Satoru Kosugi

Circulating thrombopoietin level in chronic immune thrombocytopenic purpura

The circulating thrombopoietin (TPO) level in 43 patients with chronic immune thrombocytopenic purpura (ITP) was examined by an ELISA system. The TPO level (mean±SD) in ITP patients was mildly elevated (1.86±1.17 fmol/ml) compared to that in normal subjects (0.76±0.21), and was within the normal range in 30% of ITP patients. In contrast, the TPO level in patients with aplastic anaemia was very high, 12.35±6.42 fmol/ml. There was no correlation between TPO level and platelet count in ITP patients. Splenectomy was performed in two ITP patients, after which platelet counts increased to normal levels and TPO levels showed a transient increase. These data suggest that reactive TPO production against thrombocytopenia in ITP is small when compared to that in aplastic anaemia. Relative endogenous TPO deficiency may play some role in the pathophysiology of thrombocytopenia in ITP patients.

Diagnostic Value of Tests for Reticulated Platelets, Plasma Glycocalicin, and Thrombopoietin Levels for Discriminating Between Hyperdestructive and Hypoplastic Thrombocytopenia

We measured reticulated platelets (RPs) and plasma glycocalicin (GC) and thrombopoietin (TPO) levels simultaneously in 107 thrombocytopenic patients to clarify the diagnostic value of these tests for discriminating hyperdestructive from hypoplastic thrombocytopenia. The percentage of RPs and GC index (plasma GC level normalized for the individual platelet count) were markedly elevated in patients with idiopathic thrombocytopenic purpura (ITP) but normal or slightly elevated in patients with aplastic anemia (AA) or chemotherapy-induced thrombocytopenia (ChemoT). For RP percentage for diagnosing hyperdestructive thrombocytopenia the sensitivity and specificity were excellent but were lower for the GC index. Absolute RP counts and plasma GC levels were markedly decreased and plasma TPO levels markedly elevated in patients with AA or ChemoT, but absolute RP counts and plasma GC levels were moderately decreased and plasma TPO levels only slightly elevated in patients with ITP. The sensitivity and specificity of plasma TPO levels for diagnosing hypoplastic thrombocytopenia were excellent. Using the RP percentage and plasma TPO levels in combination improved specificities. Simultaneous measurement of RP percentage and plasma TPO level may help discriminate thrombocytopenia of unknown cause in routine hematologic practice.

A Single Nucleotide Insertion in Codon 317 of the CD36 Gene Leads to CD36 Deficiency

CD36 is a multifunctional integral-membrane glycoprotein that acts as a receptor for thrombospondin, collagen, long-chain fatty acids, and oxidized LDL. Platelet CD36 deficiency can be divided into two groups. In type I, neither platelets nor monocytes/macrophages express CD36; in type II, monocytes/macrophages express CD36 but platelets do not. Two known mutations cause CD36 deficiency, ie, a 478C-->T substitution in codon 90 (proline90-->serine) and a dinucleotide deletion at nucleotide 539 in codon 110. In this study we investigated a type I Japanese subject (A.T.) and identified a new mutation, a single nucleotide insertion at nucleotide 1159 in codon 317. This mutation leads to a frameshift and the appearance of a premature stop codon. CD36 gene analysis indicated that A.T. was a compound heterozygote for a dinucleotide deletion at nucleotide 539 and the single nucleotide insertion at nucleotide 1159. RNase protection studies suggested that the new mutation as well as the dinucleotide deletion led to a marked reduction in the level of CD36 mRNA in her macrophages. However, the new mutation could be detected in macrophage but not platelet CD36 mRNA. These data suggest that the allele having the single nucleotide insertion in this subject has an additional abnormality that results in the absence of the mutated CD36 mRNA in platelets.

