Identification of a Novel Binding Site for Platelet Integrins αIIbβ3 (GPIIbIIIa) and α5β1 in the γC-domain of Fibrinogen
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m300410200
ISSN1083-351X
AutoresNataly P. Podolnikova, Valentin P. Yakubenko, Volkov Gl, Edward F. Plow, Tatiana P. Ugarova,
Tópico(s)Cell Adhesion Molecules Research
ResumoThe interactions of platelets with fibrinogen mediate a variety of responses including adhesion, platelet aggregation, and fibrin clot retraction. Whereas it was assumed that interactions of the platelet integrin αIIbβ3 with the AGDV sequence in the γC-domain of fibrinogen and/or RGD sites in the Aα chains are involved in clot retraction and adhesion, recent data demonstrated that fibrinogen lacking these sites still supported clot retraction. These findings suggested that an unknown site in fibrinogen and/or other integrins participate in clot retraction. Here we have identified a sequence within γC that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365–383 sequence in γC, designated P3, efficiently inhibited clot retraction in a dose-dependent manner. Furthermore, P3 supported platelet adhesion and was an effective inhibitor of platelet adhesion to fibrinogen fragments. Analysis of overlapping peptides spanning P3 and mutant recombinant γC-domains demonstrated that the P3 activity is contained primarily within γ370–383. Integrins αIIbβ3 and α5β1 were implicated in recognition of P3, since platelet adhesion to the peptide was blocked by function-blocking monoclonal antibodies against these receptors. Direct evidence that αIIbβ3 and α5β1 bind P3 was obtained by selective capture of these integrins from platelet lysates using a P3 affinity matrix. Thus, these data suggest that the P3 sequence in the γC-domain of fibrinogen defines a previously unknown recognition specificity of αIIbβ3 and α5β1 and may function as a binding site for these integrins. The interactions of platelets with fibrinogen mediate a variety of responses including adhesion, platelet aggregation, and fibrin clot retraction. Whereas it was assumed that interactions of the platelet integrin αIIbβ3 with the AGDV sequence in the γC-domain of fibrinogen and/or RGD sites in the Aα chains are involved in clot retraction and adhesion, recent data demonstrated that fibrinogen lacking these sites still supported clot retraction. These findings suggested that an unknown site in fibrinogen and/or other integrins participate in clot retraction. Here we have identified a sequence within γC that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365–383 sequence in γC, designated P3, efficiently inhibited clot retraction in a dose-dependent manner. Furthermore, P3 supported platelet adhesion and was an effective inhibitor of platelet adhesion to fibrinogen fragments. Analysis of overlapping peptides spanning P3 and mutant recombinant γC-domains demonstrated that the P3 activity is contained primarily within γ370–383. Integrins αIIbβ3 and α5β1 were implicated in recognition of P3, since platelet adhesion to the peptide was blocked by function-blocking monoclonal antibodies against these receptors. Direct evidence that αIIbβ3 and α5β1 bind P3 was obtained by selective capture of these integrins from platelet lysates using a P3 affinity matrix. Thus, these data suggest that the P3 sequence in the γC-domain of fibrinogen defines a previously unknown recognition specificity of αIIbβ3 and α5β1 and may function as a binding site for these integrins. The process of thrombus formation upon vascular injury is a complex series of events that involves platelets and plasma proteins, including fibrinogen (Fg). 