No abstract

Besides α-granules and dense granules, which play critical roles in and beyond hemostasis, circulating blood platelets and their precursor cells megakaryocytes contain lysosomes, the contents of which are also secreted during platelet activation. In their delivery to the lysosome, acid hydrolases bearing phosphomannosyl residues are trafficked from the trans-Golgi network to the acidic late-endosomal compartment via the mannose 6-phosphate receptor (M6PR) pathway. To determine the role of M6PR-specific targeting of lysosomal enzymes in platelet function, platelet parameters were investigated in M6pr-/- mice lacking the 46-kDa M6PR, the physiological role of which is unclear. M6pr-/- mice had normal platelet count but displayed an increased number of distinct proplatelet-like cells compared to control mice, as determined by immunofluorescent microscopy. Moreover, transmission electron microscopy revealed the presence of abnormal membrane tubulations, elongated and electron-dense granules, and large vacuole-like structures within resting M6pr-/- platelets. M6pr-/- platelets expressed normally major glycoproteins on their surface and von Willebrand factor and fibrinogen in their α-granules. M6pr-/- mice were hyper-thrombotic, as assessed by tail bleeding time, and M6pr-/- platelets adhered to type I collagen with a significantly greater propensity than control platelets under arterial shear in in vitro flow experiments. Heparanase, an endo-β-glucuronidase that cleaves extracellular matrix heparan sulfate proteoglycans, is the most abundant lysosomal enzyme in platelets. Thus, its contribution to the phenotype of M6pr-/- mice was investigated further. Heparanase expression was decreased in the bone marrow megakaryocytes and blood platelets of M6pr-/- mice and increased in M6pr-/- plasma, as evidenced by immunoblot and fluorescent microscopy analysis, consistent with its mistargeting in the absence of M6PR. Interestingly, pharmacological inhibition of heparanase with OGT 2115 normalized the adhesion of M6pr-/- platelets to collagen in vitro, indicating that increased plasma heparanase contributes to the thrombotic phenotype of M6pr-/- mice. Taken together, the data suggest that the M6PR-specific targeting of lysosomal heparanase plays a critical role in platelet function, thereby regulating hemostasis. Disclosures No relevant conflicts of interest to declare.

Dnm2fl/fl Pf4-Cre (Dnm2Plt–/–) mice lacking the endocytic GTPase dynamin 2 (DNM2) in platelets and megakaryocytes (MKs) develop hallmarks of myelofibrosis. At the cellular level, the tyrosine kinase JAK2 is constitutively active but decreased in expression in Dnm2Plt–/– platelets. Additionally, Dnm2Plt–/– platelets cannot endocytose the thrombopoietin (TPO) receptor Mpl, leading to elevated circulating TPO levels. Here, we assessed whether the hyperproliferative phenotype of Dnm2Plt–/– mice was due to JAK2 constitutive activation or to elevated circulating TPO levels. In unstimulated Dnm2Plt–/– platelets, STAT3 and, to a lower extent, STAT5 were phosphorylated, but their phosphorylation was slowed and diminished upon TPO stimulation. We further crossed Dnm2Plt–/– mice in the Mpl–/– background to generate Mpl–/–Dnm2Plt–/– mice lacking Mpl ubiquitously and DNM2 in platelets and MKs. Mpl–/– Dnm2Plt–/– platelets had severely reduced JAK2 and STAT3 but normal STAT5 expression. Mpl–/– Dnm2Plt–/– mice had severely reduced bone marrow MK and hematopoietic stem and progenitor cell numbers. Additionally, Mpl–/– Dnm2Plt–/– mice had severe erythroblast (EB) maturation defects, decreased expression of hemoglobin and heme homeostasis genes and increased expression of ribosome biogenesis and protein translation genes in spleen EBs, and developed anemia with grossly elevated plasma erythropoietin (EPO) levels, leading to early fatality by postnatal day 25. Mpl–/– Dnm2Plt+/+ mice had impaired EB development at three weeks of age, which normalized with adulthood. Together, the data shows that DNM2-dependent Mpl-mediated endocytosis in platelets and MKs is required for steady-state hematopoiesis and provides novel insights into a developmentally controlled role for Mpl in normal erythropoiesis, regulating hemoglobin and heme production.

