Calpain Cleavage of the Cytoplasmic Domain of the Integrin β2 Subunit

The cytoplasmic domains of integrin β subunits are involved in bidirectional transmembrane signaling. We report that the cytoplasmic domain of the integrin β3 subunit undergoes limited proteolysis by calpain, an intracellular calcium-dependent protease. Calpain cleavage occurs during platelet aggregation induced by agonists such as thrombin. Five cleavage sites have been identified. Four of these sites (C-terminal to Thr741, Tyr747, Phe754, and Tyr759) are utilized in intact platelets and flank two NXXY motifs (Asn744-Pro-Leu-Tyr747 and Asn756-Ile-Thr-Tyr759). The fifth site (Ala735) is accessible to calpain after EDTA treatment of the αIIbβ3 heterodimer. The NXXY motif is critical to the bidirectional signaling functions of β3 integrins and their association with the cytoskeleton. Thus, calpain cleavage of the β3 cytoplasmic domain may provide a means to regulate integrin signaling functions. The cytoplasmic domains of integrin β subunits are involved in bidirectional transmembrane signaling. We report that the cytoplasmic domain of the integrin β3 subunit undergoes limited proteolysis by calpain, an intracellular calcium-dependent protease. Calpain cleavage occurs during platelet aggregation induced by agonists such as thrombin. Five cleavage sites have been identified. Four of these sites (C-terminal to Thr741, Tyr747, Phe754, and Tyr759) are utilized in intact platelets and flank two NXXY motifs (Asn744-Pro-Leu-Tyr747 and Asn756-Ile-Thr-Tyr759). The fifth site (Ala735) is accessible to calpain after EDTA treatment of the αIIbβ3 heterodimer. The NXXY motif is critical to the bidirectional signaling functions of β3 integrins and their association with the cytoskeleton. Thus, calpain cleavage of the β3 cytoplasmic domain may provide a means to regulate integrin signaling functions. INTRODUCTIONIntegrins, a family of adhesion receptors, play important roles in cellular functions such as adhesion, migration, cell proliferation, and differentiation(1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). The functions of integrins are modulated by bidirectional transmembrane signaling as exemplified by platelet integrin αIIbβ3 (glycoprotein IIb-IIIa)(2Ginsberg M.H. Du X. O'Toole T.E. Loftus J.C. Plow E.F. Thromb. Haemostasis. 1993; 70: 87-93Crossref PubMed Scopus (106) Google Scholar). When platelets are stimulated by agonists such as thrombin, intracellular signal transduction leads to activation of the extracellular fibrinogen binding function of αIIbβ3 (inside-out signaling), resulting in platelet aggregation. Conversely, ligand binding to αIIbβ3 is involved in outside-in signals that result in its association with the cytoskeleton (3Phillips D.R. Jennings L.K. Edwards H.H. J. Cell Biol. 1980; 86: 77-86Crossref PubMed Scopus (248) Google Scholar) and biochemical changes including tyrosine phosphorylation of intracellular proteins(4Ferrell J.J. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2234-2238Crossref PubMed Scopus (204) Google Scholar, 5Golden A. Brugge J.S. Shattil S.J. J. Cell Biol. 1990; : 3117-3127Crossref PubMed Scopus (184) Google Scholar), increase in intracellular calcium level(6Pelletier A.J. Bodary S.C. Levinson A.D. Mol. Biol. Cell. 1992; 3: 989-998Crossref PubMed Scopus (90) Google Scholar), and activation of the calcium-dependent neutral protease (calpain)(7Fox J.E. Taylor R.G. Taffarel M. Boyles J.K. Goll D.E. J. Cell Biol. 1993; 120: 1501-1507Crossref PubMed Scopus (133) Google Scholar). Specific structural characteristics of the integrin cytoplasmic domains are critical for the two-way signaling mechanism. This includes a GFFKR sequence in the αIIb cytoplasmic domain (8O'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) and two sets of NXXY (NPLY and NITY) within the β3 cytoplasmic domain(9O'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, 10Ylanne J. Huuskonen J. O'Toole T.E. Ginsberg M.H. Virtanen I. Gahmberg C.G. J. Biol. Chem. 1995; 270: 9550-9557Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The NPLY sequence, in particular, is critical for controlling affinity states of the ligand-binding sites and for the interaction of the integrin with cytoskeletal elements(9O'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, 10Ylanne J. Huuskonen J. O'Toole T.E. Ginsberg M.H. Virtanen I. Gahmberg C.G. J. Biol. Chem. 1995; 270: 9550-9557Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar).Numerous cytoplasmic proteins are colocalized with integrins in focal adhesion sites and thus may be involved in intracellular signaling to and from integrins(11Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar). Calpain is among these proteins(12Beckerle M.C. Burridge K. DeMartino G.N. Croall D.E. Cell. 1987; 51: 569-577Abstract Full Text PDF PubMed Scopus (210) Google Scholar). Calpain represents a family of intracellular calcium-dependent neutral proteases(13Saido T.C. Sorimachi H. