SummaryThrombosis in paroxysmal nocturnal haemoglobinuria (PNH) has been suggested to be due to several pathophysiological states: a suppressed fibrinolytic system, increased leucocyte‐derived tissue factor, complement (C′)‐mediated damage to platelets and endothelia, or increased platelet‐ and endothelium‐derived microparticles (MPs). Because haemolytic attack is often accompanied by thrombosis in PNH, we studied the role of C′‐induced release of MPs in the thrombogenesis of PNH. C′ activation induced procoagulant alteration in PNH red blood cells (RBC), when assessed by thrombin generation in the presence of C′‐activated PNH RBC, which was abolished by their subsequent treatment with annexin V. Significant amounts of procoagulant MPs, measured by phosphatidylserine‐binding prothrombinase activity, were released from PNH RBC in association with the formation of C5b‐9, but not significantly before C5b‐8. Generation of procoagulant, annexin V‐binding, MPs from C′‐activated RBC was studied also by flow cytometry. While phorbol 12‐myristate 13‐acetate, an activator of protein kinase C (PKC), induced the release of MPs from normal RBC as well as PNH RBC, C′‐induced release of MPs from PNH RBC was Ca2+‐independent and not associated with the activation of PKC, calpain or caspase. Procoagulant properties of MPs released from PNH RBC could contribute to the thrombogenesis of PNH.
Summary. Background: There have been only seven reported cases of glycoprotein (GP) VI deficiency. However, the pathogenesis of this disorder has not been well-elucidated. Objectives: We characterized a novel patient with GPVI deficiency and used these platelets to investigate the role of GPVI in normal hemostasis. Patient: A 31-year-old female with immune thrombocytopenic purpura who had been suffering from mild bleeding diathesis even after recovery from thrombocytopenia. Results and conclusion: The patient's platelets did not aggregate in response to either convulxin or collagen-related peptide. Immunoblotting revealed complete absence of the GPVI molecule, whereas a significantly reduced but substantial amount of Fc receptor (FcR) c-chain was expressed. Platelet stimulation with convulxin did not induce tyrosine-phosphorylation of FcR c-chain, indicating a defect in GPVI-mediated signaling. Concerning the underlying pathogenesis, we found normal level of GPVI-mRNA expression, no aberration of the sequence of the entire coding region of GPVI, and presence of degraded GPVI in her plasma. However, no anti-GPVI autoantibody was detected either by the binding assay to GPVI-Fc 2 fusion protein or by immunoblotting/ immunoprecipitation using the patient's immunoglobulin. We thus consider that either a short-time exposure to anti-GPVI autoantibody or a continuous exposure to low titers of the autoantibody has resulted in persistent GPVI deficiency. Under high shear flow, the patient's platelets could not form large aggregates, although initial platelet attachment was obviously observed. These results suggest that GPVI deficiency in this patient resulted in defective platelet thrombi development, manifesting as bleeding diathesis. Furthermore, our observations indicate that coordination of GPVI with integrin a 2 b 1 is essential for physiological platelet thrombus formation.
To elucidate whether caspase activation is involved in megakaryopoiesis, we characterized megakaryocytes (MKs) in vav-bcl-2 transgenic (Tg) mice, in which Bcl-2 is overexpressed in hematopoietic cells. To exclude the effect of splenomegaly in Tg mice on megakaryopoiesis, splenectomy was performed. After splenectomy, basal platelet counts in peripheral blood were not significantly different between Tg and wild-type (WT) mice. However, when experimental thrombocytopenia was induced by injecting 5-fluorouracil into splenectomized mice, overshoot of platelet counts during the recovery phase was hardly observed in Tg mice. Analyses of MK ploidy during the recovery phase showed that MKs less than 16 N ploidy were significantly decreased in Tg mice, suggesting that MK supply from progenitors is impaired. Supporting this, differentiation of CD34À/c-kit þ /Sca-1 þ /LineageÀ stem cells into MKs was significantly hampered in Tg mice, whereas megakaryocyteerythroid progenitors (MEPs) normally differentiated into MKs. It suggests that differentiation into MKs is impaired in Tg mice before the stage of MEP. Furthermore, MK colony formation in WT cells was dose-dependently inhibited in the presence of a caspase inhibitor. Contrary, Bcl-2-overexpressing MKs showed normal ability for in vitro platelet production. We thus believe that caspase activation is involved in the differentiation of progenitors into megakaryocytic lineage but not in platelet production.
Key Points• Mucin-type O-glycans are required for terminal differentiation of megakaryocytes and platelet production.• The expression of GPIba protein is strongly reduced in O-glycans-defective megakaryocytes and platelets.C1galt1 is essential for synthesis of the core 1 structure of mucin-type O-glycans. To clarify the physiological role of O-glycans in adult hematopoiesis, we exploited the interferon-inducible Mx1-Cre transgene to conditionally ablate the C1galt flox allele (Mx1-C1).Mx1-C1 mice exhibit severe thrombocytopenia, giant platelets, and prolonged bleeding times. Both the number and DNA ploidy of megakaryocytes in Mx1-C1 bone marrow were similar to those in wild-type (WT) mice. However, there were few proplatelets in Mx1-C1 primary megakaryocytes. Conversely, bone marrow transplanted from Mx1-C1 to WT and splenectomized Mx1-C1 mice gave rise to observations similar to those described above. The expression of GPIba messenger RNA was unchanged in Mx1-C1 bone marrow, whereas flow cytometric and western blot analyses using megakaryocytes and platelets revealed that the expression of GPIba protein was significantly reduced in Mx1-C1 mice. Moreover, circulating Mx1-C1 platelets exhibited an increase in the number of microtubule coils, despite normal levels of a-and b-tubulin. Our observations suggest that O-glycan is required for terminal megakaryocyte differentiation and platelet production and that the decrease in GPIba in cells lacking O-glycan might be caused by increased proteolysis. (Blood. 2013;122(9):1649-1657
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