Structural biology of glycoprotein IIb-IIIa

Glycoprotein IIb-IIIa (GPIIb-IIIa), a calcium-dependent heterodimer whose expression is restricted to platelets and megakaryocytes, contains a binding site for protein ligands such as fibrinogen and von Willebrand factor (vWf) whose exposure by platelet stimulation is a prerequisite for platelet aggregation. GPIIb-IIIa heterodimers are assembled from nascent GPIIb and GPIIIa subunits in the calcium-rich environment of the endoplasmic reticulum, and correctly folded heterodimers are transported from the endoplasmic reticulum through the Golgi apparatus to the cell surface. Agonist stimulation of platelets produces a conformational change in GPIIb-IIIa that exposes its ligand binding site, a process termed "insideout" signaling. This signaling process, by interacting with the cytoplasmic extensions of GPIIb and GPIIIa, converts the heterodimer from an inactive to an activated state capable of binding soluble ligands.     

Decreased stability and structural heterogeneity of the residual platelet glycoprotein IIb/IIIa complex in a variant of Glanzmann''s thrombasthenia

A patient is described with a disturbance of platelet function comparable to that in Glanzmann's thrombasthenia. Platelet aggregation and binding of fibrinogen to the patient's platelets were defective and thrombin-induced clot retraction was absent. The platelet fibrinogen content was only moderately reduced. As measured by monoclonal antibody binding in the presence of divalent cations, the platelets contained about 15% of the normal amount of GPIIb and GPIIIa and only 6% of the normal amount of intact GPIIb/IIIa complex. The residual GPIIb/IIIa complex exhibited a decreased stability as shown by the lack of binding of a complex-dependent anti-GPIIb/IIIa antibody to platelets incubated with ethylene diamine tetraacetic acid (EDTA) at 22 degrees C. Crossed immunoelectrophoresis (CIE) in the presence of divalent cations showed partial dissociation of GPIIb/IIIa as well as the presence of two forms of the residual intact GPIIb/IIIa complex. In addition, both CIE in the presence of the EDTA and two-dimensional sodium dodecyl sulphate (SDS) gel electrophoresis showed the presence of two forms of GPIIb. This form of thrombasthenia is characterized by a defective platelet function, a marked reduction of GPIIb and GPIIIa, decreased stability of the residual GPIIb/IIIa complex and structural heterogeneity of GPIIb.     

Platelet-associated antibody to glycoprotein IIb/IIIa from chronic immune thrombocytopenic purpura patients often binds to divalent cation- dependent antigens

Chronic immune thrombocytopenic purpura (ITP) is a syndrome of destructive thrombocytopenia due to autoantibodies against platelet- associated antigens. These antigens are most commonly located on the platelet glycoprotein (GP) IIb/IIIa complex. In the present studies, we show that many platelet-associated anti-GPIIb/IIIa autoantibodies from chronic ITP patients depend on conformationally intact GPIIb/IIIa for maximal binding. We studied anti-GPIIb/IIIa autoantibodies from 19 ITP patients (15 platelet-associated, 8 plasma) and alloantibodies from three patients with posttransfusion purpura (anti-PIA1). Antibodies were preincubated with purified intact GPIIb/IIIa, EDTA-dissociated GPIIb/IIIa, GPIIIa, or GPIIb for 2 hours and then residual antibody was measured in an antigen capture assay. The binding results were compared with those obtained using antibody preincubated in buffer. Of the 15 platelet-associated autoantibodies studied, the intact GPIIb/IIIa complex resulted in greater inhibition of antibody binding than the EDTA-dissociated complex, with a mean inhibition ratio (intact/dissociated) of 7.9 (range, 1.4 to 30.3). Little inhibition was noted using either GPIIb or GPIIIa. Conversely, plasma anti-PIA1 alloantibodies or plasma autoantibodies from ITP patients against the c- terminal region of GPIIIa were more efficiently inhibited by the dissociated complex or purified GPIIIa. We conclude that platelet- associated anti-GPIIb/IIIa autoantibodies in chronic ITP are frequently directed to cation-dependent conformational antigens.     

