用单克隆抗体对ITP自身抗血小板抗体及其相关抗原的初步研究

本文应用三种抗血小板膜糖蛋白单克隆抗体,按ELISA 间接法、ELISA双抗体夹心法、ELISA竞争法,对ITP患者自身抗血小板抗体及其相关抗原进行了初步研究。实验结果表明,抗血小板抗体的相关抗原呈多态性,包括:GPⅠb、GPⅡb及/或 GPⅢa,以 GPⅡb及/或 GPⅢa占多数,还可能包括血小板膜蛋白以外的其它抗原成分。这提示,ITP可能是一组复杂的、不均一的自身免疫性疾病。     

Atherosclerosis and unstable angina in Bernard-Soulier syndrome.

Platelets and von Willebrand factor play pathogenetic roles in atherosclerosis and acute coronary artery ischemic syndromes. Patients with Bernard-Soulier Syndrome are deficient in several platelet membrane glycoproteins, including glycoprotein Ib (GpIb). Glycoprotein Ib is the primary platelet receptor for von Willebrand factor and plays a critical role in the initiation of thrombus formation. Glycoprotein Ib, but also GpIIb/IIIa, mediates the adhesion of platelets to damaged endothelium, particularly at the high shear stresses found in small or diseased arteries. A patient with Bernard-Soulier syndrome is described who developed coronary artery atherosclerosis and unstable angina requiring coronary artery bypass grafting. The implications of this experiment in nature on the contribution of platelets and platelet GpIb and GpIIb/IIIa receptors to the development of atherosclerosis and unstable angina are discussed.     

Localization of internal pools of membrane glycoproteins involved in platelet adhesive responses.

To investigate the existence of intracellular pools of membrane glycoproteins involved in platelet adhesive reactions, the authors have studied the distribution of glycoprotein (GP) Ib and IIb/IIIa by immunofluorescence and immunoelectron microscopy. Studies on whole cells and frozen thick sections revealed a rim pattern of fluorescence for GPIb and GPIIb/IIIa consistent with a surface distribution. In addition, extensive staining occupying the entire cell interior was observed for anti-GPIIb/IIIa, whereas anti-GPIb revealed staining of large intracellular structures that contained no stainable fibrinogen. On the ultrastructural level, the extracellular face of the plasma membrane and the intraluminal face of vacuolar structures were stained with both anti-GPIb and anti-GPIIb/IIIa. Additionally, GPIIb/IIIa antigen was localized to alpha-granule membranes. To determine whether alpha-granule GPIIb/IIIa could be transported to the cell surface, the authors employed a calcium-dependent monoclonal anti-GPIIb/IIIa antibody. Incubation of platelets with EGTA at 37 C abolished staining of plasma membrane and vacuolar but not alpha-granule GPIIb/IIIa. Recalcification of these cells failed to restore the epitope; however, thrombin treatment of recalcified cells reconstituted surface staining with a concurrent loss of internal staining. These data suggest that GPIIb/IIIa is present in alpha-granule membranes and may be transported to the cell surface in response to thrombin treatment. In addition, both GPIb and GPIIb/IIIa antigens are present in intracellular membrane-bounded vacuolar structures which are closed to antibody probes in fixed cells. Redistribution of these internal pools of adhesive protein "receptors" may participate in the regulation of platelet adhesive properties.     

Ca2+ influx and platelet membrane glycoproteins

When aequorin-loaded platelets were stimulated with thrombin, the luminescence of aequorin showed two peaks. From experiments with 1 mM external Ca2+ or 1 mM EGTA, both one-half of the first peak and the entire second peak reflected the influx of Ca2+ from the external medium, and the remaining half of the first peak reflected the mobilization of Ca2+ from its storage site. A monoclonal antibody (TM83), that recognizes the GPIIb/IIIa complex which has binding sites for fibrinogen, and synthetic peptide GRGDSP are known to inhibit fibrinogen binding and platelet aggregation. Both of them eliminated the second peak of intracellular free calcium. Similar effects were observed during activation by collagen, but not by TPA. Also dihydrocytochalasin B inhibited the second peak of Ca2+ influx by thrombin, suggesting that the signal, which was caused by fibrinogen-binding to GPIIb/IIIa (aggregation) in thrombin-activated platelets, is transferred to the inner sites of GPIIb/IIIa complex and induces the cytoskeletal reorganization such as actin polymerization. This in turn, induces the secondary increase in [Ca2+] i of platelets. It is interesting that ticlopidine inhibited the Ca2+ influx through the GPIIb/IIIa complex. This result suggests the importance of such kinds of antiplatelet drugs to prevent thrombus formation.     

