Identification of platelet glycoprotein IIb/IIIa as the major binding site for released platelet-von Willebrand factor [published erratum appears in Blood 1987 Jun;69(6):1789]

We studied the effects(s) of two monoclonal antibodies, 6D1 and 10E5 (directed against platelet glycoprotein Ib [GPIb] and the GPIIb/IIIa complex, respectively), and purified human plasma fibrinogen on the binding of released platelet-von Willebrand factor (vWf) to the platelet surface. Neither of the monoclonal antibodies nor fibrinogen had any effect on the amount of platelet-vWf expressed on unstimulated platelets or on the amount expressed on platelets stimulated in the absence of extracellular Ca++. However, the antibody directed against GPIIb/IIIa inhibited 72% of the thrombin-induced increase in the platelet-vWf bound to the platelet surface when platelets were stimulated in the presence of 5 mmol/L Ca++. The antibody against GPIb did not inhibit the surface expression of platelet-vWf on stimulated platelets in the presence of Ca++. Purified normal human fibrinogen inhibited the surface binding of platelet-vWf to thrombin-stimulated platelets to a degree similar to that observed with the monoclonal antibody directed against the GPIIb/IIIa complex. These data indicate that platelet-vWf released from platelets binds primarily to the GPIIb/IIIa complex at or near the plasma fibrinogen binding site.     

von Willebrand factor competes with fibrin for occupancy of GPIIb:IIIa on thrombin-stimulated platelets

We have investigated two major questions related to the molecular basis of interactions between the three-dimensional fibrin network and thrombin-stimulated human platelets. First, what are the roles played by glycoproteins (GP) Ib and IIb:IIIa in linking the fibrin clot tightly to the platelet surface? Second, does von Willebrand factor (vWF) modulate the extent of platelet-fibrin interactions? Quantitative fluorescence microscopy (microfluorimetry) has been used to determine the quantity of fluorescein-labeled fibrin bound to the surface of thrombin-stimulated, gel-filtered platelets (the supernatants of which contained small quantities of vWF) in the presence/absence of receptor-specific and vWF-specific monoclonal antibodies (MoAbs), as well as exogenous vWF. A MoAb specific for the GPIIb:IIIa complex exhibited a concentration-dependent inhibition of fibrin binding, whereas a MoAb specific for GPIb was ineffective in this regard. Similarly, a MoAb that recognizes the N-terminal region of vWF involved in GPIb binding did not influence fibrin binding. In contrast, a MoAb that binds to a C-terminal region of vWF involved in GPIIb:IIIa recognition caused a specific, concentration-dependent increase in the quantity of platelet-bound fibrin. We also found that exogenous vWF caused a concentration-dependent decrease in fibrin binding. These results support the hypothesis that vWF and fibrin, both of which are multimeric adhesive ligands, compete for occupancy of the GPIIb:IIIa complex on thrombin-stimulated platelets.     

The Membrane Glycoprotein IIb/IIIa Complex Mediates Deposition of Thrombin-stimulated Blood Platelets on Polystyrene Plastic Under Static Conditions

A major challenge in the use of artificial materials for implant devices, artificial organs, and extra-corporeal circulation systems, is the adhesion of platelets and the subsequent formation of platelet aggregates on the non-biological surface. The mechanism of platelet attachment to artificial surfaces is not completely understood. Using an enzyme immunoassay, we examined platelet deposition to the polystyrene plastic of microtiter plate wells under static conditions. Following thrombin stimulation, platelets adhered to the wells. This adhesion process was suppressible by the use of different substances known to interfere with the function of the platelet surface glycoprotein IIb/IIIa complex (GPIIb/IIIa). The substances we used were ethylenediaminetetraacetic acid (EDTA), tetrapeptide RGDS (Arg-Gly-Asp-Ser), and a monoclonal antibody directed against the IIIa moiety of the GPIIb/IIIa complex. Our results indicate that the GPIIb/IIIa complex is the platelet receptor which mediates platelet adhesion to polystyrene plastic under such static conditions. The GPIIb/IIIa complex should consequently be regarded as a multifunctional platelet regulator which, depending on the circumstances, may support platelet adhesion as well as platelet aggregation. By contrast, a monoclonal antibody directed against the platelet surface glycoprotein complex Ib/IX (GPIb/IX) did not under the same static conditions inhibit platelet deposition to the polystyrene plastic. In the microtiter wells, platelet alpha-granular proteins were detected either on the surface of adherent platelets or, when platelet deposition was inhibited by EDTA directly on the polystyrene plastic. In the latter case, fibrinogen and thrombospondin were definitely the dominating proteins. The presence of platelet-derived proteins in the microtiter wells significantly enhanced the adhesion of thrombin-stimulated platelets but not of non-stimulated platelets.     

