Changes in the platelet membrane glycoprotein IIb.IIIa complex during platelet activation

Platelet activation is accompanied by the appearance on the platelet surface of approximately 45,000 receptor sites for fibrinogen. The binding of fibrinogen to these receptors is required for platelet aggregation. Although it is established that the fibrinogen receptor is localized to a heterodimer complex of the membrane glycoproteins, IIb and IIIa, little is known about the changes in this complex during platelet activation that result in the expression of the receptor. In the present studies, we have developed and characterized a murine monoclonal anti-platelet antibody, designated PAC-1, that binds to activated platelets, but not to unstimulated platelets. PAC-1 is a pentameric IgM that binds to agonist-stimulated platelets with an apparent Kd of 5 nM. Binding to platelets is dependent on extracellular Ca2+ (KCa = 0.4 microM) but is not dependent on platelet secretion. Platelets stimulated with ADP or epinephrine bind 10,000-15,000 125I-PAC-1 molecules/platelet while platelets stimulated with thrombin bind 20,000-25,000 molecules/platelet. Several lines of evidence indicate that PAC-1 is specific for the glycoprotein IIb.IIIa complex. First, PAC-1 binds specifically to the IIb.IIIa complex on Western blots. Second, PAC-1 does not bind to thrombasthenic platelets or to platelets preincubated with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid at 37 degrees C, both of which lack the intact IIb.IIIa complex. Third, PAC-1 competitively inhibits the binding of 125I-A2A9, and IgG monoclonal antibody that is specific for the IIb.IIIa complex. Fourth, the antibody inhibits fibrinogen-mediated platelet aggregation. These data demonstrate that PAC-1 recognizes an epitope on the IIb.IIIa complex that is located near the platelet fibrinogen receptor. Platelet activation appears to cause a Ca2+-dependent change involving the glycoprotein IIb.IIIa complex that exposes the fibrinogen receptor and, at the same time, the epitope for PAC-1.     

A conformation-dependent epitope of human platelet glycoprotein IIIa.

This study explores conformational states of human platelet glycoprotein IIIa (GP IIIa) and possible mechanisms of fibrinogen receptor exposure. D3GP3 is an IgG1, kappa monoclonal antibody generated against purified GP IIIa and found to be specific for GP IIIa by immunoprecipitation and Western blot analysis. The binding of D3GP3 to resting platelets caused fibrinogen binding (approximately 5,000 molecules/platelet) and platelet aggregation but not secretion. Platelets express 40,000-50,000 GP IIb-IIIa molecules in their surface membranes. However, resting platelets only bound approximately 5,000 D3GP3 molecules/platelet. D3GP3 binding to platelets could be increased 2-3-fold by dissociation of the GP IIb-IIIa complex with 5 mM EDTA or by occupying the fibrinogen receptor with either RGDS peptides or fibrinogen. Platelet stimulation with ADP in the absence of fibrinogen did not cause increased D3GP3 binding above control levels. These data suggest that 1) GP IIb-IIIa can exist in multiple conformations in the platelet membrane, 2) D3GP3 binding to GP IIIa can expose the fibrinogen receptor, 3) the binding of either RGDS peptides or fibrinogen causes exposure of the D3GP3 epitope, and 4) platelet activation in the absence of ligand does not induce the same conformational changes in GP IIb-IIIa as does receptor occupancy by RGDS peptides or fibrinogen.     

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.     

Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex

Triflavin, an antiplatelet peptide containing Arg-Gly-Asp, purified from Trimeresurus flavoviridis venom, inhibits aggregation of human platelets stimulated by a variety of agonists. It blocks aggregation through interference with fibrinogen binding to its specific receptor on the platelet surface membrane in a competitive manner, but it has no apparent effect on intracellular events, such as thromboxane B2 formation, phosphoinositides breakdown and intracellular Ca2+ mobilization of thrombin-activated platelets. In this study, we determined the complete sequence of triflavin, which is composed of a single polypeptide chain of 70 amino acids. Its sequence is rich in cysteine and contains Arg-Gly-Asp at residues 49-51 in the carboxy-terminal domain. Triflavin shows about 68% identity of amino acid sequence with trigramin, which is a specific antagonist of the fibrinogen receptor associated with glycoprotein IIb/IIIa complex. [125I]Triflavin binds to unstimulated and ADP-stimulated platelets in a saturable manner and its Kd values are estimated to be 76 and 74 nM, respectively; the corresponding numbers of binding sites are 31,029 and 34,863 per platelet, respectively. [125I]Triflavin binding is blocked by Gly-Arg-Gly-Asp-Ser in a competitive manner. EDTA, the Arg-Gly-Asp-containing peptides (including naturally occurring polypeptides, trigramin and rhodostomin), and monoclonal antibody, 7E3, raised against GP IIb/IIIa complex, inhibit [125I]triflavin binding to unstimulated and ADP-stimulated human platelets. In conclusion, triflavin specifically binds to fibrinogen receptor associated with GP IIb/IIIa complex and its binding site is located at or near GP IIb/IIIa complex, overlapping with those of 7E3 and another Arg-Gly-Asp-containing polypeptide, rhodostomin.(ABSTRACT TRUNCATED AT 250 WORDS)     

An antiplatelet peptide, gabonin, from Bitis gabonica snake venom.

Interaction of fibrinogen with its receptors (glycoprotein IIb/IIIa complex) on platelet membranes leads to platelet aggregation. By means of gel filtration, CM-Sephadex C-50, and reverse-phase HPLC, an antiplatelet peptide, gabonin, was purified from the venom of Bitis gabonica. The purified protein migrates as a 21,100-Da polypeptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and as a 11,000-Da peptide in the presence of beta-mercaptoethanol, indicating that gabonin is a disulfide-linked dimer. It is a polypeptide consisting of about 84 amino acid residues, rich in Asp, Pro, and half-cystine. Gabonin dose-dependently inhibited human platelet aggregation stimulated by ADP, collagen, U46619, or thrombin in preparations of platelet-rich plasma and platelet suspension (IC50 = 340-1600 nM). It also blocked platelet aggregation of whole blood. However, it apparently did not affect the initial shape change and only slightly reduced ATP release caused by aggregation agonists. Gabonin did not inhibit the rise of cytosolic calcium in Quin-2-loaded platelets stimulated by thrombin. In addition, gabonin dose-dependently inhibited fibrinogen-induced aggregation of elastase-treated platelets. In conclusion, gabonin inhibits platelet aggregation mainly through the blockade of fibrinogen binding toward fibrinogen receptors of the activated platelets.     

Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies

Platelet membrane glycoprotein IIb-IIIa exists as a calcium-dependent complex of two large peptides (designated IIb and IIIa) in Triton X-100 solutions, but it remains unknown if these peptides are subunits of one glycoprotein or are actually two individual glycoproteins in the intact platelet membrane. We used crossed immunoelectrophoresis to define the epitopes of two monoclonal antibodies to IIb-IIIa, then used these antibodies to study the structural and functional organization of IIb and IIIa in the platelet membrane. Human platelets solubilized in Triton X-100 were electrophoresed through an intermediate gel containing 125I-monoclonal IgG, then into an upper gel containing rabbit anti-human platelet antibodies. Our previously characterized antibody. Tab, and a new monoclonal antibody, T10, both bound to the immunoprecipitate corresponding to the IIb-IIIa complex. When platelets were electrophoresed after solubilization in 5 mM EDTA, 125I-Tab bound to the dissociated IIb polypeptide, but not to IIIa. In contrast, 125-I-T10 did not react with either IIb or IIIa. Thus, Tab recognizes a determinant on IIb, while T10 recognizes a determinant created only after the association of IIb and IIIa. Gel-filtered platelets from six normal donors bound 50,600 +/- 5,600 125I-T10 molecules/platelet and 47,800 +/- 11,200 125I-Tab molecules/platelet, consistent with IIb-IIIa being a heterodimer. 125I-T10 binding was identical in unactivated platelets and platelets stimulated with 10 microM ADP. However, platelets did not aggregate or bind 125I-fibrinogen until ADP was added. T10, but not Tab or nonimmune mouse antibody, inhibited ADP-induced platelet aggregation and 125I-fibrinogen binding. Our findings suggest that IIb and IIIa exist as subunits of a single membrane glycoprotein in unstimulated platelets. Fibrinogen binding appears to require not only the interaction of IIb and IIIa, but also some additional change occurring after platelet activation.     