1The abbreviations used are: Fg, human fibrinogen; γC, globular COOH-terminal domain of the γ-chain of Fg; mAb, monoclonal antibody; CHO, Chinese hamster ovary; BSA, bovine serum albumin. Adhesive reactions of platelets with Fg are required for platelet aggregation, which triggers subsequent formation of a blood clot composed of insoluble fibrin and captured platelets. The interactions of platelets with fibrin within platelet-rich thrombi result in clot retraction, which is visually manifested in a dramatic reduction in fibrin gel volume. The mechanism and physiological significance of platelet-mediated fibrin clot retraction remain poorly understood, but it has been suggested that contraction of fibrin clots may be required for clearance of the thrombus and also may facilitate wound healing. The primary interactions of platelets with Fg and fibrin are mediated by the platelet-specific receptor αIIbβ3 (glycoprotein IIbIIIa), a member of the integrin family of receptors. αIIbβ3 is the most abundant integrin on the platelet surface and is expressed at ∼80,000 copies/cell (1Peerschke E.I.B. Lopez J.A. Loscalzo J. Schafer A.I. Thrombosis and Hemorrhage. Williams & Wilkins, Baltimore1998: 229-260Google Scholar). Numerous studies using synthetic peptides and function-blocking antibodies have demonstrated that three sites in Fg can potentially interact with αIIbβ3 upon platelet adhesion and aggregation (1Peerschke E.I.B. Lopez J.A. Loscalzo J. Schafer A.I. Thrombosis and Hemorrhage. Williams & Wilkins, Baltimore1998: 229-260Google Scholar). Because Fg consists of two identical disulfide-bonded subunits, each of which is formed by three polypeptide chains (Aα, Bβ, and γ), two copies of αIIbβ3-binding sites may reside in each subunit. They are the RGDX sequences at 95–97 and 572–575 in the Aα-chains and AGDV in the carboxyl-terminal ends of the γ-chains, γ408–411. The RGDF sequence at Aα 95–97 is cryptic and, therefore, apparently not involved in the initial binding of soluble Fg to platelets (2Ugarova T.P. Budzynski A.Z. Shattil S.J. Ruggeri Z.M. Ginsberg M.H. Plow E.F. J. Biol. Chem. 1993; 268: 21080-21087Abstract Full Text PDF PubMed Google Scholar). Direct observation of the complex between Fg and purified αIIbβ3 by electron microscopy indicated that two globular γC-domains that are formed by the carboxyl-terminal parts of the γ-chains of Fg and that contain AGDV are the primary sites for interactions with the receptor (3Weisel J.W. Nagaswami C. Vilaire G. Bennett J.S. J. Biol. Chem. 1992; 267: 16637-16643Abstract Full Text PDF PubMed Google Scholar). This conclusion has also been supported by experiments with recombinant Fg in which mutation of AGDV in γC resulted in the loss of platelet aggregation, whereas mutations of both RGD sites in the Aα chain had no effect (4Farrell D.H. Thiagarajan P. Chung D.W. Davie E.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10729-10732Crossref PubMed Scopus (301) Google Scholar, 5Farrell D.H. Thiagarajan P. J. Biol. Chem. 1994; 269: 226-231Abstract Full Text PDF PubMed Google Scholar). Several previous reports have demonstrated that αIIbβ3 plays an important role in platelet-mediated clot retraction. Platelets isolated from patients with Glanzmann's thrombasthenia, a bleeding disorder in which αIIbβ3 is dysfunctional or absent, were defective in clot retraction (6Nurden A.T. Thromb. Haemostasis. 2001; 82: 468-480Google Scholar). Furthermore, monoclonal antibodies directed against αIIbβ3 and Fg recognition peptides, which inhibit Fg binding to platelets and platelet aggregation, blocked clot retraction (7Gartner T.K. Ogilvie M.L. Thromb. Res. 1988; 49: 43-53Abstract Full Text PDF PubMed Scopus (19) Google Scholar, 8Katagiri Y. Hiroyama T. Akamatsu N. Suzuki H. Yamazaki H. Tanoue K. J. Biol. Chem. 1995; 270: 1785-1790Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 9Braaten J.V. Jerome W.G. Hantgan R. Blood. 