Differential redistribution of platelet glycoproteins Ib and IIb-IIIa after plasmin stimulation.

The subcellular localization of the platelet membrane receptors glycoproteins (GP) Ib and IIb/IIIa [corrected] has been studied within resting platelets by a combination of biochemical and cytochemical techniques. While both GPIb and GPIIb/IIIa are localized within the plasma membrane and surface-connected canalicular system (SCCS) membranes, only GPIIb/IIIa is present within the internal face of alpha-granular membranes. Previous studies demonstrated that plasmin can induce platelet stimulation and also decrease ristocetin-induced platelet aggregation; it was suggested that this was because of GPIb degradation by plasmin. In this study, the respective localizations of both GPIb and GPIIb/IIIa were visualized during in vitro plasmin stimulation of platelets. Generally, plasmin induced shape change, pseudopod formation, organelle centralization either with or without alpha-granule release depending on the conditions of stimulation. Plasmin treatment of platelets at 37 degrees C resulted in the disappearance of GPIb from the cell surface and its subsequent redistribution into the channels and vesicles of the SCCS with no significant modification of GPIIb/IIIa remaining on the plasma membrane. Within degranulated platelets, GPIIb/IIIa was expressed on the plasma membrane and within membranes of large vacuoles containing the alpha-granule proteins. GPIb was virtually absent from these structures and mainly restricted to the SCCS. Addition of cytochalasin D inhibited the migration of GPIb to the SCCS. Biochemical measurements confirmed that no important hydrolysis of GPIb had occurred because only very little amounts of glycocalicin were generated during the reaction. In conclusion, in plasmin-treated platelets GPIIb/IIIa is externalized to the plasma membrane while GPIb is internalized into the SCCS. Although previous studies have suggested that plasmin degrades GPIb, the reduction in ristocetin-induced aggregation may be explained by its apparent redistribution within the membranes of the SCCS.     

Thrombin-induced inhibition of platelet agglutination by von Willebrand factor (vWF): reversal by ionized calcium

The present study has used washed human platelets combined with ethylene diamine tetracetic acid (EDTA) to determine the influence of calcium ions on thrombin-induced down-regulation of glycoprotein (GP) Ib/IX, the platelet surface receptor for von Willebrand factor (vWF). Bovine plasma vWF (BvWF) does not require the antibiotic, ristocetin, or calcium ions to cause agglutination of platelets. Thus, EDTA platelets agglutinate as well with BvWF as platelets in the absence of the chelating agent. Thrombin treatment of EDTA platelets prevented subsequent agglutination by BvWF. However, the addition of calcium ions to the sample restores sensitivity of the six PIb/IX receptors, and irreversible agglutination occurs when BvWF is added. Monoclonal antibodies to GPIb/IX and GPIIb/IIIa demonstrated that restoration of refractory platelet sensitivity to BvWF was related to GPIb/IX, not to GPIIb/IIIa. Experimental results suggest that GPIb/IX receptors on thrombin-treated EDTA platelets can be down-regulated by thrombin, but are not cleared from the surface to internal membranes.     

A quantitative study of human platelet membrane glycoproteins using immunocolloidal gold under the electron microscope

Two human platelet membrane glucoproteins (GP) and the quantitative changes of them induced by high shear stress (HSS) were studied using SZ-2 and SZ-21 monoclonal antibodies against GPIb and GPIIb/IIIa respectively by means of immunocolloidal gold technique with electron microscopy. Exposure to HSS (50 dynes/cm2, 5 min; 100 dynes/cm2, 3 min) resulted in the transformation of platelets from native smooth discs to activated spiny spheres followed by the centralization of organelles and lysis or aggregation of platelets. The results showed that resting platelets with few gold probes were turned into activated platelets by HSS with increased gold probes which represent the quantity of GPIb or GPIIb/IIIa. HSS also caused a specific platelet reaction--lysis. In the process of platelet lysis, platelet GPIIb/IIIa were increasingly condensed. On the contrary, GPIb reduced gradually. It suggests that the increase of GPIIb/IIIa under HSS may be one of the causes inducing platelet adhesion, release and aggregation at the opening of branched vessels, curved arteries, stenosed or partially occluded arteries due to local spasm.     

