Shigenori Honda

Adiponectin Acts as an Endogenous Antithrombotic Factor

Objective—Obesity is a common risk factor in insulin resistance and cardiovascular diseases. Although hypoadiponectinemia is associated with obesity-related metabolic and vascular diseases, the role of adiponectin in thrombosis remains elusive. Methods and Results—We investigated platelet thrombus formation in adiponectin knockout (APN-KO) male mice (8 to 12 weeks old) fed on a normal diet. There was no significant difference in platelet counts or coagulation parameters between wild-type (WT) and APN-KO mice. However, APN-KO mice showed an accelerated thrombus formation on carotid arterial injury with a He-Ne laser (total thrombus volume: 13.36±4.25×107 arbitrary units for APN-KO and 6.74±2.87×107 arbitrary units for WT; n=10; P<0.01). Adenovirus-mediated supplementation of adiponectin attenuated the enhanced thrombus formation. In vitro thrombus formation on a type I collagen at a shear rate of 250 s−1, as well as platelet aggregation induced by low concentrations of agonists, was enhanced in APN-KO mice, and recombinant adiponectin inhibited the enhanced platelet aggregation. In WT mice, adenovirus-mediated overexpression of adiponectin additionally attenuated thrombus formation. Conclusion—Adiponectin deficiency leads to enhanced thrombus formation and platelet aggregation. The present study reveals a new role of adiponectin as an endogenous antithrombotic factor.

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.

Molecular basis of CD36 deficiency. Evidence that a 478C-->T substitution (proline90-->serine) in CD36 cDNA accounts for CD36 deficiency.

CD36 deficiency is divided into two subgroups: neither platelets nor monocytes express CD36 (type I deficiency), and monocytes express CD36 in spite of the lack of platelet CD36 (type II deficiency). We have already demonstrated that a 478C-->T substitution (proline90-->serine) in platelet CD36 cDNA predominates in type II deficiency (Kashiwagi, H., S. Honda, Y. Tomiyama, H. Mizutani, H. Take, Y. Honda, S. Kosugi, Y. Kanayama, Y. Kurata, and Y. Matsuzawa. 1993. Thromb. Haemostasis. 69:481-484). In this study, we revealed that monocyte CD36 cDNA from two type II deficient subjects was heterozygous for C478 and T478 form, while platelet CD36 cDNA of these subjects consisted of only T478 form. In a type I deficient subject, both platelet and monocyte CD36 cDNA showed only T478 form. Expression assay using C478 or T478 form of CD36 cDNA transfected cells revealed that there was an 81-kD precursor form of CD36, and that the maturation of the 81-kD precursor form to the 88-kD mature form of CD36 was markedly impaired by the substitution. The mutated precursor form of CD36 was subsequently degraded in the cytoplasm. These results indicate that the 478C-->T substitution directly leads to CD36 deficiency via defects in posttranslational modification, and that this substitution is the major defects underlying CD36 deficiency.

Prevalence of genetic mutations in protein S, protein C and antithrombin genes in Japanese patients with deep vein thrombosis

INTRODUCTION Genetic deficiencies of PROS1, PROC, and SERPINC1 (antithrombin) are risk factors for deep vein thrombosis (DVT). Diagnosis of the inherited deficiencies of these three genes is sometimes difficult because of the phenotypic variability. This study was undertaken to reveal the frequency of nonsynonymous mutations of these three genes in Japanese DVT patients. PATIENTS/METHODS One hundred seventy-three DVT patients were registered by the Sub-group of Blood Coagulation Abnormality, from the Study Group of Research on Measures for Intractable Diseases. We sequenced the entire coding regions of the three genes in all DNA samples and identified the nonsynonymous mutations. RESULTS AND CONCLUSIONS For PROS1 we identified 15 nonsynonymous mutations in 28 DVT patients; for PROC, 10 nonsynonymous mutations in 17 patients; and for SERPINC1, 13 nonsynonymous mutations in 14 patients. Five patients had two mutations in PROS1 and PROC, and all of them had PROS1 K196E mutation. We previously identified one patient with a large PROS1 gene deletion. Thus, 55 out of 173 patients (32%) carried at least one genetic defect in the three genes. The PROS1 K196E mutation found in 15 Japanese DVT patients was the most prevalent. Mutations of PROC K193del and V339M were the second, each found in four patients. Our data suggested that the PROC K193del mutation caused the loss of the anticoagulant activity but not the amidolytic activity. Our effort is the first DNA resequencing study to identify the genetic variations in DVT patients without any consideration of their plasma activities and antigens. To minimize selection bias in a future evaluation of the contribution of genetic deficiency to DVT, we must recruit patients consecutively.

Impaired platelet function in a patient with P2Y12 deficiency caused by a mutation in the translation initiation codon.