Analysis of platelet surface conformation in thrombin-induced aggregation

We used flow cytometry to investigate the change of platelet membrane glycoproteins (GPIb and GP IIb/IIIa) and the distributions of fibrinogen (Fbg), thrombospondin (TSP) and fibronectin (Fn) on the surface of thrombin-stimulated platelets. The binding of a monoclonal antibody directed at the von Willebrand factor binding site on GPIb decreased in thrombin-stimulated platelets. This antibody caused a reactive delay in thrombin-induced aggregation, but had little influence on aggregability. Slight thrombin-induced aggregation was observed even after blocking the binding of Fbg to GP II b/IIIa. The new expression of GP II b/IIIa was detected on the surface of thrombin-stimulated platelets, whereas there was little increase of Fbg dependent on this GP II b/IIIa. An increase of TSP after thrombin stimulation was observed on the surface of platelets of healthy controls and patients with Glanzmann's thrombasthenia (Type I). The level of on platelet surface was slightly increased by thrombin stimulation. The mechanism involved in thrombin-induced aggregation appears to differ from that in ADP-induced aggregation.     

中华眼镜蛇毒F组分抗血小板聚集的作用机制

目的探讨中华眼镜蛇毒F组分抑制血小板聚集的作用机制。方法用比浊法测定中华眼镜蛇毒F组分对二磷酸腺苷、花生四烯酸和血小板活化因子诱导血小板聚集作用的影响,流式细胞术观察中华眼镜蛇毒F组分对荧光标记的单克隆抗体CD41(FITC-CD41)和CD61(FITC-CD61)与血小板膜糖蛋白IIb/IIIa(GPIIb/IIIa)结合的影响。结果中华眼镜蛇毒F组分明显抑制二磷酸腺苷、花生四烯酸和血小板活化因子诱导的血小板聚集,其作用呈现一定程度的剂量依赖关系。中华眼镜蛇毒F组分可以明显降低单克隆抗体CD41(抗GPIIb)与血小板的结合率,而对单克隆抗体CD61(抗GPIIIa)与血小板的结合率没有影响。结论中华眼镜蛇毒F组分可以抑制多种激动剂诱导的血小板聚集,其机制和中华眼镜蛇毒F组分与血小板膜糖蛋白IIb/IIIa复合物的结合有关。     

Anti-platelet antibodies associated with the Canale-Smith syndrome bind to the same platelet glycoprotein complexes as those of idiopathic thrombocytopenic purpura patients

The Canale-Smith syndrome (CSS) is an inherited disease characterized by massive lymphadenopathy, hepatosplenomegaly and systemic autoimmunity to erythrocytes and platelets. Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disease in which approximately 60-80% of patients have anti-platelet antibodies directed against specific platelet glycoprotein complexes (GPCs) located on their membrane: GP IIb/IIIa, GPIb/IX, and GPIa/IIa. Almost all (95-100%) of the antibody-positive patients have antibodies directed against GPIIb/IIIa alone, or in combination with other glycoprotein targets. Our objective was to determine the specificities of the anti-platelet antibodies in CSS patients. The detection of anti-platelet antibodies was performed using a commercially available ELISA, the Pak-AUTO (GTI, Brookfield, WI), in which highly purified GPIIb/IIIa, GPIb/IX, and GPIa/IIa are immobilized on microtitre plates, incubated with serum or plasma, and subsequently developed with an antihuman polyclonal immunoglobulin. Of 14 CSS patients tested, 11 (79%) had anti-platelet antibodies in their serum directed toward at least one of the three major GPC, nine (82%) of which were against GPIIb/IIIa alone or in combination. Antibodies detected in the sera of ITP patients had similar specificities. No such antibodies were detected in samples from 25 consecutive normal controls. These results demonstrate that a genetically defined defect in lymphocyte apoptosis results in a humoral autoimmune response with anti-platelet specificities very similar to the common idiopathic form of autoimmune thrombocytopenia.     