Thrombin-induced inhibition of platelet agglutination by von Willebrand factor (vWF): reversal by ionized calcium

The present study has used washed human platelets combined with ethylene diamine tetracetic acid (EDTA) to determine the influence of calcium ions on thrombin-induced down-regulation of glycoprotein (GP) Ib/IX, the platelet surface receptor for von Willebrand factor (vWF). Bovine plasma vWF (BvWF) does not require the antibiotic, ristocetin, or calcium ions to cause agglutination of platelets. Thus, EDTA platelets agglutinate as well with BvWF as platelets in the absence of the chelating agent. Thrombin treatment of EDTA platelets prevented subsequent agglutination by BvWF. However, the addition of calcium ions to the sample restores sensitivity of the six PIb/IX receptors, and irreversible agglutination occurs when BvWF is added. Monoclonal antibodies to GPIb/IX and GPIIb/IIIa demonstrated that restoration of refractory platelet sensitivity to BvWF was related to GPIb/IX, not to GPIIb/IIIa. Experimental results suggest that GPIb/IX receptors on thrombin-treated EDTA platelets can be down-regulated by thrombin, but are not cleared from the surface to internal membranes.     

Characterization of the binding domains on platelet glycoproteins Ib-IX and IIb/IIIa complexes for the quinine/quinidine-dependent antibodies.

Sera of 12 patients with quinine/quinidine-induced thrombocytopenia showed drug-dependent antibody binding to glycoprotein (GP) Ib-IX complex. The reaction with GPIb-IX complex of 11 of these 12 sera was strongly inhibited by the complex-specific monoclonal antibodies (MoAbs) AK1 and SZ1. The exception was a quinine-induced serum designated BU. The reaction of the six quinidine-induced sera was also partially blocked by an anti-GPIX MoAb, FMC25. Only 3 of the 12 patient sera showed drug-dependent antibody binding to GPIIb/IIIa, which was strongly inhibited by the anti-GPIIIa MoAb 22C4, and the anti-GPIIb alpha MoAb SZ22. With detergent-solubilized Serratia metalloprotease-treated platelets, quinine/quinidine-induced sera, except BU, immunoprecipitated a membrane-bound proteolytic fragment of GPIb-IX complex. In contrast, BU immunoprecipitated glycocalicin and a 40-Kd peptide tail fragment of GPIb alpha from the cell supernatant. Using purified GPIb-IX complex or its components as the target antigen, all the quinine-induced sera, except BU, immunoprecipitated GPIb-IX complex but failed to immunoprecipitate GPIb, GPIX, or the complex reformed from GPIb and GPIX. The quinidine-induced sera strongly immunoprecipitated purified GPIb-IX complex, weakly immunoprecipitated purified GPIX and the recombined complex, but did not immunoprecipitate purified GPIb. The combined data suggest that one quinine-dependent antibody (BU) recognizes an epitope in the peptide tail region of GPIb alpha and the other five quinine-dependent antibodies react with a complex-specific epitope on the membrane-associated region of GPIb-IX complex, whereas each of the six quinidine-induced sera contain two drug-dependent antibodies, one reactive with the GPIb-IX complex-specific epitope and the other reactive with GPIX. The binding domain(s) on GPIIb/IIIa for the quinine/quinidine-dependent antibodies appear to be sterically close to the epitopes for 22C4 and SZ22.     

