Detection of circulating human platelet fragments by using monoclonal antibodies against platelet glycoprotein IIb-IIIa complex

A radioimmunoassay (RIA) for the detection of platelets and platelet fragments was developed. A sandwich of two monoclonal antibodies directed against the platelet-specific glycoprotein complex IIb-IIIa (GP IIb-IIIa) was used in this assay. A discontinuous 7.5-20% (v/v) albumin gradient was applied to separate platelets and their fragments of various sizes. In platelet suspensions fractionated in this way, we observed that particles smaller than normal platelets still carried the GP IIb-IIIa antigens. This procedure enabled us to detect platelet-derived particles in platelet-rich plasma from thrombocytopenic patients.     

Biochemical and functional consequences of dissociation of the platelet membrane glycoprotein IIb-IIIa complex

The platelet membrane glycoproteins, IIb and IIIa, form a Ca2+- dependent heterodimer complex that functions as the fibrinogen receptor in activated platelets to mediate platelet aggregation. Little is known about factors that affect the IIb-IIIa complex within the platelet membrane. It has been observed that platelets incubated with ethylene glycol tetra-acetic acid (EGTA) at 37 degrees C are unable to aggregate or to bind monoclonal antibodies specific for the IIb-IIIa complex. To determine whether this is due to a dissociation of IIb from IIIa, we developed a method for quantitating the complex on nondenaturing, polyacrylamide gradient gels. Platelets were surface-labeled with 125I and then solubilized and electrophoresed in 0.2% Triton and 10 mmol/L CHAPS. Under these conditions and in the presence of 1 mmol/L Ca2+, glycoproteins IIb and IIIa migrated on the gels as a discrete band at Rf = 0.33. Protein that was eluted from this band bound to an immunoaffinity column specific for the IIb-IIIa complex. In contrast, when the IIb-IIIa complex was solubilized and then dissociated with EGTA, the discrete band at Rf = 0.33 was no longer present, and IIb and IIIa were now found in a broad band at Rf = 0.45 to 0.50. To study IIb and IIIa within the surface membrane, the 125I-labeled platelets were first incubated with 0.5 mmol/L EGTA (1 nmol/L free Ca2+) at 22 degrees C and then solubilized in the absence of EGTA. The IIb and IIIa from these platelets migrated at Rf = 0.33, indicating the presence of the intact IIb-IIIa complex. In contrast, when the platelets were incubated at 37 degrees C for one hour with the EGTA, the discrete band at Rf = 0.33 representing the IIb-IIIa complex gradually disappeared. This phenomenon could not be reversed by adding Ca2+ back to the platelets before solubilization and electrophoresis. This loss of the IIb-IIIa complex from intact platelets was accompanied by (a) a progressive and irreversible decrease in adenosine diphosphate (ADP)-induced platelet aggregation and (b) decreased binding of a complex-dependent monoclonal antibody to the platelets. These studies demonstrate that when platelets are exposed to low Ca2+ at 37 degrees C, the IIb-IIIa heterodimer complexes in their surface membranes are irreversibly disrupted. Because intact IIb-IIIa complexes are required for platelet aggregation, the loss of these complexes may account for the failure of these platelets to aggregate in response to ADP.     

The human platelet membrane glycoprotein complex GP IIb-IIIa expresses antigenic sites not exposed on the dissociated glycoproteins

A number of recent reports have described murine monoclonal antibodies that react specifically with the complex formed by human platelet membrane glycoproteins (GP) IIb and IIIa. We show that the IgG L, a previously described human alloantibody isolated from a polytransfused thrombasthenia patient, has similar properties. When used in non-precipitating amounts in crossed immunoelectrophoresis (CIE), 125I-IgG L bound strongly to the IIb-IIIa complex. However, after dissociation of the complex with EDTA, only a weak binding to GP IIb and no binding to GP IIIa was detected. In further studies, increased amounts of IgG L were interacted with 125I-labeled membrane glycoproteins in (a) CIE and (b) classical indirect immunoprecipitation experiments. Although the antibody was able to quantitatively precipitate the IIb-IIIa complex from Triton X-100-soluble extracts of platelet membranes, no precipitation of GP IIb or GP IIIa was observed after divalent cation chelation. Addition of EDTA to immunoprecipitates containing GP IIb-IIIa resulted in dissociation and partial release of both glycoproteins. The interaction of the IgG L with electrophoretically separated GP IIb and GP IIIa was studied using a Western blot procedure in the presence of Ca2+, Mg2+, or EDTA. The presence of divalent cations did not increase the reactivity of the antibody with the individual glycoproteins. Overall, our results show that acquired antibodies to IIb-IIIa, such as the IgG L, may predominantly react with complex-dependent determinants.     

