Savignygrin, a Platelet Aggregation Inhibitor from the Soft TickOrnithodoros savignyi, Presents the RGD Integrin Recognition Motif on the Kunitz-BPTI Fold
2002; Elsevier BV; Volume: 277; Issue: 24 Linguagem: Inglês
10.1074/jbc.m112060200
ISSN1083-351X
AutoresBen J. Mans, Abraham I. Louw, A.W.H. Neitz,
Tópico(s)Ion Channels and Receptors
ResumoSavignygrin, a platelet aggregation inhibitor that possesses the RGD integrin recognition motif, has been purified from the soft tick Ornithodoros savignyi. Two isoforms with similar biological activities differ because of R52G and N60G in their amino acid sequences, indicating a recent gene duplication event. Platelet aggregation induced by ADP (IC50, 130 nm), collagen, the thrombin receptor-activating peptide, and epinephrine was inhibited, although platelets were activated and underwent a shape change. The binding of α-CD41 (P2) to platelets, the binding of purified αIIbβ3 to fibrinogen, and the adhesion of platelets to fibrinogen was inhibited, indicating a targeting of the fibrinogen receptor. In contrast, the adhesion of osteosarcoma cells that express the integrin αvβ3 to vitronectin or fibrinogen was not inhibited, indicating the specificity of savignygrin toward αIIbβ3. Savignygrin shows sequence identity to disagregin, a platelet aggregation inhibitor from the tickOrnithodoros moubata that lacks an RGD motif. The cysteine arrangement of savignygrin is similar to that of the bovine pancreatic trypsin inhibitor family of serine protease inhibitors. A homology model based on the structure of the tick anticoagulant peptide indicates that the RGD motif is presented on the substrate-binding loop of the canonical BPTI inhibitors. However, savignygrin did not inhibit the serine proteases fXa, plasmin, thrombin, or trypsin. This is the first report of a platelet aggregation inhibitor that presents the RGD motif using the Kunitz-BPTI protein fold. Savignygrin, a platelet aggregation inhibitor that possesses the RGD integrin recognition motif, has been purified from the soft tick Ornithodoros savignyi. Two isoforms with similar biological activities differ because of R52G and N60G in their amino acid sequences, indicating a recent gene duplication event. Platelet aggregation induced by ADP (IC50, 130 nm), collagen, the thrombin receptor-activating peptide, and epinephrine was inhibited, although platelets were activated and underwent a shape change. The binding of α-CD41 (P2) to platelets, the binding of purified αIIbβ3 to fibrinogen, and the adhesion of platelets to fibrinogen was inhibited, indicating a targeting of the fibrinogen receptor. In contrast, the adhesion of osteosarcoma cells that express the integrin αvβ3 to vitronectin or fibrinogen was not inhibited, indicating the specificity of savignygrin toward αIIbβ3. Savignygrin shows sequence identity to disagregin, a platelet aggregation inhibitor from the tickOrnithodoros moubata that lacks an RGD motif. The cysteine arrangement of savignygrin is similar to that of the bovine pancreatic trypsin inhibitor family of serine protease inhibitors. A homology model based on the structure of the tick anticoagulant peptide indicates that the RGD motif is presented on the substrate-binding loop of the canonical BPTI inhibitors. However, savignygrin did not inhibit the serine proteases fXa, plasmin, thrombin, or trypsin. This is the first report of a platelet aggregation inhibitor that presents the RGD motif using the Kunitz-BPTI protein fold. coagulation factor Xa tick anticoagulant peptide bovine pancreatic trypsin inhibitor dithiothreitol thrombin receptor-activating peptide fluorescein isothiocyanate size exclusion high performance liquid chromatography anion exchange high performance liquid