Biosynthesis and assembly of platelet GPIIb-IIIa in human megakaryocytes: evidence that assembly between pro-GPIIb and GPIIIa is a prerequisite for expression of the complex on the cell surface

The platelet membrane glycoproteins GPIIb and GPIIIa form a calcium- dependent heterodimer that functions as a receptor for adhesive proteins on stimulated platelets. In this study, we have investigated the kinetics of the assembly reaction that result in GPIIb-IIIa dimerization. Pulse-chase experiments analysis performed on human megakaryocytes obtained from liquid cultures of chronic myelogenous leukemic patients with antibodies specific for GPIIIa or GPIIb demonstrated the existence of a pro-GPIIb-GPIIIa complex and of a large pool (60%) of unassociated GPIIIa; nearly all the GPIIb and the pro- GPIIb molecules were found associated with GPIIIa. This free GPIIIa was not exposed on the cell surface. Pulse-chase experiments on a subclone of the human megakaryocytic cell line LAMA-84 revealed that the cells from this subclone produced only the pro-GPIIb, which was neither processed into mature GPIIb nor expressed on the cell surface. The expression of GPIIIa in PMA treated cells resulted in the production of the mature GPIIb form and the expression of the GPIIb-IIIa complex on the cell surface. These results indicate that assembly between the early forms of pro-GPIIb and GPIIIa is an obligatory step for the maturation of the heterodimer and its expression on the cell surface.     

Glycoprotein IIb and IIIa retention on fibrinogen-coated surfaces after lysis of adherent platelets

The platelet-membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex is essential for platelet aggregation and is involved in the attachment of platelets to thrombogenic surfaces. This study shows the retention of GPIIb and GPIIIa on immobilized fibrinogen after Triton X-100 (Sigma Chemical Co, St Louis, MO) lysis of adherent platelets. Glycoproteins were detected using subunit specific monoclonal antibodies in a modified enzyme-linked immunosorbent assay procedure. GPIIb-IIIa retention was judged to be specific relative to GPIb recovery, and was modulated by platelet activation. Platelet exposure to adenosine diphosphate or thrombin, but not A23187 or chymotrypsin, markedly enhanced GPIIb and GPIIIa recovery relative to that observed with unstimulated platelets, or prostaglandin E1-treated platelets. Moreover, lysis of adherent platelets in the presence of 10 mmol/L EDTA, under conditions promoting GPIIb-IIIa complex dissociation (pH 8.1, 60 minutes, 37 degrees C), had no effect on GPIIb or GPIIIa subunit recovery. Platelet activation with Zn+2 also enhanced GPIIb and GPIIIa recovery on fibrinogen-coated surfaces over that observed with unstimulated platelets, but GPIIb and IIIa retention was EDTA sensitive. This correlated with the EDTA-reversible nature of Zn+2- activated platelet adhesion to fibrinogen-coated surfaces. The data (1) show that platelet adhesion to fibrinogen is accompanied by the induction of high-affinity interactions between GPIIb-IIIa and immobilized fibrinogen that are EDTA-resistant and enhanced by platelet activation with some but not all agonists, and (2) implicate these interactions in stabilizing platelet contacts with fibrinogen-coated surfaces.     

Demonstration of a marked reduction in the amount of GPIIb in most type II patients with Glanzmann's thrombasthenia

Summary. In this study employing a sensitive immunoblot assay, we have characterized GPIIb and GPIIIa in thrombasthenic platelets from seven type II and four type I patients from 10 unrelated families. The amounts of GPIIb and GPIIIa were both markedly reduced in all these patients, and abnormal molecular weight GPIIb or GPIIIa was not detected. In all of four type I patients the amount of GPIIb was much lower than that of GPIIIa. In this study, however, we found that the amount of GPIIb was also lower even in six out of seven type II patients. Immunodepletion of patients'platelets with AP2 (a monoclonal antibody specific for the GPIIb-IIIa complex), AP3 (specific for GPIIIa) or AMF7 (specific for αv) further confirmed that GPIIIa existed in excess, and demonstrated that excess GPIIIa were mostly in free form and not associated with GPIIb or αv. The reduction of GPIIb may represent an abnormality in GPIIb processing in these type II and type I thrombasthenic platelets. It remains unclear whether these two subgroups represent distinct categories.     