Structural analysis of human platelet membrane glycoprotein I complex.

The glycoprotein I complex, consisting of two polypeptides of Mr 210,000 and 150,000, was isolated from human platelet membranes by wheat germ lectin affinity chromatography. Glycocalicin, a soluble loosely bound membrane glycoprotein of Mr 150,000 related to the glycoprotein I system, was also purified. The isolated polypeptides were radioiodinated in sodium dodecyl sulfate/polyacrylamide gels and digested with trypsin, and the labeled peptide digest was analyzed by two-dimensional high-voltage electrophoresis and thin-layer chromatography. The two polypeptides of Mr 210,000 and 150,000 in the glycoprotein I complex had essentially identical radioactive peptide maps. Glycocalicin had a completely different tryptic peptide map. These studies shed light on the molecular relationships of some of the components of the platelet membrane glycoprotein I system. The possibility is raised that the receptorlike function of the intrinsic platelet membrane glycoproteins may be related to the polymeric subunit associations of the constituent polypeptides.     

Anti-fibrinogen antibody mediates fibrinogen binding to platelet membrane glycoprotein IIb-IIIa

Summary The binding of fibrinogen to platelets requires the agonist activation of platelet membrane glycoprotein IIb/IIIa. We have now found an anti-fibrinogen polyclonal antibody (YCU-R3) that increases the fibrinogen affinity of GPIIb/IIIa-binding function (activation) and subsequent platelet aggregation. The addition of intact IgG, F(ab)₂ fragments or Fab fragments induced platelet aggregation. The antibody-mediated fibrinogen binding was specific and saturable. This binding was inhibited by native fibrinogen, the RGDS peptide, the peptide of the C-terminus γ chain of fibrinogen (γ397–411), and the anti-GPIIb/IIIa monoclonal antibody (LJ-CP8). The antibody-dependent fibrinogen binding was similar to that induced by ADP. Moreover, after pretreatment with the anti-fibrinogen antibody and fibrinogen, formalin-fixed platelets bound to fibrinogen saturably. These results suggest that this anti-fibrinogen antibody may function as partial agonist.     

A monoclonal antibody binding to human medulloblastoma cells and to the platelet glycoprotein IIb-IIIa complex

S ummary. A monoclonal antibody, designated M148, produced by the hybridoma technique from spleen cells of mice immunized with human medulloblastoma, was found by indirect immunofluorescence to bind to normal human platelets (both Pl^Al positive and Pl^Al negative) and megakaryocytes, as well as to some medulloblastoma and neuroblastoma cells and cell lines and certain other solid tumours. No binding was observed to other marrow constituents, nor to any other normal tissue examined. The antibody bound to platelets from a patient with the Bernard-Soulier syndrome but not to thrombasthenic platelets. It immunoprecipitated glycoproteins IIb and IIIa from ¹²⁵I-labelled normal platelet membranes, and completely inhibited ADP-induced fibrinogen binding and aggregation of platelets. Aggregation was also inhibited in response to adrenaline, collagen, thrombin, sodium arachidonate and the ionophore A23187; clot retraction was partially inhibited. The antibody was without effect on thromboxane formation or 5-hydroxytryptamine (5HT) secretion in response to thrombin, but inhibited 5HT secretion in response to arachidonate. It did not inhibit factor VIII binding or agglutination in response to ristocetin, but completely inhibited factor VIII binding in response to thrombin. These findings suggest that the epitopes are close to the fibrinogen and factor VIII binding sites on glycoproteins IIb/IIIa, and that the lack of these glycoproteins is sufficient explanation for the pattern of dysfunction observed in thrombasthenic platelets, without invoking any other membrane abnormality.     