chromatography reversed phase high performance liquid chromatography electrospray mass spectrometry matrix-assisted laser desorption ionization time of flight mass spectrometry untranslated region rapid amplification of cDNA ends root mean square deviation Integrins are a family of adhesion receptors that propitiates cell-cell and cell-matrix interactions. Numerous physiological processes like hemostasis, fertilization, neuron-neuron interaction, and inflammation are mediated by integrins (1Clemetson K.J. Cell. Mol. Life Sci. 1998; 54: 499-501Crossref Scopus (5) Google Scholar). The functional receptor is expressed as a transmembrane heterodimer consisting of α and β subunits. To date, 17 α and 8 β subunits have been identified and form, in various permutations, more than 20 described integrins (2Plow E.F Cierniewski C.S. Xiao Z. Haas T.A. Byzova T.V. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Different combinations of subunits convey specificity for ligands (collagen-α2β1, fibronectin-α5β1, laminin-α6β1, vitronectin-αvβ3, and fibrinogen-αIIbβ3), although αIIbβ3 can also recognize fibronectin, vitronectin, von Willebrand's factor, and prothrombin (2Plow E.F Cierniewski C.S. Xiao Z. Haas T.A. Byzova T.V. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Most ligands recognized by integrins contain the integrin recognition motif RGD (3Ruoslahti E. Pierschbacher M.D. Science. 1987; 238: 491-497Crossref PubMed Scopus (3841) Google Scholar). Some ligands may also contain other sequences recognized by integrins such as the dodecapeptide sequence HHLGGAKQAGDV from the γ-chain of fibrinogen that binds to αIIbβ3 (4Andrieux A. Hudry-Clergeon G. Ryckewaert J.J. Chapel A. Ginsberg M.H. Plow E.F. Marguerie G. J. Biol. Chem. 1989; 264: 9258-9265Abstract Full Text PDF PubMed Google Scholar).αIIbβ3 (GPIIbIIIa) is the major integrin of platelets and the only adhesion receptor capable of mediating platelet aggregation by the binding of fibrinogen or von Willebrand's factor (5Calvete J.J. Thromb. Haemostasis. 1994; 72: 1-15Crossref PubMed Scopus (177) Google Scholar, 6Calvete J.J. Proc. Soc. Exp. Biol. Med. 1995; 208: 346-360Crossref PubMed Scopus (103) Google Scholar, 7Plow E.F. Cierniewski C.S. Xiao Z. Haas T.A. Byzova T.V. Thromb. Haemostasis. 2001; 86: 34-40Crossref PubMed Scopus (38) Google Scholar). On resting platelets, αIIbβ3 exists in an inactive conformation that binds irreversibly to the γ-chain C-terminal dodecapeptide (HHLGGAKQAGDV) of immobilized fibrinogen (5Calvete J.J. Thromb. Haemostasis. 1994; 72: 1-15Crossref PubMed Scopus (177) Google Scholar). The unactivated form also has a ligand-binding site accessible to small molecules that contain RGD, KGD, RYD, or OrnGD motifs, which are presented on mobile recognition loops protruding 14–17 Å from the protein core (6Calvete J.J. Proc. Soc. Exp. Biol. Med. 1995; 208: 346-360Crossref PubMed Scopus (103) Google Scholar). The ligand-binding site can also be reached by RGD peptides, which extend 11–32 Å from the surface of polyacrylonitrile beads (8Beer J.H. Springer K.T. Coller B.S. Blood. 1992; 79: 117-128Crossref PubMed Google Scholar). These results suggest that the binding pocket in unactivated αIIbβ3 may resemble a narrow cavity buried 10–20 Å below the protein surface (6Calvete J.J. Proc. Soc. Exp. Biol. Med. 1995; 208: 346-360Crossref PubMed Scopus (103) Google Scholar). Upon platelet activation by various agonists, αIIbβ3 undergoes a conformational change that allows the binding of macromolecules that contain the RGD motif. The ligand-binding site is discontinuous and is formed by both αIIb and β3 subunits. A receptor-bound divalent cation within the ligand-binding site interacts with aspartic acid-containing ligands, as found in the RGD motif (2Plow E.F Cierniewski C.S. Xiao Z. Haas T.A. Byzova T.V. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar,5Calvete J.J. Thromb. Haemostasis. 1994; 72: 1-15Crossref PubMed Scopus (177) Google Scholar).Inhibitors specific for integrins have great potential for the study of integrin function and the development of pharmaceutical compounds (7Plow E.F. Cierniewski C.S. Xiao Z. Haas T.A. Byzova T.V. Thromb. Haemostasis. 2001; 86: 34-40Crossref PubMed Scopus (38) Google Scholar). The most extensively characterized natural inhibitors are the snake venom disintegrins that are involved in the maintenance of hemorrhage by the inhibition of platelet aggregation (9Gould R.J. Polokoff M.A. Friedman P.A. Huang T.F. Holt J.C. Cook J.J. Niewiarowski S. Proc. Soc. Exp. Biol. Med. 1990; 195: 168-171Crossref PubMed Scopus (484) Google Scholar). Platelet aggregation is compromised by the inhibition of fibrinogen binding to integrin αIIbβ3, which mediates platelet-platelet interaction. Disintegrins are a family of low molecular mass (5,400–9,000 Da) proteins that contains the RGD motif except for barbourin, which contains the sequence KGD (10Huang T.F. Cell. Mol. Life Sci. 1998; 54: 527-540Crossref PubMed Scopus (98) Google Scholar). Most disintegrins inhibit platelet aggregation with IC50 values 3,000–30,000× lower (nanomolar concentration range) than the tetrapeptide RGDS (micromolar range) because of the conformation-restricted presentation of the RGD motif (11Scarborough R.M. Rose J.W. Hsu M.A. Phillips D.R. Fried V.A. Campbell A.M. Nannizzi L. Charo I.F. J. Biol. Chem. 1991; 266: 9359-9362Abstract Full Text PDF PubMed Google Scholar). The three-dimensional structures of the snake venom disintegrins echistatin, kistrin, flavoridin, dendroaspin, and the leech-derived decorsin have been elucidated in solution with the use of nuclear magnetic resonance techniques (12Saudek V. Atkinson R.A. Pelton J.T. Biochemistry. 1991; 30: 7369-7372Crossref PubMed Scopus (200) Google Scholar, 13Adler M. Lazarus R.A. Dennis M.S. Wagner G. Science. 1991; 253: 445-448Crossref PubMed Scopus (250) Google Scholar, 14Senn H. Klaus W. J. Mol. Biol. 1993; 232: 907-925Crossref PubMed Scopus (105) Google Scholar, 15Sutcliffe M.J. Jaseja M. Hyde E.I., Lu, X. Williams J.A. Nat. Struct. Biol. 1994; 11: 802-807Crossref Scopus (46) Google Scholar, 16Krezel A.M. Wagner G. Seymour-Ulmer J. Lazarus R.A. Science. 1994; 264: 1944-1947Crossref PubMed Scopus (131) Google Scholar). Disintegrins do not possess a classical secondary structure but rather a dense core consisting of β-turns kept intact by disulfide bonds with a protruding loop region that presents the RGD motif. The sequences flanking the RGD motif as well as the spatial configuration of the RGD motif at the end of the loop have been found to be important contributors to specificity for different integrins (17Scarborough R.M. Rose J.W. Naughton M.A. Phillips D.R. Nannizzi L. Arfsten A. Campbell A.M. Charo I.F. J. Biol. Chem. 1993; 268: 1058-1065Abstract Full Text PDF PubMed Google Scholar).Inhibitors unrelated to the disintegrin family but proposed to bind to αIIbβ3 have been described for both hard and soft ticks (18Wang X. Coons L.B. Taylor D.B. Stevens S.E. Gartner T.K. J. Biol. Chem. 1996; 271: 17785-17790Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 19Karczewski J. Endris R. Connolly T.M. J. Biol. Chem. 1994; 269: 6702-6708Abstract Full Text PDF PubMed Google Scholar). The inhibitor variabilin, from the hard tickDermacentor variabilis, contains a RGD motif that is not flanked by cysteine residues, making it unique in this respect (18Wang X. Coons L.B. Taylor D.B. Stevens S.E. Gartner T.K. J. Biol. Chem. 1996; 271: 17785-17790Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Disagregin from the soft tick Ornithodoros moubata contains no RGD sequence, and it was found that the γ-fibrinogen sequence HHLGGALQAGDV competes with its binding to soluble αIIbβ3. This indicates an inhibition mechanism distinct from disintegrins (19Karczewski J. Endris R. Connolly T.M. J. Biol. Chem. 1994; 269: 6702-6708Abstract Full Text PDF PubMed Google Scholar, 20Karczewski J. Connolly T.M. Biochem. Biophys. Res. Commun. 