The platelet fibrinogen receptor: a model for the analysis of cellular adhesion mechanisms and their modification in pathology

Blood platelets are implicated in a series of cellular recognition and adhesion phenomena (adhesion to the subendothelial matrix and platelet aggregation) which are key events in the processes of haemostasis and thrombosis. Platelet aggregation is a model of homotypic cellular adhesion. It is mediated by the binding of bifunctional molecules of fibrinogen to the plasma membrane of adjacent platelets, following stimulation of the platelets by agonists such as ADP, thrombin or collagen, with the fibrinogen serving as an intercellular glue. The platelet receptor for fibrinogen is a macromolecular complex, GPIIb-IIIa, made of two transmembrane glycoproteins, GPIIb (Mr = 142,000) and GPIIIa (Mr = 99,000), assembled into a heterodimer whose conformation depends upon the binding of calcium (Ca2+) to GPIIb. Furthermore, the GPIIb-IIIa complex represents the prototype, and one of the most studied, among a large family of membrane receptors, the integrins, all implicated in various adhesion processes. Binding of fibrinogen to GPIIb-IIIa occurs through interactions between peptide sequences within the receptor and particular adhesive sites within the ligand: thus, GPIIIa can bind to a tetrapeptide sequence of the A alpha chain of fibrinogen, whereas a dodecapeptide of the gamma chain can be preferentially bound to GPIIb. On a physiopathological point of view, qualitative or quantitative defects of GPIIb-IIIa are associated with a rare haemorrhagic syndrome, Glanzmann' thrombasthenia. Its central role in platelet aggregation and thrombogenesis, together with a potential role in tumor cell-platelet interactions and in metastasis, make GPIIb-IIIa, nowadays, an important pharmacological target.     

An enzyme-linked immunosorbent assay for the detection of platelet antibodies using detergent-solubilized platelets immobilized on nitrocellulose discs

A method is detailed for the solubilization of human platelets using a dialyzable detergent, decanoyl-N-methylglucamide (Mega-10). At a detergent/protein ratio of 1:12, the efficiency of solubilization was 27%. This platelet lysate (PLy) was then bound to nitrocellulose (NC) discs to assay retention of the native immunological functions of the platelet membrane antigens. Using alkaline phosphatase-coupled anti-IgG, the major platelet membrane glycoproteins GPIb, GPIIb, and GPIIb/IIIa were detectable with as little as 20 ng of monoclonal antibody. Antisera to the class I histocompatibility antigens HLA-A1, B7, B8, the PlA1 allodeterminant, and serum from multiply transfused, alloimmunized patients were reactive even after 100 days storage of the discs at 4 degrees C, and with as little as 1.0 micrograms of NC-bound PLy. The binding of the same antisera to intact, immobilized platelets as well as specific complement-mediated lymphocytotoxicity was also inhibited by PLy. PLy from HLA-A3- or B44-positive donors, however, did not inhibit cytotoxicity of lymphocytes expressing either antigen using several different antisera. Our results indicate that Mega-10 is an excellent solubilizing agent for the immunological study of platelet membranes. The fact that clinically relevant platelet membrane antigens are preserved, immunologically reactive, and stable over long periods of storage, makes this assay amenable to a routine crossmatching procedure for platelet transfusions.     

Hemostatic evaluation in bleeding disorders from native blood. Clinical experience with the hemostatometer