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (616) Google Scholar). Of the two classic members of this family, μ-calpain and m-calpain, μ-calpain is probably the predominant form in platelets(14Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (779) Google Scholar). These two isoforms of calpain have no major difference in substrate specificity, but differ in calcium sensitivity (14Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (779) Google Scholar). Calpain can be activated during platelet aggregation by a rise in the cytoplasmic calcium level and/or by its translocation to the membrane(13Saido T.C. Sorimachi H. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (616) Google Scholar, 14Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (779) Google Scholar). Furthermore, calpain activation promotes the shedding of procoagulant membrane vesicles from aggregated platelets (15Fox J.E. Austin C.D. Boyles J.K. Steffen P.K. J. Cell Biol. 1990; 111: 483-493Crossref PubMed Scopus (131) Google Scholar). While the mechanisms of calpain regulation are still not fully understood, calpain cleavages can regulate a variety of intracellular processes(16Fox J.E. Goll D.E. Reynolds C.C. Phillips D.R. J. Biol. Chem. 1985; 260: 1060-1066Abstract Full Text PDF PubMed Google Scholar, 17Oda A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar, 18Frangioni J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (282) Google Scholar, 19Saido T.C. Mizuno K. Suzuki K. Biomed. Biochim. Acta. 1991; 50: 485-489PubMed Google Scholar). We now report that calpain may regulate the function of β3 integrins by limited cleavage of the cytoplasmic domain of β3 at specific sites flanking two sets of NXXY motifs.EXPERIMENTAL PROCEDURESPurified Proteins and PeptidesIntegrin αIIbβ3 was purified as described previously (20Du X. Plow E.F. Frelinger III, A.L. O'Toole T.E. Loftus J.C. Ginsberg M.H. Cell. 1991; 65: 409-416Abstract Full Text PDF PubMed Scopus (417) Google Scholar) by sequential chromatography of platelet lysates on heparin, concanavalin A, and gel filtration columns. μ-Calpain was purified from rabbit skeletal muscle and characterized as described previously(21Saido T.C. Shibata M. Takenawa T. Murofushi H. Suzuki K. J. Biol. Chem. 1992; 267: 24585-24590Abstract Full Text PDF PubMed Google Scholar). The specific activity of the enzyme was 680 units/mg. The synthetic peptide TIHDRKEFAKFEEERARAKWDTANNPLYKEATSTFTNITYRGT, corresponding to the cytoplasmic domain of β3, was kindly provided by Dr. E. F. Plow (Department of Cardiovascular Biology, Cleveland Clinic Foundation, Cleveland, OH). All other peptides were synthesized using an Applied Biosystems Model 430A automated peptide synthesizer and were subsequently purified by high performance liquid chromatography (HPLC). 1The abbreviations used are: HPLChigh performance liquid chromatographyPAGEpolyacrylamide gel electrophoresisPMSFphenylmethylsulfonyl fluorideAbantibody. The mass of all synthetic peptides was verified by ion-spray mass spectrometry.AntibodiesThe monoclonal antibodies PMI-1 (against the αIIb heavy and were and characterized as described III, A.L. S.C. Plow E.F. Smith Loftus J.C. Ginsberg M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, III, A.L. Du X. Plow E.F. Ginsberg M.H. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar). The rabbit antibodies (against the of the αIIb cytoplasmic (against the of the β3 cytoplasmic and (against the of the αIIb have been described J.C. Plow E.F. Jennings L.K. Ginsberg M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T.E. J. Shattil S.J. Plow E.F. Ginsberg M.H. Science. 1991; PubMed Scopus Google Scholar). The of and was verified using integrin αIIbβ3 with in the cytoplasmic domain of αIIb and β3, T.E. J. Shattil S.J. Plow E.F. Ginsberg M.H. Science. 