Autoantibodies and autoantigens in chronic immune thrombocytopenic purpura

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder in which antiplatelet autoantibodies bind to antigens on the surface of platelets, resulting in their destruction. The newer antigen-specific (phase III) assays can detect platelet-associated and plasma autoantibodies in approximately 75% and 50% of patients, respectively. Antiplatelet autoantibodies bind to both platelets and megakaryocytes and preliminary evidence suggests that they not only cause platelet destruction but can also decrease platelet production either by interfering with megakaryocyte proliferation/maturation or by causing intramedullary platelet destruction. Autoantibodies are capable of activating complement and causing platelet phagocytosis both in vitro and in vivo. Many platelet-associated and plasma autoantibodies from ITP patients are light chain-restricted, which suggests a clonal origin. Approximately 75% of platelet autoantigens are localized to either the platelet glycoprotein (GP) IIb/IIIa or Ib/IX complex. Inhibition of the binding of autoantibodies from several ITP patients by either another ITP autoantibody or by a monoclonal anti-GPIIb/IIIa antibody suggests that the antigenic repertoire in chronic ITP may be limited. Most autoantigens on GPIIb/IIIa appear to be conformational since they are dependent on the presence of divalent cations. A variety of new investigative techniques have localized a few autoantigens to specific regions of the cytoplasmic or extracellular regions of both GPIIb/IIIa and GPIb/IX.     

On the role of von Willebrand factor in promoting platelet adhesion to fibrin in flowing blood

Platelet adhesion to fibrin at high shear rates depends on both the glycoprotein (GP) IIb:IIIa complex and a secondary interaction between GPIb and von Willebrand factor (vWF). This alternative link between platelets and vWF in promoting platelet adhesion to fibrin has been examined in flowing whole blood with a rectangular perfusion chamber. Optimal adhesion required both platelets and vWF, as shown by the following observations. No binding of vWF could be detected when plasma was perfused over a fibrin surface or when coated fibrinogen was incubated with control plasma in an enzyme-linked immunosorbent assay. However, when platelets were present during perfusion, interactions between vWF and fibrin could be visualized with immunoelectron microscopy. Exposure of fibrin surfaces to normal plasma before perfusion with severe von Willebrand's disease blood did not compensate for the presence of plasma vWF necessary for adhesion. vWF mutants in which the GPIIb:IIIa binding site was mutated or the GPIb binding site was deleted showed that vWF only interacts with GPIb on platelets in supporting adhesion to fibrin and not with GPIIb:IIIa. Complementary results were obtained with specific monoclonal antibodies against vWF. Thus, vWF must first bind to platelets before it can interact with fibrin and promote platelet adhesion. Furthermore, only GPIb, but not GPIIb:IIIa is directly involved in this interaction of vWF with platelets.     

Spontaneous platelet aggregation during pregnancy in a patient with von Willebrand disease type IIB can be blocked by monoclonal antibodies to both platelet glycoproteins Ib and IIb/IIIa

Spontaneous platelet aggregation appeared in a patient with von Willebrand disease type IIB during the 37th week of pregnancy. This phenomenon was not associated with symptoms of thrombosis and the patient delivered by caesarean section with no complications. Her platelet-poor plasma (PPP) aggregated normal platelet-rich plasma (PRP) and washed platelets. Aggregation was inhibited by monoclonal antibodies with known specificity for the platelet receptors of von Willebrand factor (vWF), i.e. the glycoprotein Ib (GPIb) and the GPIIb/IIIa complex. A monoclonal antibody, which selectively inhibits the binding of vWF to the GPIIb/IIIa complex, did not block aggregation, suggesting that spontaneous aggregation is not dependent on the binding to GPIIb/IIIa of vWF from patient plasma. Aggregation induced by patient plasma could also be blocked either by two monoclonal antibodies raised against vWF or by a fragment derived from trypsin digestion of normal vWF which blocks the ristocetin-induced binding of normal vWF to platelets. These findings indicate that the spontaneous platelet aggregation in this patient results from the binding of her vWF to GPIb but is independent from the binding of her vWF to GPIIb/IIIa.     

