Signal-transducing Mechanisms Involved in Activation of the Platelet Collagen Receptor Integrin α2β1

Evidence was obtained about the mechanism responsible for platelet integrin α2β activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin α2β1 activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin α2β1, but those operating via glycoprotein Ib cannot. Activation of α2β1 induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo3,4-dpyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin α2β1 activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin α2β1 activation, while at the high agonist concentrations, there would be several pathways through which integrin α2β1 activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B max) as thrombin-induced platelets, but their affinity (K d) for soluble collagen was 3.7–12.7-fold lower, suggesting that activated integrin α2β1 induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin α2β1; these activated forms of integrin α2β1 would have different conformations that determine their ligand affinity. Evidence was obtained about the mechanism responsible for platelet integrin α2β activation by determining effects of various inhibitors on soluble collagen binding, a parameter to assess integrin α2β1 activation, in stimulated platelets. Agonists that can also activate platelet glycoprotein IIb/IIIa are able to activate integrin α2β1, but those operating via glycoprotein Ib cannot. Activation of α2β1 induced by low thrombin or collagen-related peptide concentrations was almost completely inhibited by apyrase, and the inhibitors wortmannin, 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo3,4-dpyrimidine, bisindolylmaleimide I, and SQ29548 significantly inhibited it. Activation induced by high thrombin or collagen-related peptide concentrations was far less sensitive to these inhibitors. However, only wortmannin markedly inhibited ADP-induced integrin α2β1 activation, and this was not ADP concentration-dependent. These results suggest that at the low agonist concentrations, the released ADP would be a primary inducer of integrin α2β1 activation, while at the high agonist concentrations, there would be several pathways through which integrin α2β1 activation can be induced. Kinetic analyses revealed that ADP-induced platelets had about the same number of binding sites (B max) as thrombin-induced platelets, but their affinity (K d) for soluble collagen was 3.7–12.7-fold lower, suggesting that activated integrin α2β1 induced by ADP is different from that induced by thrombin. The data are consistent with an activation mechanism involving released ADP and in which there exists two different states of activated integrin α2β1; these activated forms of integrin α2β1 would have different conformations that determine their ligand affinity. glycoprotein bisindolylmaleimide I collagen-related peptide creatine phosphate/creatine phosphokinase protein kinase C phorbol-12-myristate-13-acetate 4-amino-5-(chlorophenyl)-7-(t-butyl)pyrazolo3,4-dpyrimidine von Willebrand factor Integrins comprise a family of heterodimeric cell surface proteins that mediate intracellular and cell-to-extracellular interactions. In humans, at least 15 different α-subunits and eight different β-subunits have been identified to date. The various permutations of the α- and β-subunit complexes yield integrin dimers with diverse ligand specificities and biological activities. There is tissue-specific expression of each type of integrin; some integrins are only expressed in a certain tissue, while others are more universal.Integrin αIIbβ3 (platelet glycoprotein (GP)1 IIb/IIIa) is only expressed in platelets and megakaryocytes, but integrin α2β1 (platelet GP Ia/IIa) is known to be present in many cell types (1.Zutter M.M. Santoro S.A. Am. J. Pathol. 1990; 137: 113-120PubMed Google Scholar). The GP IIb/IIIa complex is present as a nonactive heterodimer in resting platelets and becomes activated when platelets are induced by agonists (2.Marguerie G.A. Plow E.F. Edgington T.S. J. Biol. Chem. 1979; 254: 5357-5363Abstract Full Text PDF PubMed Google Scholar, 3.Mustard J.F. Packham M.A. Kinlough-Rathbone R.L. Perry D.W. Regoeczi E. Blood. 