Primary hemostasis (PH), i.e., hemostatic platelet plug formation, and the subsequent coagulation were recorded and quantified from the same nonanticoagulated venous blood sample with the use of the Haemostatometer. In addition, platelet thrombus formation induced by interaction of flowing native blood with a collagen fiber under low shear rates (450 s-1) was simultaneously analyzed by this device. The effect of monoclonal antibodies (MoAbs) directed against von Willebrand's factor antigen (vWF:Ag), platelet glycoprotein Ib (GPIb) and the GPIIb/IIIa complex, and fibrinogen were studied. PH was significantly inhibited by MoAbs against vWF:Ag, GPIIb/IIIa, and fibrinogen but was unaffected by antibody against GPIb. Collagen-induced thrombosis was prevented by MoAbs against vWF:Ag and GPIb, slightly inhibited by antifibrinogen, and unaffected by blockage of platelet membrane GPIIb/IIIa. The effect of a single 600-mg dose of aspirin was monitored, and abnormal PH was still detectable five days later. From the 13 hemophiliacs tested, 7 showed significantly prolonged PH. In von Willebrand's disease, a characteristic defect of PH with significant inhibition or absence of collagen-platelet interaction was observed in all the 11 patients. PH was greatly prolonged in both of the two patients with storage pool deficiency. The technique detected improvement of platelet function, i.e., PH in all of six patients with bleeding disorders after replacement therapy or DDAVP infusion. The authors conclude that the Haemostatometer technique is a sensitive test for determining platelet dysfunction and monitoring efficacy of factor-replacement or DDAVP therapy.     

Plasma membrane GPIIb/IIIa. Evidence for a cycling receptor pool.

The author used immunofluorescence and digital image processing to investigate the dynamic distribution of GPIIb/IIIa in living platelets. Resting cells were incubated with AP-2, a complex-specific, monoclonal, anti-GPIIb/IIIa antibody. Examination of intact cells demonstrated a rim pattern for GPIIb/IIIa consistent with a surface localization. Permeabilization revealed a time-dependent increase in the labeling of apparent intracellular vacuoles. This pattern is distinct from the "patch-cap" pattern observed when unfixed platelets were incubated with fluoresceinated concanavalin A. Additionally, labeling of this vacuolar pool of GPIIb/IIIa was inhibited by treatment with 2% sodium azide or by incubation at 4 degrees C. Identical staining patterns were obtained with Fab fragments of AP-2. Ultrastructural examination confirmed the presence of labeled intracellular vacuolar structures. Parallel studies performed with AP-1, a monoclonal anti-GPIb antibody, failed to demonstrate internalization of GPIb. Finally, thrombin stimulation of resting platelets, which had been preincubated with AP-2, resulted in the clearing of this newly internalized pool of GPIIb/IIIa; presumably via translocation to the surface. These data suggest the presence of an actively cycling pool of GPIIb/IIIa that has not been described previously. The dynamic distribution of this pool may be important in the regulation of platelet adhesiveness.     

Freeze-fracture cytochemistry of wheat germ agglutinin and concanavalin A receptors on the plasma membrane of normal, Bernard-Soulier, and thrombasthenic platelets.

The authors report here the results of fracture-labeling of wheat germ agglutinin (WGA) and concanavalin A (Con A) receptors on the plasma membranes of normal, Bernard-Soulier, and thrombasthenic platelets. In all cases, virtually all of the label was confined to the exoplasmic half of the membrane. Despite the absence of GP Ib in Bernard-Soulier platelets and the absence or strong reduction of Gp IIb and GP IIIa in thrombasthenic platelets, their plasma membranes were strongly labeled by both Con A and WGA. These results are best accounted for by the presence of other glycoproteins and/or glycolipids at the platelet surface.     

Redistribution and hemostatic action of recombinant activated factor VII associated with platelets

Clinical evidence accumulated from hemophilic patients during prophylaxis with recombinant activated factor VII (rFVIIa) suggests that the duration of the hemostatic action of rFVIIa exceeds its predicted plasma half-life. Mechanisms involved in this outcome have not been elucidated. We have investigated in vitro the redistribution of rFVIIa in platelets from healthy donors, patients with FVII deficiency, and one patient with Bernard-Soulier syndrome. Platelet-rich plasma was exposed to rFVIIa (3 to 60 μg/mL). Flow cytometry, immunocytochemistry, and coagulation tests were applied to detect and quantify rFVIIa. The hemostatic effect of rFVIIa associated to platelets was evaluated using perfusion models. Our studies revealed a dose-dependent association of rFVIIa to the platelet cytoplasm with redistribution into the open canalicular system, and α granules. Mechanisms implicated in the internalization are multiple, involve GPIb and GPIV, and require phospholipids and cytoskeletal assembly. After platelet activation with thrombin, platelets exposed rFVIIa on their membrane. Perfusion studies revealed that the presence of 30% of platelets containing FVIIa improved platelet aggregate formation and enhanced fibrin generation (P < 0.01 versus control). Our results indicate that, at therapeutic concentrations, rFVIIa can be internalized into platelets, where it is protected from physiological clearance mechanisms and can still promote hemostatic activity. Redistribution of rFVIIa into platelets may explain the prolonged prophylactic effectiveness of rFVIIa in hemophilia.     