Elevated platelet-associated IgG in SLE patients due to anti-platelet autoantibody: differentiation between autoantibodies and immune complexes by ether elution

Summary The level of platelet‐associated IgG (PAIgG) is reported to be elevated in patients with systemic lupus erythematosus (SLE). However. the nature of PAIgG is unclear. We have investigated whether the PAIgG of SLE consists of anti‐platelet autoantibodies or immune complexes (IC). The PAIgG values measured by flow cytonietry were elevated in 11/25 patients with SLE. 3/6 SLE patients with thrombocytopenia had a high level of PAIgG (the mean fluorescence intensity >10). We used an ether elution technique to determine whether elevated PAIgG consists of anti‐platelet antibodies or IC. Preliminary experiments showed that the eluates prepared from platelets sensitized with anti‐HPA‐4a antibody reacted with normal platelets. while the eluates prepared from platelets sensitized with heat‐aggregated IgG or model IC failed to react with normal platelets. These results indicate that the reactivity of eluates can distinguish between platelet‐bound antibody and IC. We applied this technique to analysis of the PAIgG of SLE platelets. The eluates from SLE platelets (the mean fluorescence intensity > 10) reacted with normal platelets. indicating that the PAIgG of SLE platelets has the nature of anti‐platelet autoantibodies. Furthermore, we investigated the target antigens which bind PAIgGs of SLE, using the direct immunoprecipitation procedure and modified antigen capture ELISA (MACE). Both methods identified GPIIb/IIIa as the target antigens. We conclude that the ether elution technique can distinguish between anti‐platelet antibodies and TC. and that the PAIgGs of SLE with a high PAIgG value and thrombocytopenia have the nature of anti‐platelet autoantibodies.

Autoantigenic epitopes on platelet glycoproteins

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by early platelet destruction mediated by antiplatelet autoantibodies. Platelet membrane glycoproteins (GP), especially GPIIb-IIIa and GPIb-IX, contain major autoantigenic determinants in chronic ITP. Recent advances in the localization of autoantigens as well as in the detection of GP-specific antibodies have improved our understanding of the pathophysiology of the disease. The N-terminal globular head of GPIIb-IIIa, particularly the β-propeller domain in GPIIb, seems to play an important role as a hot spot for autoantigenic epitopes in chronic ITP.

Cyclic thrombocytopenia associated with IgM anti-GPIIb-IIIa autoantibodies

Summary. We studied a female patient with cyclic fluctuation in platelet count following splenectomy for autoimmune thrombocytopenia. The cyclical fluctuation appeared to be in phase with her menstrual cycle and her platelet count was low during menses. Bone marrow examinations performed at the peak as well as the bottom of the platelet count showed normal or increased numbers of megakaryocytes. The patients platelet count increased rapidly after intravenous gamma‐globulin (IVIgG) therapy, suggesting that a failure of platelet production is unlikely to account for the cycle. Platelet‐associated IgM (PAIgM) was markedly elevated, whereas PAIgG was normal at any stage of the cycle. MACE assay demonstrated that PAIgM contained IgM anti‐glycoprotein (GP) IIb‐IIIa autoantibodies. Comparison between MACE assay using untreated and EDTA‐treated platelets at 3 7°C demonstrated that the platelet‐associated IgM autoantibodies mainly recognized divalent cation‐dependent conformation(s) of GPUb‐IIIa. No antibodies were, however, detected in her serum. The levels of IgM anti‐GPIIb‐IIIa showed an inverse relationship with the platelet count. In spite of the marked increase in platelet count after IVIgG, however, the levels of IgM anti‐GPIIb‐IIIa remained elevated. These findings suggest that plateletassociated IgM anti‐GPIIb‐IIIa autoantibodies are of pathogenic significance in this patient.

Rituximab provided long-term remission in a patient with refractory relapsing thrombotic thrombocytopenic purpura.