1978; 52: 453-466Crossref PubMed Google Scholar, 4.Marguerie G.A. Ginsberg M.H. Plow E.F. MacIntyre D.E. Gordon J.L. Platelets in Biology and Pathology III. Elsevier, Amsterdam1987: 95-125Google Scholar); activated GP IIb/IIIa possesses high affinity for its ligand, fibrinogen. GP IIb/IIIa is one of the most abundant proteins in the platelet membrane, and its binding reaction with fibrinogen was shown to be one of the most important reactions in platelet aggregation. On the other hand, although integrin α2β1 was indicated to be a receptor for collagen from studies on a patient's platelets lacking this protein (5.Nieuwenhuis H.K. Akkerman J.W.N. Houdijk W.P.M. Sixma J.J. Nature. 1985; 318: 470-472Crossref PubMed Scopus (389) Google Scholar), neither soluble ligand binding to integrin α2β1 nor the activation of the integrin had not been clearly demonstrated until recently. In our previous paper, we showed that upon agonist stimulation of platelets, integrin α2β1 is activated to a form with high affinity for soluble collagen (6.Jung S.M. Moroi M. J. Biol. Chem. 1998; 273: 14827-14837Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). These results suggested that integrin α2β1 might be converted to its activated form through a mechanism similar to that responsible for the activation of GP IIb/IIIa.The activation mechanism of GP IIb/IIIa has been examined by many investigators, but is yet not fully explained. Recombinant proteins having various mutational changes in the cytoplasmic domains of GP IIb, GP IIIa, or both, with different conformational states of the extracellular portion of the integrin (7.O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (578) Google Scholar, 8.O'Toole T.E. Ylanne J. Culley B.M. J. Biol. Chem. 1995; 270: 8553-8558Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 9.Wang R. Shattil S.J. Ambruso D.R. Newman P.J. J. Clin. Invest. 1997; 100: 2393-2403Crossref PubMed Scopus (102) Google Scholar), showed different abilities to bind fibrinogen in response to activation. These results suggested that transformation of the extracellular domain to a conformation with high affinity for fibrinogen would be regulated by interactions involving the cytoplasmic domain(s) of GP IIb/IIIa (10.Shattil S.J. Ginsberg M.H. Brugge J.S. Curr. Opin. Cell Biol. 1994; 6: 695-704Crossref PubMed Scopus (188) Google Scholar,11.Shattil S.J. Gao J. Kashiwagi H. Thromb. Haemostasis. 1997; 78: 220-225Crossref PubMed Scopus (39) Google Scholar). Several proteins were indicated to interact with the cytoplasmic tails of GP IIb/IIIa, including integrin-associated protein (12.Brown E. Hooper L. Ho T. Gresham H. J. Cell Biol. 1990; 111: 2785-2794Crossref PubMed Scopus (308) Google Scholar), β3-endonexin (13.Shattil S.J. O'Toole T. Eigenthaler M. Thon V. Williams M. Babior B.M. Ginsberg M.H. J. Cell Biol. 1995; 131: 807-816Crossref PubMed Scopus (164) Google Scholar), CD98 (14.Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar), and calcium- and integrin-binding protein (15.Naik U.P. Patel P.M. Parise L.V. J. Biol. Chem. 1997; 272: 4651-4654Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). However, none of these proteins was indicated to function as a regulator of GP IIb/IIIa activity in platelets. As to the β1-integrins, the cytoplasmic domain of the α2-chain has been indicated to act as a negative regulator (16.Kawaguchi S. Bergelson J.M. Finberg R.W. Hemler M.E. Mol. Biol. Cell. 1994; 5: 977-988Crossref PubMed Scopus (33) Google Scholar), and the NPXY motif of the β-cytoplasmic domain was indicated to be critical for inside-out signaling (8.O'Toole T.E. Ylanne J. Culley B.M. J. Biol. Chem. 1995; 270: 8553-8558Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Furthermore, several proteins were reported to interact with the cytoplasmic domains of integrin α2β1 and suggested to regulate its function; these are calreticulin (17.Coppolino M. Leung-Hagesteijn C. Dedhar S. Wilkins J. J. Biol. Chem. 1995; 270: 23132-23138Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), integrin-linked kinase 1 (18.Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (960) Google Scholar), and ICAP-1 (19.Chang D.D. Wong C. Smith H. Liu J. J. Cell Biol. 1997; 138: 1149-1157Crossref PubMed Scopus (149) Google Scholar) in addition to cytoskeletal proteins. The contributions of these factors to platelet function remain to be analyzed.Phosphorylation of the cytoplasmic domain of GP IIb/IIIa was also suggested to control the affinity of the integrin (20.van Willigen G. Hers I. Gorter G. Akkerman J.