Antibodies against Platelet Membrane Glycoproteins II. INFLUENCE ON ADP- AND COLLAGEN-INDUCED PLATELET AGGREGATION, CROSSED IMMUNOELECTROPHORESIS STUDIES AND RELEVANCE TO GLANZMANN''S THROMBASTHENIA

In Glanzmann's thrombasthenia glycoproteins IIb and IIIa are missing or strongly reduced and aggregation to ADP, collagen and thrombin is impaired. Antibodies against glycoproteins IIb and IIIa did not entirely induce a thrombasthenia-like state in normal platelets. However, they did strongly inhibit collagen-induced aggregation and inhibited the second wave of aggregation induced by ADP. Crossed immunoelectrophoresis studies using Triton X-100 extracts of whole platelets with these antibodies gave a single immunoprecipitate. This immunoprecipitate was absent when similar studies were carried out with thrombasthenic platelets. Platelet antibodies gave a number of immunoprecipitates with normal platelets and differences were observed with thrombasthenic platelets, the most notable of which was a marked reduction in one of the major immunoprecipitates. These results provide further evidence that glycoproteins IIb and IIIa are involved in the latter stages of platelet aggregation.     

Further studies on the interaction between human platelet membrane glycoproteins IIb and IIIa in triton X-100

Analysis of human platelet membrane proteins by crossed immunoelectrophoresis (CIE) in the presence of Triton X-100 (TX-100) has previously shown that glycoproteins (GP) IIb and IIIa are located in a single immunoprecipitate, band 16.2 To investigate whether IIb and IIIa are associated in a complex, we have analyzed TX-100-solubilized 125I-labeled membrane proteins by density gradient ultracentrifugation using 10%-40% sucrose gradients containing the nonionic detergent. studies were performed using soluble proteins derived from membranes isolated in the presence or absence of EDTA. Analysis of gradient fractions by SDS-polyacrylamide gel electrophoresis showed that in the absence of divalent cation chelation, GP IIb and IIIa penetrated well into the gradient (fractions 15-17). Analysis of fractions 15-17 by CIE revealed the presence of band 16. In contrast, when the membrane proteins were incubated with EDTA prior to or after TX-100 solubilization, IIb and IIIa remained near the top of the gradient (fractions 8-11) and gave separate immunoprecipitates during CIE. Incubation of washed platelet lysates with leupeptin, an inhibitor of the Ca2+-dependent protease of human platelets, had no effect on the shape of the band 16 immunoprecipitate. Thus, for the first time, direct evidence has been obtained that GP IIb and IIIa may form a divalent cation-mediated complex. Calibration of the sedimentation profiles using proteins of known molecular weight suggests that the complex is of limited size. Indirect evidence suggests that the complex is a heterodimer.     

Characterization of human platelet glycoprotein antigens giving rise to individual immunoprecipitates in crossed-immunoelectrophoresis

Washed human platelets were labeled with 125I by the lactoperoxidase- catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.     

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.     

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.     

A monoclonal anti-platelet antibody with decreased reactivity for autoimmune thrombocytopenic platelets.

Two monoclonal anti-platelet antibodies, 3B2 and 8G11, have been raised that are specific for normal human platelets. 3B2 is unique in that it has decreased reactivity for platelets from 16 patients with autoimmune thrombocytopenic purpura [mean platelet count, 65,000 +/- 6,000 (SEM)]. With 8G11 in an enzyme-linked immunosorbent assay, the mean of the ratios of patient platelet OD to control platelet OD was 0.95 +/- 0.07, whereas with 3B2, the mean of the ratios of patient platelet OD to control OD was 0.24 +/- 0.04, P less than 0.001. With 3B2 the mean of the OD ratios of five patients with autoimmune thrombocytopenic purpura in remission (greater than 150,000 platelets per mm3) compared to controls was 0.80 +/- 0.14. 3B2 did not react with platelets from a patient with Glanzmann's thrombasthenia, in which membranes lack glycoproteins IIb and IIIa (GPIIb and GPIIIa). Platelet membranes were run on crossed immunoelectrophoresis against a rabbit polyclonal anti-human platelet membrane antibody with 125I-labeled purified 3B2 in an intermediate spacer gel. 3B2 reacted with the GPIIb-GPIIIa-Ca2+ complex in the presence of excess Ca2+ and with GPIIb alone in the presence of excess EGTA. When Triton X-100-solubilized platelet membranes were immunoprecipitated with 3B2 plus rabbit anti-mouse IgG, reduced, and run on NaDodSO4/polyacrylamide gel electrophoresis, a single protein band was obtained with a molecular weight of 120,000 (the molecular weight of GPIIb). Thus, the reactivity of monoclonal antibody 3B2 with GPIIb or the GPIIb-GPIIIa-Ca2+ complex appears to be inhibited by the presence of autoantibody on platelets.     

