Detection of glycoprotein IIb and IIIa by monoclonal antibodies

Murine monoclonal antibodies were produced against human platelet membranes and screened on platelets by a ¹²⁵I protein A radioimmunoassay. Several clones produced platelet specific antibodies as they showed no reaction with peripheral blood lymphocytes, neutrophils, bone marrow (excluding megakaryocytes) or several cell lines. Two antibodies (designated anti-HuPl-mla and anti-HuPl-mlb) were of particular interest in that although platelet specific they were non-reactive with platelets from a thrombasthenic patient. In functional assays these two antibodies could specifically inhibit ADP and collagen induced aggregation of platelets and release of ATP, retard platelet aggregation and ATP release induced by epinephrine, and inhibit ADP induced platelet fibrinogen binding. These two antibodies appear to recognize glycoproteins IIb and IIIa as analysis by SDS-PAGE using radiolabelled membranes revealed a two chain structure of molecular weight 112 000 and 122 000 daltons when run after reduction and 87 000 and 140 000 daltons non-reduced.     

Posttransfusion purpura associated with an autoantibody directed against a previously undefined platelet antigen

Although alloantibody against the PLA1 platelet antigen is usually found in patients with posttransfusion purpura (PTP), the mechanism of destruction of the patient's own PLA1-negative platelets is unexplained. We used a sensitive immunoblot technique to detect antiplatelet antibodies in a patient with classic PTP. The patient's acute-phase serum contained antibodies against three proteins present in control (PLA1-positive) platelets: an antibody that bound to a previously unrecognized platelet protein of mol wt 120,000 [glycoprotein (GP) 120], antibodies that bound to PLA1 (mol wt 90,000), and an epitope of GP IIb (mol wt 140,000). The antibodies against PLA1 and GP IIb did not react with the patient's own PLA1-negative platelets, control PLA1-negative platelets, or thrombasthenic platelets. In contrast, the antibody against GP 120 recognized this protein in all three platelet preparations, but not in Bernard-Soulier or Leka (Baka)-negative platelets. Antibody against GP 120 was not detected in the patient's recovery serum, although the antibodies against PLA1 and GP IIb persisted. F(ab)2 prepared from the patient's acute-phase serum also bound to GP 120. These results suggest that in PTP, transient autoantibody production may be responsible for autologous (PLA1-negative) platelet destruction. In addition, alloantibodies against more than one platelet alloantigen may be found in this disease. The nature of the GP 120 autoantigen and the GP IIb-related alloantigen defined by our patient's serum remains to be determined.     

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.     

Platelet fibrinogen and vitronectin in Glanzmann thrombasthenia: evidence consistent with specific roles for glycoprotein IIb/IIIA and alpha v beta 3 integrins in platelet protein trafficking.

To assess the individual contributions of the platelet glycoprotein (GP) IIb/IIIa receptor and the alpha v beta 3 vitronectin receptor to platelet levels of fibrinogen and vitronectin, we analyzed the platelets from two groups of Glanzmann thrombasthenic patients: Iraqi-Jews, whose platelets lack both receptors, and Arab patients in Israel, whose platelets lack GPIIb/IIIa, but have normal or increased numbers of alpha v beta 3 vitronectin receptors. The platelets from both thrombasthenic groups had profound deficiencies of fibrinogen, but the defect in the Iraqi-Jewish patients' platelets appeared to be slightly more severe. This finding indicates that GPIIb/IIIa is the major determinant of platelet fibrinogen, presumably acting by receptor-mediated uptake, and that the alpha v beta 3 vitronectin receptor plays little or no role. Arab patients' platelets have normal amounts of platelet vitronectin, whereas Iraqi-Jewish patients' platelets have nearly five times as much vitronectin as control or Arab patients' platelets. To account for these data, we propose a working hypothesis in which vitronectin is synthesized in megakaryocytes and the alpha v beta 3 vitronectin receptor is involved in transport of the protein out of megakaryocytes and/or platelets. Collectively, these observations suggest that in addition to their recognized roles in cell adhesion and in the interaction of cells with extracellular proteins, integrin receptors may be important in protein trafficking into, and perhaps out of, platelets.     