1997; 241: 744-748Crossref PubMed Scopus (22) Google Scholar). Inhibitors of the coagulation cascade serine proteases of the host have also been described for soft ticks. These include fXa1 (TAP and fXaI) and thrombin (ornithodorin and savignin) inhibitors (21Nienaber J. Gaspar A.R.M.D. Neitz A.W.H. Exp. Parasitol. 1999; 93: 82-91Crossref PubMed Scopus (80) Google Scholar, 22Law J.H. Ribeiro J.M.C. Wells M.A. Annu. Rev. Biochem. 1992; 64: 87-111Crossref Scopus (150) Google Scholar, 23Waxman L. Smith D.E. Arcuri K.E. Vlasuk G.P. Science. 1990; 248: 593-596Crossref PubMed Scopus (365) Google Scholar, 24Gaspar A.R.M.D. Joubert A.M. Crause J.C. Neitz A.W.H. Exp. Appl. Acarol. 1996; 20: 583-598Crossref PubMed Scopus (50) Google Scholar, 25Joubert A.M. Louw A.I. Joubert F. Neitz A.W.H. Exp. Appl. Acarol. 1998; 22: 603-619Crossref PubMed Scopus (54) Google Scholar, 26Van de Locht A. Stubbs M.T. Bode W. Friedrich T. Bollschweiler C. Hoffken W. Huber R. EMBO J. 1995; 15: 6011-6017Crossref Scopus (174) Google Scholar, 27Mans B.J. Louw A.I. Neitz A.W.H. Insect Biochem. Mol. Biol. 2002; (in press)PubMed Google Scholar). All possess the Kunitz bovine pancreatic trypsin inhibitor (BPTI) fold, although the mechanisms of inhibition differ from those of the canonical BPTI-like inhibitors. Whereas canonical BPTI-inhibitors present a substrate-binding loop to the active site of their respective enzymes, the tick inhibitors insert their N-terminal sequences into the active site (21Nienaber J. Gaspar A.R.M.D. Neitz A.W.H. Exp. Parasitol. 1999; 93: 82-91Crossref PubMed Scopus (80) Google Scholar, 22Law J.H. Ribeiro J.M.C. Wells M.A. Annu. Rev. Biochem. 1992; 64: 87-111Crossref Scopus (150) Google Scholar, 23Waxman L. Smith D.E. Arcuri K.E. Vlasuk G.P. Science. 1990; 248: 593-596Crossref PubMed Scopus (365) Google Scholar, 24Gaspar A.R.M.D. Joubert A.M. Crause J.C. Neitz A.W.H. Exp. Appl. Acarol. 1996; 20: 583-598Crossref PubMed Scopus (50) Google Scholar, 25Joubert A.M. Louw A.I. Joubert F. Neitz A.W.H. Exp. Appl. Acarol. 1998; 22: 603-619Crossref PubMed Scopus (54) Google Scholar, 26Van de Locht A. Stubbs M.T. Bode W. Friedrich T. Bollschweiler C. Hoffken W. Huber R. EMBO J. 1995; 15: 6011-6017Crossref Scopus (174) Google Scholar, 27Mans B.J. Louw A.I. Neitz A.W.H. Insect Biochem. Mol. Biol. 2002; (in press)PubMed Google Scholar, 28Laskowski M., Jr. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1928) Google Scholar). This study describes the platelet aggregation inhibitor savignygrin, from the soft tick Ornithodoros savignyi, that presents its RGD motif on the substrate-binding presenting loop of the canonical BPTI inhibitors. The presence of the RGD motif in the BPTI fold presents a new protein fold that can be manipulated for investigations into integrin structure/function relationships.DISCUSSIONControl of the hemostatic system of the host is essential for successful tick feeding. Efficient inhibitors of the hemostatic system are thus an important part of the feeding strategy of ticks (22Law J.H. Ribeiro J.M.C. Wells M.A. Annu. Rev. Biochem. 1992; 64: 87-111Crossref Scopus (150) Google Scholar). Soft ticks are a rich source of inhibitors of both blood coagulation and platelet aggregation. Inhibitors of the blood clotting cascade serine proteases fXa and thrombin have been characterized (21Nienaber J. Gaspar A.R.M.D. Neitz A.W.H. Exp. Parasitol. 