A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets

We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.     

Synthesis of analogs of human platelet membrane glycoprotein IIb-IIIa complex by chicken peripheral blood thrombocytes.

Human platelets and their phylogenetic counterparts, avian thrombocytes, play a key role in primary hemostasis. Based upon extensive studies in mammals, platelet cohesion resulting in the formation of the "hemostatic plug" is known to be mediated by the mammalian platelet glycoprotein IIb-IIIa complex in concert with fibrinogen and calcium. The immunological and biochemical technology already developed for the analyses of mammalian platelet glycoproteins has never been applied to avian thrombocytes. By indirect immunofluorescence, we now show that a polyclonal rabbit antibody specific for human glycoproteins IIb plus IIIa and the well-characterized murine monoclonal anti-IIb-IIIa complex antibody, AP2, both crossreact with IIb and IIIa analogs on intact chicken thrombocytes. By two-dimensional polyacrylamide gel electrophoresis, we also demonstrate that chicken thrombocytes will incorporate [35S]methionine into several proteins, including the glycoprotein IIb and IIIa analogs during short-term (4 hr) incubation in vitro. This finding indicates that peripheral blood nucleated thrombocytes of the chicken, unlike their mammalian counterparts, retain the capacity to synthesize protein. The significance of these findings is 2-fold. First, we provide biochemical and immunological evidence that those proteins responsible for platelet cohesion in humans are structurally conserved in cells of analogous function in chickens despite the fact that these species have diverged from a common ancestor more than 200-250 million years ago. Second, we identify chicken thrombocytes as a readily available source of messenger RNA encoding numerous proteins analogous to those already characterized in human platelets, including glycoproteins IIb and IIIa.     

A defect of platelet aggregation associated with an abnormal distribution of glycoprotein IIb-IIIa complexes within the platelet: the cause of a lifelong bleeding disorder.

A young Italian man (A.P.) has a lifelong history of bleeding from gums and mucocutaneous tissue. Electron microscopy showed a wide diversity of platelet size including giant forms. In citrated platelet-rich plasma (PRP), platelet aggregation induced by adenosine diphosphate (ADP) and other agonists was much reduced. Both secretion and clot retraction were normal. The aggregation of washed platelets with ADP was improved but remained subnormal, as was aggregation with collagen and thrombin. Fibrinogen-binding was analyzed by flow cytometry using platelets in whole blood or PRP and was markedly decreased. Crossed immunoelectrophoresis of Triton X-100 extracts of (A.P.) platelets showed that GP IIb-IIIa levels were 40% to 50% of normal. Glycoprotein (GP) IIb and GP IIIa were of usual migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but their labeling was much reduced during lactoperoxidase-catalyzed iodination. Binding to (A.P.) platelets of four different 125I-labeled monoclonal antibodies to GP IIb-IIIa complexes was reduced to 12% to 20% of normal levels. However, when the patient's platelets were stimulated with alpha-thrombin, monoclonal antibody binding showed the same increase (approximately 20,000 sites) as normal platelets. Both flow cytometry and immunocytochemical studies showed that the distribution of residual surface GP IIb-IIIa within the total (A.P.) platelet population was heterogeneous and not related to platelet size. Staining of ultrathin sections confirmed the presence of an internal pool of GP IIb-IIIa. Monoclonal antibodies to other membrane glycoproteins bound normally to (A.P.) platelets. The patient has a selective deficiency of the surface pool of GP IIb-IIIa complexes that is manifested clinically by a mild Glanzmann's thrombasthenia-like syndrome.     