Absence of platelet-specific alloantigens in Glanzmann''s thrombasthenia

The platelets of 11 patients with Glanzmann's thrombasthenia and their nearest family members were studied for the expression of the platelet- specific alloantigens of the Zw-, Ko- and Bak systems. The strength of the expression of the Zwa antigen was diminished on the platelets of 3 patients, and the antigen was absent from the platelets of the other 8. The platelets of none of the patients reacted with anti-Zwb serum. Therefore, Glanzmann's thrombasthenia is probably a "Zw-null disease." The expression of the Zwa antigen on the platelets of all the relatives was normal, as indicated on the cytoflurograph. Investigations on the expression of the Koa antigen were complicated by agglutinations of the platelets from genetically Koa-negative thrombasthenic patients with the anti-Koa serum. The Kob antigen was normally expressed. The Baka antigen was absent from the platelets of all thrombasthenic patients and a relatively high percentage of the relatives. A close association between the Glanzmann gene and the Bak(a-) gene is assumed on statistical grounds. Thrombasthenic platelets showed no reaction with EDTA-dependent antibodies, which are reactive with all normal platelets. Owing to immunization by multiple blood and platelet transfusions, serum samples of most patients studied contained HLA antibodies and platelet-specific alloantibodies. However, antibodies directed against the Zw-antigen-bearing glycoproteins were detected in the serum of only one patient and, therefore, seem to be rare.     

Leka, a New Platelet Antigen Absent in Glanzmann''s Thrombasthenia

The serum of a patient who developed a posttransfusion purpura contained antibodies directed against a previously undescribed platelet antigen Lek a. The antiplatelet activity was present in the IgG fraction and was detected by immunofluorescence, 51Cr lysis and 14C-serotonin release. The frequency of the Lek a phenotype in the French population is 98.18%. Lek a does not appear to be sex-linked and seems to be closely related to the Bak a antigen. The Lek a antigen is not expressed on thrombasthenic platelets but is found on platelets from patients with the Bernard-Soulier syndrome which suggests that this antigen is carried by platelet glycoproteins IIb and/or IIIa.     

Acquired IgG antibody occurring in a thrombasthenic patient: its effect on human platelet function

In subagglutinating amounts, an IgG antibody isolated from the plasma of a polytransfused thrombasthenic patient (L) inhibited ADP-, epinephrine-, collagen-, and thrombin-induced aggregation of normal human platelets. The inhibition of ADP-induced aggregation was strongly diminished following the prior incubation of the antibody with control human platelet stroma but not with the stroma prepared from the platelets of two different thrombasthenic patients. The IgG(L) did not affect the binding of 14C-ADP to control human platelet membranes and did not inhibit the ADP-induced shape change. Bovine factor VIIIVWF- induced agglutination and ristocetin-induced aggregation of control human platelets were not inhibited in the presence of the antibody. The IgG(L) strongly inhibited ADP-induced retraction of reptilase clot and thrombin-induced clot retraction. This antibody therefore induced a thrombasthenialike state in normal human platelets, suggesting that the antigenic site recognized by the antibody plays a central role in the later stages of the mechanism of platelet aggregation induced by physiologic aggregation-inducing agents.     

Requirement of VPS33B, a member of the Sec1/Munc18 protein family, in megakaryocyte and platelet alpha-granule biogenesis

Bleeding problems are associated with defects in platelet alpha-granules, yet little is known about how these granules are formed and released. Mutations affecting VPS33B, a novel Sec1/Munc18 protein, have recently been linked to arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome. We have characterized platelets from patients with ARC syndrome and observed reduced aggregation with arachidonate and adenosine diphosphate (ADP). Structural abnormalities seen in ARC platelets included increased platelet size, a pale appearance in blood films, elevated numbers of delta-granules, and completely absent alpha-granules. Soluble and membrane-bound alpha-granule proteins were significantly decreased or undetectable in ARC platelets, suggesting that both the releasable protein pools and membrane components of alpha-granules were absent. The role of VPS33B in platelet granule biogenesis was evaluated by immunofluorescence microscopy in normal human megakaryocytes. VPS33B colocalized appreciably with markers of alpha-granules, moderately with late endosomes/lysosomes, minimally with delta-granules/lysosomes, and not with cis-Golgi complexes. VPS33B protein expression determined by immunoblotting confirmed the presence of VPS33B in control fibroblasts but not in ARC fibroblasts, and in normal megakaryocytes but not in platelets. We conclude that like other Sec1/Munc18 proteins, VPS33B is involved in intracellular vesicle trafficking, being essential for the development of platelet alpha-granules but not for granule secretion.     