Aggregin: a platelet ADP receptor that mediates activation

ADP is known to induce platelet shape change, aggregation, and exposure of fibrinogen binding sites as well as inhibit stimulated adenylate cyclase. The platelet is unique in that its purinergic receptor prefers ADP over ATP, which functions as a competitive antagonist. The affinity reagent, 5'-p-fluorosulfonylbenzoyl adenosine (FSBA), has been used to covalently label a single membrane protein, aggregin, on the external platelet surface with mol wt of 100 kDa. Concomitant with incorporation of FSBA, ADP-induced shape change, aggregation, and fibrinogen binding is inhibited. FSBA is also a weak agonist at short times and high concentration, which suggests that prior noncovalent binding to aggregin takes place before covalent modification. Aggregin differs from platelet glycoprotein IIIa in its physical and immunochemical properties. Aggregin is distinct from the receptor coupled to adenylate cyclase. Using FSBA as a probe, platelet aggregation by thromboxane A2 analogs and collagen was shown to be dependent on ADP but not the shape change induced by these agonists. Binding to aggregin is required for epinephrine-induced aggregation. In turn, epinephrine increases the affinity of ADP for its receptor. Thrombin at concentrations greater than 2 nM (0.2 units/ml) stimulates platelet aggregation independent of ADP, but by raising cytoplasmic Ca2+ it activates platelet calpain, which in turn cleaves aggregin. Thus aggregin, in addition to serving as the ADP receptor linked to shape change and aggregation, plays a role in fibrinogen receptor latency that is relieved entirely by ADP binding to or proteolysis of aggregin.     

Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. II. Quantal activation parallels platelet capture in stir-associated microaggregation.

There is broad agreement that platelet aggregation is generally dependent on fibrinogen (Fg) binding to the glycoprotein (GP) IIb-IIIa receptor expressed on the activated platelet surface. We therefore compared rates and extents of aggregation and of fibrinogen receptor expression and specific Fg binding to activated platelets, as a function of ADP concentration. Human citrated platelet-rich plasma (diluted 10-fold) was stirred with adenosine diphosphate (ADP) for 10 s or 2 min to measure rates and extent of aggregation, respectively, determined from the decrease in the total number of particles. The number of fibrinogen receptors and bound Fg were measured from mean fluorescence values obtained with FITC-labeled IgM monoclonal antibody PAC1 and the IgG monoclonal antibody, 9F9, respectively, using flow cytometry as presented in part I (Frojmovic et al., 1994). Because flow cytometric and aggregation measurements were routinely determined at room temperature and 37 degrees C, respectively, we also compared and found temperature-independent initial rates of aggregation. The fraction of platelets with fluorescence values above one critical threshold value, corresponding to maximally "activated" platelets (P*), increased with increasing activator concentration and correlated linearly with the fraction of platelets recruited into aggregates for ADP (r > 0.9). Aggregation was not rate-limited by fibrinogen receptor expression or by Fg binding. It appears that each platelet expresses its maximal Fg receptors at a critical ADP concentration, i.e., occupancy of ADP receptors. This, in turn, leads to rapid Fg occupancy and capture of such "quantally activated" platelets into aggregates.     

Effect of the thiol group inhibitor monobromobimane and other inhibitors on the composition of the platelet cytoskeletal core and its association with glycoprotein IIIa

SDS-polyacrylamide gel electrophoresis was used to study the effects of the thiol inhibitor monobromobimane (MB), EDTA, and prostaglandin E1 (PGE1) on the formation and composition of the platelet cytoskeletal core (Triton-insoluble residue) and its association with glycoprotein (GP) IIIa. Stimulation or aggregation of platelets in response to ADP or thrombin increased the amount of Triton-insoluble myosin. Aggregation resulted in incorporation of [125I]GP IIIa and a new band at about 210 kDa into the cytoskeletal core. EDTA and PGE1 caused little disaggregation of platelets that were aggregated in PRP with ADP and that had secreted the contents of their granules. In contrast to EDTA, PGE1 decreased the amount of Triton-insoluble residue and its association with GP IIIa. MB added after ADP-induced aggregation caused an increase in the amount of cytoskeletal core despite marked disaggregation and a substantial decrease in core-associated GP IIIa. With aspirin-treated platelets that had not secreted, EDTA, PGE1, and MB all caused disaggregation and loss of cytoskeletal GP IIIa. MB diminished, but did not reverse, thrombin-induced aggregation of washed platelets and arrested GP IIIa incorporation into the cytoskeletal core. Concanavalin A (Con A) cross-links glycoproteins on a single platelet and induces incorporation of GP IIIa into the Triton-insoluble residue in the absence of platelet aggregation. This induction was not inhibited by MB, although this reagent, as well as aspirin, inhibited Con A-induced secretion. Since GP IIIa incorporation caused by ADP-induced aggregation differs from that caused by Con A in its susceptibility to MB, it seems unlikely that thiol groups are directly involved in the association of GP IIIa with the cytoskeletal core.     