Anti-glycoprotein IIb/IIIa autoantibodies are reversibly internalized into platelets in idiopathic (autoimmune) thrombocytopenic purpura.

We used flow cytometry to investigate the binding of platelet-binding IgG (PBIgG) to unfixed platelets in idiopathic thrombocytopenic purpura (ITP), including that of anti-glycoprotein (GP) IIb/IIIa antibodies. Anti-GPIIb/IIIa antibodies were detected in 13/64 ITP patients using antigen-capture ELISA and immunoblotting. When unfixed platelets were incubated with ITP plasma, the PBIgG level was significantly higher than after incubation with normal plasma. When 1 microM ADP was added to unfixed platelets, which were incubated with ITP plasma and washed, the PBIgG level increased additively. GMP-140 is a constituent of platelet alpha-granules, and a monoclonal antibody directed against this protein showed weak binding to platelets after 1 microM ADP stimulation. The increase of PBIgG produced by ADP was significantly greater when ITP plasma positive for anti-GPIIb/IIIa antibody was used compared with that obtained using antibody-negative ITP plasma. This increase of PBIgG was markedly inhibited by the removal of extracellular calcium with EDTA or the dissociation of the GPIIb/IIIa complex by EDTA treatment at 37 degrees C. These results suggest that anti-GPIIb/IIIa autoantibodies are internalized by unfixed ITP platelets and stored somewhere other than the alpha-granules. This stored antibody pool can be reversibly redistributed on the platelet surface by weak stimulants such as ADP and a functional GPIIb/IIIa complex appears to be necessary for this to occur.     

Identification of a human heavy chain antibody fragment directed against human platelet alloantigen la by phage display library

Abstract: The human platelet alloantigen HPA-la (Pl^A1) is responsible for most cases of post-transfusion purpura and neonatal alloimmune thrombo-cytopenia in the Caucasian population. HPA-la and HPA-lb are two allelic forms of the platelet membrane glycoprotein IIIa (GPIIIa) gene that differ by a single amino acid. In this report, we describe the development of a recom-binant heavy chain antibody fragment capable of distinguishing between the homozygous forms of HPA-la and HPA-lb. This antibody fragment was isolated from the lymphocytes of an immunized individual through the use of a phage display library system. The recombinant antibody fragment reacted with human platelet lysates from HPA-la homozygous donors, the HPA-la form of recombinant N-terminal GPIIIa and intact HPA-la platelets, but did not react with platelet lysate from HPA-1b homozygous donors, reduced HPA-la form of platelet GPIIIa or other platelet glycoproteins. This HPA-la specific human antibody fragment works well in common laboratory assays such as ELISA and flow cytometry, which can assist in identifying HPA-lb homozygous individuals who are known to have a higher risk for developing neonatal alloimmmune thrombocytopenia and post-transfusion purpura. Thus, selection of recombinant antibody fragment using phage display offers a promising alternative to hybridoma technology for the production of human antibodies against human alloantigens and holds potential as a technique in therapeutic applications.     

Identification and characterization of antibodies that bind GPIIb/IIIa: antagonist complexes

A potential limitation of anti-thrombotic therapies directed at platelet GPIIb/IIIa is immune mediated thrombocytopenia. Reagents that mimic the behavior of patient antibodies would provide a valuable tool for studies directed at understanding the basis of the immune mechanism involved in GPIIb/IIIa antagonist induced thrombocytopenia. Such reagents would bind epitopes that are exposed when the conformation of the receptor is modified in response to inhibitor binding. We describe the production and characterization of monoclonal antibodies that were raised against platelet GPIIb/IIIa bound to a potent antagonist, XP280. These antibodies have high affinity and specificity for XP280 bound GPIIb/IIIa using either purified protein or human platelets. We have demonstrated that the antibodies recognize a conformationally altered form of the receptor, that both subunits are required for binding, and that the antagonist itself does not form part of the binding epitope. Competition experiments indicate that multiple drug-dependent epitopes are exposed on the receptor in response to antagonist binding. The antibodies bind with high specificity to some but not all GP IIb/IIIa/antagonist complexes indicating that different conformational epitopes are exposed when GP IIb/IIIa is bound to different antagonists.     