1991; PubMed Scopus Google Scholar). antibodies specific calpain cleavage corresponding to by calpain of the synthetic β3 cytoplasmic domain peptide were synthesized with a at their The peptides were with by a The were used to as described T.C. M. S. I. E. T. Suzuki K. S. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar). the antibodies were using peptides on of integrin αIIbβ3 in was with calpain at for The were by and gel The were on a gel and to of in in the were with antibodies integrin αIIbβ3 at for the were with for and substrate was used to the Calpain peptides were in at of purified μ-calpain was to of peptide and at for The were by a of and the were by on a with a at a of using of in The were by at and of synthetic peptides and their was on an ion-spray of the were using of on the of the sequence of the peptide and the of the was using an automated X. S.J. Ginsberg M.H. M.C. J. Biol. Chem. 1994; Full Text PDF PubMed Google of Calpain of were from (20Du X. Plow E.F. Frelinger III, A.L. O'Toole T.E. Loftus J.C. Ginsberg M.H. Cell. 1991; 65: 409-416Abstract Full Text PDF PubMed Scopus (417) Google Scholar) and with and The platelets were at a of in X. Plow E.F. Frelinger III, A.L. O'Toole T.E. Loftus J.C. Ginsberg M.H. Cell. 1991; 65: 409-416Abstract Full Text PDF PubMed Scopus (417) Google Scholar), and at for of in an after the calcium were by with and to and with the platelets were by of an of to the lysates were with of a rabbit domain by to intact integrin β3, in the was by with an monoclonal was as described that the antibodies were with and the were using an of of αIIb and β3 was by EDTA treatment as described S.J. J.S. 1985; PubMed Google Scholar). platelets were in and at for were by an of but no calpain and at for an calpain, was and the lysates were at for to and of the by integrin αIIbβ3 is a substrate for calpain, purified αIIbβ3 and calpain were in of the integrin was by with Calpain of an the of the β3 subunit the of antibodies the subunit its extracellular domain were not by calpain was no major in mass of the β3 that the of β3 is Thus, calpain cleavage sites in β3 are within the cytoplasmic the cytoplasmic domain of αIIb not to be by calpain, as by the of with an to the of the αIIb cytoplasmic domain with an to the of the αIIb heavy was by The of the αIIb to be a with the the of the αIIb after calpain treatment This not with for the of the αIIb and of the intact αIIb was by calpain Thus, the αIIb cytoplasmic domain calpain the of the is the cytoplasmic domain of integrin β3 to be a intracellular substrate for cleavage of the β3 cytoplasmic domain by calpain occurs in intact platelets were with a calcium for at to intracellular intact β3 subunit was by sequential with and β3 was by with an to the extracellular domain of β3 that of β3 from platelets in the after by This result that of the β3 subunit in platelets the of β3 in platelets was by with that the cytoplasmic domain of β3 is intact These were platelets were in calpain and with The integrin β3 subunit from platelets with β3 from This in the was to calpain cleavage a calpain Thus, cleavage of the cytoplasmic domain of β3 occurs in intact cleavage of β3 in A, platelets in were at with calcium for with at The platelets were by of an of and with of the peptide the proteins with the of were by and with a monoclonal platelets were at for after with calpain and by of an of and of lysates were by and with of the Calpain in the calpain cleavage sites in the cytoplasmic domain of the β3 a synthetic peptide on the sequence of the cytoplasmic domain was with purified calpain, and the were by The of peptide from major were by ion-spray mass The of the were from their using the The of calpain were two were was to be the of and was the of Thus, is to for The cleavage sites are in calpain cleavage sites in the cytoplasmic domain of synthetic peptide corresponding to the cytoplasmic domain of integrin β3 was in the of purified calpain at for The were by using a The were with a of in from the were by ion-spray mass and their were as described Calpain cleavage sites were by the of these in the intact The the cleavage sites by in a in a of the cleavage site were by sequence of the of calpain of the β3 cytoplasmic domain were to peptides with from the intact and of the calpain cleavage sites by mass Thus, calpain cleavage sites were to Thr741, Tyr747, Phe754, and of the β3 in the calpain cleavage sites in were used in cleavage such with and