Recognition of distinct adhesive sites on fibrinogen by related integrins on platelets and endothelial cells

Endothelial cells and activated platelets express integrin-type receptors responsible for adhesion to fibrinogen. We have located distinct integrin-directed endothelial cell and platelet attachment sites on immobilized fibrinogen using a combination of synthetic peptides, fibrinogen fragments, and specific anti-peptide monoclonal antibodies. Endothelial cells exclusively recognize an Arg-Gly-Asp-containing site near the C-terminus of the alpha chain (alpha residues 572-574) but fail to recognize the Arg-Gly-Asp sequence in the N-terminal region of the same chain (alpha residues 95-97). In contrast, platelets do not require either Arg-Gly-Asp sequence for binding to intact fibrinogen and are capable of recognizing, in addition to the alpha 572-574 sequence, a site at the C-terminus of the gamma chain (gamma residues 400-411). These data suggest a molecular mechanism whereby platelets and endothelial cells interact with distinct sites on the fibrinogen molecule during hemostasis and wound healing.     

Identification of a novel binding site for platelet integrins alpha IIb beta 3 (GPIIbIIIa) and alpha 5 beta 1 in the gamma C-domain of fibrinogen

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 alpha IIb beta 3 with the AGDV sequence in the gamma C-domain of fibrinogen and/or RGD sites in the A alpha 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 gamma C that mediates binding of fibrinogen to platelets. Synthetic peptide duplicating the 365-383 sequence in gamma 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 gamma C-domains demonstrated that the P3 activity is contained primarily within gamma 370-383. Integrins alpha IIb beta 3 and alpha 5 beta 1 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 alpha IIb beta 3 and alpha 5 beta 1 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 gamma C-domain of fibrinogen defines a previously unknown recognition specificity of alpha IIb beta 3 and alpha 5 beta 1 and may function as a binding site for these integrins.     

Affinity modulation of the alpha IIb beta 3 integrin (platelet GPIIb-IIIa) is an intrinsic property of the receptor.

To analyze the basis of affinity modulation of integrin function, we studied cloned stable Chinese hamster ovary cell lines expressing recombinant integrins of the beta 3 family (alpha IIb beta 3 and alpha v beta 3). Antigenic and peptide recognition specificities of the recombinant receptors resembled those of the native receptors found in platelets or endothelial cells. The alpha IIb beta 3-expressing cell line (A5) bound RGD peptides and immobilized fibrinogen (Fg) but not soluble fibrinogen or the activation-specific monoclonal anti-alpha IIb beta 3 (PAC1), indicating that it was in the affinity state found on resting platelets. Several platelet agonists failed to alter the affinity state of ("activate") recombinant alpha IIb beta 3. The binding of soluble Fg and PAC1, however, was stimulated in both platelets and A5 cells by addition of IgG papain-digestion products (Fab) fragments of certain beta 3-specific monoclonal antibodies. These antibodies stimulated PAC1 binding to platelets fixed under conditions rendering them unresponsive to other agonists. Addition of these antibodies to detergent-solubilized alpha IIb beta 3 also stimulated specific Fg binding. These data demonstrate that certain anti-beta 3 antibodies activate alpha IIb beta 3 by acting directly on the receptor, possibly by altering its conformation. Furthermore, they indicate that the activation state of alpha IIb beta 3 is a property of the receptor itself rather than of the surrounding cell membrane microenvironment.     