Platelet kinetics in dogs treated with a glycoprotein IIb/IIIa peptide antagonist

Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonists have been highly effective inhibitors of platelet aggregation in preclinical studies and in clinical trials. However, decreased platelet counts have been documented in preclinical studies and in some patients receiving GPIIb/IIIa antagonists. We evaluated changes in platelet kinetics and fate in dogs receiving the GPIIb/IIIa receptor antagonist RPR 109891 orally for 4 days. Dogs receiving RPR 109891 had a 22-52% decrease in platelet count with the nadirs at 3-5 days after initiation of treatment. Platelet survival time was reduced by 19%, and platelet half-life was reduced by 63%. Indium-111-labeled platelets were rapidly cleared from the blood within 1 hour after administration of RPR 109891 on treatment days 1 and 2. This clearing was associated with a sharp increase in radioactivity in spleen but not in liver or lung. Platelet clearance was markedly attenuated on treatment days 3 and 4. Platelet counts returned to baseline within 1 week after discontinuation of treatment. These data indicate that RPR 109891 causes rapid and selective sequestration of platelets in the spleen.     

Role of platelet surface receptor abnormalities in the bleeding and thrombotic diathesis of uremic patients on hemodialysis and peritoneal dialysis

BACKGROUND: Patients with chronic renal failure suffer from bleeding diathesis and a tendency to accelerated atherosclerosis. Altered platelet function plays a well defined role in the hemorrhagic complications of these patients and has a probable impact on atherothrombotic disease in uremia. In this study we investigated the expression of platelet surface receptors, the glycoprotein GPIb (receptor for von Willebrand Factor(vWF) and GPIIb/IIIa (receptor for fibrinogen) in patient with chronic renal failure in pre-dialysis status, under hemodialysis and peritoneal dialysis treatment, in order to assess the impact of the abnormal receptorial status of uremic platelets on the clinical manifestations of hemostatic alterations in uremic patients. METHODS: Thirty-seven normal healthy subjects (controls = Group A), 18 patients with mild chronic renal failure (creatinine = 1.8 +/- 0.5 mg% - Group B), 15 patients with advanced renal failure (creatinine = 5.4 +/- 2. 1 mg% - Group C), 18 hemodialysis patients (Group D) and 11 peritoneal dialysis patients (Group E) were included in the study. The expression of platelet surface receptors GPIb and GPIIb/IIIa was investigated with monoclonal antibodies CD42 and CD41 (Immunotech, Marseille, France) and a FACScan flowcytometer (Becton-Dickinson, USA). RESULTS: Mean values of GPIb glycoprotein (mean flow +/- SD) were: group A = 48.14 +/- 9.31; group B = 40.48 +/- 8.18 (p < 0.005); group C = 34.05 +/- 7.55 (p < 0.0005) versus group A; p = 0.025 versus group B); group D = 34.51 +/- 7.22 (p < 0.0005 versus group A; p = 0.025 group B and p = ns versus group C); group E = 26.34 +/- 4.06 (p < 0.0005 versus group A, p < 0.0005 versus group B, p < 0.005 versus groups C and D). Mean values of glycoprotein GPIIb/IIIa were: group A = 375.32 +/- 90.58; group B = 398.48 +/- 54.26 (p = ns); group C = 426.86 +/- 52.78 (p < 0.025 versus group A; p = ns versus group B); group D = 425.17 +/- 75.03 (p < 0.025 versus group A; p = ns versus groups B and C); group E = 336.39 +/- 43.26 (p = ns versus group A; p < 0.005 versus group B, p < 0.0005 versus group C and p < 0.001 versus group D). CONCLUSIONS: Our data confirm the receptorial defect of glycoprotein GPIb (the receptor for vWF) on the surface of uremic platelets: a negative correlation between serum creatinine and the expression of glycoprotein GPIb was found. The defect was not corrected by hemodialysis and/or peritoneal dialysis. Hemodialysis and peritoneal dialysis have a different impact on the expression of GPIIb/IIIa glycoprotein (the receptor for vWF): peritoneal dialysis seems to have a more favourable effect by restoring normal values of the expression of this membrane integrine. Theoretically the data could be correlated to the better biocompatibility of the peritoneal dialysis and to more favorable clinical behaviour in terms of accelerated atherosclerosis and athero-thrombotic complications in the uremic patients with end stage renal disease. Finally the abnormalities of platelet surface receptors may play a main role in the hemostatic alterations of uremic patients.     

