Glanzmann Thrombasthenic Patients Research Articles

Protein Phosphatase 2A (PP2A) Associates with Integrin αIIbβ3 and Regulates Adhesion to Fibrinogen.

We have previously demonstrated that protein phosphatase 1 (PP1) associates constitutively with the integrin αIIb subunit and regulates myosin light chain phosphorylation. In this study, we considered whether other member of the serine/threonine (Ser/Thr) phosphatase family namely, protein phosphatase 2A (PP2A) associates with the integrin αIIbβ3. Co-immunoprecipitation assays using lysates from resting human platelets revealed the presence of a catalytic subunit of PP2A (PP2Ac) in the αIIb immunoprecipitates, and in a reciprocal experiment, αIIb was detected in the PP2Ac immunoprecipitates. In contrast, another platelet abundant Ser/Thr phosphatase, protein phosphatase 2C (PP2C) was not detected in the αIIb immunoprecipitates. Furthermore, the association of PP2Ac with integrin αIIbβ3 was also observed in 293 cells overexpressing αIIbβ3. These results indicate a constitutive and specific interaction of PP2Ac with the integrin αIIbβ3. Polystyrene beads coated with purified PP2Ac but not BSA supported the binding of purified integrin αIIbβ3 in a dose dependent manner, suggesting that the interaction of PP2Ac with αIIbβ3 was direct. Furthermore, purified PP2Ac as well as PP2Ac in lysates obtained from the resting platelets bound specifically to a biotinylated αIIb cytoplasmic peptide encompassing residues 985–995 but not to a scrambled peptide, suggesting that the integrin αIIb is sufficient to mediate the interaction of PP2Ac in vitro. The association of PP2Ac with the platelet integrin αIIbβ3 was not altered during platelet adhesion to fibrinogen (αIIbβ3 outside-in signaling) or during thrombin or ADP stimulation (inside-out signaling to αIIbβ3). In contrast, we have previously shown that integrin-bound PP1 dissociated from the αIIbβ3 complex upon platelet adhesion and thrombin-induced platelet activation. The association of PP2Ac with the integrin αIIbβ3 correlates well with the dephosphorylation of a PP2A substrate, extracellular-signal regulated kinase 2 (ERK2) during thrombin-induced platelet aggregation that we and others have previously demonstrated. More importantly, ERK2 dephosphorylation was not observed in platelets from Glanzmann thrombasthenic patients or in normal platelets pretreated with RGDS or integrilin, suggesting a critical role for integrin αIIbβ3 in the dephosphorylation of ERK2. To ascertain a physiological relevance for the PP2A-αIIbβ3 association, we used short interfering RNAs (siRNAs) to knock down the expression of PP2Ac in the 293/αIIbβ3 cells. Knock down was maximal (~55–70%) and specific to PP2Ac because PP1 and actin protein levels were not different between the control and PP2Ac siRNA treated cells. Consistent with the reduction in the PP2Ac protein level in the PP2Ac knock down cells there was ~70% reduction in the PP2Ac phosphatase activity, and a concomitant increased basal ERK2 phosphorylation. PP2Ac knock down significantly (P≤0.006) increased the adhesion of 293/αIIbβ3 cells to fibrinogen. The adhesion was αIIbβ3 specific because it could be abolished with an αIIbβ3 function blocking antibody (10E5). These findings supports a mechanism whereby the integrin associated Ser/Thr phosphatases might regulate αIIbβ3 adhesive functions via dephosphorylation of key cytoskeletal proteins.

Fibrinogen and von Willebrand Factor-Independent Platelet Aggregation: The Essential Roles of β3 Integrin, Thrombin, and Divalent Ca2+ Cations.