Chemical cross-linking of arginyl-glycyl-aspartic acid peptides to an adhesion receptor on platelets

A chemical cross-linking approach has been used to characterize the interaction of platelets with small peptides of 7 and 14 residues containing the arginyl-glycyl-aspartic acid (RGD) sequence recognized by a variety of cellular adhesion receptors. The radioiodinated peptides were bound to platelets, and chemical cross-linking was attained by subsequent addition of bifunctional reagents. Three different cross-linking reagents coupled the RGD-containing peptides to platelet membrane glycoprotein IIb-IIIa (GPIIb-IIIa), and both subunits of this platelet membrane glycoprotein became radiolabeled with the RGD peptides. Platelet stimulation with agonists including thrombin, phorbol myristrate acetate, and ADP increased the extent of cross-linking by predominantly enhancing the coupling of the RGD peptides to the GPIIIa subunit. Cross-linking of the labeled RGD peptides to GPIIb and GPIIIa on stimulated and nonstimulated platelets exhibited structural specificity and was inhibited by excess nonlabeled RGD peptides. The interactions were inhibited by nonlabeled RGD peptides and a peptide with an amino acid sequence corresponding to the carboxyl terminus of the gamma chain of fibrinogen but less effectively by an arginyl-glycyl-glutamic acid peptide. Cross-linking of the RGD peptides to GPIIb-IIIa was divalent ion-dependent and, on stimulated platelets, was inhibited by the adhesive proteins fibrinogen and fibronectin, but not by albumin. These results indicate that the RGD-binding sites on platelets reside in close proximity to both subunits of GPIIb-IIIa and that platelet stimulation alters the topography of these sites such that the peptides become more efficiently cross-linked to GPIIIa.     

Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIbIIIa

Platelet membrane GPIIbIIIa is a member of the family receptors named integrins that recognize RGD sequences in their ligands. GPIIbIIIa interacts with at least three different adhesive ligands: fibrinogen, fibronectin, and von Willebrand factor. These interactions are inhibited by RGD-containing peptides and by peptides corresponding to a sequence unique to fibrinogen in the COOH-terminal domain of its gamma chain (HLGGAKQAGDV). Two RGD sequences are present in fibrinogen A alpha chain: an RGDS sequence at A alpha 572-575, and an RGDF sequence at A alpha 95-98. Polyclonal antibodies raised against the RGDF sequence and the gamma COOH-terminal domain both reacted specifically with fibrinogen in solid phase enzyme-linked immunosorbent assays and immunoprecipitated the protein in solution. The Fab fragments prepared from these antibodies inhibited fibrinogen-platelet interaction and aggregation. These results demonstrate that these two sequences are both accessible within the fibrinogen molecule and are both implicated in ligand binding and cell-cell interaction. In addition, by further examining the interaction of the gamma chain peptide with platelets, it was found that RGDF and the gamma peptide produced a similar dose-dependent inhibition of the binding of the labeled gamma peptide to ADP-stimulated platelets. These results provide evidence that the RGDF sequence present at the A alpha 95-98 constitutes with the gamma 401-411 sequence two recognition sites interacting with the same site or with mutually exclusive sites on GPIIbIIIa.     

The ligand binding site of the platelet integrin receptor GPIIb-IIIa is proximal to the second calcium binding domain of its alpha subunit

The extreme carboxyl-terminal amino acid sequence of the gamma chain of fibrinogen is involved in the binding of this adhesive protein to the platelet integrin glycoprotein (GP) IIb-IIIa, and synthetic peptides corresponding to this region inhibit fibrinogen as well as fibronectin and von Willebrand factor binding to platelets. A chemical cross-linking approach was used to characterize the interaction of a 16-amino acid fibrinogen gamma chain peptide with platelets and to localize the site of its binding to GPIIb-IIIa. This peptide became specifically cross-linked to GPIIb, and platelet stimulation selectively enhanced its cross-linking to this alpha subunit. The cross-linking reaction was specifically inhibited by fibrinogen and an Arg-Gly-Asp peptide but not by an unrelated protein or a substituted peptide. Utilizing a combination of immunochemical mapping, enzymatic and chemical digestions, and amino acid sequencing, the cross-linking site of the gamma chain peptide in GPIIb was localized to a stretch of 21 amino acids. The identified region, GPIIb 294-314, contains the second putative calcium binding domain within GPIIb. The primary structure of this region is highly conserved among alpha subunits of other integrin adhesion receptors. These results identify a discrete region of GPIIb that resides in close proximity to a ligand binding site within GPIIb-IIIa. The homologous region may be involved in the functions of other integrin receptors.     