Summary.  In this study, we have identified a patient (OSP‐1) with a congenital P2Y12 deficiency showing a mild bleeding tendency from her childhood and examined the role of P2Y12 in platelet function. At low concentrations of agonists OSP‐1 platelets showed an impaired aggregation to several kinds of stimuli, whereas at high concentrations they showed a specifically impaired platelet aggregation to adenosine diphosphate (ADP). ADP normally induced platelet shape change and failed to inhibit PGE1‐stimulated cAMP accumulation in OSP‐1 platelets. Molecular genetic analysis revealed that OSP‐1 was a homozygous for a mutation in the translation initiation codon (ATG to AGG) in the P2Y12 gene. Heterologous cell expression of wild‐type or mutant P2Y12 confirmed that the mutation was responsible for the deficiency in P2Y12. OSP‐1 platelets showed a markedly impaired platelet spreading onto immobilized fibrinogen. Real‐time observations of thrombogenesis under a high shear rate (2000 s−1) revealed that thrombi over collagen were small and loosely packed and most of the aggregates were unable to resist against high shear stress in OSP‐1. Our data suggest that secretion of endogenous ADP and subsequent P2Y12‐mediated signaling are critical for platelet aggregation, platelet spreading, and as a consequence, for stabilization of thrombus.

Analysis of genetic and predisposing factors in Japanese patients with atypical hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS) is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. Approximately 10% of cases are classified as atypical due to the absence of Shiga toxin-producing bacteria as a trigger. Uncontrolled activation of the complement system plays a role in the pathogenesis of atypical HUS (aHUS). Although many genetic studies on aHUS have been published in recent years, only limited data has been gathered in Asian countries. We analyzed the genetic variants of 6 candidate genes and the gene deletion in complement factor H (CFH) and CFH-related genes, examined the prevalence of CFH autoantibodies and evaluated the genotype-phenotype relationship in 10 Japanese patients with aHUS. We identified 7 causative or potentially causative mutations in CFH (p.R1215Q), C3 (p.R425C, p.S562L, and p.I1157T), membrane cofactor protein (p.Y189D and p.A359V) and thrombomodulin (p.T500M) in 8 out of 10 patients. All 7 of the mutations were heterozygous and four of them were novel. Two patients carried CFH p.R1215Q and 3 other patients carried C3 p.I1157T. One patient had 2 causative mutations in different genes. One patient was a compound heterozygote of the 2 MCP mutations. The patients carrying mutations in CFH or C3 had a high frequency of relapse and a worse prognosis. One patient had CFH autoantibodies. The present study identified the cause of aHUS in 9 out of 10 Japanese patients. Since the phenotype-genotype correlation of aHUS has clinical significance in predicting renal recovery and transplant outcome, a comprehensively accurate assessment of molecular variation would be necessary for the proper management of aHUS patients in Japan.

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.

Integrin-linked kinase associated with integrin activation

Platelet integrin alphaIIbbeta3 activation is tightly controlled by intracellular signaling pathways, and several molecules, including talin, have been identified as critical for alphaIIbbeta3 activation. However, the whole pathway associated with alphaIIbbeta3 activation remains to be determined. To address this issue, we established a Chinese hamster ovary cell line (parental cells) that expresses constitutively activated chimeric integrin alphaIIbalpha6Bbeta3, and then obtained mutant cells expressing inactivated alphaIIbalpha6Bbeta3 by genome-wide mutagenesis. We have performed expression cloning to isolate signaling molecules responsible for integrin activation in the mutant cells. We show that integrin-linked kinase (ILK) complements defective integrin activation in the mutant cells. ILK mRNAs in the mutant cells contained 2 nonsense mutations, R317X and W383X, in a compound heterozygous state, resulting in a complete loss of ILK expression. Moreover, the mutant cells showed partially impaired activation of endogenous beta1 integrins. Knockdown of ILK in parental cells significantly suppressed the activated state of alphaIIbalpha6Bbeta3. However, ILK overexpression did not rescue the impaired integrin activation in talin knocked-down parental cells, whereas overexpression of talin-F3, a subdomain of the talin head domain, restored the function. Our present data suggest that ILK contributes to inside-out integrin activation.

Demonstration of the heterogeneity of epitopes of the platelet-specific alloantigen, Baka

It is well known that the platelet‐specific alloantigen, Baka is carried on glycoprotein (GP) IIb, but little is known about the biochemical characteristics of its epitopes. To clarify the characteristics of the epitopes, we examined the interaction of four anti‐Baka sera (Yam, Lin, Kl and MO) with their epitopes, either with or without modifications by sodium dodecyl sulphate (SDS) and/or neuraminidase. By immunoprecipitation, all four antisera bound to the intact GP IIb/IIIa complex from a Baka‐positive subject. In contrast, immunoblotting demonstrated that Yam, Lin and Kl bound to SDS‐denatured GP IIb, while MO did not. When blotted GP IIb was treated with neuraminidase, Yam and Lin did not bind to desialylated GP IIb, while Kl still did. When the purified GP IIb/IIIa complex or washed platelets were treated first with neuraminidase followed by immunoblotting, the molecular weight of GP IIb decreased from 145 kD to 138 kD; Yam did not bind to desialylated GP IIb, but Kl did. Furthermore, to eliminate the effect of SDS, we examined the interaction of Yam and Lin with neuraminidase‐treated platelets using flow cytometry. The results were the same as those obtained using immunoblotting. Our results thus demonstrate that the expression of the Baka epitopes is not uniform and that sialic acid contributes to the expression of some actual allogenic epitopes.