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J. Biol. Chem., Vol. 278, Issue 34, 32251-32258, August 22, 2003

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Identification of a Novel Binding Site for Platelet Integrins _IIb3 (GPIIbIIIa) and ₅₁ in the C-domain of Fibrinogen^*

Nataly P. Podolnikova  , Valentin P. Yakubenko , George L. Volkov , Edward F. Plow  and Tatiana P. Ugarova  ¶

From the Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Lerner Research Institute, Cleveland, Ohio 44195 and the Palladin Institute of Biochemistry, Kiev 01601, Ukraine

Received for publication, January 14, 2003 , and in revised form, April 30, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 _IIb3 with the AGDV sequence in the C-domain of fibrinogen and/or RGD sites in the A 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 C that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365–383 sequence in C, 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 C-domains demonstrated that the P3 activity is contained primarily within 370–383. Integrins _IIb3 and ₅₁ 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 _IIb3 and ₅₁ 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 C-domain of fibrinogen defines a previously unknown recognition specificity of _IIb3 and ₅₁ and may function as a binding site for these integrins.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The process of thrombus formation upon vascular injury is a complex series of events that involves platelets and plasma proteins, including fibrinogen (Fg).¹ 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 _IIb3 (glycoprotein IIbIIIa), a member of the integrin family of receptors. _IIb3 is the most abundant integrin on the platelet surface and is expressed at 80,000 copies/cell (1). Numerous studies using synthetic peptides and function-blocking antibodies have demonstrated that three sites in Fg can potentially interact with _IIb3 upon platelet adhesion and aggregation (1). Because Fg consists of two identical disulfide-bonded subunits, each of which is formed by three polypeptide chains (A, B, and ), two copies of _IIb3-binding sites may reside in each subunit. They are the RGDX sequences at 95–97 and 572–575 in the A-chains and AGDV in the carboxyl-terminal ends of the -chains, 408–411. The RGDF sequence at A 95–97 is cryptic and, therefore, apparently not involved in the initial binding of soluble Fg to platelets (2). Direct observation of the complex between Fg and purified _IIb3 by electron microscopy indicated that two globular C-domains that are formed by the carboxyl-terminal parts of the -chains of Fg and that contain AGDV are the primary sites for interactions with the receptor (3). This conclusion has also been supported by experiments with recombinant Fg in which mutation of AGDV in C resulted in the loss of platelet aggregation, whereas mutations of both RGD sites in the A chain had no effect (4, 5).

Several previous reports have demonstrated that _IIb3 plays an important role in platelet-mediated clot retraction. Platelets isolated from patients with Glanzmann's thrombasthenia, a bleeding disorder in which _IIb3 is dysfunctional or absent, were defective in clot retraction (6). Furthermore, monoclonal antibodies directed against _IIb3 and Fg recognition peptides, which inhibit Fg binding to platelets and platelet aggregation, blocked clot retraction (7–10). However, in contrast to platelet aggregation, the AGDV sequence in the C-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 (11). In addition, mice in which the -chain gene was targeted to eliminate the C terminus of the -chain of Fg manifested bleeding associated with impaired platelet aggregation, but clot retraction was normal (12). 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 A-chains can mediate clot retraction (13). However, when this hypothesis was tested directly, using recombinant Fg in which RGDs were mutated, this mutant Fg exhibited normal clot retraction (13). It is noteworthy that when two RGD sites and AGDV in the C-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 (13). Taken together, these findings suggested that clot retraction is a two-step process, such that AGDV sites in the C-domains are important for initial binding to _IIb3 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 _IIb3 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 C-domain of Fg, 370–383, and demonstrated that two platelet integrins, _IIb3 and ₅₁, bind this sequence during clot retraction and platelet adhesion.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (14, 15). Fg was labeled with ¹²⁵Ibythe Chloramine T procedure. Thrombin was obtained from Enzyme Research Laboratories. The peptide duplicating the Fg sequence 365–383, NGIIWATWKTRWYSMKKTT, a series of overlapping peptides spanning this sequence, and a scrambled 370–383 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 400–411 (H12) was synthesized. Peptides duplicating 340–357, 351–370, and 383–395 of Fg (designated H19, H20, and P2-C, respectively) and the IIICS-1 peptide of fibronectin were previously described (16, 17).