Detection of antigenic determinants of autoantibodies in idiopathic thrombocytopenic purpura using anti-platelet monoclonal antibodies and flow cytometry in whole blood]

To detect antigenic sites of platelet-bound autoantibodies in chronic ITP, whole blood or platelet-rich plasma from patients was stained with monoclonal antibodies (MoAbs) directed against platelet membrane glycoproteins (GPs) and with FITC-conjugated anti-mice IgG in an unwashed system followed by flow cytometric analysis. Platelets were identified by light-scattering profile and the amount of anti GP MoAb bound to platelets was measured and expressed as mean fluorescence intensity. This system enabled to quantitate the amount of intact GPs on platelet surface, regardless of activation of platelet. As a result, decreased amount of platelet-bound anti GPIIb/IIIa MoAb was noted in 5 of 22 patients with chronic ITP comparing to those with normal subjects. On the contrary, none of these patients revealed any demonstrable decrease in the amount of anti GPIb MoAb binding. From these observations, we suggest that the decreased MoAb binding to platelets in these five patients indicates the binding of autoantibody directed toward antigenic determinants, which are on or close to the epitope of this MoAb.     

Membrane glycoproteins and platelet cytoskeleton in immune complex- induced platelet activation

To clarify the mechanism of platelet activation by immune complexes and the possible involvement of surface glycoproteins (GPs), we studied platelet activation induced by heat-aggregated IgG (HAG). We examined the effects of monoclonal antibodies (MoAbs) against GPIb, GPIIb/IIIa, and the Fc receptor on resting platelets and on platelets stimulated by HAG. HAG increased the cytosolic ionized calcium concentration ([Ca2+]i) and stimulated protein (P47 and P20) phosphorylation, phosphatidic acid (PA) synthesis, serotonin secretion, and platelet aggregation. IV.3, an anti-Fc gamma RII receptor MoAb, inhibited HAG binding to platelets and all subsequent platelet responses. Like IV.3, MoAbs against GPIIb/IIIa (Tab, 10E5, AP-3) or GPIb (AP-1, 6D1) strongly inhibited platelet activation by HAG. However, while anti-GPIIb/IIIa MoAbs inhibited binding of IV.3 and HAG to platelets, anti-GPIb MoAbs had little effect on platelet binding of IV.3 or HAG. These observations suggest a close topographical and functional association of GPIIb/IIIa with Fc gamma RII in the platelet response to HAG. Cytochalasin B, an inhibitor of actin polymerization, also inhibited platelet activation but not HAG or IV.3 binding. Measurement of the fluorescence of 7-nitrobenz-2-oxa-1,3-(NBD)-phallacidin, a specific marker for filamentous actin (F-actin), showed that both cytochalasin B and AP-1 blocked the increase of F-actin induced by HAG. The common effects of anti-GPIb MoAbs and of cytochalasin B suggest that unlike the activity of GPIIb/IIIa, the ability of anti-GPIb to inhibit the activation of platelets by immune complexes is associated with perturbation of the cytoskeleton.     

Localization of GPIb/IX and GPIIb/IIIa on Discoid Platelets

The organization and reorganization of mobile receptors, GPIIb/IIIa and GPIb/IX, on surface- and suspension-activated platelets have been studied in detail, but their distribution on resting, discoid platelets is uncertain. The present study has treated platelets in suspension with cytochalasin E before mounting on formvar grids or glass slide fragments in order to preserve their discoid appearance, then probed the organization of GPIIb/IIIa with fibrinogen coupled to gold particles (Fgn/Au) and GPIb/IX with bovine or ristocetin-activated human plasma detected by combined anti-vWF antibody and protein A coupled to gold particles. Multimers of vWF had the same tortuous, linear distribution from edge to edge observed previously on surface-activated platelets. However, the gold particles marking the complex of vWF-anti-vWF bound to GPIb/M were closer together on the discoid cells. Fgn/Au particles bound to GPIIb/IIIa receptors were uniformly distributed from edge to edge on many discoid platelets. On others they tended to clump or cluster in strips or patches. The latter organization of Fgn/Au-GPIIb/IIIa receptors may be due to the rugose nature of the discoid platelet surface or an influence of cytochalasin E. Definition of mobile receptor organization on discoid cells provides a useful baseline for determining their fate following surface or suspension activation.     

