Differentiation between Bernard-Soulier syndrome and immune thrombocytopenia by immunostaining of peripheral blood.
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Peripheral blood smears from seven patients with Bernard-Soulier syndrome were examined by an immunocytochemical staining procedure using a monoclonal antibody specific for platelet glycoprotein Ib. No platelet staining was observed except for very slight staining of the large sized platelets from one of the patients. Application of the assay to blood smears from 12 patients with immune thrombocytopenia showed that their peripheral platelets stained normally, so the assay can be used to differentiate between immune thrombocytopenia and Bernard-Soulier syndrome and to confirm a diagnosis of the syndrome. (+info)
Acquired Bernard-Soulier syndrome. Evidence for the role of a 210,000-molecular weight protein in the interaction of platelets with von Willebrand factor.
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A patient with a lymphoproliferative disorder developed bleeding associated with a prolonged bleeding time and a selective defect of platelet aggregation in response to ristocetin. The patient's purified IgG was shown to inhibit aggregation of washed normal platelets by ristocetin and von Willebrand factor (F VIII:vWF). By Western blotting, it was shown that antibody bound specifically to an antigen of Mr 210,000 present on normal platelets but missing on platelets from patients with congenital Bernard-Soulier syndrome (BSS). Binding was effected by the F(ab)2 portion of the IgG, indicating the presence of an autoantibody rather than an immune complex. These results suggest that the 210,000-Mr protein is involved in the interaction of F VIII:vWF with platelets. Furthermore, we have demonstrated the apparent absence of an additional protein on congenital BSS platelets. Heat-aggregated IgG was also shown to bind to the 210,000-Mr protein, suggesting that this protein may function as an Fc receptor on platelets. The relationship of the 210,000-Mr protein to glycoprotein Ib and the precise role of this protein in the interaction of platelets with F VIII:vWF need to be characterized. (+info)
Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb-IIIa, Ib, and storage granules.
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Patients whose platelets are deficient in glycoprotein (GP) Ib, IIb-IIIa (thrombasthenia), or granule substances (storage pool deficiency, SPD) were studied to define further the properties of platelets that mediate platelet adhesion and thrombus formation on subendothelium. Both nonanticoagulated and citrated blood were exposed to everted, de-endothelialized rabbit vessel segments under controlled flow conditions and shear rates varying from 650 to 3,300 sec-1. Morphometry was used to measure platelet thrombus dimensions and the percentage of the subendothelial surface covered with contact (C) or spread (S) platelets. Adhesion was defined as C + S. The results in SPD demonstrated (1) reduced thrombus dimensions in delta-SPD (pure dense granule deficiency) in proportion to the magnitude of the dense granule defect; (2) an even greater reduction in thrombus dimensions in patients with combined deficiencies of alpha and dense granules (alpha delta-SPD); and (3) impaired platelet adhesion at several conditions in alpha delta-SPD and, in delta-SPD, a hematocrit-dependent impairment of adhesion in citrated blood at 2,600 sec-1. In thrombasthenia, platelets were present as a monolayer on the subendothelial surface in both nonanticoagulated and citrated blood, indicating an absolute requirement for GPIIb-IIIa in promoting platelet-platelet interaction at all shear rates and perfusion times. Two types of abnormalities in platelet-vessel wall interactions were observed. In nonanticoagulated blood, the percentage of platelets in the C phase was consistently increased at all shear rates, but C + S values were normal. These observations indicate that platelets deficient in GPIIb-IIIa do not spread normally on the subendothelial surface exposed to nonanticoagulated blood. With citrated blood, the C + S value in thrombasthenia was reduced at both 800 and 2,600 sec-1, as in von Willebrand's disease, and a similar degree of reduction (about 50%) was observed in normal blood treated with a monoclonal antibody to GPIIb-IIIa. The findings, together with theoretical considerations, are consistent with an hypothesis that GPIIb-IIIa mediates the spreading of platelets on subendothelium following the initial attachment through GPIb and that GPIIb-IIIa may be considered an adhesion site on the platelet membrane. Abnormalities of GPIIb-IIIa may, depending on the conditions of study, result in either increased values of C platelets or decreased values of C + S. The results of the study further suggest that a complex interaction of platelet granule factors and membrane GP mediate platelet adhesion and thrombus formation. (+info)
Identification of the thrombin receptor on human platelets by chemical crosslinking.
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To identify the molecular site of thrombin binding to the platelet membrane, we covalently linked 125I-thrombin to platelets by using the bifunctional chemical cross-linking agents disuccinimidyl suberate and dithiobis(succinimidyl propionate). The proteins cross-linked to 125I-thrombin by this method were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by autoradiography. Two radiolabeled thrombin complexes were identified, a major species of Mr approximately 200,000 and a minor one of Mr approximately 400,000. Hirudin prevented the formation of both complexes. The radioactivity of the approximately 200,000-Mr complex was always 7-10-fold greater than the radioactivity of the approximately 400,000-Mr complex regardless of the thrombin concentration to which the platelets were exposed (0.1-29 nM). Although 125I-thrombin complexes generated with thrombasthenic platelets (lacking glycoprotein IIb/IIIa) were indistinguishable from normal, no complexes appeared when Bernard-Soulier platelets (lacking glycoprotein Ib [GPIb]) were used. Complex formation was blocked by rabbit antiglycocalicin antiserum, but not by the monoclonal antibody 6D1, which is directed against the site on GPIb where von Willebrand factor (vWf) binds in the presence of ristocetin. Although cross-linking studies suggested that vWf might partially inhibit thrombin binding to platelets, this was not confirmed by equilibrium binding studies in the presence of vWf and ristocetin. The data suggest, therefore, that at all thrombin concentrations binding occurs at the same membrane site, despite evidence from equilibrium studies for high and low affinity classes of receptors, and that the approximately 400,000-Mr complex is simply a dimer of the approximately 200,000-Mr species. We conclude that the membrane site to which thrombin binds is the glycocalicin portion of platelet GPIb at a site remote from the point of ristocetin-dependent vWf binding. (+info)
Identification of glycoprotein Ib beta as one of the major proteins phosphorylated during exposure of intact platelets to agents that activate cyclic AMP-dependent protein kinase.