Diagnosis of Glanzmann's thrombasthenia and carrier detection using monoclonal antibodies to platelet glycoprotein IIb and IIIa in immunoblotting.

Glanzmann's thrombasthenia is a rare hemorrhagic syndrome, characterized by a quantitative or functional defect of the platelet glycoprotein GPIIb-IIIa complex. The authors describe a method to diagnose thrombasthenic patients and identify carrier subjects by using monoclonal antibodies specific for GPIIb and GPIIIa in an immunoblotting technique. The immunoreaction patterns of two thrombasthenic patients lacking GPIIb or GPIIIa, respectively, are shown. The described method produces further evidence concerning the biochemical heterogeneity of Glanzmann's thrombasthenia.     

Autoantibodies against the platelet glycoprotein IIb/IIIa complex in patients with chronic ITP

Chronic idiopathic thrombocytopenic purpura (ITP) is caused by an antibody reactive with platelet-associated antigens. The present studies provide direct evidence that some patients with chronic ITP have autoantibodies against the platelet glycoprotein (GP) IIb/IIIa complex. Microtiter wells, coated with a monoclonal antibody (2G12) specific for GPIIb/GPIIIa were reacted with GPIIb/GPIIIa contained in a platelet extract. Control wells containing the same antibody were reacted with a cell extract containing no GPIIb/GPIIIa. After washing, the wells were reacted with patient or control plasma, and IgG binding was detected using 125I-Fab2-anti-human IgG. Assay values were expressed as binding ratios (cpm GPIIb/GPIIIa wells/cpm control wells). Plasma from 5 of 56 patients with chronic ITP had ratios (1.36-3.14) greater than 3 standard deviations above the mean (+/- SD) of control plasmas--0.93 +/- 0.12. Elevated values were also noted in two patients with anti-P1A1 antibody (ratios greater than 30) and in one patient with Hodgkin's disease and an ITP-like syndrome (ratio 1.53). Normal values were noted in 34 patients with a variety of immune and nonimmune diseases. Plasma from two of the positive ITP patients was reacted with 125I-surface-labeled platelets and, after solubilization, the IgG and bound antigen were precipitated with Staph-A. Autoradiographs from SDS- PAGE electrophoresis of the Staph-A-bound proteins shows two radioactive bands consistent in size with GPIIb and GPIIIa.     

Detection of an epitope specific for the dissociated form of glycoprotein IIIa of platelet membrane glycoprotein IIb-IIIa complex and its expression on the surface of adherent platelets

Summary Glycoproteins (GPs) IIb and IIIa form a Ca²⁺-dependent complex in platelet membrane and change their conformation upon platelet activation and dissociation of the complex. A new anti-GPIIIa monoclonal antibody (mAb). CRC54, is described which could distinguish different conformational states of GPIIIa. This antibody (i) precipitated GPIIb-IIIa from platelet Triton X-100-lysate. (ii) recognized the GPIIIa band in Western blotting of platelet SDS-lysate, and (iii) did not react with platelets from a Glanzmann's thrombasthenia patient lacking GPIIb-IIIa. Immunoblotting of chymotryptic digestion products of purified GPIIb-IIIa has shown that CRC54 epitope is located within residues 1–100 at the N-terminus of GPIIIa. CRC54 bound weakly to platelets in the presence of Ca²⁺ and Mg²⁺, 2.34 ± 0.28 ± 10³ molecules per platelet at saturation. The same level of binding was observed without any divalent cations in the medium. However, binding of CRC54 was increased by several times after treatment of platelets with EDTA, 10.04 ± 0.28 ± 10³ molecules per platelet. Increase of CRC54 binding correlated with the dissociation of GPIIb-IIIa complex which was followed by the decrease of the binding of another mAb, CRC64, directed against complex-specific epitope of GPIIb-IIIa. Binding of CRC54 to platelets was changed neither by platelet activation in suspension with thrombin or ADP nor by the occupancy of GPIIb-IIIa ligand binding site with GRGDSR peptide. However. binding was significantly stimulated by platelet adhesion to polystyrene plastic. As measured using ⁵¹Cr-labelled platelets, binding of ^l25I-CRC54 to adherent platelets in the presence of divalent cations was about 4 times higher than to platelets in suspension, 8.68 ± 0.48 ± 10³ per platelet. This increase was not due to the dissociation of GPIIb-IIIa since complex-specific antibody CRC64 still bound effectively to the surface of adherent platelets. The data obtained indicated that: (1) CRC54 recognized an epitope specific for the dissociated form of GPIIIa: (2) the CRC54-reactive epitope of GPIIIa is also expressed on the surface of adherent platelets.     