-W.N. Biochem. J. 1996; 314: 769-779Crossref PubMed Scopus (65) Google Scholar), but other studies suggested that the phosphorylation of GP IIb/IIIa is related to the interaction with the cytoskeleton; i.e. outside-in signaling (21.Lerea K.M. Cordero K.P. Sakariassen K.S. Kirk R.I. Fried V.A. J. Biol. Chem. 1999; 274: 1914-1919Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22.Jenkins A.L. Nannizzi-Alaimo L. Silver D. Sellers J.R. Ginsberg M.H. Law D.A. Phillips D.R. J. Biol. Chem. 1998; 273: 13878-13885Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The activated GP IIb/IIIa binds with fibrinogen, and this interaction also stimulates platelets (outside-in signaling), which severely complicates the analyses of the activation mechanism of GP IIb/IIIa. However, this is not the case for integrin α2β1, where collagen is not secreted from platelets after they are activated; thus, this allows us to neglect the effect of outside-in signaling, making it particularly amenable to the analysis of the integrin activation mechanism.Our previous study demonstrated that platelet integrin α2β1 is activated to a form with high affinity for soluble collagen after platelets are stimulated by various agonists (6.Jung S.M. Moroi M. J. Biol. Chem. 1998; 273: 14827-14837Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Although many cells were observed to increase their adhesive activity to the integrin ligands after cell activation with stimuli (23.Shimizu Y. van Seventer G.A. Hogan K.J. Shaw S. Nature. 1990; 345: 250-253Crossref PubMed Scopus (536) Google Scholar, 24.Chan B.M. Wong J.G. Rao A. Hemler M.E. J. Immunol. 1991; 147: 398-404PubMed Google Scholar), the activation of integrins, especially of β1-integrins, was ascribed to avidity changes, since there had not been any clear evidence for the affinity change of β1-integrins using soluble ligand binding (25.Bazzoni G. Hemler M.E. Trends Biochem. Sci. 1998; 23: 30-34Abstract Full Text PDF PubMed Scopus (229) Google Scholar). Our demonstration of the activation of integrin α2β1 associated with affinity change suggested the existence of an activation mechanism that would induce a conformational change in the integrin. A similar activation mechanism was indicated for integrin GP IIb/IIIa (integrin αIIbβ3) of platelets, and many investigations have been performed to describe this activation mechanism, designated as inside-out signaling (4.Marguerie G.A. Ginsberg M.H. Plow E.F. MacIntyre D.E. Gordon J.L. Platelets in Biology and Pathology III. Elsevier, Amsterdam1987: 95-125Google Scholar, 26.Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar).In this paper, we analyzed the effects of various inhibitors and agonists on the activation of integrin α2β1. The results indicated the following. 1) All of the agonists that induce GP IIb/IIIa-dependent platelet aggregation induced integrin α2β1activation. 2) An ADP scavenger, apyrase, almost completely inhibited integrin α2β1 when platelets were stimulated with a low concentration of an agonist (thrombin or collagen-related peptide (CRP)); and other inhibitors, wortmannin, PP2, bisindolylmaleimide I (BIMI), and SQ29548, inhibited the activation significantly under this condition. 3) When platelets were stimulated with higher concentrations of agonists, these inhibitors had no significant effect, except for the case of wortmannin, which had an inhibitory effect on ADP-induced activation. 4) Integrin α2β1 activated with ADP and integrin α2β1 activated by a high concentration of thrombin showed different K d values but had the same number of binding sites per platelet. These results suggest that released ADP participates in the activation of integrin α2β1 and suggest the presence of two different states of activated integrin α2β1that have different conformations. Integrins comprise a family of heterodimeric cell surface proteins that mediate intracellular and cell-to-extracellular interactions. In humans, at least 15 different α-subunits and eight different β-subunits have been identified to date. The various permutations of the α- and β-subunit complexes yield integrin dimers with diverse ligand specificities and biological activities. There is tissue-specific expression of each type of integrin; some integrins are only expressed in a certain tissue, while others are more universal. Integrin αIIbβ3 (platelet glycoprotein (GP)1 IIb/IIIa) is only expressed in platelets and megakaryocytes, but integrin α2β1 (platelet GP Ia/IIa) is known to be present in many cell types (1.Zutter M.M. Santoro S.A. Am. J. Pathol. 