Characterization of platelet-specific alloantigens by immimoblotting: localization of Zw and Bak antigens

The glycoprotein localization of the platelet-specific antigens Zwa, Zwb and Baka and their presence on tryptic fragments of glycoproteins was studied by immunoblotting. Human platelets were solubilized and pre-cleared from platelet-associated IgG. The glycoproteins were separated on SDS polyacrylamide gels, transferred to nitrocellulose and incubated with platelet antibodies, followed by 125I-radiolabelled anti-human Ig antibodies. Glycoprotein IIb/IIIa were isolated from platelet lysates by immuno-affinity chromatography. These proteins were subjected to trypsin digestion, and then used for the immunoblot procedure with platelet antibodies. A glycoprotein specifically reacting with either anti-Zwa or anti-Zwb was found, with an apparent molecular weight of 88 kDa. This protein co-migrated, and was probably identical with, glycoprotein IIIa. After trypsin digestion the smallest fragment, reactive with IgG anti-Zwa or IgM anti-Zwb, had a molecular weight of approximately 23 kDa. IgG anti-Baka and anti-Leka antibodies reacted with a protein of 130 kDa from platelets of Bak(a+) donors. This protein was identified as glycoprotein IIb.     

Platelet glycoproteins IIb and IIIa associated with blood monocytes are derived from platelets

Platelet glycoprotein IIb/IIIa (GP IIb/IIIa), the receptor complex for fibrinogen, has been regarded as a megakaryocyte/platelet lineage- restricted antigen. Recently, however, it has been reported that GP IIb/IIIa is expressed in blood monocytes. Studies were performed to establish the origin and immunological characteristics of monocyte- associated glycoproteins IIb and IIIa (GPs IIb and IIIa). Preparations of blood monocytes containing varying platelet-monocyte ratios were metabolically labeled with [35S]methionine with the expectation that any newly synthesized GPs IIb and IIIa would be monocyte-derived, since platelets have only rudimentary protein synthetic apparatuses. Analyses of sodium dodecyl sulfate (SDS) gels of homogenates of cell preparations containing from 200 to 5:1 platelet-monocyte ratios revealed that unlabeled GPs IIb and IIIa were readily immunoisolated using protein A-Sepharose immunobeads. However, fluorographic analyses of the same cell preparations pulse-labeled with [35S]methionine failed to demonstrate synthesis of GP IIb or IIIa. Additionally, no GP IIb or IIIa was detected when immunoisolation was carried out in pure preparations of monocytes containing less than 1:100 platelet-monocyte ratios and SDS acrylamide gels were stained by the sensitive silver stain method. Furthermore, heterologous polyspecific antisera and two monoclonal antibody preparations against GPs IIb and IIIa, which bound to platelets, failed to bind to monocyte membranes. Thus, evidence was presented that indicated that monocytes do not synthesize platelet GPs IIb and IIIa and that detection of these molecules in blood monocyte preparations reflects platelet contamination.     

Immunochemical characterization of platelet-specific alloantigens

Immunoprecipitation was performed with platelet-specific alloantisera (anti-Zwa, -Zwb, -Baka and antiserum Luc) and 125I-labelled platelets of a panel of donors typed for these platelet-specific alloantigens. This was done by sensitization of intact, radiolabelled platelets with the antisera, solubilization of the sensitized platelets with Nonidet P-40 and recovery of the immune complexes from the lysate with heat-killed, formalin-fixed Staphylococcus aureus, strain Cowan I. In the case of platelet antibodies of the IgM class, bacteria also preincubated with rabbit-IgG anti-human IgM before treatment of the lysate were applied. Radiolabelled antigens were then eluted from the bacteria by boiling in an SDS-mercaptoethanol-containing buffer. Subsequently, the eluted antigens were analyzed by SDS-polyacrylamide gel electrophoresis. It revealed that both the Zwa and the Baka antigens are located on the glycoprotein-IIb/IIIa complex. The location of the Zwb antigen could not be established in this way. From the intensity of the precipitated glycoprotein bands, it appeared that Zwa is probably a marker of glycoprotein IIIa, Baka or glycoprotein IIb. However, immunoprecipitates of anti-Zwa and anti-Baka sera both carried, to some extent, complexes of glycoproteins IIb and IIIa. Iodinated platelets of a Glanzmann thrombasthenia patient failed to show any material precipitated by these antisera or the antiserum Luc, known to contain antibodies against both glycoproteins.     