Effects of platelet glycoprotein IIb-IIIa number and glycoprotein IIIa Leu33Pro polymorphism on platelet aggregation and sensitivity to glycoprotein IIb-IIIa antagonists

We investigated the influence of glycoprotein (GP) IIIa Leu33Pro polymorphism, platelet GP IIb-IIIa number, and plasma fibrinogen concentration on platelet aggregation and antiaggregatory action of GP IIb-IIIa antagonists. Healthy volunteers with GP IIIa Pro33(-) (Leu33Leu33, n = 20) and Pro33(+) (Leu33Pro33, n = 13, and Pro33Pro33, n = 2) genotypes were included into the study. GP IIIa Leu33Pro substitution was associated with the increase of the level and rate of platelet microaggregate formation induced by GP IIb-IIIa activating antibody CRC54 (100, 200, 400 microg/ml) against the epitope within 1-100 residues of GP IIIa N-terminal part (p from 0.001 to 0.047). No significant differences were detected between parameters of platelet aggregation induced by ADP (1.25, 2.5, 5.0, 20 microM) in GP IIIa Pro33(+) and Pro33(-) donors. GP IIb-IIIa antagonist Monafram (F(ab')(2) fragment of GP-IIb-IIIa blocking antibody CRC64) (1, 2, 3 microg/ml), but not eptifibatide (50, 100, 150 ng/ml) inhibited ADP-induced aggregation slightly less efficiently in GP IIIa Pro33(+) group (p < 0.05 at 1 and 2 microg/ml Monafram). GP IIb-IIIa number (evaluated as maximal binding of (125)I-labelled antibody CRC64) varied from 40.5 to 80.8 x 10(3) per platelet with no significant influence of GP IIIa genotype. Consistent correlations were revealed between GP IIb-IIIa quantity and the level and rate of ADP-induced aggregation (r from 0.353 to 0.583, p from <0.001 to 0.037) as well as resistance (level of residual aggregation) to both GP IIb-IIIa antagonists (r from 0.345 to 0.602, p from <0.001 to 0.042). ADP-induced aggregation was considerably increased and efficiency of GP IIb-IIIa antagonists decreased in donors with high in comparison with low GP IIb-IIIa quantity (>60 and 40-50 x 10(3) per platelet respectively, p < 0.01 for most tests). No correlations were observed between all tested parameters and plasma fibrinogen concentration. Our results indicate that inter-individual variability of platelet GP IIb-IIIa number significantly affects platelet aggregation and antiaggregatory effects of GP IIb-IIIa antagonists. Contribution of this factor is higher than that of GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration.     

Effects of platelet glycoprotein IIb-IIIa number and glycoprotein IIIa Leu33Pro polymorphism on platelet aggregation and sensitivity to glycoprotein IIb-IIIa antagonists

We investigated the influence of glycoprotein (GP) IIIa Leu33Pro polymorphism, platelet GP IIb-IIIa number, and plasma fibrinogen concentration on platelet aggregation and antiaggregatory action of GP IIb-IIIa antagonists. Healthy volunteers with GP IIIa Pro33(?) (Leu33Leu33, n?=?20) and Pro33(+) (Leu33Pro33, n?=?13, and Pro33Pro33, n?=?2) genotypes were included into the study. GP IIIa Leu33Pro substitution was associated with the increase of the level and rate of platelet microaggregate formation induced by GP IIb-IIIa activating antibody CRC54 (100,?200,?400?µg/ml) against the epitope within 1–100 residues of GP IIIa N-terminal part (p from 0.001 to 0.047). No significant differences were detected between parameters of platelet aggregation induced by ADP (1.25,?2.5,?5.0,?20?µM) in GP IIIa Pro33(+) and Pro33(?) donors. GP IIb-IIIa antagonist Monafram (F(ab’)₂ fragment of GP-IIb-IIIa blocking antibody CRC64) (1,?2,?3?µg/ml), but not eptifibatide (50,?100,?150?ng/ml) inhibited ADP-induced aggregation slightly less efficiently in GP IIIa Pro33(+) group (p?<?0.05 at 1 and 2?µg/ml Monafram). GP IIb-IIIa number (evaluated as maximal binding of ¹²⁵I-labelled antibody CRC64) varied from 40.5 to 80.8?×?10³ per platelet with no significant influence of GP IIIa genotype. Consistent correlations were revealed between GP IIb-IIIa quantity and the level and rate of ADP-induced aggregation (r from 0.353 to 0.583, p from <0.001 to 0.037) as well as resistance (level of residual aggregation) to both GP IIb-IIIa antagonists (r from 0.345 to 0.602, p from <0.001 to 0.042). ADP-induced aggregation was considerably increased and efficiency of GP IIb-IIIa antagonists decreased in donors with high in comparison with low GP IIb-IIIa quantity (>60 and 40–50?×?10³ per platelet respectively, p?<?0.01 for most tests). No correlations were observed between all tested parameters and plasma fibrinogen concentration. Our results indicate that inter-individual variability of platelet GP IIb-IIIa number significantly affects platelet aggregation and antiaggregatory effects of GP IIb-IIIa antagonists. Contribution of this factor is higher than that of GP IIIa Leu33Pro polymorphism and variations of fibrinogen concentration.     

