules in vitro (32Kroll M.H. Harris T.S. Moake J.L. Handin R.I. Schafer A.I. J. Clin. Invest. 1991; 88: 1568-1573Crossref PubMed Scopus (242) Google Scholar, 33Du X. Harris S.J. Tetaz T.J. Ginsberg M.H. Berndt M.C. J. Biol. Chem. 1994; 269: 18287-18290Abstract Full Text PDF PubMed Google Scholar, 34Andrews R.K. Shen Y. Gardiner E.E. Dong J.F. Lopez J.A. Berndt M.C. Thromb. Haemostasis. 1999; 82: 357-364Crossref PubMed Scopus (116) Google Scholar) and that dimerization of GPIbα by certain mAbs affects platelet function by yet undefined mechanisms in vitro and in vivo (18Bergmeier W. Rackebrandt K. Schroder W. Zirngibl H. Nieswandt B. Blood. 2000; 95: 886-893Crossref PubMed Google Scholar, 35Cadroy Y. Hanson S.R. Kelly A.B. Marzec U.M. Evatt B.L. Kunicki T.J. Montgomery R.R. Harker L.A. Blood. 1994; 83: 3218-3224Crossref PubMed Google Scholar, 36Cauwenberghs N. Meiring M. Vauterin S. van Wyk V Lamprecht S. Roodt J.P. Novak L. Harsfalvi J. Deckmyn H. Kotze H.F. Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1347-1353Crossref PubMed Scopus (126) Google Scholar). Our results do not exclude the possibility that rhodocytin interacts with an epitope on the GPIb-V-IX complex that is distinct from the 45-kDa N-terminal region of GPIbα. However, in studies using mAbs against different epitopes on either GPIX, GPV, or GPIbα/β, we were unable to alter rhodocytin-induced activation/aggregation. In addition, GPV-deficient mouse platelets respond normally to rhodocytin (not shown). These results suggest that the GPIb-V-IX complex has no essential role in rhodocytin-induced platelet activation. Using genetic ablation of α2β1, antibody-mediated depletion of GPVI, and proteolytic digestion of GPIb, we show that platelets lacking all three major receptors that directly or indirectly interact with collagen (α2β1, GPVI, and GPIb) respond normally to rhodocytin. Because rhodocytin-induced aggregation of human and mouse platelets occurs with a dose-dependent lag time and independently of the FcRγ chain and both processes are sensitive to acetylsalicylic acid, it is unlikely that species-specific differences explain the contrasting results presented here 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. 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