1990; 137: 113-120PubMed Google Scholar). The GP IIb/IIIa complex is present as a nonactive heterodimer in resting platelets and becomes activated when platelets are induced by agonists (2.Marguerie G.A. Plow E.F. Edgington T.S. J. Biol. Chem. 1979; 254: 5357-5363Abstract Full Text PDF PubMed Google Scholar, 3.Mustard J.F. Packham M.A. Kinlough-Rathbone R.L. Perry D.W. Regoeczi E. Blood. 1978; 52: 453-466Crossref PubMed Google Scholar, 4.Marguerie G.A. Ginsberg M.H. Plow E.F. MacIntyre D.E. Gordon J.L. Platelets in Biology and Pathology III. Elsevier, Amsterdam1987: 95-125Google Scholar); activated GP IIb/IIIa possesses high affinity for its ligand, fibrinogen. GP IIb/IIIa is one of the most abundant proteins in the platelet membrane, and its binding reaction with fibrinogen was shown to be one of the most important reactions in platelet aggregation. On the other hand, although integrin α2β1 was indicated to be a receptor for collagen from studies on a patient's platelets lacking this protein (5.Nieuwenhuis H.K. Akkerman J.W.N. Houdijk W.P.M. Sixma J.J. Nature. 1985; 318: 470-472Crossref PubMed Scopus (389) Google Scholar), neither soluble ligand binding to integrin α2β1 nor the activation of the integrin had not been clearly demonstrated until recently. In our previous paper, we showed that upon agonist stimulation of platelets, integrin α2β1 is activated to a form with high affinity for soluble collagen (6.Jung S.M. Moroi M. J. Biol. Chem. 1998; 273: 14827-14837Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). These results suggested that integrin α2β1 might be converted to its activated form through a mechanism similar to that responsible for the activation of GP IIb/IIIa. The activation mechanism of GP IIb/IIIa has been examined by many investigators, but is yet not fully explained. Recombinant proteins having various mutational changes in the cytoplasmic domains of GP IIb, GP IIIa, or both, with different conformational states of the extracellular portion of the integrin (7.O'Toole T.E. Katagiri Y. Faull R.J. Peter K. Tamura R. Quaranta V. Loftus J.C. Shattil S.J. Ginsberg M.H. J. Cell Biol. 1994; 124: 1047-1059Crossref PubMed Scopus (578) Google Scholar, 8.O'Toole T.E. Ylanne J. Culley B.M. J. Biol. Chem. 1995; 270: 8553-8558Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 9.Wang R. Shattil S.J. Ambruso D.R. Newman P.J. J. Clin. Invest. 1997; 100: 2393-2403Crossref PubMed Scopus (102) Google Scholar), showed different abilities to bind fibrinogen in response to activation. These results suggested that transformation of the extracellular domain to a conformation with high affinity for fibrinogen would be regulated by interactions involving the cytoplasmic domain(s) of GP IIb/IIIa (10.Shattil S.J. Ginsberg M.H. Brugge J.S. Curr. Opin. Cell Biol. 1994; 6: 695-704Crossref PubMed Scopus (188) Google Scholar,11.Shattil S.J. Gao J. Kashiwagi H. Thromb. Haemostasis. 1997; 78: 220-225Crossref PubMed Scopus (39) Google Scholar). Several proteins were indicated to interact with the cytoplasmic tails of GP IIb/IIIa, including integrin-associated protein (12.Brown E. Hooper L. Ho T. Gresham H. J. Cell Biol. 1990; 111: 2785-2794Crossref PubMed Scopus (308) Google Scholar), β3-endonexin (13.Shattil S.J. O'Toole T. Eigenthaler M. Thon V. Williams M. Babior B.M. Ginsberg M.H. J. Cell Biol. 1995; 131: 807-816Crossref PubMed Scopus (164) Google Scholar), CD98 (14.Fenczik C.A. Sethi T. Ramos J.W. Hughes P.E. Ginsberg M.H. Nature. 1997; 390: 81-85Crossref PubMed Scopus (254) Google Scholar), and calcium- and integrin-binding protein (15.Naik U.P. Patel P.M. Parise L.V. J. Biol. Chem. 1997; 272: 4651-4654Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar). However, none of these proteins was indicated to function as a regulator of GP IIb/IIIa activity in platelets. As to the β1-integrins, the cytoplasmic domain of the α2-chain has been indicated to act as a negative regulator (16.Kawaguchi S. Bergelson J.M. Finberg R.W. Hemler M.E. Mol. Biol. Cell. 1994; 5: 977-988Crossref PubMed Scopus (33) Google Scholar), and the NPXY motif of the β-cytoplasmic domain was indicated to be critical for inside-out signaling (8.O'Toole T.E. Ylanne J. Culley B.M. J. Biol. Chem. 1995; 270: 8553-8558Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Furthermore, several proteins were reported to interact with the cytoplasmic domains of integrin α2β1 and suggested to regulate its function; these are calreticulin (17.Coppolino M. Leung-Hagesteijn C. Dedhar S. Wilkins J. J. Biol. Chem. 1995; 270: 23132-23138Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar), integrin-linked kinase 1 (18.Hannigan G.E. Leung-Hagesteijn C. Fitz-Gibbon L. Coppolino M.G. Radeva G. Filmus J. Bell J.C. Dedhar S. Nature. 