1994; 83: 982-993Crossref PubMed Google Scholar, 10Osdoit S. Rosa J.-P. J. Biol. Chem. 2001; 276: 6703-6710Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). However, in contrast to platelet aggregation, the AGDV sequence in the γC-domain is not absolutely required for clot retraction. Recombinant human Fg, which lacks AGDV sequences, did not support platelet aggregation but still supported normal clot retraction that was indistinguishable from retraction mediated by normal recombinant or plasma Fg (11Rooney M.M. Parise L.V. Lord S.T. J. Biol. Chem. 1996; 271: 8553-8555Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In addition, mice in which the γ-chain gene was targeted to eliminate the C terminus of the γ-chain of Fg manifested bleeding associated with impaired platelet aggregation, but clot retraction was normal (12Holmback K. Danton M.J. Suh T.T. Daugherty C.C. Degen J.L. EMBO J. 1996; 15: 5760-5771Crossref PubMed Scopus (125) Google Scholar). These results suggested that the sites in Fg that are required for platelet aggregation differ from the sites that are required for clot retraction. Therefore, it was proposed that RGD sites in the Aα-chains can mediate clot retraction (13Rooney M.M. Farrell D.H. Van Hemel B.M. de Groot P.G. Lord S.T. Blood. 1998; 92: 2374-2381Crossref PubMed Google Scholar). However, when this hypothesis was tested directly, using recombinant Fg in which RGDs were mutated, this mutant Fg exhibited normal clot retraction (13Rooney M.M. Farrell D.H. Van Hemel B.M. de Groot P.G. Lord S.T. Blood. 1998; 92: 2374-2381Crossref PubMed Google Scholar). It is noteworthy that when two RGD sites and AGDV in the γC-domain were all mutated, only the rate of clot retraction mediated by Fg containing a triple mutation was delayed, whereas the final extent of clot retraction was similar to that produced by wild-type recombinant Fg (13Rooney M.M. Farrell D.H. Van Hemel B.M. de Groot P.G. Lord S.T. Blood. 1998; 92: 2374-2381Crossref PubMed Google Scholar). Taken together, these findings suggested that clot retraction is a two-step process, such that AGDV sites in the γC-domains are important for initial binding to αIIbβ3 and may be involved in the initial step of clot retraction. The second step, the development of clot tension, does not depend exclusively on either AGDV or RGD sites. Thus, such a model suggests involvement of a novel binding site in Fg that is engaged by αIIbβ3 and/or other integrin(s) in the second step of clot remodeling. In this study, we have sought to localize the binding site in Fg that participates in platelet-mediated clot retraction. Guided by a lead that mAb 2G5 inhibited clot retraction, we have identified a novel recognition sequence in the γC-domain of Fg, γ370–383, and demonstrated that two platelet integrins, αIIbβ3 and α5β1, bind this sequence during clot retraction and platelet adhesion. Proteins, Peptides, and Monoclonal Antibodies—Human Fg was obtained from Enzyme Research Laboratories (South Bend, IN). The D100 (M r 100,000) and D98 (M r 100,000) fragments of Fg were prepared by digestion of Fg with plasmin (Enzyme Research Laboratories) and purified as described previously (14Ugarova T.P. Budzynski A.Z. J. Biol. Chem. 1992; 267: 13687-13693Abstract Full Text PDF PubMed Google Scholar, 15Lishko V.K. Kudryk B. Yakubenko V.P. Yee V.C. Ugarova T.P. Biochemistry. 