The Arg-Gly-Asp (RGD) recognition site of platelet glycoprotein IIb-IIIa on nonactivated platelets is accessible to high-affinity macromolecules.

We have characterized a murine IgG monoclonal antibody, OP-G2, specific for platelet glycoprotein (GP) IIb-IIIa (alpha IIb beta 3). OP-G2 Fab fragments inhibit fibrinogen-mediated platelet aggregation and competitively inhibit adenosine diphosphate-induced binding of 125I-fibrinogen to washed platelets. OP-G2 binding to GPIIb-IIIa is specifically inhibited by RGD-containing peptides but not the fibrinogen gamma-chain carboxy-terminal peptide, and OP-G2 Fab fragments, like RGD-containing peptides, alter the conformation of GPIIb-IIIa resulting in the expression of a ligand-induced binding site (LIBS) recognized by PMI-1. OP-G2 fails to bind to the recombinant Cam variant of GPIIb-IIIa (alpha III beta 3Cam) wherein an Asp119 to Tyr119 substitution in GPIIIa abrogates the ability to recognize RGD. These data indicate that OP-G2 recognizes an epitope at or in very close proximity to the RGD recognition site of GPIIb-IIIa and that, in every aspect tested, OP-G2 behaves like a macromolecular RGD ligand. Interestingly, two-color flow cytometry shows that OP-G2 IgG can bind to nonactivated platelets. Quantitative binding assays indicate that nonactivated platelets bind approximately 50,000 125I-OP-G2 molecules/platelet. Furthermore, the affinity of OP-G2 for platelets activated with thrombin is roughly fivefold higher (nonactivated, kd = 24.8 nmol/L; activated, kd = 4.9 nmol/L). These results suggest that the RGD recognition site of GPIIb-IIIa is available to macromolecules that contain RGD even on nonactivated platelets, provided that the affinity of the ligand is adequate.     

Combined defect in membrane expression and activation of platelet GPIIb--IIIa complex without primary sequence abnormalities in myeloproliferative disease

Defects in glycoprotein (GP)IIb-IIIa or in its activation may cause abnormal platelet aggregation and a bleeding diathesis. We report studies in a 67-year-old man with a myeloproliferative disease and markedly abnormal platelet responses. By flow cytometry, platelet binding of two complex-specific anti-GPIIb-IIIa monoclonal antibodies (mAbs), A2A9 and 10E5, was approximately 50% of normal. An enzyme-linked immunosorbent assay (ELISA) using immobilized kistrin showed 18% of normal membrane GPIIb-IIIa complex. By immunoblot analysis, GPIIb and GPIIIa levels in platelet lysates and membranes were near normal. Activation of GPIIb-IIIa, monitored with mAb PAC-1, was markedly decreased (< 20% of normal) in response to ADP, thrombin and platelet-activating factor (PAF); expression of ligand-induced binding sites (LIBS) was < or = 30% of normal. Signal transduction-independent LIBS expression, induced by echistatin, was approximately 60% of normal, suggesting that the integrin present had intact ligand-binding capability. Sequence analysis of GPIIb and GPIIIa cDNA, and platelet mRNA levels for both subunits, were normal. These findings document an acquired combined defect in membrane expression (secondary to a defect in post-translational processing of the complex) and inside-out signalling-dependent activation of the GPIIb-IIIa complex.     

