Kazunobu Kiyomizu

Mcl-1 and Bcl-xL regulate Bak/Bax-dependent apoptosis of the megakaryocytic lineage at multistages

Anti-apoptotic Bcl-2 family proteins, which inhibit the mitochondrial pathway of apoptosis, are involved in the survival of various hematopoietic lineages and are often dysregulated in hematopoietic malignancies. However, their involvement in the megakaryocytic lineage is not well understood. In the present paper, we describe the crucial anti-apoptotic role of Mcl-1 and Bcl-xL in this lineage at multistages. The megakaryocytic lineage-specific deletion of both, in sharp contrast to only one of them, caused apoptotic loss of mature megakaryocytes in the fetal liver and systemic hemorrhage, leading to embryonic lethality. ABT-737, a Bcl-xL/Bcl-2/Bcl-w inhibitor, only caused thrombocytopenia in adult wild-type mice, but further induced massive mature megakaryocyte apoptosis in the Mcl-1 knockout mice, leading to severe hemorrhagic anemia. All these phenotypes were fully restored if Bak and Bax, downstream apoptosis executioners, were also deficient. In-vitro study revealed that the Jak pathway maintained Mcl-1 and Bcl-xL expression levels, preventing megakaryoblastic cell apoptosis. Similarly, both were involved in reticulated platelet survival, whereas platelet survival was dependent on Bcl-xL due to rapid proteasomal degradation of Mcl-1. In conclusion, Mcl-1 and Bcl-xL regulate the survival of the megakaryocytic lineage, which is critically important for preventing lethal or severe hemorrhage in both developing and adult mice.

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.

Demonstration of novel gain‐of‐function mutations of αIIbβ3: association with macrothrombocytopenia and glanzmann thrombasthenia‐like phenotype

Integrin αIIbβ3 is indispensable for normal hemostasis, but its role for thrombopoiesis is still controversial. Recently, αIIb and β3 mutations have been identified in patients with congenital macrothrombocytopenia. We analyzed three unrelated Japanese families with congenital macrothrombocytopenia. Expression and activation state of αIIbβ3 in platelets was examined by flow cytometry and immunoblotting. Sequence of whole coding region and exon–intron boundaries of ITGA2B and ITGB3 genes was performed. The effects of mutations on αIIbβ3 activation state and phosphorylation of FAK were analyzed in transfected cells. We newly identified three mutations: two mutations in highly conserved Gly‐Phe‐Phe‐Lys‐Arg sequence in juxtamembrane region of αIIb, p.Gly991Cys and p.Phe993del, and one donor site mutation of intron 13 of ITGB3 leading to 40 amino acids deletion, p.(Asp621_Glu660del), in the membrane proximal β‐tail domain of β3. One patient, who showed Glanzmann thrombasthenia‐like marked reduction in surface αIIbβ3 expression (3–11% of normal control), was a compound heterozygote with ITGA2B p.Gly991Cys and a novel nonsense mutation, ITGA2B p.Arg422*. All three mutations, ITGA2B p.Gly991Cys, ITGA2B p.Phe993del, and ITGB3 p.(Asp621_Glu660del), led to highly activated conformation of αIIbβ3 and spontaneous tyrosine phosphorylation of FAK in transfected cells. These results suggest that gain‐of‐function mutations around membrane region of αIIbβ3 lead to abnormal platelet number and morphology with impaired surface αIIbβ3 expression.

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.

Trans-fatty acid promotes thrombus formation in mice by aggravating antithrombogenic endothelial functions via Toll-like receptors

SCOPE Since excessive intake of trans-fatty acid (TFA) increases the risk of myocardial infarction, we investigated the effects of TFA on thrombus formation using animal and cell culture experiments. METHODS AND RESULTS C57BL/6 mice were fed a diet containing TFA or cis-fatty acid (5% each of total calories) or a chow diet for 4 weeks, and thrombus formation was induced in the carotid artery by He-Ne laser irradiation. The high-TFA diet significantly promoted thrombus formation in the carotid artery compared to the chow or cis-fatty acid diet. TFA activated the inflammatory signaling pathway in cultured endothelial cells and in mice; aortic gene expression levels of antithrombogenic molecules, including thrombomodulin and tissue factor pathway inhibitor, were decreased, and the expression levels of prothrombogenic molecules were increased in TFA-treated mice. TFA markedly upregulated the prothrombogenic molecules and downregulated the antithrombogenic molecules in endothelial cells. In addition, TFA induced phosphorylation of c-Jun N-terminal kinase, extracellular signal-regulated kinase, and nuclear factor-κB. The TFA-activated signal pathways and prothrombogenic phenotypic changes of endothelial cells were inhibited by genetic or pharmacological inactivation of Toll-like receptors 2 and 4. CONCLUSION TFA aggravates the antithrombogenic phenotypes of vascular endothelial cells via Toll-like receptors and promotes thrombus formation in mice.