2002; 41: 12942-12951Crossref PubMed Scopus (97) Google Scholar). Fg was labeled with 125Ibythe Chloramine T procedure. Thrombin was obtained from Enzyme Research Laboratories. The peptide duplicating the Fg sequence γ365–383, NGIIWATWKTRWYSMKKTT, a series of overlapping peptides spanning this sequence, and a scrambled γ370–383 peptide (P3′-scr) (Table I) were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry and purified by high pressure liquid chromatography on a preparative C18 Vydac column using a 5–90% linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Authenticity and purity of the peptides were verified by mass spectroscopy. In addition, the Fg peptide γ400–411 (H12) was synthesized. Peptides duplicating γ340–357, γ351–370, and γ383–395 of Fg (designated H19, H20, and P2-C, respectively) and the IIICS-1 peptide of fibronectin were previously described (16Ugarova T.P. Solovjov D.A. Zhang L. Loukinov D.I. Yee V.C. Medved L.V. Plow E.F. J. Biol. Chem. 1998; 273: 22519-22527Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 17Ugarova T. Ljubimov A.V. Deng L. Plow E.F. Biochemistry. 1996; 35: 10913-10921Crossref PubMed Scopus (37) Google Scholar).Table IKinetic parameters of clot retraction in the presence of P3 and P3-derived peptidesPeptideSequenceIC50Lag phaseaLag phase of clot retraction in the presence of the concentration of each peptide that produced 50% inhibition of retraction.V maxbV max was determined for the concentration of each peptide that produced 50% inhibition of retraction.μMminaLag phase of clot retraction in the presence of the concentration of each peptide that produced 50% inhibition of retraction.% retraction / minControlNo peptide20 ± 0.51.72 ± 0.16γ365-383NGIIWATWKTRWYSMKKTT51 ± 744 ± 41.20 ± 0.11γ365-377NGIIWATWKTRWY8 ± 2108 ± 50.94 ± 0.02γ373-383KTRWYSMKKTT272 ± 1229 ± 31.44 ± 0.05γ370-380ATWKTRWYSMK202 ± 932 ± 11.36 ± 0.06γ351-374.. NGIIWATWKT275 ± 1223 ± 21.50 ± 0.10γ377-395YSMKKTT...≥2000NDNDγ383-395TTMKIIPFNRLTIG20 ± 0.51.68 ± 0.02γ340-357HAGHLNGVYYQGSTYSKA20 ± 0.51.74 ± 0.08P3′-ScrKMTATKSWRTYTKW20 ± 0.51.70 ± 0.05a Lag phase of clot retraction in the presence of the concentration of each peptide that produced 50% inhibition of retraction.b V max was determined for the concentration of each peptide that produced 50% inhibition of retraction. Open table in a new tab The following antibodies directed to different integrin subunits were purchased from Chemicon International (Temecula, CA): anti-β1 mAb 1965 (clone JB1A), anti-β1 mAb 1957z (clone 25E11), anti-α5β1 mAb 1969 (clone JBS5), anti-α5 mAb 1956z (clone P1D6), anti-αv mAb 2021z (clone AV1), anti-αvβ3 mAb 1976z (LM609), anti-α2β1 mAb 1998 (clone BHA2.1), and polyclonal antibody to integrin α5, 1928, directed against the cytoplasmic tail. mAb CD41 against αIIbβ3 was purchased from Immunotech (Marseille, France). mAb GTI-N4P (clone AP3) against αIIbβ3 was from GTI (Brookfield, WI). Chimeric Fab 7E3 (abciximAb), which recognizes integrins αIIbβ3 and αvβ3, was a generous gift from Dr. B. Coller (Rockefeller University). mAbs 4F10 and 2G12 directed against αIIbβ3 were from Dr. V. Woods (University of California, San Diego). mAb 1413 (clone R7.1), which recognizes the αL subunit of leukocyte integrin αLβ2, mAb w6/32 directed against major histocompatibility complex class I, and purified IgG were used as controls. The anti-Fg mAbs were mAb 2G5, mAb 3G11, mAb 2F10, mAb 4-2, and mAb 4A5. mAb 2G5 was raised using human fragment DD and recognizes the Fg γ373–385 sequence (18Zamarron C. Ginsberg M.H. Plow E.F. J. Biol. Chem. 1991; 266: 16193-16199Abstract Full Text PDF PubMed Google Scholar). mAbs 3G11 and 2F10 cross-compete with mAb 2G5, suggesting that they recognize the epitopes within γCin the vicinity of γ365–383 (19Zamarron C. Ginsberg M.H. Plow E.F. Thromb. Haemostasis. 1990; 64: 41-46Crossref PubMed Scopus (82) Google Scholar). mAb 4–2 recognizes the Fg sequence γ390–402 (20Moskowitz K.A. Kudryk B. Coller B.S. Thromb. Haemostasis. 