1999; 93: 82-91Crossref PubMed Scopus (80) Google Scholar, 22Law J.H. Ribeiro J.M.C. Wells M.A. Annu. Rev. Biochem. 1992; 64: 87-111Crossref Scopus (150) Google Scholar, 23Waxman L. Smith D.E. Arcuri K.E. Vlasuk G.P. Science. 1990; 248: 593-596Crossref PubMed Scopus (365) Google Scholar, 24Gaspar A.R.M.D. Joubert A.M. Crause J.C. Neitz A.W.H. Exp. Appl. Acarol. 1996; 20: 583-598Crossref PubMed Scopus (50) Google Scholar, 25Joubert A.M. Louw A.I. Joubert F. Neitz A.W.H. Exp. Appl. Acarol. 1998; 22: 603-619Crossref PubMed Scopus (54) Google Scholar, 26Van de Locht A. Stubbs M.T. Bode W. Friedrich T. Bollschweiler C. Hoffken W. Huber R. EMBO J. 1995; 15: 6011-6017Crossref Scopus (174) Google Scholar, 27Mans B.J. Louw A.I. Neitz A.W.H. Insect Biochem. Mol. Biol. 2002; (in press)PubMed Google Scholar). Apyrase, which inhibits platelet aggregation by the hydrolysis of ADP, has also been investigated (29Mans B.J. Louw A.I. Gaspar A.R.M.D. Neitz A.W.H. Exp. Appl. Acarol. 1998; 22: 353-366Crossref PubMed Scopus (55) Google Scholar, 31Mans B.J. Coetzee J. Louw A.I. Gaspar A.R.M.D. Neitz A.W.H. Exp. Appl. Acarol. 2000; 24: 271-282Crossref PubMed Scopus (34) Google Scholar, 32Mans B.J. Louw A.I. Gaspar A.R.M.D. Neitz A.W.H. Comp. Biochem. Physiol. 1998; 120B: 617-624Crossref Scopus (37) Google Scholar, 54Ribeiro J.M.C. Endris T.M. Endris R. Comp. Biochem. Physiol. 1991; 100A: 109-112Crossref Scopus (63) Google Scholar). Collagen-specific platelet aggregation inhibitors and αIIbβ3 agonists have been described (19Karczewski J. Endris R. Connolly T.M. J. Biol. Chem. 1994; 269: 6702-6708Abstract Full Text PDF PubMed Google Scholar, 20Karczewski J. Connolly T.M. Biochem. Biophys. Res. Commun. 1997; 241: 744-748Crossref PubMed Scopus (22) Google Scholar, 55Waxman L. Connolly T.M. J. Biol. Chem. 1993; 268: 5445-5449Abstract Full Text PDF PubMed Google Scholar, 56Karczewski J. Waxman L. Endris R.G. Connolly T.M. Biochem. Biophys. Res. Commun. 1995; 208: 532-541Crossref PubMed Scopus (53) Google Scholar). This study describes savignygrin, a potent platelet aggregation inhibitor, that is thought to inhibit platelets by targeting the platelet integrin αIIbβ3. This is the first described platelet aggregation inhibitor from soft ticks with an RGD integrin recognition motif that is presented on the canonical substrate-binding presenting loop of the Kunitz-BPTI fold.The different isoforms from O. savignyi were detected in 20 individual ticks analyzed, which indicate that the (+/−) forms are gene duplicates and not allelic variants (results not shown). Interestingly, no isoforms were observed for disagregin, and no difference could be observed in the electrophoretic mobilities under reduced and non-reduced conditions (19Karczewski J. Endris R. Connolly T.M. J. Biol. Chem. 1994; 269: 6702-6708Abstract Full Text PDF PubMed Google Scholar). This suggests that the (+/−) gene duplication is a fairly recent event that occurred after the divergence of these two tick species from a common ancestor. The presence of the A/B conformational isoforms is more problematic, as no sequences were obtained that could explain the differences. However, it is not uncommon to find separation of a single protein into two peaks during RPHPLC. Such 2-peak separations are generally observed for proteins that are stable under reversed-phase chromatography conditions (low pH and high concentrations of organic mobile phase) so that native as well as unfolded forms are present (57Kunitani M. Johnson D. J. Chromatogr. 