Antiplatelet "hybrid" peptides analogous to receptor recognition domains on gamma and alpha chains of human fibrinogen

Platelet receptor recognition domains are located on the gamma and alpha chains of human fibrinogen. The former encompasses residues 400-411 [Kloczewiak, M., Timmons, S., Lukas, T. J., & Hawiger, J. (1984) Biochemistry 23, 1767], and the latter is present in two loci on the alpha chain (alpha 95-97 and alpha 572-574) [Hawiger, J., Kloczewiak, M., Bednarek, M. A., & Timmons, S. (1989) Biochemistry (first of three papers in this issue)]. Peptide gamma 400-411 (HHLGGAKQAGDV) inhibited aggregation of ADP-treated platelets mediated not only by gamma-chain but also by alpha-chain multimers. Peptide alpha 572-575 (RGDS) inhibited aggregation of platelets mediated by alpha-chain as well as gamma-chain multimers. These results indicate that the platelet receptor for fibrinogen is isospecific with regard to the domain present on alpha and gamma chains. Subsequent "checkerboard" analysis of combinations of gamma 400-411 and alpha 572-575 showed that the inhibitory effect toward binding of 125I-fibrinogen was additive rather than synergistic. Next, a series of "hybrid" peptides was constructed in which the alpha-chain sequence RGDF (alpha 95-98) replaced the carboxy-terminal segment of gamma 408-411. The dodecapeptide HHLGGAKQRGDF was inhibitory with concentration, causing 50% inhibition of binding (IC50) at 6 microM, 5 times more potent than gamma 400-411. The shorter peptides AKQRGDF and KQRGDF were also more inhibitory than gamma 400-411. The second series of hybrid peptides was constructed with the alpha-chain sequence RGDS preceding the sequence of gamma 400-411 or sequence RGDV following it.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of integrins alpha v beta 3 and glycoprotein IIb-IIIa with fibrinogen. Differential peptide recognition accounts for distinct binding sites

Glycoprotein (GP) IIb-IIIa is the major fibrinogen receptor on platelets and participates in platelet aggregation at the site of a wound. Integrin alpha v beta 3, which contains an identical beta-subunit, is expressed on endothelial cells and also serves as a fibrinogen receptor. Here, we demonstrate by several criteria that purified GPIIb-IIIa and integrin alpha v beta 3 bind to distinct sites on fibrinogen. First, a plasmin-generated fragment of fibrinogen lacking the RGD sequence at residues 572-574 retained the ability to bind GPIIb-IIIa, but failed to bind integrin alpha v beta 3. Second, a monoclonal antibody which exclusively recognizes the RGD sequence at fibrinogen A alpha chain residues 572-574 abolished interaction between integrin alpha v beta 3 and fibrinogen, but had only a minimal effect on fibrinogen binding to GPIIb-IIIa. Finally, we show that the difference in recognition of sites on fibrinogen by these two integrins is probably a consequence of their remarkably different ligand binding properties. Peptides corresponding to fibrinogen gamma chain residues 400-411 effectively blocked RGD sequence and fibrinogen binding by GPIIb-IIIa, but had no effect on the ability of integrin alpha v beta 3 to bind these ligands. We also show that integrin alpha v beta 3 has a higher affinity than GPIIb-IIIa for a synthetic hexapeptide containing the RGD sequence. In fact, this RGD-containing peptide was 150-fold more effective at blocking fibrinogen binding to integrin alpha v beta 3 than to GPIIb-IIIa. Collectively, our results demonstrate that integrins alpha v beta 3 and GPIIb-IIIa display qualitative and quantitative differences in their ligand binding properties, as is evident by their ability to interact with synthetic peptides. The ultimate result of these differences is the recognition of distinct sites on fibrinogen by the two integrins. These observations may have relevance in the processes of hemostasis and wound healing.     

Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.     

Interaction of fibrinogen with its platelet receptor. Differential effects of alpha and gamma chain fibrinogen peptides on the glycoprotein IIb-IIIa complex

The platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) recognizes peptides containing the amino acid sequence Arg-Gly-Asp, a sequence present at two locations in the alpha chain of fibrinogen. GPIIb-IIIa also interacts with peptides containing the carboxyl-terminal 10-15 residues of the fibrinogen gamma chain. We found that the alpha chain tetrapeptide, Arg-Gly-Asp-Ser (RGDS), and the gamma chain peptide, Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (LGGAKQAG-DV), each inhibited fibrinogen binding to ADP-stimulated platelets with Ki values of 15.6 +/- 2.7 and 46.2 +/- 8.2 microM, respectively. Furthermore, the inhibitory effect of the peptides was additive, indicating that they interact with GPIIb-IIIa in a mutually exclusive manner. Mutually exclusive binding suggests that either the alpha and gamma chain peptides bind to identical or overlapping sites on the GPIIb-IIIa complex or that one peptide induces a change in the complex that excludes the other. To differentiate between these possibilities, we compared the ability of RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and PAC-1, to ADP-stimulated platelets. A2A9 and PAC-1 appear to bind to different sites on GPIIb-IIIa because A2A9 binds to both stimulated and unstimulated platelets while PAC-1 only binds to stimulated platelets. RGDS specifically inhibited fibrinogen and PAC-1 binding with nearly identical Ki values of 15.6 +/- 2.7 and 20.2 +/- 3.5 microM, respectively. In contrast, LGGAKQAGDV had a differential effect on fibrinogen and PAC-1 binding, inhibiting PAC-1 binding with a Ki of 116.1 +/- 12.9 microM and fibrinogen binding with a Ki of 46.2 +/- 8.2 microM (p less than 0.005). Furthermore, while RGDS had no effect on the binding of the monoclonal antibody A2A9, LGGAKQAGDV was a partial inhibitor of A2A9 binding to activated platelets. These results suggest that the bindings sites for RGDS and LGGAKQAGDV are spatially distinct. They also suggest that ligand-induced changes in GPIIb-IIIa conformation are likely to be responsible for the mutually exclusive nature of alpha and gamma chain peptide binding.     

Evidence that three adhesive proteins interact with a common recognition site on activated platelets

Synthetic peptides corresponding to the extreme COOH terminus of the gamma chain of fibrinogen gamma 400-411, (400)HHLGGAKQAGDV(411), have been used to analyze recognition specificities of the platelet-binding sites for fibrinogen, fibronectin, and von Willebrand factor. gamma 403-411 did not inhibit 125I-fibrinogen binding to platelets. In contrast, gamma 402-411 produced dose-dependent inhibition of fibrinogen binding to ADP and thrombin-stimulated living or fixed cells and was a competitive antagonist. Inhibitory activity was not modified by addition of one (gamma 401-411) or two (gamma 400-411) histidinyl residues to the NH2 terminus, but peptides with a trifluoroacetyl group on the epsilon-amino group of lysine 406 were inactive. 125I-Fibronectin and 125I-von Willebrand factor binding to thrombin-stimulated living or fixed cells was inhibited in the same dose range by the same set of peptides which inhibited fibrinogen binding and not by gamma 403-411 or trifluoroacetate-blocked peptides. The capacity of the peptides to inhibit binding to cells with an expressed receptor, i.e. fixed cells, excludes an effect on receptor induction. Thus, these results suggest that the three adhesive glycoproteins share a common site on thrombin-activated platelets, and a 10-amino acid peptide, corresponding to gamma 402-411, defines the recognition specificity of this site.     