Characterization of a monoclonal antibody ITI-Pl 1 directed against human platelet membrane glycoprotein IIb using extracts of whole platelets and platelet surface and intracellular membranes

A monoclonal antibody (mAb) termed ITI-Pl 1 has been prepared by the hybridoma procedure. Using immuno-absorption and crossed immunoelectrophoresis of Triton X-100 extracts of untreated and EDTA-treated human platelets it was shown to be directed against the surface membrane glycoprotein IIb (GP IIb). This mAb binds to whole platelets independently of ADP-stimulation and the presence of Ca2+-ions. It saturates at around 870 ng/10(8) cells corresponding to approximately 35,800 molecules/platelet. ITI-Pl 1 did not significantly inhibit GP IIb-IIIa dependent functions such as platelet aggregation or fibrinogen binding. Immunofluorescence could be demonstrated using ITI-Pl 1 and intact normal platelets, but not with platelets from a Glanzmann's thrombasthenia patient. Crossed immuno-electrophoresis with platelet extracts from four different thrombasthenic patients gave a line precipitate in the intermediate gel with 125I-labelled ITI-Pl 1 and autoradiography indicating trace amounts of free GP IIb or the GP IIb-IIIa complex. The epitope on GP IIb detected by ITI-Pl 1 is not destroyed by neuraminidase treatment. Thus the mAb also interacts with neuraminidase-treated GP IIb-IIIa complex in highly purified platelet surface membrane fractions as well as with GP IIb-IIIa from untreated internal membranes isolated by continuous flow electrophoresis.     

Cultured human endothelial cells synthesize a plasma membrane protein complex immunologically related to the platelet glycoprotein IIb/IIIa complex

We have previously demonstrated that endothelial cells synthesize a plasma membrane protein indistinguishable from platelet glycoprotein (GP) IIa. The present study provides evidence for a further analogy between the platelet and the endothelial cell membrane by showing that cultured endothelial cells also synthesize a membrane protein complex immunologically related to the platelet GP IIb/GP IIIa complex. This evidence is based on the following observations: (1) C17, a murine monoclonal antiplatelet GP IIIa antibody, consistently precipitates two proteins, apparent molecular weights, respectively, 115,000 and 125,000 reduced and 95,000 and 135,000 nonreduced, from metabolically (35S- methionine) as well as surface 125I-labeled cultured human endothelial cells; (2) upon crossed immunoelectrophoresis of solubilized endothelial cells against a polyclonal rabbit antiplatelet antiserum and 125I-labeled C17 IgG, a single precipitate of the protein(s) recognized by C17 is observed. As judged by their mobility in 9% polyacrylamide gels, both endothelial proteins appear to have a somewhat larger molecular weight than their platelet counterparts. Patterns obtained by crossed immunoelectrophoresis are also indicative of a difference in electrophoretic behavior of the platelet GP IIb/IIIa complex and the endothelial cell protein complex.     

Disturbed platelet aggregation to collagen associated with an antibody against an 85- to 90-Kd platelet glycoprotein in a patient with prolonged bleeding time.

We studied a 5-year-old girl presenting with a markedly prolonged bleeding time. Her platelets were refractory to collagen stimulation, but the response to other agonists was normal. There were no coagulation abnormalities as measured by standard tests. Two-dimensional electrophoresis showed no abnormalities of the patient's platelet membrane glycoproteins. When the patient's plasma or purified plasma IgG was mixed with normal platelets, collagen-induced platelet aggregation was blocked. Western blotting showed the presence of an antibody in the patient's plasma directed against a protein of molecular weight 85 to 90 Kd under both reducing and nonreducing conditions. This protein comigrated with glycoprotein (GP) IV immunoprecipitated by OKM5 from 125I-labeled platelets. Immunoprecipitation of 125I-labeled normal platelet glycoproteins with the patient's IgGs also yielded an 85- to 90-Kd protein that migrated on the diagonal following nonreduced/reduced two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Despite similarities in electrophoretic behavior, the antigen was not demonstrated to be GPIV, since purified GPIV was not recognized by the antibody.     