A role for glycoprotein IIb-IIIa complex in the binding of IgE to human platelets and platelet IgE-dependent cytotoxic functions

A possible relationship between binding sites for Immunoglobulin E (IgE) on human platelets, involved in IgE-dependent cytotoxic functions of platelets against helminth parasites, and well-characterized platelet constituents involved in haemostasis, was investigated. We first explored the interaction with IgE of platelets from patients with rare inherited deficiencies of defined platelet constituents and functions: Glanzmann's thrombasthenia, Bernard-Soulier and grey platelet syndromes. We report that only type I and II thrombasthenic platelets, which lack the membrane glycoproteins (GP) IIb and IIIa, failed to bind IgE and to exhibit IgE-dependent effector functions. Since thrombasthenic monocytes, however, showed normal interaction with IgE, this defect appeared restricted to platelets. Polyclonal and monoclonal antibodies directed against GP IIb-IIIa complex, but not monoclonal antibody directed against GP Ib, inhibited the binding of IgE to normal platelets, and their IgE-dependent cytotoxicity. Taken together, these findings indicate a relation between the GP IIb-IIIa complex and the expression of IgE binding sites and IgE-dependent effector functions in human platelets.     

Cell-lineage antigens of the stem cell-megakaryocyte-platelet lineage are associated with the platelet IIb-IIIa glycoprotein complex

The stem cell-platelet lineage is uniquely defined by platelet cell- lineage antigens. These antigens are present on all stem cells measured by the spleen colony assay and become restricted to the platelet cell lineage as differentiation proceeds. In this study, anti-platelet serum (APS) has been used to identify cells in the bone marrow that express platelet cell-lineage antigens and to identify platelet cell surface molecules expressing these antigens. Anti-platelet IgG extensively absorbed with brain, thymus, and peritoneal cells bound selectively to stem cells, megakaryocyte progenitor cells (Mk-CFC), and megakaryocytes in CBA mouse bone marrow and to blood platelets. No other hemopoietic cell type, tissue, cell line, or tumor cell bound significant amounts of antibody against platelet cell-lineage antigens as determined by ability to absorb the anti-stem cell activity in APS. Studies with lactoperoxidase-labeled platelets showed that two major iodinated proteins of Mr = 114,000 and 138,000 were immunoprecipitated with APS and with antiserum that had been extensively absorbed. These proteins correspond to the platelet IIb-IIIa glycoprotein complex, which is known to express receptors for collagen and fibrinogen, molecules known to influence hemopoietic cell proliferation and tumor cell growth. A panel of six monoclonal antibodies against human IIb-IIIa inhibited spleen colony formation by 17% to 100%, J15 and A5.15 also being cytotoxic for granulocyte-macrophage progenitor cells and Mk-CFC. Other platelet monoclonal antibodies did not inhibit spleen colony formation. Although APS inhibited fibrinogen binding to platelets and platelet aggregation, these activities were greatly reduced with absorbed antiserum. Furthermore, fibrinogen treatment of bone marrow did not block the anti-stem cell activity in APS. Thus the evidence is consistent with expression of platelet cell-lineage antigens on the platelet IIb-IIIa glycoprotein complex at a site removed from the fibrinogen binding site.     

Platelet membrane topography: colocalization of thrombospondin and fibrinogen with the glycoprotein IIb-IIIa complex

The distribution of platelet thrombospondin (TSP), fibrinogen, and glycoproteins IIb-IIIa (GPIIb-IIIa) and GPIb were studied in resting and activated human platelets using frozen thin-section immunoelectron microscopy. In resting platelets, TSP and fibrinogen were found within alpha granules and not on the platelet surface. In unstimulated platelets, GPIIb-IIIa and GPIb were distributed diffusely over the platelet membrane as well as within the body of the platelets. Upon thrombin or A23187 stimulation, TSP, fibrinogen, and GPIIb-IIIa colocalized on the platelet membrane and the canalicular system as well as on pseudopodia and between adherent platelets. GPIb distribution was unchanged by platelet activation. The findings support the hypothesis that a macromolecular complex of TSP-fibrinogen and GPIIb-IIIa forms on the activated platelet membrane.     

The human platelet membrane glycoprotein IIb/IIIa complex: a multi functional adhesion receptor.