Platelet receptor recognition domains on the alpha chain of human fibrinogen: structure-function analysis

We have previously shown that the alpha chain of human fibrinogen interacts directly with ADP-activated human platelets [Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D. D., & Doolittle, R. F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2068]. Now, we report that platelet receptor recognition domains are localized on two CNBr fragments of the human fibrinogen alpha chain. They encompass residues 92-147 and 518-584, which inhibit 125I-fibrinogen binding to ADP-stimulated platelets. The inhibitory CNBr fragment alpha 92-147 contains the RGD sequence at residues 95-97. Synthetic peptides encompassing this sequence were inhibitory while peptide 99-113 lacking the RGD sequence was inactive. The synthetic peptide RGDF, corresponding to residues alpha 95-98, inhibited the binding of 125I-fibrinogen to ADP-treated platelets (IC50 = 2 microM). However, the peptides containing sequence RGDF, with residues preceding Arg95 or following Phe98, were less inhibitory. It appears that the sequence alpha 95-98 constitutes a platelet receptor recognition domain which is constrained by flanking residues. The second inhibitory CNBr fragment, alpha 518-584, also contains the sequence RGD at positions 572-574. Synthetic peptides overlapping this sequence were inhibitory, while peptides lacking the sequence RGDS were not reactive. Thus, another platelet reactive site on the alpha chain encompasses residues 572-575 containing sequence RGDS. In conclusion, the platelet receptor recognition domains on the human fibrinogen alpha chain in the amino-terminal and in the carboxy-terminal zones contain the ubiquitous cell recognition sequence RGD shared with other known adhesive proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the cross-linking sites of RGD and KQAGDV peptides to the isolated fibrinogen receptor, the human platelet integrin glycoprotein IIb/IIIa. Influence of peptide length.

The non-covalent and Ca(2+)-dependent heterodimer GPIIb/IIIa, formed by platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa), also known as the integrin alpha IIb beta 3, is the inducible receptor for fibrinogen and other adhesive proteins on the surface of activated platelets. A fraction of the isolated GPIIb/IIIa in solution binds RGD or KQAGDV inhibitory peptides and, upon peptide removal, apparently acquires the capacity to bind fibrinogen ('activated' GPIIb/IIIa) [Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C. & Ginsberg, M. H. (1991) Cell 65, 409-416]. Photoaffinity labelling was used here to study the ligand binding site(s) of GPIIb/IIIa in solution, for which the peptides CKRKRKRKRRGDV (alpha 1), CGRGDF (alpha 2), CYHHLGGAKQAGDV (gamma 1) and CGAKQAGDV (gamma 2) were synthesized with a photoactivable cross-linker group and a fluorescent reporter group attached to the N-terminal cysteine residue. Contrary to the situation in activated platelets, both GPIIb and GPIIIa were equally labelled by the four peptides and the cross-linking sites were localized by protein chemical analyses of the fluorescently labelled tryptic peptides of both subunits. Thus, the localization of the cross-linking sites in GPIIb varies considerably with the peptide length and is very different from that localization observed in activated platelets: alpha 2 and gamma 2 were found cross-linked to the N-terminal of both the heavy (GPIIbH 42-73) and the light (GPIIbL2 30-75) chains of GPIIb; while the longer peptides alpha 1 and gamma 1 were cross-linked to the C-terminal of GPIIbH within the 696-724 and 752-768 peptide stretches, respectively. On the other hand, the cross-linking sites of the four inhibitory peptides in GPIIIa were found mainly within the proteolysis susceptible region, between the N-terminal (GPIIIa 1-52) and the core (GPIIb 423-622) highly disulphide-bonded domains, observing that the longer the peptide the closer the cross-linking site is to the N-terminal of GPIIIa: alpha 1 at GPIIIa 63-87 and 303-350; gamma 1 at GPIIIa 9-37; alpha 2 at GPIIIa 151-191; and gamma 2 at GPIIIa 303-350. These results led us to the following conclusions. (a) The GPIIIa 100-400 region contributes to the ligand-binding domain in GPIIb/IIIa both in solution and in activated platelets.(ABSTRACT TRUNCATED AT 400 WORDS)     