The peptide Glu-His-Ile-Pro-Ala binds fibrinogen and inhibits platelet aggregation and adhesion to fibrinogen and vitronectin.

Antiplatelet agents are clinically useful as antithrombotic entities. The importance of antiplatelet agents led us to design, synthesize, and characterize a new antiplatelet peptide. This peptide is a presumptive mimic of a ligand binding site on the platelet fibrinogen receptor. Unlike peptides related to Arg-Gly-Asp-Ser and His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val that bind to the fibrinogen receptor, this peptide binds to fibrinogen. The anticomplementarity hypothesis was used to design this presumptive peptide mimic of the vitronectin binding site on the fibrinogen receptor, glycoprotein IIb/IIIa complexes. The resulting peptide (Glu-His-Ile-Pro-Ala) has the characteristics of a fibrinogen binding site mimic: It binds fibrinogen and inhibits both the adhesion of platelets to fibrinogen and platelet aggregation. The peptide also inhibits the adhesion of platelets to vitronectin. The antiplatelet activity of this mimic peptide was dependent on its amino acid sequence, since closely related analogues were either inactive or less active inhibitors of platelet function than the original peptide. These results demonstrate that the peptide Glu-His-Ile-Pro-Ala has the characteristics expected of a mimic of a glycoprotein IIb/IIIa ligand binding site.     

Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture

The receptor for ADP on the platelet membrane, which triggers exposure of fibrinogen-binding sites and platelet aggregation, has not yet been identified. Two enzymes with which ADP interacts on the platelet surface, an ecto-ATPase and nucleosidediphosphate kinase, have been proposed as possible receptors for ADP in ADP-induced platelet aggregation. In the present study, experiments were conducted with washed human platelets to examine if a relationship existed between platelet aggregation, fibrinogen binding and the enzymatic degradation of ADP. With 12 different platelet suspensions, a good correlation (P less than 0.01) was found between the extent of platelet aggregation and the amount of 125I-fibrinogen bound to platelets after ADP stimulation. No correlation was found between these parameters and the rate or extent of transformation of [14C]ADP to [14C]ATP or [14C]AMP. The binding of fibrinogen to platelets was inhibited in parallel with aggregation when ADP stimulation was impaired by the enzymatic degradation of ADP by the system creatine phosphate/creatine phosphokinase, or by the use of specific antagonists, such as ATP and AMP. These antagonists also influenced the enzymatic degradation of ADP. This effect occurred at lower concentrations of ATP or AMP than those required to inhibit ADP-induced platelet aggregation and fibrinogen binding. Our results demonstrate that ATP and AMP may be used as specific antagonists of the ADP-induced fibrinogen binding to platelets. They do not provide evidence to suggest that enzymes which metabolize ADP on the platelet surface are involved in the mechanism of ADP-induced platelet aggregation.     

Differences between platelet and microparticle glycoprotein IIb/IIIa.

Glycoprotein (GP) IIb and IIIa are major constituents of the platelet membrane which are involved in forming the fibrinogen receptor on activated platelets. We used flow cytometry to study the effects of ethylene-diamine tetraacetic acid (EDTA) on the membrane GPIIb/IIIa complexes of platelets and microparticles, and to study the effects of cations on dissociated GP complexes. Microparticles were detected by both the volume signal and by fluorescence using an FITC-conjugated anti-GPIb antibody (NNKY5-5). When platelets were stimulated with ADP, calcium ionophore A23187, or thrombin, fibrinogen binding to the platelet surface increased markedly. However, fibrinogen binding to microparticles showed little increase in response to such agonists. Microparticle GPIIb/IIIa complexes were dissociated by incubation with EDTA at 37 degrees C but did not reassociate after treatment with divalent cations (Ca2+, Mg2+, and Mn2+) in contrast to platelet GPIIb/IIIa complexes. These results suggest that some interaction of GPIIb/IIIa and linked structures like the platelet cytoskeleton may be involved in the reassociation of dissociated GPIIb and GPIIIa, perhaps explaining the failure of reassociation of microparticle GPIIb/IIIa (i.e., the fibrinogen binding to microparticles).     