It has been documented that fibrinogen (Fg) is required for platelet aggregation. However, we recently found that platelet rich thrombi still formed in mice lacking either Fg (Fg−/−) or both Fg and vWF (Fg/vWF−/−) (Ni et al, JCI 106:385–392, 2000). To explore the potential mechanisms of Fg/vWF-independent platelet aggregation, we studied platelet aggregation in vitro in platelet rich plasma (PRP). We found no platelet aggregation in Fg−/− or Fg/vWF−/− PRP induced by adenosine diphosphate (ADP) in the presence of anticoagulant reagents including divalent cation chelators (ACD, sodium citrate, and EDTA), and thrombin inhibitors (heparin, hirudin, PPACK). Since no fibrin formation occurs in Fg/vWF−/− plasma, we then induced Fg/vWF−/− platelet aggregation in non-anticoagulated platelet poor plasma (PPP). Surprisingly, robust aggregation occurred after ADP treatment. We further demonstrated that directly triggering the thrombin receptor PAR4 with thrombin receptor activation peptide (TRAP, AYPGKF-NH2) induced platelet aggregation in thrombin-inhibitor treated Fg/vWF−/− PRP and gel-filtered Fg/vWF-platelets in 1mM Ca2+ PIPES buffer. Thus Fg/vWF-independent aggregation can be induced in vitro and both divalent cations and thrombin are required for this novel platelet aggregation pathway. It is likely that ADP induced thrombin generation in Fg/vWF-/- PRP. Considering platelet aggregation has been observed in type I Glanzmann Thrombasthenic patients lacking αIIbβ3 protein, we hypothesized that Fg/vWF−/− platelet aggregation may occur in either a β3 integrin-dependent or β3 integrin independent manners. To test this hypothesis, β3 integrin deficient (β3−/−) platelets were added into the same non-anticoagulated Fg/vWF−/− plasma and no aggregation was observed after ADP treatment. β3−/− platelets also did not co-aggregate with Fg/vWF−/− platelets in vitro in aggregation assays, which were analyzed by flow cytometry with an anti-CD61 (anti-β3 integrin) antibody. Furthermore, when fluorescently labeled β3−/− platelets were injected into Fg/vWF−/− mice, β3−/− platelets did not significantly incorporate into Fg/vWF−/− thrombi under confocal intravital microscopy. The inability of β3−/− platelets to aggregate is not due to a deficiency in other adhesive receptors, since no reduction of GPIbα, β1 integrins, and P-selectin was observed on β3−/− platelets. Thus, although the GPIb complex, P-selectin, and β1 integrins may be involved in platelet aggregation, β3 integrin is essential for this Fg/vWF-independent platelet aggregation pathway. This also indicates that other alternative ligands of β3 integrin from either the plasma or platelet granules are capable of mediating platelet aggregation independent of both Fg and vWF under more physiologically relevant conditions (i.e. non anti-coagulated blood) where divalent cations and thrombin are present.

Critical Residues of Integrin αIIb Subunit for Binding of αIIbβ3 (Glycoprotein IIb-IIIa) to Fibrinogen and Ligand-mimetic Antibodies (PAC-1, OP-G2, and LJ-CP3)

Integrin alphaIIbbeta3 plays a critical role in platelet aggregation through its interaction with fibrinogen. Elucidation of the mechanisms of alphaIIbbeta3-fibrinogen interaction is critical to understanding hemostasis and thrombosis. Here we report that mutations of Gly-184, Tyr-189, Tyr-190, Phe-191, and Gly-193 within the predicted turn structure of the third amino-terminal repeat of alphaIIb significantly block binding of alphaIIbbeta3 to soluble fibrinogen. These mutations also block binding of alphaIIbbeta3 to ligand-mimetic monoclonal antibodies PAC-1, OP-G2, LJ-CP3, which have an RGD-related RYD sequence in their antigen-binding sites. These mutations do not significantly affect the expression of alphaIIbbeta3, in contrast to most of the natural alphaIIb mutations occurring in Glanzmann's thrombasthenic patients. The data suggest that these residues are critically involved in alphaIIbbeta3-ligand interactions.

A single amino acid substitution flanking the fourth calcium binding domain of alpha IIb prevents maturation of the alpha IIb beta 3 integrin complex.

To define specific structural domains involved in the biosynthesis and processing of integrin subunits, we examined the biosynthesis of normal and mutated forms of the platelet-specific integrin alpha IIb beta 3. Platelet mRNA was isolated from a Glanzmann thrombasthenic patient who failed to express significant levels of the glycoprotein (GP) IIb-IIIa complex on the platelet surface. Sequence analysis of polymerase chain reaction-amplified platelet GPIIb mRNA revealed a Gly418-->Asp amino acid substitution in GPIIb. Gly418 is a highly conserved residue that flanks the fourth calcium binding domain of GPIIb. Cotransfection of Asp418 GPIIb and GPIIIa plasmid constructs into COS-7 cells resulted in the accumulation of a pre-GPIIb-IIIa complex that failed to reach the cell surface, in effect recreating the thrombasthenic phenotype. Pulse-chase and endoglycosidase studies demonstrated that the biosynthetic blockade occurred in a pre-Golgi compartment. Removal of the negatively charged carboxyl group of Asp418 GPIIb, creating Ala418 GPIIb, rescued intracellular transport and surface expression of the integrin complex. Mutagenesis of a homologous Gly within the integrin alpha subunit alpha v also resulted in the failure to express alpha v beta 3 on the cell surface. These results suggest that the presence of a small, uncharged amino acid 6-8 residues amino-terminal to the calcium coordination complex is crucial for the proper folding and maturation of integrin complexes.