Homozygous Cys542-->Arg substitution in GPIIIa in a Swiss patient with type I Glanzmann's thrombasthenia

Glanzmann's thrombasthenia (GT) arises from a qualitative or quantitative defect in the GPIIb-IIIa complex (integrin alphaIIbbeta3), the mediator of platelet aggregation. We describe a patient in whom clinical and laboratory findings typical of type I GT were found together with a second pathology involving neurological and other complications symptomatic of tuberous sclerosis. Analysis of platelet proteins by Western blotting revealed trace amounts of normally migrating GPIIb and equally small amounts of GPIIIa of slightly slower than normal migration. Flow cytometry confirmed a much decreased binding to platelets of monoclonal antibodies to GPIIb, GPIIIa or GPIIb-IIIa, and an antibody to the alphav subunit also showed decreased binding. Nonradioactive PCR single-strand conformation polymorphism analysis followed by direct sequencing of PCR-amplified DNA fragments showed a homozygous point mutation (T to C) at nucleotide 1722 of GPIIIa cDNA and which led to a Cys542-->Arg substitution in the GPIIIa protein. The mutation gave rise to a HinP1 I restriction site in exon 11 of the GPIIIa gene and allele-specific restriction enzyme analysis of family members confirmed that a single mutated allele was inherited from each parent. This amino acid substitution presumably changes the capacity for disulphide bond formation within the cysteine-rich core region of GPIIIa and its study will provide new information on GPIIb-IIIa and alphavbeta3 structure and biosynthesis.     

Plasma autoantibodies against platelet glycoprotein IIb/IIIa from patients with autoimmune thrombocytopenic purpura may recognize different antigenic determinants

Abstract: Autoantibodies against platelet glycoprotein (GP) GPIIb/IIIa have been demonstrated in patients with autoimmune thrombocytopenic purpura. Recently, it has been shown that plasma autoantibodies from some patients bind to the cytoplasmic domain of GPIIIa. Our aim was to evaluate further the binding specificity of these plasma autoantibodies. From 7 patients with detectable plasma antibodies against intact GPIIb/IIIa, 1 showed strong antibody binding to a synthetic C-terminal peptide of GPIIIa. Ig class analysis of affinity purified anti-GPIIb/IIIa autoantibodies from this patient revealed an IgM antibody that reacted with intact GPIIb/IIIa as well as with recombinant GPIIb/IIIa lacking the C-terminal domains, and an IgG antibody that bound to intact GPIIb/IIIa but not to GPIIb/IIIa lacking the C-terminal region. These data indicate that this patient has at least 2 autoantibodies, an IgG directed against the cytoplasmic domain of GPIIIa and an IgM reacting with the extracellular part of GPIIIa. This may support the hypothesis that plasma IgG antibodies directed against the C-terminal domain of GPIIIa may be due to the exposition of cytoplasmic epitopes of GPIIIa as a result of increased cell lysis by IgM autoantibodies.     

Platelet-associated anti-GPIIb-IIIa autoantibodies in chronic immune thrombocytopenic purpura recognizing epitopes close to the ligand-binding site of glycoprotein (GP) IIb

Localization of epitopes for platelet-associated (PA) anti-GPIIb-IIIa (alpha(IIb)beta(3)) autoantibodies in chronic immune thrombocytopenic purpura remains elusive. Previous studies suggest that PA antibodies recognize the tertiary structure of intact glycoprotein (GP) IIb-IIIa. To localize their epitopes using antigen-capture enzyme-linked immunosorbent assay (ELISA), the reactivity of 34 PA anti-GPIIb-IIIa antibodies was examined with recombinant GPIIb-IIIa having a defect in ligand-binding sites in either GPIIb or GPIIIa, and no major conformational change was induced: KO variant GPIIb-IIIa was attributed to a 2-amino acid insertion between residues 160 and 161 in the W3 4-1 loop in GPIIb, and CAM variant GPIIb-IIIa was attributed to D119Y in GPIIIa. In one third (11 of 34) of the patients, PA antibodies showed a marked decrease (less than 50%) in reactivity with KO compared with wild-type GPIIb-IIIa. Their reactivity was also impaired against GPIIbD163A-IIIa. In sharp contrast, they reacted normally with CAM GPIIb-IIIa. OP-G2, a ligand-mimetic monoclonal antibody, markedly inhibited their binding to GPIIb-IIIa in patients with impaired binding to KO GPIIb-IIIa, but small GPIIb-IIIa antagonists did not. In addition, a newly developed sensitive ELISA indicated that autoantibodies showing impaired binding to KO are more potent inhibitors for fibrinogen binding. The present data suggest that certain PA anti-GPIIb-IIIa autoantibodies recognize epitopes close to the ligand-binding site in GPIIb, but not in GPIIIa.     