Function of fibrinogen gamma-chain dodecapeptide-conjugated latex beads under flow

In order to perform a fundamental study of platelet substitutes, novel particles that bound to activated platelets were prepared using two oligopeptides conjugated to latex beads. The oligopeptides were CHHLGGAKQAGDV (H12), which is a fibrinogen gamma-chain carboxy-terminal sequence (gamma 400-411), and CGGRGDF (RGD), which contains a fibrinogen alpha-chain sequence (alpha 95-98 RGDF). Both peptides contained an additional amino-terminal cysteine to enable conjugation. Human serum albumin was adsorbed onto the surface of latex beads (average diameter 1microm) and pyridyldisulfide groups were chemically introduced into the adsorbed protein. H12 or RGD peptides were then chemically linked to the modified surface protein via disulfide linkages. H12- or RGD-conjugated latex beads prepared in this way enhanced the in vitro thrombus formation of activated platelets on collagen-immobilized plates under flowing thrombocytopenic-imitation blood. Based on the result of flow cytometric analyses of agglutination, PAC-1 binding, antiP-selectin antibody binding, and annexin V binding, the H12-conjugated latex beads showed minimal interaction with non-activated platelets. These results indicate the excellent potential of H12-conjugated particles as a candidate for a platelet substitute.     

Mapping the binding domains of the alpha(IIb) subunit. A study performed on the activated form of the platelet integrin alpha(IIb)beta(3).

alpha(IIb)beta(3), a member of the integrin family of adhesive protein receptors, is the most abundant glycoprotein on platelet plasma-membranes and binds to adhesive proteins via the recognition of short amino acid sequences, for example the ubiquitous RGD motif. However, elucidation of the ligand-binding domains of the receptor remains controversial, mainly owing to the fact that integrins are conformationally labile during purification and storage. In this study, a detailed mapping of the extracellular region of the alpha(IIb) subunit is presented, using overlapping 20-peptides, in order to identify the binding sites of alpha(IIb) potentially involved in the platelet-aggregation event. Regions alpha(IIb) 313-332, alpha(IIb) 265-284 and alpha(IIb) 57-64 of alpha(IIb)beta(3) were identified as putative fibrinogen-binding domains because the corresponding peptides inhibited platelet aggregation and antagonized fibrinogen association, possibly by interacting with this ligand. The latter is further supported by the finding that the above peptides did not interfere with the binding of PAC-1 to the activated form of alpha(IIb)beta(3). Furthermore, alpha(IIb) 313-332 was found to bind to fibrinogen in a solid-phase binding assay. It should be emphasized that all the experiments in this study were carried out on activated platelets and consequently on the activated form of this integrin receptor. We hypothesize that RAD and RAE adhesive motifs, encompassed in alpha(IIb) 313-332, 265-284 and 57-64, are capable of recognizing complementary domains of fibrinogen, thus inhibiting the binding of this ligand to platelets.     

Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen- dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.     

Isolation and characterization of a platelet membrane protein related to the vitronectin receptor