Platelet aggregation induced by type IIb platelet von Willebrand factor

Platelet lysates from five patients with a form of type IIb von Willebrand's disease (vWd), associated with spontaneous platelet aggregation and thrombocytopenia, induced platelet aggregation of normal and other vWd's platelet-rich plasma (PRP). Platelet lysate from normals, type I or type IIa vWd did not cause platelet aggregation of normal PRP. When polyclonal monospecific antibodies directed against plasma von Willebrand factor (vWf) were incubated with the type IIb platelet lysate, they inhibited the platelet aggregation. Monoclonal antibodies directed against the glycoprotein (GP) Ib binding domain of plasma vWf incubated with the type IIb platelet lysate did not inhibit the platelet aggregation. Normal platelets suspended in afibrinogenaemic plasma did not aggregate when type IIb vWd platelet lysate was added. Normal platelets incubated with monoclonal antibodies directed against the fibrinogen and vWf binding site(s) on the GPIIb/IIIa were not aggregated by the type IIb platelet lysate. Bernard-Soulier PRP aggregated when type IIb vWd platelet lysate was added, while Glanzmann's thrombasthenic platelets did not. Peptides containing the RGDS sequence or the sequence of the carboxy terminal 15 amino acids of the gamma chain of fibrinogen inhibited the type IIb vWd platelet lysate-induced platelet aggregation. These data suggest that type IIb platelet vWf can cause platelet aggregation of PRP without the addition of any agonist. This interaction is different from that observed with the plasma vWf from these patients.     

Presence of cross-reactive antibody between human immunodeficiency virus (HIV) and platelet glycoproteins in HIV-related immune thrombocytopenic purpura.

We previously reported the presence in platelet eluates of autoantibodies directed against epitopes of the platelet glycoprotein (GP)IIb/IIIa complex in acquired immunodeficiency syndrome (AIDS)-free human immunodeficiency virus (HIV)-infected patients with immunologic thrombocytopenic purpura (ITP). We investigated whether HIV antibodies recognized platelet membrane antigens to determine whether the virus might be directly or indirectly responsible for the thrombocytopenia in this context. Direct eluates of platelets from 25 patients with HIV-related ITP contained IgG reacting with HIV-GP160/120 and also, in 45% of patients, detectable antiplatelet antibodies, immunochemically characterized as anti-GPIIb and/or anti-GPIIIa in 5 patients. Furthermore, serum HIV-GP160/120 antibodies could be absorbed on and eluted from platelets from normal non-HIV-infected healthy blood donors (indirect eluates). In contrast, GP160/120 antibodies present in the serum of nonthrombocytopenic HIV-infected patients were not absorbable on normal platelets in most patients, suggesting a pathogenic role in HIV-related ITP. We performed detailed studies of a patient with the highest titer of both HIV-GP160/120 and GPIIb/IIIa antibodies in direct and indirect platelet eluates. No antibody binding to GPIIb/IIIa-deficient Glanzmann thrombasthenic platelets was detected. Furthermore, binding/elution experiments conducted with insoluble recombinant GP160 (expressed in baculovirus) and purified platelet GPIIb/IIIa demonstrated that the patient's IgG bound specifically, through the F(ab')2 portion, to a common epitope of HIV-GP160/120 and platelet GPIIb/IIIa. This common epitope was present on a recombinant GP160 expressed in baculovirus but absent from another recombinant GP160 expressed in vaccinia virus, suggesting that the cross-reactivity is dependent on the glycosylation or conformational structure of the GP. We conclude that molecular mimicry between HIV-GP160/120 and platelet GPIIb/IIIa may explain at least some cases of ITP in AIDS-free HIV-infected patients.     

Topographical Association of the Platelet Fc-receptor with the Glycoprotein IIb-IIIa Complex