Quantitation of membrane glycoprotein IIIa on intact human platelets using the monoclonal antibody, AP-3

A murine monoclonal antibody specific for glycoprotein (GP)IIIa was prepared by immunization with a GPIIb- and GPIIIa-enriched Triton X-114 extract of platelet membranes. This antibody, designated AP-3, was shown by indirect immunoprecipitation to react solely with GPIIIa derived from either P1A1-positive or -negative individuals. The epitope on GPIIIa recognized by AP-3 is expressed on dissociated GPIIIa as well as on Ca+2-dependent complexes of GPIIb and GPIIIa, as shown by crossed immunoelectrophoresis in the presence or absence of EDTA. A previously described monoclonal antibody specific for the GPIIb/IIIa complex (AP- 2) inhibited platelet aggregation induced by ADP, thrombin, collagen, or arachidonic acid (Pidard et al, J Biol Chem 258:12582-12586, 1983). In contrast, AP-3 had no effect on aggregation induced by any of these reagents, a finding similar to that previously reported for the GPIIb- specific monoclonal antibody, Tab (McEver et al, J Clin Invest 66:1311- 1318, 1980). At saturation, 40,200 AP-3 molecules were bound per platelet, a value similar to that obtained for AP-2 or Tab. Thus, data derived using AP-3 indicate that significant amounts of free GPIIIa are not present, thereby supporting the hypothesis that GPIIb and GPIIIa exist complexed in a 1:1 stoichiometry in the plasma membrane of intact, nonactivated platelets.     

Reassociation and translocation of glycoprotein IIB-IIIA in EDTA-treated human platelets

Platelet membrane glycoproteins IIb and IIIa form a calcium-dependent heterodimer that plays a key role in platelet adhesion and aggregation. The present objective was to measure the dissociation and reassociation of GPIIb-IIIa by flow cytometric analysis of platelets labelled with mAbs specific for the glycoprotein complex or each monomer. In agreement with previous studies, EDTA chelation of extracellular calcium, [Ca2+]o, dissociated the heterodimer in a time and temperature dependent manner. Agonist stimulation of EDTA-treated platelets induced subunits to reassociate with the following order of potency: thrombin > collagen > ADP. Two-fold increases in GPIIb-IIIa and GPIIb indicate that thrombin caused reassociation of surface subunits and concurrent translocation of complexes from intracellular pools. The latter was partially inhibited by cytochalasin B thus indicating that a subpopulation of GPIIb-IIIa required cytoskeletal remodelling for translocation. Surface GPIIIa as reported by anti-CD61 declined more and upregulated less compared with GPIIb-IIIa or GPIIb. Results suggest that EDTA incubation might have altered the conformation of this epitope and decreased mAb binding. Collagen induced GPIIb-IIIa reassociation but not translocation of cryptic complexes. BAPTA suppression of rises in cytosolic calcium concentration or low [Ca2+]o inhibited GPIIb-IIIa reassociation, thus indicating that this reaction was driven by signal transduction. Thrombin and collagen induced a comparable level of aggregation of EDTA-treated platelets despite a 3-fold difference in cell surface GPIIb-IIIa. It is concluded that the effects of EDTA on GPIIb-IIIa dissociation and loss of adhesive functions are largely but not completely reversible.     