The following antibodies directed to different integrin subunits were purchased from Chemicon International (Temecula, CA): anti-₁ mAb 1965 (clone JB1A), anti-₁ mAb 1957z (clone 25E11), anti-₅₁ mAb 1969 (clone JBS5), anti-₅ mAb 1956z (clone P1D6), anti-_v mAb 2021z (clone AV1), anti-_v3 mAb 1976z (LM609), anti-₂₁ mAb 1998 (clone BHA2.1), and polyclonal antibody to integrin ₅, 1928, directed against the cytoplasmic tail. mAb CD41 against _IIb3 was purchased from Immunotech (Marseille, France). mAb GTI-N4P (clone AP3) against _IIb3 was from GTI (Brookfield, WI). Chimeric Fab 7E3 (abciximAb), which recognizes integrins _IIb3 and _v3, was a generous gift from Dr. B. Coller (Rockefeller University). mAbs 4F10 and 2G12 directed against _IIb3 were from Dr. V. Woods (University of California, San Diego). mAb 1413 (clone R7.1), which recognizes the _L subunit of leukocyte integrin _L2, 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 373–385 sequence (18). mAbs 3G11 and 2F10 cross-compete with mAb 2G5, suggesting that they recognize the epitopes within Cin the vicinity of 365–383 (19). mAb 4–2 recognizes the Fg sequence 390–402 (20). mAb 4A5 recognizes the C terminus of C, 406–411 (21), 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 _IIb3 (22) 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 _IIb3 and ₅₁ on _IIb3-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 ₅₁ in wild-type and _IIb3-transfectants was similar, and the level in _IIb3-transfectants was 8-fold lower than that of _IIb3 as assessed from the ratio of mean fluorescence intensities.

Expression of Recombinant C-domains and Mutagenesis—The recombinant C-domains were expressed as fusion proteins with glutathione S-transferase as described previously. The coding region for the wild-type C-domain (residues Ile¹⁴⁵–Val⁴¹¹) was amplified using as template plasmids p674 (23) consisting of full-length cDNA encoding the human Fg -chain that was provided by Dr. S. Lord (University of North Carolina). The primers used for the C-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 C 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--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 C 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 C-domain was confirmed by Western blot analysis using mAb 4A5 directed against 406–411. A series of mutants with truncations in the C-terminal part of C were produced using the QuikChange^TM 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 x 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 t₀)] x 100, where area t₀ 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 (24). 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 (14). 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 x 10⁸/ml in the same buffer supplemented with 1% BSA, 1 mM MgCl₂, and 1 mM CaCl₂. Calcein-labeled wild-type and the _IIb3-expressing CHO cells were resuspended in Dulbecco's modified Eagle's medium/F-12 medium at 1 x 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 365–383, 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 _IIb (CD41, 3 µg/ml), ₃ (AP3, 0.5 µg/ml), and ₁ (1965, 1 µg/ml) and polyclonal anti-₅ 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Inhibition of Platelet-mediated Fibrin Clot Retraction by mAb 2G5—Previous studies have demonstrated that mAb 2G5 inhibited agonist-induced platelet aggregation (18). This mAb recognizes the Fg sequence 365–383 in the C-domain and, thus, does not appear to compete with AGDV at 408–411, the binding site for platelet integrin _IIb3 (18). In fact, previous data have indicated that mAb 2G5 had no effect on binding of radiolabeled Fg to stimulated platelets (18). 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 _IIb3 and _V3 and inhibits clot retraction (10, 25, 26). In addition, the effect of mAb 2G5 was similar to that of mAb 4A5 (IC₅₀ 10 µg/ml) directed against the binding site for _IIb3 at 408–411 (21), which inhibits platelet adhesion (27) and clot retraction (26). Two other anti-Fg mAbs, 3G11 and 2F10, which have specificity overlapping with that of mAb 2G5 (19) also efficiently blocked clot retraction.

Effect of 365–383 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 365–383 (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 (365–383), H19 (340–357), and P2-C (383–395) 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 (18).