the is by the of the by the that these antibodies not and with β3 from platelets When the platelets were with the calcium was a increase in with and This the of in β3 corresponding to calpain cleavage sites to and Furthermore, the of these antibodies with β3 was by of the calpain that calpain is for the of these in These that calpain β3 at sites to and in intact cleavage site in integrin β3 in intact platelets in were with in the of no was at platelets were by of an of and The proteins were by and with the calpain cleavage binding was by antibodies and the site to not to be by calpain in the cellular that β3, platelets were with EDTA at for to αIIb from was to the to such of with β3 was that cleavage after in of β3 on a to that of β3, was This that cleavage may in the extracellular to the of an extracellular within S. A. H. E. Biochim. Acta. 1989; PubMed Scopus Google Scholar). Thus, the calpain cleavage site to is utilized to the αIIb and β3 cleavage of β3 at in platelet platelets were with EDTA in the of at for and by an of but no calpain an at for a of was to platelet lysates to The lysates were at for a and by and with that the the platelets were with EDTA calpain of calpain the β3 subunit during platelet activation induced by platelets were with and to for of in an were by and with antibodies specific for the calpain sites of in the cleavage not to β3 from platelets binding of the was as as after the platelets were fully with with and was by not that calpain cleavage of the antibodies the other calpain cleavage sites and were during platelet aggregation not cleavage of β3 integrin in platelets activated by thrombin. platelets were at in the of for with at The were by an of and Proteins in the platelet lysates were by and with the calpain cleavage have that the cytoplasmic domain of the integrin β3 subunit is by calpain and have cleavage Four of these sites (C-terminal to Thr741, Tyr747, Phe754, and Tyr759) are utilized in intact The fifth site is accessible after treatment to the αIIbβ3 heterodimer. These calpain cleavages critical for the of the integrin to the cytoskeleton and bidirectional transmembrane signaling. Thus, calpain cleavage may regulate functions of β3 that calpain the cytoplasmic domain of integrin β3 from of in cleavage of the β3 cytoplasmic domain of purified integrin αIIbβ3 by purified cleavage of synthetic β3 cytoplasmic domain peptides by purified and limited cleavage of the cytoplasmic domain of the integrin in intact platelets stimulated by thrombin. the differ the from these are that of the β3 cytoplasmic domain are by Calpain cleavages peptide from the of β3, resulting in no in its on This may calpain cleavage of the β3 subunit was not J.E. Reynolds C.C. Phillips D.R. J. Biol. Chem. Full Text PDF PubMed Google cleavage of β3 occurs during platelet that may regulate platelet with an specific for the β3 β3 from of β3 in platelets not with this This that the of β3 in platelets is calpain cleavage of β3 was as as after a platelet and with a to that of calpain activation in platelets J.E. Taylor R.G. Taffarel M. Boyles J.K. Goll D.E. J. Cell Biol. 1993; 120: 1501-1507Crossref PubMed Scopus (133) Google Scholar). aggregation leads to calpain cleavage of cytoskeletal proteins such as J.E. Goll D.E. Reynolds C.C. Phillips D.R. J. Biol. Chem. 1985; 260: 1060-1066Abstract Full Text PDF PubMed Google Scholar) and signaling such as J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (282) Google Scholar), A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar), and T.C. Mizuno K. Suzuki K. Biomed. Biochim. Acta. 1991; 50: 485-489PubMed Google Scholar). the β subunit cytoplasmic domain is to with A. K. M.C. Burridge K. PubMed Scopus Google Scholar, A. K. 1989; Google Scholar), and integrins regulate the functions of the other signaling E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar, 17Oda A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar, 18Frangioni J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (282) Google Scholar). Thus, is that calpain cleavage of the cytoplasmic domain of an integrin and its signaling may be a of the purified integrin by calpain cleavage at the domain of the αIIb is with an that αIIb may be J.C. Plow E.F. Jennings L.K. Ginsberg M.