The glycoprotein GPIIb/IIIa complex is a major constituent of the platelet membrane; it plays an important role in platelet adhesion and aggregation. The complex is a member of the integrin superfamily. Integrins are related membrane receptors which mediate the adhesive interactions of a variety of cells; they specifically recognize the arginine-glycine-aspartic acid (RGD) sequence present in several adhesive proteins. The GPIIb/IIIa complex of activated platelets can bind fibrinogen, von Willebrand factor, fibronectin, vitronectin and thrombospondin. Platelets are activated by a variety of signals including extracellular matrix molecules and soluble factors; upon platelet activation the complex undergoes a conformational change, thus permitting the macromolecular ligands access to their binding sites. In turn, fibrinogen binding results in a receptor modification and neoantigens exposure; such events may participate in signal transduction. The adhesive proteins compete reciprocally for binding to GPIIb/IIIa, and the complex binds to different domains of them, thus creating multiple interactions with the ligands.     

Thrombin-induced changes in platelet membrane glycoproteins Ib, IX, and IIb-IIIa complex

Platelet membrane glycoprotein Ib (GPIb) and the GPIIb-IIIa complex have central roles in the interaction of platelets with the plasma coagulation system, damaged vessel walls, and other platelets. We investigated the effects of thrombin on these glycoproteins. Monoclonal antibodies were used to assess platelet surface glycoproteins by flow cytometry, total platelet glycoprotein content by immunoassay, and glycoproteins released from platelets, also by immunoassay. Five new observations were made with regard to thrombin-induced changes in platelet membrane glycoproteins: (a) The marked decrease in platelet surface binding of antibodies directed at GPIb was not confined to antibodies directed at the von Willebrand factor binding site. (b) There was a marked decrease in platelet surface binding of an antibody directed at GPIX, with maintenance of the 1:1 ratio of platelet surface binding of antibodies directed at GPIb and GPIX. (c) Changes in platelet surface binding of antibodies were not restricted to a distinct subpopulation of platelets. (d) There was no associated platelet release of glycocalicin (a proteolytic fragment of GPIb). (e) There was no associated platelet release of the GPIIb-IIIa complex. These thrombin-induced changes may be important in modulating the reactivity of platelets with the damaged vessel wall and with each other.     

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 role of platelet membrane glycoproteins Ib and IIb-IIIa in platelet adherence to human artery subendothelium

Platelet adherence to human artery subendothelium in blood from eight normal subjects, four patients with Glanzmann's thrombasthenia (deficiency of platelet membrane glycoproteins IIb and IIIa: GPIIb-IIIa), two patients with Bernard-Soulier syndrome (deficiency of platelet membrane glycoprotein Ib: GPIb) and one patient with von Willebrand's disease (VWD subtype III. deficient in factor VIII-von Willebrand factor: FVIII-VWF) was compared at various wall shear rates (300, 500, 1000, 1800 and 2500 s-1). Platelet adherence in blood from the patients with Glanzmann's thrombasthenia was within the normal range at shear rates below 1000 s-1. There was some decrease in adhesion at higher shear rates and platelets were less spread out on the subendothelium than normally at all shear rates. Platelet aggregate formation was almost totally absent. Platelet adherence in blood from patients with the Bernard-Soulier syndrome was strongly impaired at all shear rates. Platelet adherence in blood from the patient with VWD subtype III was normal at shear rates of 300 and 500 s-1, but impaired at shear rates above 1000 s-1. Aggregate formation was also decreased at these shear rates. Platelet adhesion was strongly inhibited by a monoclonal antibody against glycoprotein Ib, which had previously been shown to inhibit ristocetin-induced aggregation, at shear rates of 500 and 1800 s-1 but not at 300 s-1. Platelet adhesion at 1800 s-1 was also inhibited, though to a lesser extent, by two antibodies against GPIIb-IIIa. These antibodies also inhibited platelet aggregate formation. The data indicates that GPIb is involved in adhesion at the same shear rates as von Willebrand factor. Absence or inhibition of GPIIb-IIIa primarily causes a defect of aggregate formation but GPIIb-IIIa may also play a role in adhesion, particularly at high shear rates. The defect of adhesion in the Bernard-Soulier syndrome may be dependent on factors other than a deficiency of GPIb alone.     