View larger version (25K): [in this window] [in a new window]   FIG. 1. Effect of the P3 peptide on platelet-mediated fibrin clot retraction. A, platelets were mixed with 0.25 mg/ml Fg in isotonic HEPES buffer containing 1 mM CaCl2 and different concentrations of P3. Fibrin clots were formed by adding of 1 unit/ml thrombin at 22 °C. Clot retraction was observed by taking photographs at different times (0–120 min). The left lane of tubes (0) shows clot retraction in the absence of P3 (control). The tubes in each lane (from 1 to 6) contain increasing concentrations of P3 (20, 50, 100, 200, 300, and 400 µg/ml). B, kinetics of clot retraction. Clot areas in each tube were measured from images presented in A, and a percentage of clot retraction was calculated. Kinetic curves of retraction in the presence of different concentrations of P3 were generated by plotting clot areas versus time. A representative experiment is shown. C, dose dependence of P3 effect on clot retraction. Clot retraction in the presence of selected concentrations of P3 was determined. The percentage of clot retraction was measured at 160 min. At this time, clot retraction in control (in the absence of P3) was complete, and clot retraction in the presence of P3 was at different stages of completion depending on the peptide concentration.

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, 365–377 was more active than the parental P3. The reason for the enhanced inhibitory activity of 365–377 is not clear. Truncation of the NH₂-terminal part of P3 resulted in a 4-fold decrease of the inhibitory activities of 370–380 and 373–383 (IC₅₀ = 202 ± 32 and 272 ± 12 µM, respectively) compared with 51 ± 7 µM for P3. The activity of 377–395, which spans the C-terminal part of P3, was low to the point that its kinetic parameters could not be estimated. Based on these results, the majority of P3 activity is probably contained within 365–377, although the C-terminal part, 377–383, may contribute to function.

P3 Directly Supports Platelet Adhesion—To test the possibility that P3 inhibits platelet-mediated fibrin clot retraction by directly interacting with platelets, we have examined whether P3 within the C-domain of Fg can support platelet adhesion. In these experiments, the D₉₈ fragment of Fg, which lacks the binding site for _IIb3 at ⁴⁰⁸AGDV⁴¹¹ (15) but contains P3, was compared with the D₁₀₀ fragment, which possesses both -chain sequences. As shown in Fig. 2A, D₉₈ supported efficient platelet adhesion, which was about 53% that of D₁₀₀ (maximal adhesion), suggesting that the P3 sequence may contribute to adhesion. Adhesion of platelets to D₉₈ was not activation-dependent, since similar numbers of platelets adhered in the presence of ADP and epinephrine or in the absence of stimulation. This is in contrast to D₁₀₀; platelet activation increased adhesion to D₁₀₀ by 1.7-fold. P3 inhibited platelet adhesion to D₉₈ in a dose-dependent manner (Fig. 2B), but a control peptide (H19) was not active. P3 also inhibited (40%) adhesion to D₁₀₀ (not shown). Since D₁₀₀ contains 408–411, which supports adhesion via _IIb3, this may account for partial inhibition of adhesion. The ability of P3 and P3-derived peptides to directly support platelet adhesion was next tested. Peptides were coated onto the plastic at different concentrations, and the maximal adhesion for each peptide was determined (Fig. 3). All P3-based peptides supported efficient adhesion, which was comparable with or higher (in the case of P3) than that to 400–411 (peptide H12) containing ⁴⁰⁸AGDV⁴¹¹. The adhesion-promoting capacity of P3 and P3-derived peptides correlated with their abilities to inhibit platelet adhesion to D₉₈ and P3 (not shown). The Fg control peptides 340–357 and 351–374 and the scrambled P3' peptide supported platelet adhesion poorly (Fig. 3).

View larger version (14K): [in this window] [in a new window]   FIG. 2. Adhesion of platelets to Fg fragments D100 and D98. A, gel-filtered platelets were labeled with calcein in isotonic HEPES buffer supplemented with 1 mM MgCl2 and 1 mM CaCl2, and aliquots (0.1 ml of 1 x 108 cells/ml) were added to the wells coated with 20 µg/ml D100 and D98 fragments. Cells that were not stimulated (black bars) or that were activated with 10 µM each ADP and epinephrine (white bars) were examined. The nonadherent cells were removed by two washes with phosphate-buffered saline, and fluorescence was measured. Data are expressed as a percentage of added cells. The result shown is representative of five independent experiments. B, inhibition of platelet adhesion to D98 by P3. Calcein-labeled platelets were incubated with different concentrations of P3 (•) or control H19 () peptides for 20 min at 22 °C. Data are expressed as a percentage of control adhesion (in the absence of peptides) and are the mean ± S.D. of six individual experiments performed with triplicate determinations in each experiment.