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). This cleavage is not by platelet J.C. Plow E.F. Jennings L.K. Ginsberg M.H. J. Biol. Chem. Full Text PDF PubMed Google cleavage site in intact was with antibodies specific for the cleavage peptide a sequence a for an corresponding to the by a calpain The antibodies with β3 described previously T.C. M. S. I. E. T. Suzuki K. S. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar) and in this this can thus be used to protease cleavage site in αIIb subunit may calpain to cleavage sites in the β3 cytoplasmic Four of calpain cleavage sites in β3 were by calpain in intact the cleavage site (Ala735) to calpain cleavage after of the integrin with EDTA is to the calcium-dependent of αIIb and β3 S.J. J.S. 1985; PubMed Google Scholar), this result that the was from calpain cleavage in the αIIbβ3 heterodimer. Thus, the interaction of αIIb with β3 may regulate the of the β3 cytoplasmic the cytoplasmic domain of αIIb may with the cytoplasmic domain of β3 at a site to is not by the flanking the D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (779) Google Scholar). Thus, the and of the in the of the may be important of calpain such as and the to an T. T. I. T. J. PubMed Scopus Google Scholar). a to and to an a at the cleavage site is a in calpain (14Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (779) Google Scholar). The calpain cleavage sites in the β3 cytoplasmic domain in intact platelets and flank two NXXY The two NXXY motifs in the β3 cytoplasmic domain a at of the to a tyrosine at the calpain cleavage Thus, the NXXY motifs are to cleavage sites in other calpain Calpain cleavage occurs on the of the NXXY the the motif a that and are A. Cell. 1991; Full Text PDF PubMed Scopus Google Scholar). is that such in the β3 cytoplasmic of the β3 cytoplasmic domain by calpain two NXXY sites may be an important for the regulation of its bidirectional signaling and αIIbβ3 to the cytoskeleton ligand The NXXY motif of the β3 cytoplasmic domain the sequence to the signal in the J. Biol. Chem. 1990; Full Text PDF PubMed Google Scholar). this motif the of the β3 cytoplasmic domain to regulate the affinity of the (inside-out T.E. Ylanne J. Culley B.M. J. Biol. Chem. 1995; 270: 8553-8558Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), to with the cytoskeleton at focal adhesion J. Huuskonen J. O'Toole T.E. Ginsberg M.H. Virtanen I. Gahmberg C.G. J. Biol. Chem. 1995; 270: 9550-9557Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, J. Y. O'Toole T.E. Loftus J.C. Y. Ginsberg M.H. J. Cell Biol. 1993; PubMed Scopus Google Scholar), and to cell J. Cell Biol. 1995; PubMed Scopus Google Scholar). Furthermore, NXXY motifs at of the are in the cytoplasmic domain of integrin β including β3, and and have been in the functions of of these Y. J. Cell Biol. 1992; PubMed Scopus Google Scholar, H. M. J. 1993; Google Scholar). Thus, is that calpain cleavage may to these integrins the NXXY motif and regulate the functions of these the cytoplasmic domain been to be a substrate for Suzuki S. E. E. J. Cell Biol. 1992; PubMed Scopus (121) Google Scholar, A.J. Croall D.E. Cell 1994; PubMed Scopus Google Scholar). The cytoplasmic domain is from that of other integrin β subunits in sequence, and F. I. A. EMBO J. 1990; PubMed Scopus Google Scholar, S. Y. EMBO J. 1990; PubMed Scopus Google Scholar). the cytoplasmic domain an NXXY F. I. A. EMBO J. 1990; PubMed Scopus Google Scholar), calpain cleavage sites in the cytoplasmic domain have not been and thus be with cleavage sites in β3 as described in this the of calpain cleavage of an integrin cytoplasmic cleavage of the β3 subunit platelet aggregation and of may be important to the shedding of procoagulant membrane vesicles during platelet J.E. 1994; PubMed Scopus Google Scholar). calpain with integrins in focal adhesion calpain cleavage at these sites may as a to from the extracellular an integrin J. Cell Biol. 1993; PubMed Scopus Google Scholar). INTRODUCTIONIntegrins, a family of adhesion receptors, play important roles in cellular functions such as adhesion, migration, cell proliferation, and differentiation(1Hynes R.O. Cell. 1992; 69: 11-25Abstract Full Text PDF PubMed Scopus (8966) Google Scholar). The functions of integrins are modulated by bidirectional transmembrane signaling as exemplified by platelet integrin αIIbβ3 (glycoprotein IIb-IIIa)(2Ginsberg M.H. Du X. O'Toole T.E. Loftus J.C. Plow E.F. Thromb. Haemostasis. 