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Platelet function is inhibited by prostaglandin E1, prostaglandin I2, or forskolin, agents that increase the intracellular concentration of cyclic AMP. The inhibition appears to result from cyclic AMP-stimulated phosphorylation of specific intracellular proteins. One of the major increases in phosphorylation occurs in a polypeptide of Mr = 24,000 (P24). In this study, an effort was made to identify P24. Platelets prelabeled with [32P]phosphate were incubated with prostaglandin E1, prostaglandin I2, or forskolin. Proteins that became phosphorylated were detected by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. Several lines of evidence indicated that P24 was the beta-subunit of the plasma membrane glycoprotein (GP) Ib, a glycoprotein that is essential for the adhesion of platelets to damaged subendothelium, for the rapid response of platelets to thrombin, and for the attachment of the membrane skeleton to the cytoplasmic face of the plasma membrane. P24 co-migrated with GP Ib beta on reduced gels (Mr = 24,000) and also on nonreduced gels (when GP Ib beta is disulfide-linked to GP Ib alpha and migrates with Mr = 170,000). Like GP Ib beta, P24 was associated with actin filaments in Triton X-100 lysates. Like GP Ib beta, it was selectively associated with filaments of the membrane skeleton and was released from filaments when the Ca2+-dependent protease was active. Antibodies against GP Ib immunoprecipitated P24 from platelet lysates. Finally, exposure of Bernard-Soulier platelets (which lack GP Ib) to prostaglandin E1 resulted in phosphorylation of other polypeptides, but not of P24. These studies show that P24, one of the major polypeptides phosphorylated when platelets are exposed to agents that inhibit platelet function by increasing the concentration of cyclic AMP, is the beta-subunit of GP Ib. (+info)
Residual amounts of glycoprotein Ib concomitant with near-absence of glycoprotein IX in platelets of Bernard-Soulier patients.
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A study of the Bernard-Soulier syndrome in two unrelated families using different polyclonal antibodies in a sensitive immunoblot assay showed residual amounts of platelet membrane glycoprotein (GP) lb in the eight homozygotes, as well as the near-absence of GPlb beta and GPIX. The eight heterozygotes studied showed a double band pattern for GPlb and about half the normal level of GPlb beta and GPIX. Therefore, we conclude that the Bernard-Soulier syndrome is heterogeneous and is probably not due to gene deletions. (+info)
This report describes studies into the pathophysiology of heparin-induced thrombocytopenia. The IgG fraction from each of nine patients with heparin-induced thrombocytopenia caused heparin-dependent platelet release of radiolabeled serotonin. Both the Fc and the Fab portions of the IgG molecule were required for the platelet reactivity. The platelet release reaction could be inhibited by the Fc portion of normal human or goat IgG, and patient F(ab')2, but not F(ab')2 from healthy controls. These results suggested that the Fab portion of IgG binds to heparin forming an immune complex and the immune complexes initiate the platelet release reaction by binding to the platelet Fc receptors. To directly challenge this hypothesis, we preincubated the serotonin-labeled platelets with the monoclonal antibody against the platelet Fc receptor (IV.3). This monoclonal antibody completely inhibited the release reaction caused by heparin and patient sera, as well as heat aggregated IgG, but did not block collagen or thrombin-induced platelet release. Heparin-dependent platelet release also could be inhibited in vitro by the addition of monocytes and neutrophils, but not by red cells, presumably because the Fc receptors on the phagocytic cells have a higher binding affinity for IgG complexes than do platelets. Platelets from patients with congenital deficiencies of specific glycoproteins Ib and IX (Bernard-Soulier syndrome) and IIb and IIIa (Glanzmann's thrombasthenia) displayed normal heparin-dependent release indicating that the release reaction did not require the participation of these glycoproteins. These studies indicate that heparin-induced thrombocytopenia is an IgG-heparin immune complex disorder involving both the Fab and Fc portion of the IgG molecule. (+info)
Evidence that a 210,000-molecular-weight glycoprotein (GP 210) serves as a platelet Fc receptor.
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We previously identified a 210,000-mol-wt platelet glycoprotein (GP 210) that is missing from Bernard-Soulier platelets, and found that an antibody against GP 210 inhibits ristocetin-induced platelet agglutination. We now show by immunoblotting that GP 210 binds heat-aggregated rabbit and human IgG, as well as keyhole limpet hemocyanin (KLH)-anti-KLH and ovalbumin (OA)-anti-OA immune complexes. Immune complex binding to GP 210 was preserved on chymotrypsin-treated platelets that lacked glycoprotein Ib (GP Ib). In contrast, ristocetin-induced platelet agglutination resulted in disappearance of immunologically detectable GP 210 and loss of immune complex binding, even though GP Ib remained intact. Purified Fc fragments inhibited binding of anti-GP 210 antibody to intact platelets and to GP 210 on immunoblots. The Fc fragments also blocked immune complex binding to GP 210. Conversely, anti-GP 210 antiserum and F(ab)2 fragments inhibited binding of fluorescein-labeled Fc fragments to intact platelets. We conclude that GP 210 functions as a platelet Fc receptor. (+info)