Tsuyoshi Kamae

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.

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 potential role for α-actinin in inside-out αIIbβ3 signaling.

Many different biochemical signaling pathways regulate integrin activation through the integrin cytoplasmic tail. Here, we describe a new role for α-actinin in inside-out integrin activation. In resting human platelets, α-actinin was associated with αIIbβ3, whereas inside-out signaling (αIIbβ3 activation signals) from protease-activated receptors (PARs) dephosphorylated and dissociated α-actinin from αIIbβ3. We evaluated the time-dependent changes of the αIIbβ3 activation state by measuring PAC-1 binding velocity. The initial velocity analysis clearly showed that PAR1-activating peptide stimulation induced only transient αIIbβ3 activation, whereas PAR4-activating peptide induced long-lasting αIIbβ3 activation. When αIIbβ3 activation signaling dwindled, α-actinin became rephosphorylated and reassociated with αIIbβ3. Compared with control platelets, the dissociation of α-actinin from αIIbβ3 was only transient in PAR4-stimulated P2Y(12)-deficient platelets in which the sustained αIIbβ3 activation was markedly impaired. Overexpression of wild-type α-actinin, but not the mutant Y12F α-actinin, increased its binding to αIIbβ3 and inhibited PAR1-induced initial αIIbβ3 activation in the human megakaryoblastic cell line, CMK. In contrast, knockdown of α-actinin augmented PAR-induced αIIbβ3 activation in CMK. These observations suggest that α-actinin might play a potential role in setting integrins to a default low-affinity ligand-binding state in resting platelets and regulating αIIbβ3 activation by inside-out signaling.

Bleeding tendency and impaired platelet function in a patient carrying a heterozygous mutation in the thromboxane A2 receptor.

Summary.  Background: Thromboxane A2 receptor (TXA2R) abnormality appears to dominantly disturb platelet function. Objectives: To reveal a molecular genetic defect in a patient with TXA2R abnormality and investigate the mechanism for the impaired response to TXA2. Patient: The proband (OSP‐2, PT) was a 7‐year‐old Japanese girl, suffering from repeated mucocutaneous bleeding. Methods and results: U46619 (2.5 and 10 μm)‐induced platelet aggregation was remarkably impaired in the proband and her father. Immunoblots showed that TXA2R expression levels in their platelets were approximately 50% of controls, and nucleotide sequence analysis revealed that they were heterozygous for a novel mutation, c.167dupG in the TXA2R cDNA. Expression studies using Chinese hamster ovary (CHO) cells indicated that the mutation is responsible for the expression defect in TXA2R. We then examined αIIbβ3 activation by employing an initial velocity analysis and revealed that U46619 failed to induce a sustained αIIbβ3 and Rap1B activation in the proband. In addition, platelet secretion as monitored by P‐selectin expression was markedly impaired in response to U46619 but not to ADP. The interaction between secreted ADP and P2Y12 has been shown to play a critical role in the sustained αIIbβ3 activation (Kamae et al. J Thromb Haemost 2006; 4: 1379). As expected, small amounts of exogenous ADP (0.5 μm) partially restored the sustained αIIbβ3 activation induced by U46619. Conclusion: Our present data strongly suggest that the impaired platelet activation in response to U46619 in the heterozygous subject for the TXA2R mutation is, at least in part, as a result of the decrease in ADP secretion.

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.

New variant of acute promyelocytic leukemia with IRF2BP2-RARA fusion.

We present an acute promyelocytic leukemia (APL) patient with two subtypes of IRF2BP2–RARA, in which the IRF2BP2 gene showed completely new breakpoints. Bone marrow examination revealed morphologic features indicative of APL. However, promyelocytic leukemia–RARA fusion was not detected. A paired‐end mRNA sequencing followed by RT‐PCR and direct sequencing revealed two types of fusion transcripts between exon 1B of IRF2BP2 and exon 3 of RARA. The patient received all‐trans retinoic acid and conventional chemotherapy, but showed resistance. This is the second report of IRF2BP2 involvement in APL, and we describe various breakpoints for the IRF2BP2–RARA fusion gene.