A human monoclonal autoantibody to platelet glycoprotein IIb derived from normal human lymphocytes

Tonsillar lymphocytes from an otherwise healthy nonthrombocytopenic male child were fused with the lymphoblastoid cell line GM 4672. Twenty of 472 (4%) hybridomas had antiplatelet reactivity detected using intact platelets in an enzyme-linked immunosorbent assay. One hybridoma (STO 171) reacted to platelet glycoprotein IIb (integrin alpha IIb) as determined by radioimmunoprecipitation and immunoblotting. Antibody specificity was confirmed using immunodepletion experiments with isotypic antibodies derived from a mutlitransfused Glanzmann's thrombasthenic patient. The antibody reactivity was restricted to platelets and did not react with other integrin alpha-chain proteins expressed on granulocytes or cultured human brain-derived microvascular endothelial cells. These studies indicate that lymphocytes of normal, nonthrombocytopenic individuals have the genetic potential to produce antiplatelet autoantibodies.

A Human Monoclonal Autoantibody to Platelet Glycoprotein lib Derived From Normal Human Lymphocytes

Tonsillar lymphocytes from an otherwise healthy nonthrombocytopenic male child were fused with the lymphoblastoid cell line GM 4672. Twenty of 472 (4%) hybridomas had antiplatelet reactivity detected using intact platelets in an enzyme-linked immunosorbent assay. One hybridoma (STO 171) reacted to platelet glycoprotein IIb (integrin alpha IIb) as determined by radioimmunoprecipitation and immunoblotting. Antibody specificity was confirmed using immunodepletion experiments with isotypic antibodies derived from a mutlitransfused Glanzmann's thrombasthenic patient. The antibody reactivity was restricted to platelets and did not react with other integrin alpha-chain proteins expressed on granulocytes or cultured human brain-derived microvascular endothelial cells. These studies indicate that lymphocytes of normal, nonthrombocytopenic individuals have the genetic potential to produce antiplatelet autoantibodies.

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 [see comments

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.

Platelet Fibrinogen and Vitronectin in Glanzmann Thrombasthenia: Evidence Consistent With Specific Roles for Glycoprotein IIb/IIIa and αvβ3 Integrins in Platelet Protein Trafficking

AbstractTo assess the individual contributions of the platelet glycoprotein (GP) IIb/IIIa receptor and the αvβ3 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 αvβ3 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 αvβ3 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 αvβ3 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 the interaction of cells with extracellular proteins, integrin receptors may be important in protein trafficking into, and perhaps out of, platelets.

Aggregation to Thrombin and Collagen of Platelets from a Glanzmann Thrombasthenic Patient Lacking Glycoproteins IIb and IIIa

The aim of this study was to investigate the platelets of a Glanzmann thrombasthenic patient, which in citrated PRP failed to respond to various agonists, but aggregated and secreted to high concentrations of thrombin (0.36, 0.72 and 1 U/ml) and collagen (4, 10 and 20 micrograms/ml) when washed and resuspended in a Tyrode-albumin solution (containing 2 mM Ca2+). Aggregation of the patient platelets was not affected by anti-IIb/IIIa monoclonal antibody (P18) which strongly inhibits thrombin or collagen induced aggregation of normal platelets. Washed platelets of this patient did not aggregate to ADP (10-100 microM) in the presence of added fibrinogen (2 mg/ml) nor bind 125I-labelled fibrinogen (40 to 320 micrograms/ml) when thrombin-stimulated. Different anti-IIb/IIIa monoclonal antibodies (P2, P18) when used in binding or crossed immunoelectrophoretic studies showed a complete absence of the IIb-IIIa glycoprotein complex on the patient platelets. Moreover, glycoproteins IIb or IIIa were absent on silver-stained two-dimensional (non-reduced/reduced) polyacrylamide gel separations of the patient platelets and were not detected by Western blots used in combination with anti-PLA1 (antigen present on IIIa), anti-Leka (antigen present on IIb). This study shows that platelets lacking glycoproteins IIb or IIIa can aggregate in response to high concentrations of collagen or thrombin when resuspended in the presence of physiological concentrations of calcium. Results obtained in this study could indicate the existence of other mechanisms (other than the IIb-IIIa glycoprotein complex) involving glycolipids, heparans, proteoglycans, and/or unknown membrane glycoproteins to mediate platelet aggregation of stimulated thrombasthenic platelets.