View larger version (19K): [in this window] [in a new window]   FIG. 3. Adhesion of platelets to P3 and P3-derived peptides. Aliquots (1 x 107) of calcein-labeled platelets in isotonic HEPES buffer containing 1 mM MgCl2 and 1 mM CaCl2 were added to the wells coated with 20 µg/ml of D100, D98, P3, P3-derived peptides, H12 (400–411), H19 (340–357), or P3'-scr. Platelet adhesion was quantitated as above. Results are expressed as a percentage of added cells and are the mean ± S.D. of three or four individual experiments.

The capacity of P3 to function as a platelet binding site was verified by using the recombinant C-domains in which portions of P3 were deleted. The C-domain without the C-terminal part 391–411 supported 62 ± 7.3% of the adhesion of nonstimulated platelets compared with that of wild-type C (Fig. 4). The sequential truncation of C resulted in a further decline in the ability of C(377–411) to support platelet adhesion, and C(373–411) and C(370–411) mutants were completely inactive.

View larger version (13K): [in this window] [in a new window]   FIG. 4. Adhesion of platelets to recombinant wild-type and mutant C-domains. Calcein-labeled platelets were added to wells coated with increasing concentrations of recombinant C-domains. Adhesion was performed as described under "Experimental Procedures." Maximal adhesion to 10 µg/ml of each recombinant C-domain is shown. Results are expressed as the percentage of cell adhesion to wild-type C.

Function-blocking mAbs to _IIb3 and ₅₁ Inhibit Platelet Adhesion to P3—The possibility that P3 supports adhesion by interacting with platelet integrins was examined. Several function-blocking mAbs directed to integrins expressed on platelets were tested. The effect of each mAb on platelet adhesion to P3 or D₉₈ was measured using increasing mAb concentrations to determine maximal inhibition. The results of inhibition of adhesion to P3 are summarized in Fig. 5. Several anti-_IIb3 mAbs, including 2G12, 4F10, and 7E3, produced 80–90% inhibition of adhesion. In addition, anti-₃ mAb AP3 also inhibited platelet adhesion to P3 (70 ± 5%). Likewise, a panel of anti-₁ and anti-₅ blocking mAbs inhibited adhesion in a dose-dependent manner and produced 60–70% inhibition. The mAb against integrin ₅₁ (clone JBS5) also was a potent inhibitor of adhesion (75% inhibition). In contrast, two anti-_v3 mAbs, mAb 2021z (clone AV1) and mAb 1976z (LM609), were not inhibitory, and a mAb against ₂₁ was not effective. Several control mAbs raised against ₂ integrins expressed on leukocytes (shown for anti-_L) and anti-major histocompatibility complex mAb w6/32 did not affect platelet adhesion to P3. Thus, these results suggest that platelets can interact with P3 via _IIb3 and ₅₁ integrins or that there is a cross-talk between the two integrins, which controls P3 recognition.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Effect of mAbs on adhesion of platelets to P3. Calcein-labeled platelets in isotonic HEPES buffer containing 1 mM MgCl2 and 1 mM CaCl2 were incubated with maximal concentrations of different mAbs for 20 min at 22 °C, and then 0.1 ml (1 x 107) of cells were added to wells coated with 20 µg/ml P3. Concentrations of each mAb producing maximal inhibition were determined in the preliminary experiments. Platelet adhesion was quantitated as above. Data are expressed as a percentage of adhesion to P3 and are the mean ± S.D. of three individual experiments.

To substantiate the findings that integrins _IIb3 and ₅₁ are responsible for recognition of P3, adhesion of wild-type CHO cells, which express ₅₁ naturally, and CHO cells stably transfected with _IIb3 was compared. As shown in Fig. 6A, both cell lines adhered to P3, albeit adhesion of the _IIb3-expressing cells was 3-fold more efficient. Adhesion of the _IIb3-CHO cells was reduced by anti-₅₁ mAb 1969 (30%) and by anti-_IIb3 mAb 7E3 (75%), indicating that _IIb3 was primarily responsible for the interaction with P3 (Fig. 6B). Adhesion of wild-type CHO cells was partially inhibited by anti-₅₁ mAb (55%), and mAb 7E3 was not effective. In parallel experiments, P3 supported strong adhesion of HEK 293 cells, which express endogenous ₅₁ (not shown).

