[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Platelet integrin α(IIb)β(3) activation is regulated by inside-out signaling via agonist stimulation. However, when α(IIb)β(3) was exogenously expressed in cell lines such as Chinese hamster ovarian cells, physiological agonists hardly induced α(IIb)β(3) activation. To overcome this disadvantage, we characterized the functional regulation of endogenously expressed α(IIb)β(3) in a megakaryoblastic cell line, CMK, employing an initial velocity assay for PAC-1 binding. We firstly demonstrated that protease-activated receptor 1-activating peptide induced robust, but transient α(IIb)β(3) activation in CMK cells with high glycoprotein-Ib expression. Stable talin-1 or kindlin-3 knockdown cells confirmed that the protease-activated receptor 1-activating peptide-induced α(IIb)β(3) activation was dependent on talin-1 and kindlin-3 expression. In sharp contrast to exogenously expressed α(IIb)β(3) in Chinese hamster ovarian cells, transient overexpression of full-length talin (FL-talin) or talin-head domain (THD) alone did not activate α(IIb)β(3) in CMK cells, but required agonist stimulation. Similarly, kindlin-3 overexpression alone did not induce α(IIb)β(3) activation, but it significantly augmented agonist-induced α(IIb)β(3) activation. Several mutants of FL-talin and THD suggested that the head-rod interaction was critical for autoinhibition of talin-1, and the interaction between the THD and the membrane-proximal region of the β(3) cytoplasmic tail was essential for talin-mediated α(IIb)β(3) activation. In addition, THD and kindlin-3 cooperatively augmented protease-activated receptor 1-induced α(IIb)β(3) activation. We proposed that the CMK cell line is an attractive platform for investigating agonist-, talin-1-, and kindlin-3- dependent α(IIb)β(3) activation.
[Show abstract][Hide abstract] ABSTRACT: 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.
Cell death and differentiation 07/2012; 19(11):1856-69. · 8.24 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelet-associated (PA) IgG autoantibodies play an essential role in primary immune thrombocytopenia (ITP). However, little is known about the epitopes of these Abs. This study aimed to identify critical binding regions for PA anti-αIIbβ3 Abs. Because PA anti-αIIbβ3 Abs bound poorly to mouse αIIbβ3, we created human-mouse chimera constructs. We first examined 76 platelet eluates obtained from patients with primary ITP. Of these, 26 harbored PA anti-αIIbβ3 Abs (34%). Further analysis of 15 patients who provided sufficient materials showed that the epitopes of these Abs were mainly localized in the N-terminal half of the β-propeller domain in αIIb (L1-W235). We could identify 3 main recognition sites in the region; 2 eluates recognized a conformation formed by the W1:1-2 and W2:3-4 loops, 5 recognized W1:2-3, and 4 recognized W3:4-1. The remaining 4 eluates could not be defined by the binding sites. Within these regions, we identified residues critical for binding, including S29 and R32 in W1:1-2; G44 and P45 in W1:2-3; and P135, E136, and R139 in W2:3-4. Of 11 eluates whose recognition sites were identified, 5 clearly showed restricted κ/λ-chain usage. These results suggested that PA anti-αIIbβ3 Abs in primary ITP tended to recognize highly restricted regions of αIIb with clonality.
[Show abstract][Hide abstract] ABSTRACT: Thromboxane A(2) receptor (TXA(2)R) abnormality appears to dominantly disturb platelet function.
To reveal a molecular genetic defect in a patient with TXA(2)R abnormality and investigate the mechanism for the impaired response to TXA(2).
The proband (OSP-2, PT) was a 7-year-old Japanese girl, suffering from repeated mucocutaneous bleeding.
U46619 (2.5 and 10 μm)-induced platelet aggregation was remarkably impaired in the proband and her father. Immunoblots showed that TXA(2)R 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 TXA(2)R cDNA. Expression studies using Chinese hamster ovary (CHO) cells indicated that the mutation is responsible for the expression defect in TXA(2)R. We then examined α(IIb)β(3) activation by employing an initial velocity analysis and revealed that U46619 failed to induce a sustained α(IIb)β(3) 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 P2Y(12) has been shown to play a critical role in the sustained α(IIb)β(3) activation (Kamae et al. J Thromb Haemost 2006; 4: 1379). As expected, small amounts of exogenous ADP (0.5 μm) partially restored the sustained α(IIb)β(3) activation induced by U46619.
Our present data strongly suggest that the impaired platelet activation in response to U46619 in the heterozygous subject for the TXA(2)R mutation is, at least in part, as a result of the decrease in ADP secretion.
Journal of Thrombosis and Haemostasis 02/2011; 9(5):1040-8. · 6.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The occurrence of transfusion-related alloimmunization against αIIbβ3 is a major concern in patients with Glanzmann thrombasthenia (GT). However, few data are available about molecular defects of GT patients with anti-αIIbβ3 alloantibodies as well as clinical impact of these antibodies on platelet transfusion. Here, we report a case of type I GT with anti-HLA and anti-αIIbβ3 alloantibodies, who underwent laparoscopic total gastrectomy due to gastric cancer. We found a novel β3 nonsense mutation, 892C > T (Arg272X), and the patient was homozygous for the mutation. Laparoscopic gastrectomy was successfully performed with continuous infusion of HLA-matched platelet concentrates and bolus injection of recombinant factor VIIa at 2 h intervals. Total bleeding was 370 mL and no red-cell transfusion was necessary. Flow cytometric analysis employing anti-αIIbβ3 monoclonal antibody revealed that the transfused platelet count was maintained around 20-30 × 10⁹/L during the operation and 10 × 10⁹/L on the following day. Flow cytometric analysis also showed that transfused platelets retained normal reactivity to ADP stimulation. These results indicate that flow cytometry is useful to assess survival and function of transfused platelets in GT patients with anti-αIIbβ3 antibodies.
International journal of hematology 01/2011; 93(1):106-11. · 1.17 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.