1998; 79: 824-831Crossref PubMed Scopus (45) Google Scholar). mAb 4A5 recognizes the C terminus of γC, γ406–411 (21Matsueda G.R. Bernatowicz M.S. Mosesson M.W. Amrani D. Siebenlist K.R. DiOrio P. Fibrinogen 3: Biochemistry, Biological Functions, Gene Regulation, and Expression. Elsevier Science Publishers B.V., Amsterdam1988: 133-136Google Scholar), and was a gift from Dr. G. Matsueda (Bristol-Meyers Squibb). Cells—Platelets were collected from aspirin-free human blood, anti-coagulated with acid/citrate/dextrose, and isolated by differential centrifugation followed by gel filtration on Sepharose 2B-CL. CHO cells expressing αIIbβ3 (22(1998) J. Biol. Chem., 273, 35039–35047Google Scholar) were provided by Dr. J. Fox (Cleveland Clinic). The cells were maintained in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% fetal bovine serum and 25 mm HEPES. Surface expression levels of αIIbβ3 and α5β1 on αIIbβ3-expressing and wild-type CHO cells were detected by fluorescence-activated cell sorting analysis using integrin subunit-specific mAbs. The cells were stained with mAbs and with anti-mouse IgG conjugated with Alexa-488 (Molecular Probes, Inc., Eugene, OR) and analyzed with a FACScan flow cytometer (Beckton Dickinson). The level of α5β1 in wild-type and αIIbβ3-transfectants was similar, and the level in αIIbβ3-transfectants was ∼8-fold lower than that of αIIbβ3 as assessed from the ratio of mean fluorescence intensities. Expression of Recombinant γC-domains and Mutagenesis—The recombinant γC-domains were expressed as fusion proteins with glutathione S-transferase as described previously. The coding region for the wild-type γC-domain (residues Ile145–Val411) was amplified using as template plasmids p674 (23Bolyard M.G. Lord S.T. Gene (Amst.). 1988; 66: 183-192Crossref PubMed Scopus (27) Google Scholar) consisting of full-length cDNA encoding the human Fg γ-chain that was provided by Dr. S. Lord (University of North Carolina). The primers used for the γC-domain were 5′-GGAACCTTGCAAAGACACGGGATCCATCCATGATATC-3′ (forward), 5′-CTCTTTTGAAACGGATCCTTAAACGTCTCC-3′ (reverse). The underlined region is the BamHI recognition sequence that was introduced in primers for the γC cloning. The fragments were digested and cloned in the expression vector pGEX-4T-1 (Amersham Biosciences). The accuracy of the DNA sequence was verified by sequencing. The plasmids were transformed in Escherichia coli strain BL-21(DE3)pLysS, and expression was induced by adding 0.5 mm isopropyl-1-thio-β-d-galactopyranoside for 3–4 h at 30 °C. The recombinant proteins were purified from soluble fractions of E. coli lysates by affinity chromatography using glutathione-agarose. The analyses of purified γC proteins by SDS-PAGE showed a major band migrating as expected (60 kDa) and a minor band (5–10% of the level of the major bands in different preparations) of ∼30 kDa. The intactness of the COOH-terminal end of the γC-domain was confirmed by Western blot analysis using mAb 4A5 directed against γ406–411. A series of mutants with truncations in the C-terminal part of γC were produced using the QuikChange™ mutagenesis kit (Stratagene, San Diego, CA). Fibrin Clot Retraction Assays—Whole blood was collected with informed consent from healthy volunteers and anticoagulated by adding acid/citrate/dextrose in the presence of 2.8 μm prostaglandin E1. Platelets were isolated by differential centrifugation followed by gel filtration on Sepharose 2B in divalent cation-free Tyrode's buffer, pH 7.2, containing 0.1% BSA and were resuspended in isotonic HEPES buffer (20 mm HEPES, pH 7.3, 137 mm NaCl, 2.7 mm KCl, 1 mm MgCl2, 3.3 mm NaH2PO4), containing 35 mg/ml BSA (Sigma) and 1 mg/ml glucose. The reaction mixture (total volume 1.0 ml) consisted of 3 × 108 platelets, 0.25 mg/ml Fg, 1 mm CaCl2 in glass tubes coated with Sigmacote (Sigma). Clot retraction was initiated by adding 1 