View larger version (14K): [in this window] [in a new window]   FIG. 6. Adhesion of the IIb3-expressing and wild-type CHO cells to P3. A, calcein-labeled wild-type () and the IIb3-expressing (•) cells were added to the wells coated with different concentrations of P3. Data are expressed as a percentage of added cells and are from three separate adhesion assays, performed with triplicates at each experimental point. B, cells were pretreated with 40 µg/ml anti-IIb3 mAb 7E3 or 10 µg/ml anti-51 mAb 1969 for 15 min before the addition to the wells coated with 20 µg/ml P3. Results are expressed as a percentage of adhesion in the absence of antibodies (control).

Integrins _IIb3 and ₅₁ Bind to the P3-bound Affinity Matrix—To confirm further that P3 interacts with _IIb3 and ₅₁, affinity chromatography on a P3 affinity matrix was utilized. Platelet lysates were applied to P3-agarose, and bound material was sequentially eluted with a starting buffer, control peptide H19, 2 mg/ml P3, and 4 M urea. The eluted proteins were analyzed by SDS-PAGE and by Western blotting using mAbs specific for _IIb, ₃, ₅, and ₁ subunits and a mAb against Fg. As shown in Fig. 7A, the P3 peptide eluted proteins that migrated as two bands with molecular masses of 120 kDa (_IIb) and 104 kDa (₃). Western blot analyses of the P3-eluted material demonstrated that it contained both _IIb3 (Fig. 7B) and ₅₁ (Fig. 7C) integrin complexes. A control peptide was not effective, and no Fg was detected in the material eluted with P3, suggesting that the P3 affinity matrix did not retain platelet Fg (Fig. 7A, lane 5). Additional material, containing both _IIb3 and ₅₁, was eluted with 4 M urea. In separate experiments, the lysate of HEK 293 cells, which express endogenous ₅₁, was applied to P3-agarose, and eluted material was found to be ₅₁ (not shown). Thus, these data confirmed that both integrins _IIb3 and ₅₁ are capable of binding to P3.

View larger version (46K): [in this window] [in a new window]   FIG. 7. Affinity chromatography of platelet lysates on a P3-agarose. Platelets were lysed in a buffer containing 2% reduced Triton X-100 as described under "Experimental Procedures" and then incubated with a P3 affinity matrix. The affinity matrix was extensively washed with buffer A followed by 3 mg/ml control peptide H19, 2 mg/ml P3, and then Tris-buffered saline buffer containing 4 M urea. A, the initial lysate and proteins eluted with P3 and urea were subjected to SDS-PAGE and Coomassie Brilliant Blue staining (lanes 1–3) or analyzed by Western blotting (lanes 4–6) using anti-Fg mAb 4–2 (lanes 4–6). Lanes 2 and 4, initial platelet lysate; lanes 3 and 5, elution with P3; lane 6, elution with 4 M urea; lane 1, molecular weight markers. B, Western blot with anti-IIb and anti-3 mAbs. Lanes 1 and 5, initial platelet lysate; lanes 2 and 6, elution with H19; lanes 3 and 7, elution with P3; lanes 4 and 8, elution with 4 M urea. C, Western blot with anti-5 and anti-1 Abs. Lanes 1 and 3, initial platelet lysate; lane 4, elution with H19; lanes 2 and 5, elution with P3; lane 6, elution with 4 M urea. Samples for analyses with anti-5 mAb were concentrated 3-fold.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In this study, we have identified a sequence in the C-domain of Fg, 365–383, which is recognized by platelet integrins _IIb3 and ₅₁. P3 synthetic peptide duplicating this sequence inhibited platelet-mediated fibrin clot retraction and platelet adhesion. Furthermore, P3 directly supported adhesion of nonstimulated and activated platelets. Structure-function analyses of the P3-derived peptides and recombinant C-domains indicated that P3 activity is contained within 370–383. The experiments with function-blocking mAbs and affinity chromatography demonstrated that two platelet integrins, _IIb3 and ₅₁, bind P3. Thus, the P3 sequence represents a new binding motif, which may mediate the interaction of Fg with platelet integrins.