Glycoprotein IIb-IIIa is the most prominent Arg-Gly-Asp (RGD)-binding adhesion receptor on platelets. By affinity chromatography on an immobilized RGD peptide, we have investigated the possible existence of other platelet-associated adhesion receptors that bind RGD peptides. When an octyl glucoside extract of surface-radioiodinated platelets was applied to an affinity matrix of KYGRGDS-coupled Sepharose 4B, a 160-kDa-labeled protein (P160) and GPIIb-IIIa bound and were specifically eluted by soluble GRGDSP peptide, but not by the variant GRGESP peptide. Furthermore, a dodecapeptide corresponding to fibrinogen gamma 400-411 eluted only GPIIb-IIIa but not P160 from the RGD affinity matrix. Characterization of P160 by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by the O'Farrell gel electrophoresis system indicated that P160 is a component of platelet GPIc. GoH3, a monoclonal antibody recognizing the alpha subunit of the very late antigen-6, failed to immunoprecipitate P160 from the RGD eluate, indicating that it did not contain the very late antigen-6 alpha subunit. In immunoblots, P160 reacted specifically with a polyclonal anti-peptide antibody recognizing the alpha subunit of the vitronectin receptor (VnR), but not with the monoclonal anti-GPIIb antibody PMI-1, suggesting that P160 is the alpha subunit of platelet VnR. This possibility was further substantiated by the complete identity between the determined amino-terminal sequence of P160 and the known sequence of the VnR alpha subunit. Moreover, direct association of P160 with a beta subunit having an apparent molecular weight similar to that of GPIIIa was demonstrated by immunoprecipitation with LM609, an anti-VnR complex monoclonal antibody. These results indicate that the VnR complex is present on platelets and may play a functional role in platelet adhesive reactions.     

Ligands "activate" integrin alpha IIb beta 3 (platelet GPIIb-IIIa)

Integrin alpha IIb beta 3 (platelet GPIIb-IIIa) binds fibrinogen via recognition sequences such as Arg-Gly-Asp (RGD). Fibrinogen binding requires agonist activation of platelets, whereas the binding of short synthetic RGD peptides does not. We now find that RGD peptide binding leads to changes in alpha IIb beta 3 that are associated with acquisition of high affinity fibrinogen-binding function (activation) and subsequent platelet aggregation. The structural specificities for peptide activation and for inhibition of ligand binding are similar, indicating that both are consequences of occupancy of the same site(s) on alpha IIb beta 3. Thus, the RGD sequence is a trigger of high affinity ligand binding to alpha IIb beta 3, and certain RGD-mimetics are partial agonists as well as competitive antagonists of integrin function.     

Integrin alpha IIb beta 3-dependent calcium signals regulate platelet-fibrinogen interactions under flow. Involvement of phospholipase C gamma 2

Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin alpha IIb beta 3 engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP3) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLC gamma 2-/- mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.     

Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb-IIIa on nonactivated platelets

We demonstrate that unstimulated platelets attach to immobilized fibrinogen in a selective process mediated by the membrane glycoprotein (GP) complex IIb-IIIa (alpha IIb beta 3). The initial attachment, independent of platelet activation, is followed by spreading and irreversible adhesion even in the presence of activation inhibitors. Using fibrinogen fragments derived from plasmin digestion, we found that unstimulated platelets do not attach to immobilized fragment E, which contains an Arg-Gly-Asp sequence at A alpha 95-97, and adhere to fragments X and D, both containing the gamma 400-411 dodecapeptide adhesion sequence, less efficiently than to intact fibrinogen. Thus, the carboxyl terminus of the A alpha chain, missing in the "early" fragment X used in these studies, appears to be involved in the interaction of fibrinogen with unstimulated platelets. In contrast, activated platelets adhere to immobilized fibrinogen and fragments X, D, and E in a time-dependent and equivalent manner. Although activated platelets adhere to immobilized vitronectin, fibronectin, and von Willebrand factor through GP IIb-IIIa, unstimulated platelets fail to adhere to vitronectin and have only a limited capacity to adhere to fibronectin and von Willebrand factor. These results demonstrate that GP IIb-IIIa on unstimulated platelets displays a recognition specificity for attachment to immobilized adhesive proteins that is distinct from that seen following platelet activation. Thus, unstimulated platelets selectively interact with fibrinogen, and the initial attachment is followed by spreading and irreversible adhesion in the absence of exogenous agonists. This process may be regulated by plasmin cleavage of the fibrinogen A alpha chain and may play an important role during normal hemostasis and during the pathological development of thrombotic vascular occlusions.