In this study, we have examined whether the platelet Fc-receptor, FcγRII (CD32), is associated with either of the two major platelet membrane glycoproteins, the GPIb-IX complex and the GPIIb-IIIa complex. Monoclonal and polyclonal anti-GPIb-IX complex antibodies inhibited to only a moderate degree (< 40%) the binding of the anti-FcγRII monoclonal antibody, IV.3, to platelets. In contrast, 6 of 12 anti-GPIIb-IIIa monoclonal antibodies and a polyclonal, affinity-purified rabbit anti-GPIIb-IIIa antibody strongly cross-blocked the binding of IV.3 to platelets. This inhibition was dependent upon the Fab-mediated binding of these antibodies to the GPIIb-IIIa complex since they did not inhibit the binding of IV.3 to Glanzmann's thrombasthenic platelets which have normal levels of FcγRII but lack the GPIIb-IIIa complex. The anti-GPIIb-IIIa monoclonal antibodies, AP3 and VM16a, had no effect on platelet aggregation induced by ADP or thrombin but inhibited Fc-receptor-dependent platelet aggregation as induced by either acetone-aggregated human IgG or by activating monoclonal antibodies against GPIV, PTA1 or CD9. F(ab')₂ fragments of these two anti-GPIIb-IIIa monoclonal antibodies also inhibited Fc-receptor-dependent platelet aggregation indicating that the observed interference by intact antibody was not due to the direct interaction of the Fc-portion of the antigen-antibody complex with FcγRII. In addition, the inhibitory anti-GPIIb-IIIa antibodies cross-blocked the binding of IV.3 to platelets at 0°C as well as at 22°C suggesting that the observed inhibition was not dependent on the lateral mobility of either GP IIb-IIIa or FcγRII in the platelet membrane. The combined results therefore strongly suggest that the platelet Fc-receptor, FcγRII, is topographically associated with the GPIIb-IIIa complex in the intact platelet membrane.     

Localization of platelet osteonectin at the internal face of the alpha-granule membranes in platelets and megakaryocytes.

Osteonectin is a 32-Kd phosphoglycoprotein originally described in bone but also found in platelets. Platelet and bone osteonectin are different both structurally and immunologically. We have previously shown that platelet osteonectin, by binding to thrombospondin, is involved in the secretion-dependent phase of the platelet aggregation process. In this study, we used antiosteonectin antibodies in combination with immunogold labeling to investigate by electron microscopy the fine localization of osteonectin within normal and gray platelets. Using both a polyclonal and monoclonal antibody ON3, osteonectin was specifically located at the internal face of alpha-granule membranes within normal platelets. Osteonectin was not distributed within all alpha-granules, probably because of its low platelet content. In addition, using immunofluorescence, osteonectin could also be detected in immature and mature megakaryocytes with a granular pattern of staining, suggesting that osteonectin is synthesized by megakaryocytes. Using platelets from two patients with gray platelet syndrome, osteonectin was absent within all abnormal small alpha-granules, but was detected in some rare normal-sized alpha-granules. In separate double-label studies, thrombospondin and von Willebrand factor did not colocalize with osteonectin in resting platelets. However, osteonectin was located at the inner face of the alpha-granules, as it is for alpha-granule membrane protein GMP-140 and glycoprotein IIb-IIIa. These results, taken together with the fact that monoclonal antibodies to osteonectin bind only to the surface of activated platelets, suggest that platelet osteonectin is redistributed to the cell surface during fusion of alpha-granule membranes with the plasma membrane.     

A unique talin antigenic determinant and anomalous megakaryocyte talin distribution associated with abnormal platelet formation in the Wistar Furth rat.

Rats of the Wistar Furth (WF) strain have hereditary macrothrombocytopenia with decreased platelet alpha-granule proteins. The autosomal recessive pattern of inheritance of the large mean platelet volume (MPV) phenotype and platelet alpha-granule protein deficiencies suggest that a component common to both formation of platelet alpha-granules and subdivision of megakaryocyte cytoplasm into platelets is quantitatively or qualitatively abnormal in WF megakaryocytes and platelets. We examined WF platelets for such an abnormality using electrophoretic and immunologic analyses. Rabbit antiserum prepared against WF rat platelets and absorbed with Wistar rat platelets recognized a major 235-Kd band, and minor bands of WF rat platelets ranging from 200 to 130 Kd, not present in immunoblots of Wistar, Sprague-Dawley, or Long-Evans rat platelets. The minor bands were labeled with affinity-isolated antibody to the 235-Kd band, indicating that all bands contained the same unique antigenic site. The 235-Kd antigen had the same mobility as rat platelet talin identified with a platelet antitalin antibody. Activation of calcium-dependent proteases during Triton X-100 extraction caused conversion of the 235-Kd antigen into a major fragment of 200 Kd and minor fragments ranging to 115 Kd, identical in mobility to fragments of rat platelet talin produced in the same samples. The absorbed anti-WF platelet antiserum also detected a 235-Kd antigen in WF lung, kidney, and small intestine by immunoblotting. Finally, the 235-Kd antigen unique to WF rats was immunoprecipitated from Triton X-100 supernatants of WF platelets with an antitalin monoclonal antibody (MoAb). These data indicate that the unique antigenic site is on WF talin. Examination of talin distribution in Wistar megakaryocytes showed localization beneath the plasma membrane, on the cytosolic face of demarcation membranes, associated with alpha-granule membranes, and diffusely throughout the cytoplasm. Although WF megakaryocytes showed the same general distribution pattern, some differences were apparent. In contrast to membrane systems of the Wistar rat, the large membrane complexes in WF megakaryocytes contained little or no talin. In addition, approximately half of WF megakaryocytes showed an increased peripheral localization of talin, often associated with membrane blebs, with decreased talin in the cytoplasmic interior. The association of the unique talin antigenic determinant and anomalous megakaryocyte talin distribution with abnormal platelet formation in WF rats suggests that talin is abnormal in this rat strain and that talin plays an important role in subdivision of megakaryocyte cytoplasm into platelets.     