Purification and characterization of a new RGD/KGD-containing dimeric disintegrin, piscivostatin, from the venom of Agkistrodon piscivorus piscivorus: the unique effect of piscivostatin on platelet aggregation

Piscivostatin, a novel dimeric disintegrin containing Arg-Gly-Asp (RGD) and Lys-Gly-Asp (KGD) sequences, was isolated from the venom of Agkistrodon piscivorus piscivorus. The molecule consisted of two chains designated as the alpha and beta chains, comprising 65 and 68 amino acid residues, respectively. Piscivostatin had two binding motifs recognized by platelet glycoprotein IIb/IIIa (GPIIb/IIIa), and the biological activity of dimeric disintegrin piscivostatin toward platelet aggregation differed from those of other monomeric disintegrins such as trimestatin and echistatin. We measured platelet aggregation by the laser light scattering method during the process of ADP-induced platelet aggregation. Both dimeric and monomeric disintegrins inhibited the formation of small (9 to 25 microm in diameter), medium-sized and large aggregates (25 to 70 microm in diameter) in a dose-dependent manner. The platelet aggregates disaggregated after reaching a maximal number on either treatment with ADP alone or monomeric disintegrin/ADP. However, the small aggregates did not disaggregate on treatment with piscivostatin/ADP even when applied over time. When washed platelets were incubated with an anti-GPIIb/IIIa monoclonal antibody, PT25-2, which induces conformational changes of GPIIb/IIIa to a form accessible to fibrinogen and other adhesion proteins without platelet activation, piscivostatin induced a platelet shape change alone with no aggregate formation. The present study indicated that piscivostatin has two unique contradictory activities; acting as a double inhibitor of platelet aggregation and platelet aggregate dissociation.     

Association of pp60src with Triton X-100-insoluble residue in human blood platelets requires platelet aggregation and actin polymerization.

Protein-tyrosine phosphorylation during platelet activation is inhibited under conditions that inhibit platelet binding of fibrinogen and aggregation. We suggested that pp60src, a major platelet tyrosine kinase, or its protein substrates might become associated with the cytoskeleton upon platelet stimulation, and that this might be related to aggregation. By Western blotting with an anti-Src monoclonal antibody, we found time-dependent association of pp60src with the cytoskeleton (10,000 x g Triton X-100-insoluble matrix) but not the "membrane" cytoskeleton (100,000 x g Triton X-100-insoluble matrix) in platelets activated by U46619 (PGH2 analog). Cytoskeletal association and platelet aggregation were inhibited by the peptide Arg-Gly-Asp-Ser (RGDS) (but not by Arg-Gly-Glu-Ser (RGES)), by 10E5 antibody against glycoprotein (Gp) IIb/IIIa, and by EGTA. U46619-induced association of pp60src with cytoskeleton but not secretion or aggregation was inhibited by cytochalasin D (2 microM). Both cytochalasin D and RGDS inhibited "slow" tyrosine phosphorylation of platelet proteins. Association of pp60src with cytoskeleton induced by U46619 or ADP was not blocked by aspirin. Aspirin blocked epinephrine-induced association of pp60src with the cytoskeleton during a second phase of aggregation when an initial phase had occurred without shape change or secretion. Association of GpIIb/IIIa with the cytoskeleton also accompanied platelet aggregation, shape change, and actin polymerization; this was shown with anti-GpIIb and anti-GpIIIa antibodies. Association of pp60src and GpIIb/IIIa with the cytoskeleton and slow tyrosine phosphorylation are related phenomena.     

