Masamichi Shiraga

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.

Endogenous adenosine inhibits P-selectin-dependent formation of coronary thromboemboli during hypoperfusion in dogs.

The activation of platelets and the formation of neutrophil- platelet conjugates may lead to the development of thromboemboli. We studied whether blockade of adenosine receptors during coronary hypoperfusion may cause thromboemboli via P-selectin-dependent mechanisms in 30 open-chest dogs. When coronary blood flow was reduced to 20% of the control, it was stable at low levels with increases in adenosine levels. When 8-p-sulfophenyltheophylline, an adenosine receptor antagonist, was infused during coronary hypoperfusion, coronary blood flow decreased gradually and approached almost zero 20 min after its administration. Histological examination revealed thromboemboli in the small coronary vessels. During hypoperfusion in the presence of 8-p-sulfophenyltheophylline, the mAb against P-selectin attenuated both the reduction in coronary blood flow and the formation of thromboemboli, and improved contractile and metabolic dysfunction of the myocardium. Flow cytometric analysis indicated that the expression of P-selectin on platelet and neutrophil-platelet adhesion were increased during coronary hypoperfusion, and that both were further augmented by 8-p-sulfophenyltheophylline. Immunohistochemical examination showed no staining of P-selectin in the ischemic myocardium. Adenosine inhibited the thrombin-induced expression of P-selectin on platelet and neutrophil- platelet adhesion via adenosine A2 receptors. Adenosine appears to inhibit the formation of thromboemboli during coronary hypoperfusion by suppressing the expression of P-selectin on platelets and neutrophil-platelet adhesion.

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.

The tetraspanin CD9 modulates epidermal growth factor receptor signaling in cancer cells

CD9 is a member of the tetraspanins, and has been shown to be involved in a variety of cellular activities such as migration, proliferation, and adhesion. In addition, it has been known that CD9 can associate with other proteins. Here we demonstrated the physical and functional association of CD9 with epidermal growth factor receptor (EGFR) on MKN‐28 cells. Double‐immunofluorescent staining and immunoprecipitation demonstrated the complex formation of CD9‐EGFR and CD9‐β1 integrin, and that both complexes are colocalized on the cell surface especially at the cell–cell contact site. Anti‐CD9 monoclonal antibody ALB6 induced a dotted or patch‐like aggregation pattern of both CD9‐EGFR and CD9‐β1 integrin. The internalization of EGFR after EGF‐stimulation was significantly enhanced by the treatment with ALB6. CD9 can associate with EGFR in hepatocellular carcinoma cells (HepG2/CD9) and Chinese hamster ovary cancer cells (CHO‐HER/CD9), which were transfected with pTJ/human EGFR/CD9. Furthermore expression of CD9 specifically attenuated EGFR signaling in CHO‐HER/CD9 cells through the down regulation of surface expression of EGFR. These results suggest that CD9 might have an important role that attenuates EGFR signaling. Therefore, CD9 not only associates EGFR but also a new regulator, which may affect EGF‐induced signaling in cancer cells. J. Cell. Physiol. 216: 135–143, 2008.

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.

Critical role of ADP interaction with P2Y12 receptor in the maintenance of alpha(IIb)beta3 activation: association with Rap1B activation.

Summary.  Objective: Platelet integrin αIIbβ3 plays a crucial role in platelet aggregation, and the affinity of αIIbβ3 for fibrinogen is dynamically regulated. Employing modified ligand‐binding assays, we analyzed the mechanism by which αIIbβ3 maintains its high‐affinity state. Methods and results: Washed platelets adjusted to 50 × 103 μL−1 were stimulated with 0.2 U mL−1 thrombin or 5 μm U46619 under static conditions. After the completion of αIIbβ3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated αIIbβ3 was then detected by fluorescein isothiocyanate (FITC)‐labeled PAC1. The addition of 1 μm AR‐C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained αIIbβ3 activation by ∼92% and ∼38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 μL−1 also disrupted the sustained αIIbβ3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5′‐diphosphate (ADP) in a dose‐dependent fashion. The amounts of ADP released from activated platelets determined by high‐performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL−1 thrombin. Thrombin induced long‐lasting PKC and Rap1B activation. AR‐C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. Conclusions: Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of αIIbβ3 activation.

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.

A two-amino acid insertion in the Cys146- Cys167 loop of the alphaIIb subunit is associated with a variant of Glanzmann thrombasthenia. Critical role of Asp163 in ligand binding.

The ligand binding site(s) of the alpha subunit of integrin alphaIIb beta3 (GPIIb-IIIa), a prototypic non-I domain integrin, remains elusive. In this study, we have characterized a Japanese variant of Glanzmann thrombasthenia, KO, whose platelets express normal amounts of alphaIIb beta3. KO platelets failed to bind the activation-independent ligand-mimetic mAb OP-G2 and did not bind fibrinogen or the activation-dependent ligand-mimetic mAb PAC-1 following activation of alphaIIb beta3 under any condition examined. Sequence analysis of PCR fragments derived from KO platelet mRNA revealed a 6-bp insertion leading to a 2-amino-acid insertion (Arg-Thr) between residues 160 and 161 of the alphaIIb subunit. Introduction of the insertion into wild-type recombinant alphaIIb beta3 expressed in 293 cells led to the normal expression of alphaIIb beta3 having the defect in ligand binding function. The insertion is located within the small loop (Cys146-Cys167) in the third NH2-terminal repeat of the alphaIIb subunit. Alanine substitution of each of the oxygenated residues within the loop (Thr150, Ser152, Glu157, Asp159, Ser161, and Asp163) did not significantly affect expression of alphaIIbbeta3, and only Asp163AlaalphaIIb beta3 abolished the ligand binding function. In addition, Asp163AlaalphaIIb beta3 as well as KO mutant alphaIIb beta3 constitutively expressed the PMI-1 epitope. Our present data suggest that Asp163 of the alphaIIb subunit is one of the critical residues for ligand binding.

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.