Platelet aggregometry and anti-platelet isoantibodies.

102 sera from polytransfused patients have been screened in platelet aggregometry. Anti-human platelet isoantibodies, tested against 'responsive' human platelets in PRP, give in the aggregometer a decrease in optical density recorded as a sigmoidal curve. In the first step of reaction PF3 availability suggests that the primary action of antibody is to damage the platelet membrane. The second step might be a true aggregation (an ADP-mediated platelet clumping) or a platelet lysis, depending on the type and the potency of antibody, on the type of platelets (normal or thrombasthenic) and on some experimental conditions. These conclusions, confirmed by electron microscopy findings, suggest that aggregometry is a very rapid and simple method of detecting platelet antibodies.     

Topographical Association of the Platelet Fc-receptor with the Glycoprotein IIb-IIIa Complex

In this study, we have examined whether the platelet Fc-receptor, FcγRII (CD32), is associated with either of the two major platelet membrane glycoproteins, the GPIb-IX complex and the GPIIb-IIIa complex. Monoclonal and polyclonal anti-GPIb-IX complex antibodies inhibited to only a moderate degree (< 40%) the binding of the anti-FcγRII monoclonal antibody, IV.3, to platelets. In contrast, 6 of 12 anti-GPIIb-IIIa monoclonal antibodies and a polyclonal, affinity-purified rabbit anti-GPIIb-IIIa antibody strongly cross-blocked the binding of IV.3 to platelets. This inhibition was dependent upon the Fab-mediated binding of these antibodies to the GPIIb-IIIa complex since they did not inhibit the binding of IV.3 to Glanzmann's thrombasthenic platelets which have normal levels of FcγRII but lack the GPIIb-IIIa complex. The anti-GPIIb-IIIa monoclonal antibodies, AP3 and VM16a, had no effect on platelet aggregation induced by ADP or thrombin but inhibited Fc-receptor-dependent platelet aggregation as induced by either acetone-aggregated human IgG or by activating monoclonal antibodies against GPIV, PTA1 or CD9. F(ab')(2) fragments of these two anti-GPIIb-IIIa monoclonal antibodies also inhibited Fc-receptor-dependent platelet aggregation indicating that the observed interference by intact antibody was not due to the direct interaction of the Fc-portion of the antigen-antibody complex with FcγRII. In addition, the inhibitory anti-GPIIb-IIIa antibodies cross-blocked the binding of IV.3 to platelets at 0°C as well as at 22°C suggesting that the observed inhibition was not dependent on the lateral mobility of either GP IIb-IIIa or FcγRII in the platelet membrane. The combined results therefore strongly suggest that the platelet Fc-receptor, FcγRII, is topographically associated with the GPIIb-IIIa complex in the intact platelet membrane.     