Ligand inhibition of the platelet glycoprotein IIb-IIIa complex function as a calcium channel in liposomes

Platelet glycoproteins IIb and IIIa function as a fibrinogen receptor on the activated platelet. We have shown that these glycoproteins can be incorporated onto the surface of phosphatidylcholine vesicles with retention of fibrinogen and antibody binding properties and can permit Ca2+ transit across the phospholipid bilayer. In the current study we demonstrate that this apparent Ca2+ channel function is specifically inhibited by the synthetic analogue of the fibrinogen gamma COOH-terminal peptide, His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (His-12-Val), but not by the adhesive protein sequence Arg-Gly-Asp-Ser (RGDS). Prior incubation of IIb-IIIa liposomes with RGDS prevented Ca2+ transit inhibition by 25 microM His-12-Val, analogous to RGDS inhibition of His-12-Val binding to platelets. His-12-Val inhibited a minor component of transmembrane Ca2+ influx into ADP and thrombin-activated human platelets but had no effect on steady-state platelet 45Ca flux. These data indicate that ligand binding may exert a regulatory influence on transmembrane Ca2+ influx into activated platelets. The difference in inhibitory potency of the peptides studied may be related to differences in conformational changes in the glycoprotein IIb-IIIa complex induced by His-12-Val and RGDS, steric considerations, or differences in interactions with glycoprotein IIb Ca2+ binding domains.     

Localization of fibrinogen during ADP- or thrombin-induced aggregation of washed rabbit platelets

In contrast to human platelets, which aggregate poorly in response to ADP unless fibrinogen is present in the external medium, washed rabbit platelets form large aggregates in response to ADP without fibrinogen in the suspending medium. Addition of fibrinogen to the suspending medium of rabbit platelets frequently has little or no effect on the extent of ADP-induced platelet aggregation. We examined washed rabbit platelets by immunocytochemistry during ADP-induced aggregation and deaggregation and during thrombin-induced aggregation when the external medium did not contain added fibrinogen to determine if (a) fibrinogen was expressed on the surface of rabbit platelets that could support aggregation when the platelets were stimulated, or (b) fibrinogen secreted from the alpha granules supports platelet aggregation. Glutaraldehyde-fixed samples were prepared at different times after addition of ADP or thrombin, embedded in Lowicryl K4M, sectioned, incubated with sheep anti-rabbit fibrinogen, washed, reacted with gold-labeled anti-sheep IgG, and prepared for electron microscopy. The alpha granules of rabbit platelets were heavily labeled with immunogold; the platelet membrane was not labeled. During platelet aggregation and deaggregation in response to ADP, fibrinogen was not detectable on the platelet surface. In response to thrombin, large aggregates formed before fibrinogen was secreted from the alpha granules; fibrinogen was detectable focally at sites of granule discharge by 30-60 sec and fibrin formed by 3 min. Therefore, stimulated washed rabbit platelets can adhere to each other without large amounts of fibrinogen taking part in the close platelet-to-platelet contact, since aggregation occurs before detectable secretion, and large areas where the platelets are in contact are devoid of fibrinogen between the adherent membranes. Adhesion mechanisms not involving fibrinogen may support the aggregation of washed rabbit platelets.     

Activation affects access to the platelet receptor for adhesive glycoproteins

Blood platelets have a receptor for macromolecular adhesive glycoproteins, located on a heteroduplex membrane glycoprotein complex (GPIIb/IIIa) that only becomes "exposed" when platelets are activated. Binding of the adhesive glycoproteins, in particular fibrinogen, to the receptor is required for platelet aggregation, which in turn is required to arrest bleeding. A murine monoclonal antibody whose rate of binding to the receptor is affected by platelet activation was both cross-linked and fragmented to assess the effects of changes in molecular size on its rate of binding to unactivated and activated platelets. The results indicate that small molecules can bind more rapidly to the receptors on unactivated platelets than can large molecules and that activation involves a conformational and/or microenvironmental change that permits the large molecules to bind more rapidly.     