1993; 70: 87-93Crossref PubMed Scopus (106) Google Scholar). When platelets are stimulated by agonists such as thrombin, intracellular signal transduction leads to activation of the extracellular fibrinogen binding function of αIIbβ3 (inside-out signaling), resulting in platelet aggregation. Conversely, ligand binding to αIIbβ3 is involved in outside-in signals that result in its association with the cytoskeleton (3Phillips D.R. Jennings L.K. Edwards H.H. J. Cell Biol. 1980; 86: 77-86Crossref PubMed Scopus (248) Google Scholar) and biochemical changes including tyrosine phosphorylation of intracellular proteins(4Ferrell J.J. Martin G.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2234-2238Crossref PubMed Scopus (204) Google Scholar, 5Golden A. Brugge J.S. Shattil S.J. J. Cell Biol. 1990; : 3117-3127Crossref PubMed Scopus (184) Google Scholar), increase in intracellular calcium level(6Pelletier A.J. Bodary S.C. Levinson A.D. Mol. Biol. Cell. 1992; 3: 989-998Crossref PubMed Scopus (90) Google Scholar), and activation of the calcium-dependent neutral protease (calpain)(7Fox J.E. Taylor R.G. Taffarel M. Boyles J.K. Goll D.E. J. Cell Biol. 1993; 120: 1501-1507Crossref PubMed Scopus (133) Google Scholar). Specific structural characteristics of the integrin cytoplasmic domains are critical for the two-way signaling mechanism. This includes a GFFKR sequence in the αIIb cytoplasmic domain (8O'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) and two sets of NXXY (NPLY and NITY) within the β3 cytoplasmic domain(9O'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, 10Ylanne J. Huuskonen J. O'Toole T.E. Ginsberg M.H. Virtanen I. Gahmberg C.G. J. Biol. Chem. 1995; 270: 9550-9557Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). The NPLY sequence, in particular, is critical for controlling affinity states of the ligand-binding sites and for the interaction of the integrin with cytoskeletal elements(9O'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, 10Ylanne J. Huuskonen J. O'Toole T.E. Ginsberg M.H. Virtanen I. Gahmberg C.G. J. Biol. Chem. 1995; 270: 9550-9557Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar).Numerous cytoplasmic proteins are colocalized with integrins in focal adhesion sites and thus may be involved in intracellular signaling to and from integrins(11Clark E.A. Brugge J.S. Science. 1995; 268: 233-239Crossref PubMed Scopus (2809) Google Scholar). Calpain is among these proteins(12Beckerle M.C. Burridge K. DeMartino G.N. Croall D.E. Cell. 1987; 51: 569-577Abstract Full Text PDF PubMed Scopus (210) Google Scholar). Calpain represents a family of intracellular calcium-dependent neutral proteases(13Saido T.C. Sorimachi H. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (616) Google Scholar). Of the two classic members of this family, μ-calpain and m-calpain, μ-calpain is probably the predominant form in platelets(14Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (779) Google Scholar). These two isoforms of calpain have no major difference in substrate specificity, but differ in calcium sensitivity (14Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (779) Google Scholar). Calpain can be activated during platelet aggregation by a rise in the cytoplasmic calcium level and/or by its translocation to the membrane(13Saido T.C. Sorimachi H. Suzuki K. FASEB J. 1994; 8: 814-822Crossref PubMed Scopus (616) Google Scholar, 14Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (779) Google Scholar). Furthermore, calpain activation promotes the shedding of procoagulant membrane vesicles from aggregated platelets (15Fox J.E. Austin C.D. Boyles J.K. Steffen P.K. J. Cell Biol. 1990; 111: 483-493Crossref PubMed Scopus (131) Google Scholar). While the mechanisms of calpain regulation are still not fully understood, calpain cleavages can regulate a variety of intracellular processes(16Fox J.E. Goll D.E. Reynolds C.C. Phillips D.R. J. Biol. Chem. 1985; 260: 1060-1066Abstract Full Text PDF PubMed Google Scholar, 17Oda A. Druker B.J. Ariyoshi H. Smith M. Salzman E.W. J. Biol. Chem. 1993; 268: 12603-12608Abstract Full Text PDF PubMed Google Scholar, 18Frangioni J.V. Oda A. Smith M. Salzman E.W. Neel B.G. EMBO J. 1993; 12: 4843-4856Crossref PubMed Scopus (282) Google Scholar, 19Saido T.C. Mizuno K. Suzuki K. Biomed. Biochim. Acta. 1991; 50: 485-489PubMed Google Scholar). We now report that calpain may regulate the function of β3 integrins by limited cleavage of the cytoplasmic domain of β3 at specific sites flanking two sets of NXXY