Evidence for a role of glycoprotein IIb-IIIa, distinct from its ability to support aggregation, in platelet activation by ionophores in the presence of extracellular divalent cations

Ionophore A23187-induced 14C-serotonin secretion and thromboxane B2(TxB2) formation were found to be absent in citrated platelet-rich plasma (PRP) from thrombasthenic subjects and in normal PRP treated with glycoprotein (GP) IIb-IIIa complex-specific monoclonal antibodies. Both responses were restored to normal levels when 5 mmol/L EDTA was present, indicating that their absence was not caused by the absence of aggregation per se. In gel-filtered platelets (GFP) incubated with various additions, the blockade or absence of GPIIb-IIIa resulted in reduced A23187-induced secretion and TxB2 formation in media containing 1 mmol/L Ca2+ with or without fibrinogen and 1 mmol/L Mg2+ plus fibrinogen, but not when Ca, Mg-free buffer alone, 1 mmol/L EDTA, or fibrinogen alone were present. In contrast, no such dependence or GPIIb- IIIa was seen in GFP stimulated with thrombin or phorbol myristate acetate in the presence of 1 mmol/L Ca2+, 1 mmol/L EDTA, or buffer alone. The inhibition of ionophore-induced responses seen in both normal GFP treated with antibodies and thrombasthenic GFP was not associated with any significant alteration of the ionophore-mediated [Ca2+]i increase, as measured in both aequorin-loaded GFP stimulated with A23187 and fura-2-loaded GFP stimulated with ionomycin. Incubation of normal GFP with either the monoclonal antibodies or the ligand binding site peptide RGDS in the presence of 1 mmol/L Ca2+ caused virtually complete inhibition of A23187-induced aggregation, measured as the loss of single platelets, but RGDS, in contrast to the antibodies, did not inhibit secretion or TxB2 formation. We conclude that platelet activation induced by ionophores in the presence, but not in the absence, of extracellular divalent cations involves a GPIIb-IIIa- dependent process that most likely involves a property of the ligand- occupied form of the complex distinct from its ability to support aggregation. This could represent another example of an aggregation- independent activity of the receptor-occupied state of the GPIIb-IIIa complex in signal transduction.     

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.     

Brb, a Platelet Alloantigen Involved in Neonatal Alloimmune Thrombocytopenia

Serum from a pregnant woman with the May-Hegglin anomaly contained a platelet-specific antibody. The serum reacted in the platelet indirect immunofluorescence test (PIIFT) with 97.6% of random donor platelets and those of the father but not with the mother's own platelets. This antibody induced a moderate thrombocytopenia in the infant that responded to infusion of intravenous immunoglobulin concentrates. The platelet phenotypes were PLA1+, Baka+, Bra+/Brb- for the mother, PLA1+, Baka+, Bra-/Brb+ for the father, and PLA1+, Bra+/Brb+ for the neonate. Analysis of the maternal serum with an immunoassay based on monoclonal antibody immobilization of platelet antigens (MAIPA) and immunoprecipitation techniques demonstrated the absence of antibodies directed against HLA class I antigens and that the antigen recognized was located on the platelet-GpIa/IIa complex. This antigen was present on 113/115 random donor platelets, in 7 of the 7 unrelated May-Hegglin platelets, and only absent in 3/24 Bra+ individuals, including the mother. No platelet-specific antibody was present in the serum of the 7 unrelated May-Hegglin subjects. The antigen recognized by this platelet-specific antibody thus meets the criteria defining the antithetic allele of Bra, i.e. the Brb alloantigen.     

Platelet interaction with subendothelial extracellular matrix: platelet- fibrinogen interactions are essential for platelet aggregation but not for the matrix-induced release reaction

Cultured endothelial cells produce an extracellular matrix (ECM) to which platelets adhere and spread, ultimately resulting in platelet aggregation, thromboxane B2 production, and serotonin release. We have investigated the role of fibrinogen binding to the platelet GPIIb/IIIa complex in these reactions by comparing normal platelet-rich plasma (PRP), PRP from patients with Glanzman's thrombasthenia (whose platelets lack the GPIIb/IIIa complex), PRP in the presence of a monoclonal antibody that blocks the binding of fibrinogen to the GPIIb/IIIa complex, platelets washed free of fibrinogen, and washed platelets to which fibrinogen was added. Although platelet aggregation was virtually completely inhibited in the samples in which the normal interaction between fibrinogen and GPIIb/IIIa was impaired, adhesion of platelets to the matrix, spreading, and release of [14C]-serotonin were not affected. All of the platelet preparations released significant amounts of T X B2 with time, but there was a decrease in the amount produced by both the thrombasthenic and antibody-treated platelets. We conclude that the interaction of fibrinogen with platelet GPIIb/IIIa is not required for platelet adhesion to ECM or for adhesion-induced shape change or serotonin release. On the other hand, the platelet-fibrinogen interaction may play some role in augmenting adhesion-induced T X B2 production, and it is absolutely required for adhesion-induced platelet aggregation.