Analysis of human platelet glycoproteins IIb-IIIa and Glanzmann's thrombasthenia in whole blood by flow cytometry

Antibodies that bind to human platelet membrane glycoproteins IIb and IIIa were used to develop methods for analyzing platelet membrane components by flow cytometry. Platelets were tentatively identified by their low-intensity light scatter profiles in whole blood or platelet- rich plasma preparations. Identification of this cell population as platelets was verified by using platelet-specific antibodies and fluorescein-conjugated antiimmunoglobulin. Two-parameter analysis of light scatter versus fluorescence intensity identified greater than 98% of the cells in the "platelet" light scatter profile as platelets due to their acquired fluorescence. Both platelet-rich plasma and whole blood were used to study platelet membrane glycoproteins IIb and IIIa on a single cell basis in an unwashed system. Prostacycline was included in these preparations as a precautionary step to inhibit platelet aggregation during analysis. Flow cytometry is a successful technique for rapid detection of platelet membrane defects such as Glanzmann's thrombasthenia. Platelets from Glanzmann's thrombasthenic individuals were readily distinguished from platelets with normal levels of glycoprotein IIb and IIIa and from platelets with glycoprotein levels characteristic of heterozygote carriers of this disorder. This technique provides a sensitive tool for investigating platelet functional defects due to altered expression or deficiency of platelet surface proteins.     

Isolation and structural characterization of the polypeptide subunits of membrane glycoprotein IIb-IIIa from human platelets

We have previously demonstrated the isolation of platelet membrane glycoprotein IIb-IIIa by affinity chromatography with a specific monoclonal antibody. We have now separated the polypeptide subunits IIb and IIIa of the isolated glycoprotein by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and have compared their structural features. Both IIb and IIIa contain approximately 15% carbohydrate, but IIIa contains a larger percentage of mannose residues, suggesting the presence of high mannose as well as complex N- linked oligosaccharide chains. The amino acid compositions are sufficiently similar to imply areas of sequence homology between the two subunits. To examine further the relationship between the subunits, we digested a mixture of 125I-IIb and 131I-IIIa with trypsin and then separated the radiolabeled peptides by high performance liquid chromatography. The resultant peptide maps of IIb and IIIa are completely different. This indicates that neither subunit is derived from the other and suggests that polypeptides IIb and IIIa are products of separate genes.     

Effects of leukocyte-derived cathepsin G on platelet membrane glycoprotein Ib-IX and IIb-IIIa complexes: a comparison with thrombin

Cathepsin G is a serine, chymotrypsin-like protease released by activated polymorphonuclear leukocytes (PMN) that may act as a platelet agonist. The effect of this enzyme on platelet surface glycoproteins (Gp) Ib and IIb-IIIa was evaluated by means of a cytofluorimetric assay, using fluorescein isothiocyanate-labeled monoclonal antibodies (MoAbs) directed at the alpha chain of Gp Ib (SZ2), at Gp IX or at the complex Gp IIb-IIIa (P2), and the fibrinogen-receptor-specific MoAb PAC- 1. In human washed platelets, cathepsin G increased the binding of P2 and PAC-1, decreased the binding of SZ2, but only slightly affected the binding of anti-Gp IX. SZ2 binding decrease was more rapid in cathepsin G- than in thrombin-stimulated platelets, whereas the increase of P2 and PAC-1 binding occurred to a comparable extent with either agonist. In paraformaldehyde (PFA)-fixed and energy-depleted platelets, no effect on either Gp Ib or Gp IIb-IIIa complex was observed with thrombin. At variance, cathepsin G was still able to reduce binding of SZ2, whereas increased binding of P2 or PAC-1 antibodies was not observed. Triton X-100 permeabilization of cathepsin G-treated, PFA- fixed platelets did not restore SZ2 binding at variance with thrombin. Moreover, platelet incubation with cathepsin G resulted in the loss of ristocetin-induced agglutination in the presence of the von Willebrand factor and in the appearance of Gp Ib-derived proteolytic products in supernatants. After dissociation by EDTA pretreatment of surface Gp IIb- IIIa complexes, cathepsin G still induced increased binding of P2. Aspirin and an adenosine diphosphate scavenger system had only a slight but not significant effect on changes in antibody binding induced by cathepsin G. All these data would indicate that cathepsin G, like thrombin, interacts with platelet-surface Gp, inducing the exposure of the intracellular pool of the Gp IIb-IIIa complex with concomitant expression of a functional fibrinogen receptor. Moreover, it induces a loss of antigenic sites on Gp Ib, but the mechanism involved, a proteolytic cleavage of Gp Ib, is substantially different from that of thrombin. These changes, induced by a product of activated PMN, might reduce the reactivity of platelets to the subendothelium, while increasing their ability to undergo aggregation and release reaction.