1986; 371: 313-333Crossref PubMed Scopus (75) Google Scholar).Biological activity is not affected by sequence or conformation differences as indicated by similar IC50 values (∼130 nm) for all four isoforms. This could be accounted for in part by the RGD motif that is present in all four isoforms. Platelets are activated by various agonists via specific receptors and distinct pathways culminating in the activation of αIIbβ3, which mediates aggregation through the binding of fibrinogen (6Calvete J.J. Proc. Soc. Exp. Biol. Med. 1995; 208: 346-360Crossref PubMed Scopus (103) Google Scholar). Platelet activation is accompanied by a shape change from a discoid (resting) to a spherical form (activated), the extension of pseudopods, and the aggregation-independent release of platelet granules (58Cattaneo M. Kinlough-Rathbone R.L. Lecchi A. Bevilacqua C. Packham M.A. Mustard J.F. Blood. 1987; 70: 221-226Crossref PubMed Google Scholar). The activation of platelets preincubated with savignygrin was indicated by a decrease in transmittance during the inhibition of platelet aggregation induced by various agonists. Electron microscopic analysis of platelets incubated with savignygrin before activation with ADP confirmed the discoid to spherical shape change associated with activation (59Zucker M.B. Methods Enzymol. 1989; 169: 117-133Crossref PubMed Scopus (75) Google Scholar). This suggests postactivation inhibition by the savignygrins and implicates the common denominator of platelet aggregation, the integrin αIIbβ3. The inhibition of the binding of α-CD41-FITC in the presence or absence of ADP strongly suggests that savignygrins bind to resting as well as to activated αIIbβ3. The targeting of αIIbβ3 was further supported by the inhibition of αIIbβ3 binding to fibrinogen. The discrimination observed for savignygrin between αvβ3 and αIIbβ3was also found for disagregin (19Karczewski J. Endris R. Connolly T.M. J. Biol. Chem. 1994; 269: 6702-6708Abstract Full Text PDF PubMed Google Scholar). This could indicate that BPTI-like inhibitors of platelet aggregation are integrin specific. The BPTI fold might thus be useful to design integrin-specific antagonists.It was shown that disagregin inhibits the binding of echistatin (which contains the RGD motif) to platelets, suggesting an interaction with the RGD-binding site (19Karczewski J. Endris R. Connolly T.M. J. Biol. Chem. 1994; 269: 6702-6708Abstract Full Text PDF PubMed Google Scholar). This is strongly supported by the presence of an RGD motif in the sequence of savignygrin. Although disagregin has a RED motif, and peptide studies showed that the peptide REDV does not inhibit platelet aggregation, a restricted conformation induced by the three-dimensional structure of disagregin could account for inhibitory activity (60Chen C.S. Papayannopoulos I.A. Timmons S. Chou S.H. Thiagarajan P. Biochim. Biophys. Acta. 1991; 1075: 237-247Crossref PubMed Scopus (3) Google Scholar). This is supported by the dependence of savignygrin on intact disulfide bonds for the maintenance of an active but restricted biological conformation. BPTI has also been shown to occur as a very disordered polymer in its reduced form, even in the absence of denaturants (61Creighton T.E. Prog. Biophys. Mol. Biol. 1978; 33: 231-297Crossref PubMed Scopus (443) Google Scholar). The sequence adjacent to the RGD motif of savignygrin (YGCRGDEDA) is similar to that of disagregin (YGCREDDDS), which suggests that the sequence around the RGD motif could have an important effect on its function. Of particular interest is the fact that two downstream residues are negatively charged. It is possible that these residues interact with the receptor-bound cation localized inside the αIIbβ3 ligand-binding site (2Plow E.F Cierniewski C.S. Xiao Z. Haas T.A. Byzova T.V. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar, 6Calvete J.J. Proc. Soc. Exp. Biol. Med. 1995; 208: 346-360Crossref PubMed Scopus (103) Google Scholar). It was also shown that disagregin can inhibit the binding of the dodecapeptide sequence of γ-fibrinogen to αIIbβ3 (20Karczewski J. Connolly T.M. Biochem. Biophys. Res. Commun. 1997; 241: 744-748Crossref PubMed Scopus (22) Google Scholar). There might thus be other binding sites for αIIbβ3 apart from the RGD motif in the savignygrins.Secondary structure elements are normally conserved in proteins with the same structural folds. This is exemplified by the conserved areas observed for the predicted β-sheet and α-helix elements in the alignment of disagregin and savignygrin. Of interest is the fact that the region surrounding the RGD/RED motif is the largest conserved stretch between disagregin and savignygrin. However, no consensus secondary structure is predicted in this region. As residues are normally conserved either for structural or functional purposes, this suggests that this region is important for activity. The surface models corroborate this view, as the RGDED forms a continuous binding epitope.The BPTI fold is generally associated with serine protease inhibitors (28Laskowski M., Jr. Kato I. Annu. Rev. Biochem. 1980; 49: 593-626Crossref PubMed Scopus (1928) Google Scholar). This study indicated that the savignygrins do not inhibit serine proteases. This fits with the specialization of the substrate-binding presenting loop of the savignygrins to target the platelet fibrinogen receptor. Other BPTI-like proteins that do not inhibit serine proteases include a rather large group of toxins found in snake venom, which function by targeting the ion channels of the cardiac and nervous systems (62Pritchard L. Dufton M.J. J. Mol. Biol. 1999; 285: 1589-1607Crossref PubMed Scopus (59) Google Scholar).To have any biological significance during tick feeding, bioactive components need to be secreted (22Law J.H. Ribeiro J.M.C. Wells M.A. Annu. Rev. Biochem. 1992; 64: 87-111Crossref Scopus (150) Google Scholar). Secretory proteins are targeted to the endoplasmic reticulum via a hydrophobic signal peptide in their N terminus from where they are transported to the Golgi network and finally secreted by either constitutive or regulated secretion in secretory granules. Extracellular proteins in general are disulfide rich, and disulfide bonds are absent in intracellular proteins because of the reducing environment in the cytoplasm (63Gierasch L.M. Biochemistry. 1989; 28: 923-930Crossref PubMed Scopus (423) Google Scholar, 64von Heijne G. J. Membr. Biol. 1990; 115: 195-201Crossref PubMed Scopus (854) Google Scholar, 65Fahey R.C. Hunt J.S. Windham G.C. J. Mol. Evol. 1977; 10: 155-160Crossref PubMed Scopus (118) Google Scholar). The presence of a signal peptide in the full-length sequences of savignygrin and the fact that all cysteines are involved in disulfide bonds indicate that the savignygrins are targeted to the secretory pathway. No evidence suggesting secretion has yet been described for variabilin or disagregin (18Wang X. Coons L.B. Taylor D.B. Stevens S.E. Gartner T.K. J. Biol. Chem. 1996; 271: 17785-17790Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 19Karczewski J. Endris R. Connolly T.M. J. Biol. Chem. 1994; 269: 6702-6708Abstract Full Text PDF PubMed Google Scholar).Variabilin, a 40 amino acid platelet aggregation inhibitor from the hard tick D. variabilis contains a RGD motif in the last third of its sequence (18Wang X. Coons L.B. Taylor D.B. Stevens S.E. Gartner T.K. J. Biol. Chem. 1996; 271: 17785-17790Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). There is no amino acid sequence similarity between variabilin and savignygrin, and the position of the RGD motif is completely different. This suggests that platelet aggregation inhibitors with RGD-like motifs have evolved after the divergence of hard and soft ticks. This implies that the main tick families have adapted to their blood feeding environments independently.New strategies to investigate integrin-ligand interaction are essential to gain a comprehensive view of αIIbβ3antagonism. This would be useful in the design of a new generation of antithrombotic therapies (7Plow E.F. Cierniewski C.S. Xiao Z. Haas T.A. Byzova T.V. Thromb. Haemostasis. 