種々の抗血小板膜抗体の血小板機能におよぼす影響

Antibodies I and II (heterologous anti-human platelet membrane antisera) were produced in rabbit against human platelet membrane dissolved in 10mM Tris/HCl buffer, pH 7.4, containing 1% Triton X-100, 0.25M sucrose, 2mM CaCl2, and in 38mM Tris/HCl buffer, pH 8.6, containing 1% Triton X-100, 0.1M glycine, 5mM EDTA, respectively. Antibody III (mouse monoclonal antibody) was raised against crude human platelet membrane of normal platelets. By crossed immunoelectrophoresis (CIE), it was shown that antibody I could bind to a single protein. Since the immunoprecipitate on CIE was not detectable by using platelets from a Glanzmann's thrombasthenic patient, antibody I was considered to be specific antibody against GP IIb/IIIa complex. Avidin biotin complex system (ABC)-immunoblot showed the strong binding of antibody II to electrophoretically separated GP IIb and GP IIIa. ABC-immunoblot revealed no binding of antibody III to platelet proteins after SDS-PAGE, but two immunoprecipitates were obtained with an apparent molecular weight of 130K and 110K under reduced conditions by double antibody immunoprecipitate, thus indicating that antibody III was a monoclonal antibody against GP IIb/IIIa complex. Fluorescence flow cytometric analysis showed that the activity of antibody I to sensitize human platelets was less than that of antibody II. In contrast, antibody I was more potent in inhibiting ADP-and collagen-induced platelet aggregation than antibody II. Antibody III inhibited ADP-and collagen-induced aggregation in a dose dependent manner. The addition of 6.3μg/ml of antibody III to platelets sensitized 68% of platelets, and caused complete inhibition of ADP-induced aggregation, while its addition of 3.1μg/ml sensitized 50% of platelets, and caused complete inhibition of collagen-induced aggregation. The results give further support to the proposal that platelet GP IIb/IIIa complex, not dissociated GP IIb and GP IIIa, plays an important role in platelet aggregation.

Identification of a Glycoprotein III a Dimer in Polyacrylamide Gel Separations of Human Platelet Membranes

Membrane glycoproteins IIb and IIIa play a major role in human blood platelet aggregation. The absence or the severe reduction of these two membrane glycoproteins, as observed in platelets of Glanzmann's thrombasthenic patients, is related to a lack of platelet aggregation. Separation of Glanzmann's thrombasthenic platelet samples by two-dimensional polyacrylamide O'Farrell gels show the absence of a high and several low molecular mass glycoproteins, in addition to the loss of glycoproteins IIb and IIIa (McGregor J. L. et al. Eur. J. Biochem. 1981; 116: 379-388). The aim of this study was to identify the nature of the high molecular mass component, absent in thrombasthenic platelets. A high molecular mass glycoprotein (200 kDa), present in two-dimensional SDS-polyacrylamide O-Farrell gel separations, was recognized by a monoclonal antibody (MP37) directed against glycoprotein IIIa. Moreover, the tryptic peptide map of this high molecular mass glycoprotein was nearly identical to that of glycoprotein IIIa. These results indicate that this high molecular mass glycoprotein present in SDS-polyacrylamide gels is a dimer of glycoprotein IIIa. This work raises the possibility that the high molecular mass glycoprotein, absent in two-dimensional O'Farrell gel separations of thrombasthenic platelets, is a dimer of glycoprotein IIIa.

Membrane proteins on human megakaryocytes and platelets identified by monoclonal antibodies.

We describe five monoclonal antibodies that react with four discrete antigens present on human platelets. Antibodies B2.12 and B59.2 precipitate the glycoprotein IIb-IIIa complex from radiolabeled platelet membrane extracts and inhibit platelet aggregation induced by adenosine diphosphate (ADP), collagen, or epinephrine. The antigen recognized by the two antibodies is present on megakaryocytes but either absent entirely or expressed in small amounts on platelets from Glanzmann's thrombasthenic patients. The antigen recognized by antibody B37.3 is absent from thrombasthenic platelets. Antibody B1.12 reacts with an antigen shared by platelets and 20% of peripheral blood lymphocytes and is a potent inducer of platelet aggregation. Antibody B2.10 reacts specifically with platelets and megakaryocytes but does not affect platelet functions. Thus, these reagents are useful tools in diagnostic and functional studies of both normal and abnormal platelets.