Platelet IgG Fc receptor

The glycoprotein localization of the platelet binding site for the Fc IgG has been the subject of debate. We attempted to resolve this issue by relating the binding of radiolabeled IgG immune complexes composed of heat-aggregated IgG to platelets from healthy individuals; an individual with Bernard-Soulier syndrome lacking glycoproteins IIb and IX; and a patient with Glanzmann's thrombasthenia lacking glycoproteins IIb and IIIa. The binding of IgG complexes to platelets was determined by measuring the specific binding of radiolabeled heat-aggregated IgG to washed platelets in a plasma-free mileu. 125I aggregated IgG bound to normal platelets in a saturable and concentration-dependent fashion. Specific binding could be inhibited by a 50-fold excess of purified Fc, but not by F(ab')2. Identical binding curves were obtained by using platelets from a patient with Glanzmann's thrombasthenia and a patient with Bernard-Soulier syndrome, indicating that the platelet Fc receptor is not carried on glycoproteins Ib, IIb, IIIa, or IX. We then measured the binding of radiolabeled detergent-solubilized platelets to IgG fixed to a solid matrix. A 40-kD platelet fragment bound to the immobilized IgG following passage across a density gradient. Confirmation of the Fc specificity of the interaction was shown by inhibition of platelet glycoprotein binding by excess IgG or purified Fc but not F(ab')2. The electrophoretic mobility decreased slightly after reduction, which indicated the existence of at least one intrachain disulfide bond. Treatment with high salt solutions or urea did not solubilize the receptor, which indicated that it was an integral protein. Enzyme studies showed that the platelet Fc receptor was not digested by neuraminidase, but neuraminidase treatment altered mobility by about 3%. In addition, treatment of platelets with trypsin or pronase did not affect its function as measured by the binding of 125I-IgG aggregates to enzyme-treated platelets, but did prevent its detection when using radioimmunoprecipitation studies. The platelet Fc receptor is a 40-kD, integral protein without interchain disulfide bonds.     

Platelet membrane glycoprotein abnormalities in patients with myeloproliferative disorders and secondary thrombocytosis

Carbohydrate-specific surface labelling and 125I-labelled lectin binding techniques, in combination with one- or two-dimensional (non-reduced/reduced) SDS-polyacrylamide gel electrophoresis have been used with platelets from patients with myeloproliferation disorders and secondary thrombocytosis and from healthy donors. In essential thrombocythaemia platelet membrane glycoproteins were significantly less sialylated than in normals (particularly GP Ib and IIIa). Increased binding of 125I-labelled Lens culinaris lectin to thrombospondin and GP IIIa indicated a defect in the glucose/mannose glycosylation of the platelet glycoproteins in essential thrombocythaemia. In polycythaemia vera and in chronic myeloid leukaemia the terminal sialic acid of glycoprotein IIIb was labelled slightly more than normal. In chronic myeloid leukaemia there was increased labelling of the penultimate galactose/N-acetylgalactosamine residues of GP Ib, IIb, IIIa and IIIb. In comparison to myeloproliferative disorders, platelets from patients with secondary thrombocytosis showed no significant changes, except for platelets from two patients with idiopathic thrombocytopenic purpura which showed an increased sialylation of all surface glycoproteins.     

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.     

A monoclonal antibody (LYP18) directed against the blood platelet glycoprotein IIb/IIIa complex inhibits human melanoma growth in vivo

A monoclonal antibody (MoAb) (LYP18), generated against human platelet glycoprotein IIb/IIIa (GPIIb/IIIa), immuno-precipitated a IIb/IIIa-like GP complex from a highly tumorigenic human melanoma cell line (M3Dau). The M3Dau melanoma cells specifically bound 125I-labeled LYP18. To study the biologic role of these IIb/IIIa-like glycoproteins, M3Dau melanoma cells were incubated with LYP18 or a control MoAb directed against another melanoma cell-surface antigen and implanted subcutaneously (SC) in nude mice. LYP18 dramatically inhibited the growth of tumor in vivo. LYP18 was not directly cytotoxic to the melanoma cells. These results demonstrate that the IIb/IIIa-like GPs are present on melanoma cells and play a crucial role in tumor cell growth. MoAbs directed against tumor cytoadhesive receptors may represent a novel approach in tumor treatment.     

Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.