Previous data provided compelling evidence that the recognition site(s), in addition to the previously identified ⁴⁰⁸AGDV⁴¹¹ in the C-domain and RGDX sites in the A-chains of Fg, are involved in the interaction of platelets with Fg (11–13). Rooney et al. (11) demonstrated that recombinant Fg with AGDV deleted supported normal clot retraction. Recently, the same group reported that the Fg variant in which all three putative _IIb3 binding sites were mutated was still capable of mediating clot retraction (13). Although the initiation of clot retraction mediated by the triple Fg mutant was slightly delayed, the rate of retraction and the final clot size were the same as a clot formed with normal recombinant Fg. Based on these data, it was proposed that clot retraction is a two-step process, such that one or more of the three putative platelet binding sites is important for the initial step in clot retraction but not for the subsequent step. The second step of clot retraction, the development of clot tension, requires the involvement of a novel site. The findings of the present study are consistent with the possibility that the 365–383 sequence is the second putative binding site in the C-domain that contributes to the interaction of Fg with platelets during clot retraction.

Another important finding of the present study is that the P3 sequence is able to mediate platelet adhesion. Previous studies have demonstrated the essential role of the C-terminal AGDV sequences in C in platelet adhesion; deletion of this sequence in recombinant Fg or replacement with the 20-residue sequence occurring in the '-chain of a human Fg variant impaired platelet adhesion under static (5) and flow (28) conditions. However, the removal of AGDV did not eliminate adhesion completely (5, 29), and the binding of purified _IIb3 to Fg-agarose was not inhibited by 400–411 (H12) or RGDS peptides (30), suggesting that other sites in Fg interact with platelets. The ability of the D₉₈ fragment and recombinant C(391–411) mutant, which lack AGDV, to support adhesion; the inhibition of platelet adhesion by P3; and the inhibition of platelet adhesion to P3 by function blocking anti-_IIb3 are all consistent with a model in which P3 is a second site within C that interacts with _IIb3 on platelets. Furthermore, since deletion of 372–411 in C resulted in the complete loss of platelet adhesion, these data suggest that AGDV and P3 are responsible for the full adhesive function of C.

The relationship between AGDV and P3 in binding of Fg to _IIb3 remains to be determined. The contribution of P3 and AGDV to various platelet adhesive reactions appears to be different. Adhesion of platelets to Fg depends on AGDV, especially that of activated platelets and under conditions of flow (5, 28). Consistent with this idea, our data demonstrated that platelets adhered to D₁₀₀ better than to D₉₈. In addition, adhesion to D₁₀₀ was activation-dependent, whereas adhesion to D₉₈ was not, suggesting that the binding of the two sequences is differentially regulated. On the other hand, AGDV does not appear to have a critical role in fibrin clot retraction. Therefore, it is possible that the interaction of _IIb3 with P3 within fibrin might be responsible for the development of the tensile force, which leads to clot retraction. Notably, differences in the binding of platelets to Fg and fibrin have been reported (31). Further experiments involving mutational analyses of recombinant Fg should provide insights into the contributions of the two sequences to clot retraction and adhesion.

Numerous studies have suggested that the binding of Fg to _IIb3 involves multiple contacts in each integrin subunit (32–38). The presence of two recognition sequences in close proximity to each other within the C-terminal part of C suggests that they could contribute to a ligand binding interface between C and _IIb3. Although the crystal structure of C is solved (39, 40), the exact spatial relationship between ⁴⁰⁸AGDV⁴¹¹ and P3 is not clear. Within C, P3 is part of the C-terminal subdomain and forms an extended loop (Fig. 8). The N-terminal part of P3, 365–370, is hidden within the fibrin polymerization cavity and does not appear to be accessible for interaction with receptor, whereas the remaining region, 370–383, is exposed. The conformation of the C-terminal 402–411 segment was not determined, and the preceding 392–402 segment was observed in different conformations (39). Previous studies have demonstrated that the entire 392–411 segment is flexible (41–43), and some observations are consistent with the possibility that 400–411 may fold back such that P3 and AGDV would come in proximity (44).² Also, in electron micrographs (3), purified _IIb3 interacted closely with C, which would not have been possible were 392–411 an extended structure with ⁴⁰⁸AGDV⁴¹¹, being 19 residues (66 Å) away from the core of C. The transformation of Fg to fibrin might change the relationships between C and its C-terminal part. However, the nature of these alterations is not known, and further structural studies will be required to determine the conformation of the C-terminal tail in C and its relationship with P3.