We describe a 69-year-old man with refractory relapsing thrombotic thrombocytopenic purpura (TTP) successfully treated with rituximab. The patient had once been successfully treated with plasmapheresis and vincristine, but he had relapsed after a short period. Although plasmapheresis, vincristine, and splenectomy could not achieve a consistent elevation of the platelet count, rituximab administration provided sustained remission for more than 7 months. Rituximab should be considered as a therapeutic alternative for refractory TTP.

Activation of integrin αIIbβ3 in the glycoprotein Ib-high population of a megakaryocytic cell line, CMK, by inside-out signaling

Summary.  Affinity/avidity state of integrin αIIbβ3 is regulated by intracellular inside‐out signaling. Although several megakaryocytic cell lines have been established, soluble ligand binding to αIIbβ3 expressed in these cells by cellular agonists has not been demonstrated. We have re‐examined agonist‐induced αIIbβ3 activation on megakaryocytic cell lines with a marker of the late stage of megakaryocytic differentiation, glycoprotein Ib (GPIb). Activation of αIIbβ3 was assessed by PAC1 and soluble fibrinogen binding to the cells. We found that αIIbβ3 expressed in CMK cells with high GPIb expression was activated by a phorbor ester, phorbol myristate acetate (PMA). Although the population of the GPIbhigh cells was <0.5% of the total cells, incubation with a nucleoside analog, ribavirin, efficiently increased the PMA‐reactive GPIbhigh cells. Not only PMA but also a calcium ionophore, A23187, induced αIIbβ3 activation, and PMA and A23187 had an additive effect on αIIbβ3 activation. Ligand binding to the activated αIIbβ3 in the GPIbhigh CMK cells is totally abolished by an αIIbβ3‐specific antagonist, and inhibited by wortmannin, cytochalasin‐D and prostaglandin E1, and the effects of these inhibitors on αIIbβ3 activation in the GPIbhigh CMK cells were compatible with those in platelets. We have also demonstrated that the ribavirin‐treated CMK cells express PKC‐α, ‐β, ‐δ and ‐θ, and suggested that PKC‐α and/or ‐β appear to be responsible for PMA‐induced activation of αIIbβ3 in CMK cells.

Localized relapse in bone marrow in a posttransplantation patient with t(6;9) acute myeloid leukemia

We report a 38-year-old woman with t(6;9) acute myeloid leukemia who relapsed with localized leukemic cell growth in the bone marrow after she had undergone allogeneic bone marrow transplantation. The localized cell growth was first recognized by an apparent discrepancy in the DEK-CAN fusion transcript levels between the aspirates from the left and right iliac bone marrow. Magnetic resonance imaging of the iliac bone revealed localized cell accumulation in the left side. The nonhomogeneous and localized leukemic cell growth in this case may have been due to the graft-versus-leukemia effect following allogeneic transplantation with donor lymphocyte infusion. Serial monitoring of molecular markers for leukemia at different sites or magnetic resonance imaging of the bone marrow may be of value in detecting this type of relapse.

Demonstration of a marked reduction in the amount of GPIIb in most type II patients with Glanzmann's thrombasthenia

Summary. In this study employing a sensitive immunoblot assay, we have characterized GPIIb and GPIIIa in thrombasthenic platelets from seven type II and four type I patients from 10 unrelated families. The amounts of GPIIb and GPIIIa were both markedly reduced in all these patients, and abnormal molecular weight GPIIb or GPIIIa was not detected. In all of four type I patients the amount of GPIIb was much lower than that of GPIIIa. In this study, however, we found that the amount of GPIIb was also lower even in six out of seven type II patients. Immunodepletion of patients’platelets with AP2 (a monoclonal antibody specific for the GPIIb‐IIIa complex), AP3 (specific for GPIIIa) or AMF7 (specific for αv) further confirmed that GPIIIa existed in excess, and demonstrated that excess GPIIIa were mostly in free form and not associated with GPIIb or αv. The reduction of GPIIb may represent an abnormality in GPIIb processing in these type II and type I thrombasthenic platelets. It remains unclear whether these two subgroups represent distinct categories.