Platelet glycoprotein IIb as a target antigen in two patients with chronic idiopathic thrombocytopenic purpura

We have investigated the target antigens recognized by anti-platelet antibodies in patients with chronic idiopathic thrombocytopenic purpura (ITP) using an immunoblot procedure which could electrically separate the glycoprotein (GP) IIb/IIIa complex into GPIIb and GPIIIa. Various platelet proteins, having molecular weights of 167, 160, 145, 135, 124, 102, 92 and 80 kD, were recognized by circulating antibodies in 11 of 40 ITP patients. We identified the 145 kD antigen band, seen in two ITP patients, as GPIIb using thrombasthenic platelets as a source of target antigens. In one patient the anti-GPIIb antibody reacted with autologous GPIIb. These studies provide direct evidence for the presence of autoantibodies against GPIIb in some ITP patients.     

Reassociation and translocation of glycoprotein IIB-IIIA in EDTA-treated human platelets

Platelet membrane glycoproteins IIb and IIIa form a calcium-dependent heterodimer that plays a key role in platelet adhesion and aggregation. The present objective was to measure the dissociation and reassociation of GPIIb-IIIa by flow cytometric analysis of platelets labelled with mAbs specific for the glycoprotein complex or each monomer. In agreement with previous studies, EDTA chelation of extracellular calcium, [Ca²⁺]_o, dissociated the heterodimer in a time and temperature dependent manner. Agonist stimulation of EDTA-treated platelets induced subunits to reassociate with the following order of potency: thrombin?>?collagen?>?ADP. Two-fold increases in GPIIb-IIIa and GPIIb indicate that thrombin caused reassociation of surface subunits and concurrent translocation of complexes from intracellular pools. The latter was partially inhibited by cytochalasin B thus indicating that a subpopulation of GPIIb-IIIa required cytoskeletal remodelling for translocation. Surface GPIIIa as reported by anti-CD61 declined more and upregulated less compared with GPIIb-IIIa or GPIIb. Results suggest that EDTA incubation might have altered the conformation of this epitope and decreased mAb binding. Collagen induced GPIIb-IIIa reassociation but not translocation of cryptic complexes. BAPTA suppression of rises in cytosolic calcium concentration or low [Ca²⁺]_o inhibited GPIIb-IIIa reassociation, thus indicating that this reaction was driven by signal transduction. Thrombin and collagen induced a comparable level of aggregation of EDTA-treated platelets despite a 3-fold difference in cell surface GPIIb-IIIa. It is concluded that the effects of EDTA on GPIIb-IIIa dissociation and loss of adhesive functions are largely but not completely reversible.     

A 1063G-->A mutation in exon 12 of glycoprotein (GP)IIb associated with a thrombasthenic phenotype: mutation analysis of [324E]GPIIb

We report the molecular, genetic and functional analysis of a case of thrombasthenic phenotype. The proband showed absence of platelet glycoprotein (GP)IIb and very low content of GPIIIa, and both his parents showed a marked reduction in the levels of platelet GPIIb-IIIa. Single-stranded conformational polymorphism-polymerase chain reaction (SSCP-PCR) analysis and direct sequencing of PCR-amplified GPIIb exon-12 revealed the presence of a G-->A transition at position 1063 with the expected substitution of glutamate 324 with lysine (K). This mutation did not alter the level of GPIIb mRNA. Co-expression of normal or mutant [324K] GPIIb with normal human GPIIIa in Chinese hamster ovary (CHO) cells failed to show surface exposure of [324K]GPIIb-IIIa complexes. Pulse-chase and immunoprecipitation analysis demonstrated that [324K]GPIIb cDNA was translated into proGPIIb, but neither mutant GPIIb heavy chain (GPIIbH) nor [324K]GPIIb-GPIIIa complexes were detected, suggesting that this mutation is the underlying molecular basis for the thrombasthenic phenotype. Mutation analysis demonstrated that 324E of GPIIb could be replaced by other negatively charged or polar amino acids (AAs) without impairing the surface expression of GPIIb-IIIa. However, substitution of 324E of GPIIb for a positively charged AA other than K prevented the expression of GPIIb-IIIa complexes. These observations suggest that a domain encompassing 324E of GPIIb is essential for heterodimerization with GPIIIa and its substitution for a positively charged residue precludes normal subunit association.