1996; 379: 91-96Crossref PubMed Scopus (960) Google Scholar), and ICAP-1 (19.Chang D.D. Wong C. Smith H. Liu J. J. Cell Biol. 1997; 138: 1149-1157Crossref PubMed Scopus (149) Google Scholar) in addition to cytoskeletal proteins. The contributions of these factors to platelet function remain to be analyzed. Phosphorylation of the cytoplasmic domain of GP IIb/IIIa was also suggested to control the affinity of the integrin (20.van Willigen G. Hers I. Gorter G. Akkerman J.-W.N. Biochem. J. 1996; 314: 769-779Crossref PubMed Scopus (65) Google Scholar), but other studies suggested that the phosphorylation of GP IIb/IIIa is related to the interaction with the cytoskeleton; i.e. outside-in signaling (21.Lerea K.M. Cordero K.P. Sakariassen K.S. Kirk R.I. Fried V.A. J. Biol. Chem. 1999; 274: 1914-1919Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 22.Jenkins A.L. Nannizzi-Alaimo L. Silver D. Sellers J.R. Ginsberg M.H. Law D.A. Phillips D.R. J. Biol. Chem. 1998; 273: 13878-13885Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The activated GP IIb/IIIa binds with fibrinogen, and this interaction also stimulates platelets (outside-in signaling), which severely complicates the analyses of the activation mechanism of GP IIb/IIIa. However, this is not the case for integrin α2β1, where collagen is not secreted from platelets after they are activated; thus, this allows us to neglect the effect of outside-in signaling, making it particularly amenable to the analysis of the integrin activation mechanism. Our previous study demonstrated that platelet integrin α2β1 is activated to a form with high affinity for soluble collagen after platelets are stimulated by various agonists (6.Jung S.M. Moroi M. J. Biol. Chem. 1998; 273: 14827-14837Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Although many cells were observed to increase their adhesive activity to the integrin ligands after cell activation with stimuli (23.Shimizu Y. van Seventer G.A. Hogan K.J. Shaw S. Nature. 1990; 345: 250-253Crossref PubMed Scopus (536) Google Scholar, 24.Chan B.M. Wong J.G. Rao A. Hemler M.E. J. Immunol. 1991; 147: 398-404PubMed Google Scholar), the activation of integrins, especially of β1-integrins, was ascribed to avidity changes, since there had not been any clear evidence for the affinity change of β1-integrins using soluble ligand binding (25.Bazzoni G. Hemler M.E. Trends Biochem. Sci. 1998; 23: 30-34Abstract Full Text PDF PubMed Scopus (229) Google Scholar). Our demonstration of the activation of integrin α2β1 associated with affinity change suggested the existence of an activation mechanism that would induce a conformational change in the integrin. A similar activation mechanism was indicated for integrin GP IIb/IIIa (integrin αIIbβ3) of platelets, and many investigations have been performed to describe this activation mechanism, designated as inside-out signaling (4.Marguerie G.A. Ginsberg M.H. Plow E.F. MacIntyre D.E. Gordon J.L. Platelets in Biology and Pathology III. Elsevier, Amsterdam1987: 95-125Google Scholar, 26.Shattil S.J. Kashiwagi H. Pampori N. Blood. 1998; 91: 2645-2657Crossref PubMed Google Scholar). In this paper, we analyzed the effects of various inhibitors and agonists on the activation of integrin α2β1. The results indicated the following. 1) All of the agonists that induce GP IIb/IIIa-dependent platelet aggregation induced integrin α2β1activation. 2) An ADP scavenger, apyrase, almost completely inhibited integrin α2β1 when platelets were stimulated with a low concentration of an agonist (thrombin or collagen-related peptide (CRP)); and other inhibitors, wortmannin, PP2, bisindolylmaleimide I (BIMI), and SQ29548, inhibited the activation significantly under this condition. 3) When platelets were stimulated with higher concentrations of agonists, these inhibitors had no significant effect, except for the case of wortmannin, which had an inhibitory effect on ADP-induced activation. 4) Integrin α2β1 activated with ADP and integrin α2β1 activated by a high concentration of thrombin showed different K d values but had the same number of binding sites per platelet. These results suggest that released ADP participates in the activation of integrin α2β1 and suggest the presence of two different states of activated integrin α2β1that have different conformations. We thank Dr. T. Morita, Meiji College of Pharmacy (Tokyo, Japan) for the kind gifts of alboaggregin B and botrocetin.