The thrombopoietin receptor Mpl plays a critical role in thrombopoiesis, as it is the primary signaling constituent in megakaryocyte (MK) differentiation from hematopoietic stem and progenitor cells (HSPCs) and maintains circulating blood thrombopoietin levels via clathrin-mediated endocytosis. Thus, thrombopoiesis is tightly controlled by Mpl cell-surface expression on HSPCs, MKs, and platelets. Previous work by our group has shown that Dnm2fl/flPF4-Cre (Dnm2Plt-/-) mice, lacking the large endocytic GTPase dynamin 2 (DNM2) specifically within MKs and platelets, develop a variety of clinical phenotypes that closely resemble myelofibrosis, such as bone marrow fibrosis, marked HSPC expansion, MK hyperplasia, extramedullary hematopoiesis, and severe splenomegaly (Bender, Giannini, et al. Blood. 2015;125(6):1014-1024). Dnm2Plt-/- mice also displayed elevated plasma TPO levels and constitutive JAK2 activation in platelets, due to defective Mpl endocytosis. Here, the role of Mpl endocytosis in the maintenance of normal hematopoiesis was assessed by generating Dnm2Plt-/- mice in the Mpl-/- background. At 3 weeks of age, Mpl-/- Dnm2Plt-/- mice displayed significantly reduced HSPCs, a near complete depletion of bone marrow MKs, similar to Mpl-/- mice, indicating that Mpl is the primary receptor contributing to the aberrant hyperproliferative phenotype of Dnm2Plt-/- mice. However, Mpl-/- Dnm2Plt-/- mice also showed severe anemia, defects in erythroblast maturation, grossly elevated plasma erythropoietin (EPO) levels, and splenomegaly, resulting in early fatality by 3 to 4 weeks of age, suggesting that Mpl contributes to normal erythropoiesis in young mice. Mpl-/- and Mpl+/- Dnm2Plt-/- mice displayed reduced erythroblast development at 3 weeks of age, which returned to normal with adulthood. Taken together, the data shows that DNM2-dependent Mpl endocytosis is required for steady-state hematopoiesis and provides novel insights into a developmentally-controlled role for Mpl in normal erythropoiesis. Disclosures Sola-Visner: Sysmex America, Inc.: Other: Laboratory equipment on loan, Research Funding.

Sickle cell disease (SCD) results from a sequence defect in the β‐globin chain of adult hemoglobin (HbA) leading to expression of sickle hemoglobin (HbS). It is traditionally diagnosed by cellulose‐acetate hemoglobin electrophoresis or high‐performance liquid chromatography. While clinically useful, these methods have both sensitivity and specificity limitations. We developed a novel mass spectrometry (MS) method for the rapid, sensitive and highly quantitative detection of endogenous human β‐globin and sickle hβ‐globin, as well as lentiviral‐encoded therapeutic hβAS3‐globin in cultured cells and small quantities of mouse peripheral blood. The MS methods were used to phenotype homozygous HbA (AA), heterozygous HbA–HbS (AS) and homozygous HbS (SS) Townes SCD mice and detect lentiviral vector‐encoded hβAS3‐globin in transduced mouse erythroid cell cultures and transduced human CD34+ cells after erythroid differentiation. hβAS3‐globin was also detected in peripheral blood 6 weeks post‐transplant of transduced Townes SS bone marrow cells into syngeneic Townes SS mice and persisted for over 20 weeks post‐transplant. As several genome‐editing and gene therapy approaches for severe hemoglobin disorders are currently in clinical trials, this MS method will be useful for patient assessment before treatment and during follow‐up.