View larger version (61K): [in this window] [in a new window]   FIG. 8. Positioning of 365–383 in the three-dimensional structure of C. The ribbon diagram of the Fg C-domain (144–411) is based upon its crystal structure (39), Protein Data Bank identifier 1FIC [PDB] . 365–383 is colored in green. The 365–370 segment is located within the fibrin polymerization cavity and is not accessible for interaction with receptors. The position of 393–403 was not solved definitively in the crystal of C and is shown in one of its possible conformations (39). The 404–411 segment was absent in the crystal and is shown as a dashed line in one possible conformation.

This study has revealed that integrin ₅₁ binds P3 and is involved in platelet adhesion. Thus, the P3 sequence defines a new recognition specificity for ₅₁. Previous studies have demonstrated that ₅₁ binds ligands mainly through the RGD-dependent specificity (45), and the RGD-inhibitable binding of Fg to ₅₁ on endothelial cells has been shown (46). ₅₁ on other cells has also been reported to bind Fg (47–49). However, the role of ₅₁ in platelet adhesive reactions with Fg is not clear, since this integrin is present on the platelet surface at a considerably lower density than _IIb3. One possibility is that ₅₁ is not required during the initial stages of Fg binding to platelets but instead can cooperate with _IIb3 in engaging P3 within fibrin during the advanced stages of clot tension. On the other hand, ₅₁ can potentially play an important role in clot retraction mediated by nucleated cells. Fibroblasts, endothelial cells, smooth muscle cells, and tumor cells express this integrin abundantly and are all known to interact with a fibrin matrix and induce the retraction of fibrin clots (8, 26, 50–52). Previous data have demonstrated that, similar to platelets, recombinant Fg lacking RGD residues supported endothelial cell-mediated clot retraction and adhesion (26, 53). These data would be consistent with the presence of an additional site on fibrin that is involved in retraction mediated by nucleated cells. Whether P3 binds ₅₁ and/or other integrins in these cells during clot retraction and adhesion remains to be determined.

In summary, we have identified the sequence 370–383, which together with ⁴⁰⁸AGDV⁴¹¹ accounts for the full recognition of the C-domain of Fg by platelet integrin _IIb3. Furthermore, we show that 370–383 is a binding site for platelet integrin ₅₁. Three questions then remain. How and when does _IIb3 engage the two C sequences? What postfibrinogen binding events do they trigger? What is the role of ₅₁ in thrombus formation and remodeling? Synthesis of recombinant Fg molecular with selected specificities provides the approach to answer these questions.

	FOOTNOTES

 
^* This work was supported by the American Heart Association Established Investigator Award (to T. P. U.), National Institutes of Health Grants HL 63199 (to T. P. U.) and HL 54924 (to E. F. P.), and a predoctoral fellowship (to N. P. P.) from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Cleveland Clinic, 9500 Euclid Ave., Mail Code NB-50, Cleveland, OH 44195. Tel.: 216-445-8209; Fax: 216-445-8204; E-mail: ugarovt{at}ccf.org.

¹ The abbreviations used are: Fg, human fibrinogen; C, globular COOH-terminal domain of the -chain of Fg; mAb, monoclonal antibody; CHO, Chinese hamster ovary; BSA, bovine serum albumin.

² N. P. Podolnikova, V. P. Yakubenko, G. L. Volkov, E. F. Plow, and T. P. Ugarova, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Dr. S. Lord for providing cDNA for the - and -chains of fibrinogen, Dr. B. Coller for Fab 7E3, Dr. G. Matsueda for mAb 4A5, and Dr. J. Fox for providing the _IIb3-expressing cells. We thank Dr. V. Yee for useful discussions of the C structure and Tim Burke for critical reading of the manuscript.

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