unit of thrombin at 22 °C. Fibrin clot retraction triggered by activated platelets was monitored by taking photographs of clots at several time intervals using a digital camera. The images were analyzed, and the areas occupied by clots were calculated using Scion Image software. Clot retraction was expressed as a percentage of retraction defined as [1 - (area t/area t 0)] × 100, where area t 0 is the cross-sectional area occupied by fibrin clot in the absence of platelets and area t is the area occupied by the retracted clot. Thus, 0% is defined as no retraction, and 100% would be hypothetical full retraction. Maximal retraction attained in these experiments is typically ∼80–90% after ∼2 h. To characterize the process of clot retraction in the presence of inhibitors (mAbs and peptides) and to compare their potency, several parameters were derived from the kinetic curves of retraction. They are the lag phase, V max, and IC50. The lag phase is defined as the time from the onset of the process until the first visible changes in clot morphology. It was determined from the interception of the steepest part of the kinetic curve with the abscissa, which reflects the time spanned after adding thrombin. The maximal slope of the curve reflects the rate (V max) of retraction at a given concentration of the inhibitor and was measured as percentage of retraction/min. The value of IC50 is defined as the concentration of the inhibitor that produces 50% of maximal inhibition. Effect of Fg Peptides on Platelet Function, on Binding of Fg to Platelets, and on Fibrin Polymerization—To assess the effect of Fg peptides on platelet function, secretion of ATP by thrombin-activated platelets was measured using a Lumi-aggregometer (Chromo-Log Corp., Havertown, PA) according to the manufacturer's protocol. Briefly, to 0.45 ml of platelet-rich plasma, 50 μl of CHRONO-LUME reagent containing luciferin-luciferase was added, and the mixture was preincubated for 5 min at 37 °C. Different concentrations (0–150 μm) of the Fg peptides were added, and aggregation was initiated by the addition of 2 units/ml thrombin. A change in luminescence that indicates the amount of ATP released was measured in the absence or presence of peptides. 125I-Fg binding to platelets was measured with the ligand at 0.3 μm as described (24Marguerie G.A. Edgington T.S. Plow E.F. J. Biol. Chem. 1980; 255: 154-161Abstract Full Text PDF PubMed Google Scholar). The platelet-bound Fg was separated from the free ligand by centrifugation of 50-μl aliquots of the reaction mixture through 20% sucrose, and the number of Fg molecules bound was calculated based on specific activity. The effect of peptides on fibrin polymerization was assessed in fibrin polymerization assays using fibrin monomer as described (14Ugarova T.P. Budzynski A.Z. J. Biol. Chem. 1992; 267: 13687-13693Abstract Full Text PDF PubMed Google Scholar). Fibrin monomer was prepared by clotting of Fg with thrombin and dissolving the fibrin clot in 0.02 m acetic acid at 4 °C. Adhesion Assays—The wells of 96-well tissue culture plates (Costar, Cambridge, MA) were coated with different concentrations of proteins or peptides for 3 h at 37 °C or overnight at 4 °C. The coated wells were postcoated with 1% BSA inactivated at 75 °C for platelet adhesion assays or 1% polyvinyl alcohol for CHO cell assays. Platelets were labeled with 10 μm Calcein AM (Molecular Probes, Inc., Eugene, OR) for 30 min at 37 °C, washed in isotonic HEPES buffer, and resuspended at 1 × 108/ml in the same buffer supplemented with 1% BSA, 1 mm MgCl2, and 1 mm CaCl2. Calcein-labeled wild-type and the αIIbβ3-expressing CHO cells were resuspended in Dulbecco's modified Eagle's medium/F-12 medium at 1 × 105 cells/ml. Aliquots (100 μl) of cells