Platelet membrane abnormalities in myeloproliferative disorders: decrease in glycoproteins Ib and IIb/IIIa complex is associated with deficient receptor function

The number and functional activity of membrane glycoproteins (GP) Ib and IIb/IIIa were investigated in platelets from 11 patients with myeloproliferative disorders (MPD). Three patients had essential thrombocythaemia, two had chronic myeloid leukaemia and six had polycythaemia vera. The numbers of GPIb and GPIIb/IIIa molecules were detected on the platelet surface using different 125I-labelled monoclonal antibodies. The functional properties of GPIb and GPIIb/IIIa were evaluated using purified 125I-labelled asialo von Willebrand factor (vWF) and purified 125I-labelled fibrinogen, respectively, in a binding assay. Binding of the anti-GPIIb/IIIa antibody was decreased by 40% in almost all patients studied and, when measured, it was accompanied by decreased fibrinogen binding to activated platelets. Binding of anti-GPIb antibodies to platelets was also slightly decreased or virtually the same in eight out of 11 patients. The decrease correlated with decreased binding of asialo vWF. The increased plasma glycocalicin levels, measured in four patients, depended on the high platelet count. Scatchard analysis revealed normal receptor binding affinity for all ligands tested in all but one patient. In this report we demonstrate that abnormalities in the concentrations of GPIIb/IIIa membrane proteins are commonly present in patients with MPD, while a decrease in GPIb concentration is also seen, although in fewer patients. These abnormalities are accompanied by a concurrent decrease in the respective receptor functions. These findings may explain part of the haemorrhagic tendency often encountered in MPD.     

Interaction of porcine von Willebrand factor with the platelet glycoproteins Ib and IIb/IIIa complex

Porcine von Willebrand factor (PvWF) induces platelet aggregation which is thought to be responsible for the thrombocytopenia that occurs in haemophilic patients treated with commercial preparations of porcine factor VIII. This study demonstrates that such aggregation can be completely inhibited by a monoclonal antibody against human platelet glycoprotein GPIb and partially inhibited by an antibody directed against platelet GPIIb/IIIa. The interaction of PvWF with GPIb is also demonstrated by the inhibitory effect of purified glycocalycin on aggregation. The binding site of PvWF to GPIb is very close to that of human vWF, since a recombinant peptide blocks the binding of both molecules to GPIb. When platelets are incubated with PvWF, the GPIIb/IIIa receptor is activated and binds fibrinogen. PvWF also binds to GPIIb/IIIa when platelets are stimulated with thrombin, suggesting that the molecule has the same RGD sequence as other adhesive proteins (human vWF, fibrinogen, fibronectin and vitronectin). These findings identify the dual mechanisms responsible for in vivo platelet aggregation induced by PvWF, i.e. binding to GPIb and activation of the GPIIb/IIIa receptor.     

Vitronectin inhibits blood platelet aggregation

The effect of vitronectin on platelet aggregation has been investigated. Vitronectin inhibited both thrombin- and ADP-induced platelet aggregation in a dose-dependent manner. A monoclonal antibody (MoAb) to vitronectin increased thrombin-induced platelet aggregation. This effect of the MoAb was not mediated via the platelet Fc-receptor, suggesting that the antibody directly counteracted the inhibitory effect of vitronectin on platelet aggregation. Like some other adhesive proteins such as fibrinogen, fibronectin, and von Willebrand factor, vitronectin contains the amino-acid sequence Arg-Gly-Asp (RGD) which enables binding to the platelet membrane glycoprotein complex IIb/IIIa (GPIIb/IIIa). The results of this study indicate that vitronectin can modulate the function of fibrinogen on platelet aggregation by interfering with the binding of fibrinogen to GPIIb/IIIa in activated platelets.     