MHC class I-associated peptides identified from normal platelets and from individuals with idiopathic thrombocytopenic purpura

Major histocompatibility complex (MHC) class I molecules bind and display peptide antigens on the cell surface. CD8(+) T lymphocytes recognize peptides in association with class I proteins to initiate a cytotoxic immune response. To understand the specificity of such immune responses and to facilitate the development of therapies for disease, it is important to identify MHC-presented peptides. In this study, platelets, easily obtainable and often associated with immune-mediated disease, were selected to identify MHC class I-associated peptides. MHC-associated peptides presented on platelets of normal individuals and individuals with idiopathic thrombocytopenic purpura (ITP) were characterized. ITP is characterized by the premature immune destruction of platelets. It is associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane GPIIb/IIIa or GPIb/IX. In addition to characterizing five fully and several partially sequenced peptides from platelets, the peptide GPRGA(L/I)S(L/I)(L/I) was identified from four of the five ITP patients. The anchor motif of this peptide correlates with the presence of the HLA-B7 allele. A BLAST search identified this peptide as GPIb (4-12). In conclusion, platelets from normal and ITP individuals can present peptides from general cellular proteins and platelet specific proteins, such as GPIb, to the immune system via MHC class I.     

Dynamic redistribution of major platelet surface receptors after contact-induced platelet activation and spreading. An immunoelectron microscopy study.

The authors used an immunogold labeling procedure to investigate the redistribution of platelet receptors and their ligands on the surface of contact-activated adherent platelets before and after thrombin stimulation. During the initial stage of platelet adhesion, a typical segregation of receptors occurred. Gold particles identifying glycoprotein (GP) Ib (CD42b) and GPIIb-IIIa (CD41a) remained distributed over the entire platelet surface, whereas gold particles identifying GPIa-IIa (CDw 49b) and GPIV (CD36) were found essentially overlying the granulomere; p24 (CD9) was present at the peripheral platelet rim and over the cell body. An increased labeling of GPIIb-IIIa, GPIV and p24 was also observed on pseudopods, with GPIIb-IIIa and GPIV concentrated at the enlarged extremities and at sites of contact between two platelets, whereas GPIb was absent from pseudopods. After thrombin stimulation of adherent platelets, GPIb underwent a relocation to the cell center, in contrast to GPIIb-IIIa which still remained randomly distributed over the cell body. To investigate whether ligand distribution paralleled this receptor segregation, platelet released von Willebrand factor (vWF), fibrinogen (Fg) and thrombospondin (TSP) were visualized. During the early stages of platelet activation, surface labeling for all three adhesive proteins was minimal and almost undetectable. Occasionally, intragranular Fg and vWF was accessible to gold-coupled antibodies, with vWF exhibiting the typical eccentric alpha-granular localization. At later stages of activation and especially after thrombin stimulation, no surface labeling for vWF was observed, whereas immunogold particles identifying vWF were still present inside enlarged clear vacuoles. In contrast, labeling of Fg and TSP was increased over the granulomere and extended to the cell periphery and the pseudopods, but was absent from the hyalomere, despite the presence of GPIIb-IIIa molecules. Double labeling experiments showed colocalization of Fg and TSP, GPIV and TSP, as well as Fg and GPIIb-IIIa, although no typical coclustering of GPIIb-IIIa and GPIV or GPIIb-IIIa and p24 was apparent. Our results further suggest that 1) on surface activated adherent platelets, not all GPIIb-IIIa molecules become competent to bind Fg, 2) GPIa-IIa is not anchored to the platelet membrane skeleton, and 3) during the early stage of platelet activation, a communication exists between the alpha granules and the platelet surface.