2001; 86: 34-40Crossref PubMed Scopus (38) Google Scholar). Savignygrin and related BPTI-like inhibitors present a new protein fold that can be used in integrin-ligand investigations. The presentation of RGD on the BPTI fold could be useful to engineer novel scaffolds that exhibit both blood coagulation and platelet aggregation inhibitory capabilities. Integrins are a family of adhesion receptors that propitiates cell-cell and cell-matrix interactions. Numerous physiological processes like hemostasis, fertilization, neuron-neuron interaction, and inflammation are mediated by integrins (1Clemetson K.J. Cell. Mol. Life Sci. 1998; 54: 499-501Crossref Scopus (5) Google Scholar). The functional receptor is expressed as a transmembrane heterodimer consisting of α and β subunits. To date, 17 α and 8 β subunits have been identified and form, in various permutations, more than 20 described integrins (2Plow E.F Cierniewski C.S. Xiao Z. Haas T.A. Byzova T.V. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Different combinations of subunits convey specificity for ligands (collagen-α2β1, fibronectin-α5β1, laminin-α6β1, vitronectin-αvβ3, and fibrinogen-αIIbβ3), although αIIbβ3 can also recognize fibronectin, vitronectin, von Willebrand's factor, and prothrombin (2Plow E.F Cierniewski C.S. Xiao Z. Haas T.A. Byzova T.V. J. Biol. Chem. 2000; 275: 21785-21788Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Most ligands recognized by integrins contain the integrin recognition motif RGD (3Ruoslahti E. Pierschbacher M.D. Science. 1987; 238: 491-497Crossref PubMed Scopus (3841) Google Scholar). Some ligands may also contain other sequences recognized by integrins such as the dodecapeptide sequence HHLGGAKQAGDV from the γ-chain of fibrinogen that binds to αIIbβ3 (4Andrieux A. Hudry-Clergeon G. Ryckewaert J.J. Chapel A. Ginsberg M.H. Plow E.F. Marguerie G. J. Biol. Chem. 1989; 264: 9258-9265Abstract Full Text PDF PubMed Google Scholar). αIIbβ3 (GPIIbIIIa) is the major integrin of platelets and the only adhesion receptor capable of mediating platelet aggregation by the binding of fibrinogen or von Willebrand's factor (5Calvete J.J. Thromb. Haemostasis. 1994; 72: 1-15Crossref PubMed Scopus (177) Google Scholar, 6Calvete J.J. Proc. Soc. Exp. Biol. Med. 1995; 208: 346-360Crossref PubMed Scopus (103) Google Scholar, 7Plow E.F. Cierniewski C.S. Xiao Z. Haas T.A. Byzova T.V. Thromb. Haemostasis. 2001; 86: 34-40Crossref PubMed Scopus (38) Google Scholar). On resting platelets, αIIbβ3 exists in an inactive conformation that binds irreversibly to the γ-chain C-terminal dodecapeptide (HHLGGAKQAGDV) of immobilized fibrinogen (5Calvete J.J. Thromb. Haemostasis. 1994; 72: 1-15Crossref PubMed Scopus (177) Google Scholar). The unactivated form also has a ligand-binding site accessible to small molecules that contain RGD, KGD, RYD, or OrnGD motifs, which are presented on mobile recognition loops protruding 14–17 Å from the protein core (6Calvete J.J. Proc. Soc. Exp. Biol. Med. 1995; 208: 346-360Crossref PubMed Scopus (103) Google Scholar). The ligand-binding site can also be reached by RGD peptides, which extend 11–32 Å from the surface of polyacrylonitri
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