Quinine- and quinidine platelet antibodies can react with GPIIb/IIIa

Quinine- and quinidine-dependent antiplatelet antibodies are generally believed to bind to the membrane glycoprotein complex, GPIb/IX. However, we and others have found that some drug-dependent antibodies bind to platelets from patients with the Bernard-Soulier syndrome which lack these glycoproteins. We therefore studied the reactions of a group of these antibodies with normal and Bernard-Soulier platelets and their membrane proteins. As indicated by rosette formation of the sensitized platelets around protein A-Sepharose beads, two quinine- and two quinidine-dependent antibodies reacted with both normal and Bernard-Soulier syndrome platelets at a high (300 microM) concentration of drug. At a pharmacologic drug concentration (10 microM), all four antibodies reacted with normal platelets but only the two quinine-induced antibodies reacted with Bernard-Soulier platelets. Immunoprecipitation studies with solubilized, tritium-labelled normal platelets, at both high and low drug concentrations, showed that each of the four antibodies precipitated proteins corresponding to GPIb and GPIX. Fainter bands corresponding to glycoproteins IIb and IIIa, which do not label well with tritium, were also detected. With radioiodinated normal platelets, it was found that each of the four antibodies was capable of precipitating GPIIb/IIIa, but only in the presence of drug. The four antibodies also promoted drug-dependent precipitation of GPIIb and GPIIIa from lysates of radioiodinated Bernard-Soulier platelets. The two quinine-dependent antibodies precipitated these glycoproteins at both high and low drug concentrations, while the quinidine-dependent antibodies reacted much more strongly at the higher drug concentration. Precipitation of GPIb/IX was not observed with BSS platelets. Absorption of a quinine-induced antibody with Bernard-Soulier platelets in the presence of drug eliminated its ability to precipitate GPIIb and GPIIIa. However, the absorbed antibody retained the ability to precipitate GPIb from solubilized normal platelets. Thus, at least two drug-dependent antibodies were present, one specific for GPIb/IX and the other for GPIIb/IIIa. These findings indicate that glycoproteins IIb and/or IIIa, in addition to the GPIb/IX complex, can serve as targets for drug-dependent antibodies in both intact and detergent-solubilized platelet preparations.     

Immunologic and biochemical characterization of homozygous and heterozygous Glanzmann thrombasthenia in the Iraqi-Jewish and Arab populations of Israel: comparison of techniques for carrier detection

To define biochemically and immunologically the platelet defect in Iraqi-Jews and Arabs with Glanzmann thrombasthenia in Israel, we tested the platelets of 32 thrombasthenics and 37 obligate carriers from 19 families with affected members. Thrombasthenic platelets were devoid of glycoprotein IIb (GPIIb) as judged by polyacrylamide gel electrophoresis and devoid of the GPIIb/IIIa complex as judged by radio-electroimmunoassay. Binding of a murine monoclonal antibody directed at GPIIb and/or GPIIIa to intact thrombasthenic platelets averaged less than 2% of the control value. Evaluation of the number of molecules of antibody bound per platelet permitted discrimination between controls and obligate carriers with a high degree of accuracy (sensitivity = 91.9%, specificity = 92.3%). Obligate carriers could also be discriminated from controls by determining the ratio of GPIIb to GPIb by polyacrylamide gel electrophoresis and by quantifying the GPIIb/IIIa complex by radio-electroimmunoassay. These studies indicate that the thrombasthenics in Israel have the severe form of the disease (type I) and that the platelets of heterozygotes have significantly reduced amounts of both total and surface-exposed GPIIb and/or GPIIIa.     

Analysis of GPIIb/IIIa receptor number by quantification of 7E3 binding to human platelets

A large number of glycoprotein (GP) IIb/IIIa receptors are present on the surface of platelets. Studies to define precisely the number of GPIIb/IIIa receptors using specific monoclonal antibodies (MoAbs) or fibrinogen binding have, however, yielded varying estimates of receptor number. To refine the quantitative estimation of GPIIb/IIIa receptors on resting platelets, we have used the MoAb 7E3, which has high affinity for GPIIb/IIIa. Quantitative binding studies were performed using radiolabeled conjugates of 7E3 IgG, as well as fragments and derivatives of 7E3. For platelets obtained from any single individual, the numbers of 7E3 F(ab')2 and IgG molecules bound per platelet were equivalent (approximately 40,000), whereas the number of Fab molecules bound per platelet was consistently approximately twofold higher (approximately 80,000). To investigate the basis of the quantitative disparity in binding of intact 7E3 and 7E3 F(ab')2 versus 7E3 Fab, we studied the binding of a newly constructed, bispecific (Fab')2 molecule containing only a single 7E3 combining site. Because this construct bound to the same extent as the Fab species, the larger size of the intact 7E3 and 7E3 F(ab')2 molecules could not explain the reduced number of molecules that bound per platelet compared to the Fab fragment. Rather, it appears that the valency of the antibody is the critical factor determining the number of antibody molecules bound per platelet. Thus, we conclude that the binding of 7E3 Fab corresponds most closely with surface GPIIb/IIIa number and that the number of GPIIb/IIIa receptors is approximately 80,000 per platelet.