The effect of glycoprotein IIb-IIIa receptor occupancy on the cytoskeleton of resting and activated platelets

The platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa), serves as the receptor for fibrinogen. This study examined what effect GPIIb-IIIa receptor occupancy had on the cytoskeleton of resting and activated platelets. Triton X-100-insoluble residues (cytoskeletons) were isolated from resting washed platelets incubated with either 500 microM RGDS or 500 microM RGES and examined for protein content. RGDS did not increase the amount of GPIIb-IIIa associated with the cytoskeletal residues which sedimented at either 15,800 x g or 100,000 x g. To determine the effect of receptor occupancy on the formation of the activated platelet cytoskeleton, stirred and nonstirred RGDS-treated platelets in plasma were activated with ADP. Triton X-100-insoluble residues were isolated and examined for both protein content and retention of GPIIb-IIIa. Further, morphological studies were performed on the RGDS-ADP-stimulated platelets. The results of this study suggest that 1) RGDS peptide receptor occupancy does not lead to GPIIb-IIIa linkage to the cytoskeleton, 2) ADP-stimulated platelet shape change, polymerization of actin, and association of myosin with the cytoskeleton are unaffected by RGDS peptide receptor occupancy. 3) RGDS inhibits an aggregation-dependent incorporation of ABP, alpha-actinin, talin, and GPIIb-IIIa into the Triton-insoluble residue.     

Variations of glycoprotein IIb–IIIa (αIIb/β3-integrin) content in healthy donors. The influence on platelet aggregation activity and efficacy of aspirin action

The effects of content of a fibrinogen receptor, glycoprotein (GP) IIb–IIIa (αIIb/β3-integrin), GP IIIa genetic polymorphism (substitution Leu33Pro), and fibrinogen concentration in blood plasma on platelet aggregation activity have been investigated in a group of healthy volunteers. In 35 examined donors the GP IIb–IIIa content on platelet surface varied from 40 to 71 × 10³ per platelet. Repeated measurements revealed that the GP IIb–IIIa content coefficient of variation was 9.5%, and deviations from mean levels did not exceed 20%. The level and the rate of platelet aggregation induced by ADP (1.25–20 μM) correlated with GP IIb–IIIa number (r from 0.315 to 0.591) and were higher in the group of donors with high in comparison with low GP IIb–IIIa content (>60 and (40–50) × 10^?3 per platelet, respectively). Aspirin, the inhibitor of thromboxane A₂ synthesis, partially suppressed ADP-induced platelet aggregation. The level of residual aggregation in the presence of aspirin also correlated with GP IIb–IIIa content and increased in subjects with high receptor content. Parameters of ADP-induced aggregation did not differ in donors with genotypes GP IIIa Pro33(?) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotype. GP IIb–IIIa content was also not affected by GP IIIa polymorphism. No significant correlations were found between the level and rate of platelet aggregation and fibrinogen concentration in blood plasma. The data obtained indicate that the effects of variations of GP IIb–IIIa content on platelet aggregation are higher than GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration. High GP IIb–IIIa content is associated with increased platelet aggregation activity and decreased efficacy of aggregation inhibition by aspirin.     

Characteristics of an ADP receptor mediating platelet activation

Summary Adenosine diphosphate (ADP) is known to induce platelet shape change, aggregation and fibrinogen binding, followed by secretion. These processes are mediated by the binding of ADP to an externally oriented protein of the platelet plasma membrane. An affinity analog of ATP, a competitive inhibitor of the action of ADP, has been utilized to probe the structure and function of this receptor. FSBA (5-p-fluorosulfonylbenzoyl adenosine) covalently modifies a single protein in intact platelets with M_r = 100 000 and concomitantly inhibits platelet shape change, aggregation and fibrinogen binding. Studies on platelet membranes demonstrate non-covalent association of ADP-binding protein with actin which is also labeled by FSBA but only in isolated membranes. This finding suggests a structural and functional coupling of the receptor to the contractile process. The putative ADP receptor covalently modified with FSBA is cleaved by chymotrypsin, a process that reverses the inability of the platelets to bind fibrinogen. Thus, the M_r = 100 000 polypeptide may be involved in the proteolytic exposure of fibrinogen binding sites on the platelet surface. The ability of FSBA to inhibit platelet aggregation and fibrinogen binding by prostaglandin H₂ derivatives and epinephrine suggest that ADP is involved in these processes. However, the interaction is not at the receptor level since shape change, stimulated by PGH₂ derivatives and yohimbine (epinephrine antagonist) binding are unaffected by FSBA. Finally, the action of ADP to inhibit PGE₁- or PGI₂-stimulated adenylate cyclase appears to be mediated by a receptor distinct for the protein modified by FSBA.Abbreviation 5FSBA 5-p-fluorosulfonylbenzoyl adenosine