were added to the wells and incubated at 37 °C for 50 and 30 min for platelets and CHO cells, respectively. The nonadherent cells were removed by two washes with phosphate-buffered saline, and fluorescence was measured in a fluorescence plate reader (Applied Biosystems, Framingham, MA). The number of adherent cells was determined using the fluorescence of aliquots with a known number of labeled cells. In inhibition experiments, platelets were mixed with different concentrations of peptides or mAbs for 20 min at 22 °C before they were added to the wells coated with adhesive substrates. Affinity Chromatography of Platelet Lysates—To identify the integrins that bind to γ365–383, the P3 peptide was coupled to ECH-Sepharose (Amersham Biosciences) according to the manufacturer's protocol. Platelet lysates were prepared from outdated platelets by lysing cells in 20 mm Tris, 150 mm NaCl, 1 mm CaCl2, 2 mm benzamidine, 1 mm PMSF, 10 μm leupeptin, 2% Triton X-100 reduced, pH 7.4, and applied onto the affinity matrix. The columns were washed first with buffer A (20 mm Tris, 150 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, 1 mm MnCl2, containing 0.2% Triton X-100 reduced, pH 7.4), and bound material was eluted with buffer A containing 2 mg/ml P3. Proteins strongly bound to the affinity matrix were eluted with Tris-buffered saline buffer containing 4 m urea. The samples were subjected to SDS-PAGE on 7.5% gels under nonreducing conditions followed by Western blotting using anti-integrin subunit specific and anti-Fg mAbs. Proteins in the gels were transferred to Immobilon-P membranes (Millipore Corp.), and the membranes were incubated with mAbs against αIIb (CD41, 3 μg/ml), β3 (AP3, 0.5 μg/ml), and β1 (1965, 1 μg/ml) and polyclonal anti-α5 antibody (1928) at 1:5000 dilution and anti-Fg mAb 4-2 (5 μg/ml). Bound antibodies were detected by reaction with a peroxidase-conjugated second antibody (Bio-Rad) followed by the addition of SuperSignal chemiluminescent substrate (Pierce). The integrin subunits were identified on the basis of positive staining and characteristic molecular weight. Inhibition of Platelet-mediated Fibrin Clot Retraction by mAb 2G5—Previous studies have demonstrated that mAb 2G5 inhibited agonist-induced platelet aggregation (18Zamarron C. Ginsberg M.H. Plow E.F. J. Biol. Chem. 1991; 266: 16193-16199Abstract Full Text PDF PubMed Google Scholar). This mAb recognizes the Fg sequence γ365–383 in the γC-domain and, thus, does not appear to compete with AGDV at γ408–411, the binding site for platelet integrin αIIbβ3 (18Zamarron C. Ginsberg M.H. Plow E.F. J. Biol. Chem. 1991; 266: 16193-16199Abstract Full Text PDF PubMed Google Scholar). In fact, previous data have indicated that mAb 2G5 had no effect on binding of radiolabeled Fg to stimulated platelets (18Zamarron C. Ginsberg M.H. Plow E.F. J. Biol. Chem. 1991; 266: 16193-16199Abstract Full Text PDF PubMed Google Scholar). We have further examined whether mAb 2G5 affects platelet-mediated fibrin clot retraction. The mAb inhibited clot retraction in a dose-dependent manner; 50% inhibition was attained at ∼15 μg/ml mAb, and 50 μg/ml produced complete inhibition. The potency of mAb 2G5 was similar to that of Fab 7E3, which binds platelet integrins αIIbβ3 and αVβ3 and inhibits clot retraction (10Osdoit S. Rosa J.-P. J. Biol. Chem. 2001; 276: 6703-6710Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 25Cohen I. Burk D. White J.G. Blood. 1989; 73: 1880-1887Crossref PubMed Google Scholar, 26Smith R.A. Mosesson M.W. Rooney M.M. Lord S.T. Daniels A.U. Gartner T.K. J. Biol. Chem. 1997; 272: 22080-22085Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). In addition, the effect of mAb 2G5 was similar to that of mAb 4A5 (IC50 ∼10 μg/ml) directed against the binding site for αIIbβ3 at γ408–411 (21Matsueda G.R. Bernatowicz M.S. Mosesson M.W. Amrani D. Siebenlist K.R. DiOrio P. Fibrinogen 3: Biochemistry, Biological Functions, Gene Regulation, and Expression. Elsevier Science Publishers B.V., Amsterdam1988: 133-136Google Scholar), which inhibits platelet adhesion (27Gartner T.K. Amrani D.L. Derrick J.M. Kirschbaum N.E. Matsueda G.R. Taylor D.B. Thromb. Res. 1993; 71: 47-60Abstract Full Text PDF PubMed Scopus (49) Google Scholar) and clot retraction (26Smith R.A. Mosesson M.W. Rooney M.M. Lord S.T. Daniels A.U. Gartner T.K. J. Biol. Chem. 1997; 272: 22080-22085Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Two other anti-Fg mAbs, 3G11 and 2F10, which have specificity overlapping with that of mAb 2G5 (19Zamarron C. Ginsberg M.H. Plow E.F. Thromb. Haemostasis. 1990; 64: 41-46Crossref PubMed Scopus (82) Google Scholar) also efficiently blocked clot retraction. Effect of γ365–383 on Clot Retraction—Based on the recognition specificity of mAb 2G5, we hypothesized that a peptide duplicating its epitope might block clot retraction. Accordingly, we synthesized a peptide, corresponding to γ365–383 (designated P3), and tested its ability to inhibit clot retraction. Fig. 1A shows that P3 was a strong inhibitor of retraction. Increasing concentrations of peptide progressively blocked retraction; at 300 μg/ml, the process was inhibited completely, and fibrin clots did not retract after 24 h. The effect of P3 on clot retraction was characterized in detail by using a sensitive assay in which temperature was decreased to 22 °C, which retarded the process of retraction and allowed accurate quantification of several kinetic parameters, including the lag phase, V max, and IC50 (see "Experimental Procedures"). Fig. 1B shows the rate of clot retraction in the presence of different concentrations of P3. The lag phase, V max, and IC50 values were calculated from the progress curves of retractions (Table I). The IC50 value, defined as the concentration of peptide that produced 50% of maximal clot retraction after 2–3 h, was 51 ± 7 μm (Fig. 1C). The specificity of the P3 effect was verified by testing several control peptides. Fg peptides corresponding to sequences flanking P3 (γ365–383), H19 (γ340–357), and P2-C (γ383–395) and the peptide duplicating the IIICS-1 sequence in fibronectin did not inhibit clot retraction. In addition, a scrambled P3′ peptide was completely inactive. As other essential controls, 1) the P3 peptide did not inhibit fibrin polymerization and did not change the fibrin clot morphology in the absence of platelets; 2) platelet function was not affected by P3, as tested by the ATP release reaction; and 3) P3 did not inhibit the binding of soluble Fg to activated platelets, as determined by using 125I-Fg. The last finding is consistent with the previous data indicating that mAb 2G5 did not inhibit Fg binding to stimulated platelets (18Zamarron C. Ginsberg M.H. Plow E.F. J. Biol. Chem. 1991; 266: 16193-16199Abstract Full Text PDF PubMed Google Scholar). To define the active determinants within P3, several overlapping peptides spanning P3 and its flanking regions were synthesized (Table I) and tested for their ability to inhibit clot retraction. Kinetic parameters derived from progression curves of clot retraction in the presence of different peptides allowed the comparison of peptide potencies. Shown in Table I are the concentrations of each peptide that produced 50% inhibition of the lag phase and V max of clot retraction. Peptides derived from the NH2-terminal and central parts of P3 were the most active inhibitors. In fact, γ365–377 was more active than the parental P3. The reason for the enha
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