Circulating megakaryocytes: Delivery of large numbers of intact, mature megakaryocytes to the lungs

Macrothrombocytopenia in MYH9-related disease (MYH9-RD) results from defects in nonmuscular myosin-IIA function. Thrombopoietin receptor agonists (eltrombopag; romiplostim) seem to improve hemostasis, but little is known about their biologic effects in MYH9-RD. We administered romiplostim to Myh9 ؊/؊ mice (100 g/kg, every 3 days, during 1 month). MKs increased to similar numbers in Myh9 ؊/؊ and wild-type (WT) mice (with an increase in immature MKs), but Myh9 ؊/؊ platelet count response was much less (2.5-fold vs 8-fold increase). A strong increase in MK nuclei emboli in the lung, in WT and Myh9 ؊/؊ mice, indicates increased transmigration of MKs from the BM. Prolonged (but not acute) treatment with romiplostim decreased expression of GPIb-IX-V complex and GPVI, but not of GPIIbIIIa, and bleeding time increased in WT mice. Microcirculation was not altered by the increased number of large platelets in any of the assessed organs, but in Myh9 ؊/؊ mice a much stronger increase in BM reticulin fibers was present after 4 weeks of romiplostim treatment vs WT mice. These data further encourage short-term use of thrombopoietic agents in patients with MYH9-RDs; however, myelofibrosis has to be considered as a potential severe adverse effect during longer treatment. Reduction of GPIbIX/GPVI expression by romiplostim requires further studies. IntroductionMYH9-related disorders (MYH9-RDs) are a group of rare diseases characterized by congenital macrothrombocytopenia that results from mutations in the MYH9 gene encoding the nonmuscle myosin IIA, the only isoform of myosin present in platelets. 1 Thrombocytopenia ranges from mild to severe and remains relatively stable in a person throughout life. Patients may experience easy bruising, epistaxis, and menorrhagia, in some rare occasions requiring blood transfusion. Other manifestations may occur later in life such as cataract, hearing loss, and nephropathy; the mechanism leading to these additional symptoms is presently unknown. 2,3 In these patients, thrombocytopenia results from defective platelet production, probably resulting from impairment in marrow MK maturation because of decreased or abnormal myosin IIA, leading to a strong decrease in their capacity to extend proplatelets. 4,5 Second-generation thrombopoietic agents, which stimulate megakaryocytopoiesis by binding to the thrombopoietin (TPO) receptor, 6 may be an option for increasing the platelet count in MYH9-RDs. Two of these TPO receptor agonists have completed phase 3 trials in primary immune thrombocytopenia (eltrombopag, a nonpeptide TPO receptor agonist, and romiplostim, a peptide TPO receptor agonist 7,8 ) and have been recently approved in several countries for treatment of certain patients with immune thrombocytopenia.Eltrombopag administration has recently been tested in patients with MYH9-RDs in a phase 2, multicenter trial, showing moderate increase in platelet counts and reduction in bleeding tendency, 9 suggesting that these new thrombopoietic agents could also represent an interesting therapeutic opti...

Section: Org Fromsupporting

confidence: 92%

Romiplostim administration shows reduced megakaryocyte response-capacity and increased myelofibrosis in a mouse model of MYH9-RD

Léon

Evert²,

Dombrowski³

et al. 2012

“…[7][8][9] More recently, using intravital microscopy to visualize platelet generation in mice, Junt et al 10 have provided evidence that MKs extend voluminous processes into the lumen of sinusoids, which are sheared off by the flowing blood and thereby produce proplatelets that subsequently fragment into individual platelets. 2,[11][12][13] Although the DMS has been well characterized at the end stages of MK maturation, little is known about the biogenesis of this unique membrane system during the initial stages of MK development. Various subcellular origins have been proposed as a source of DMS biogenesis: (1) the MK plasma membrane (PM), (2) specializations of the endoplasmic reticulum (ER) or the Golgi apparatus, and (3) de novo membrane formation.…”

Section: Introductionmentioning

confidence: 99%

Biogenesis of the demarcation membrane system (DMS) in megakaryocytes

Eckly

Heijnen

Pertuy

et al. 2014

119

104

Key Points• Using state-of-the-art three-dimensional electron microscopy approaches, we show that the onset of the DMS formation is at the megakaryocyte plasma membrane.• A pre-DMS structure is formed in the perinuclear region, through a PM invagination process that resembles cleavage furrow formation.The demarcation membrane system (DMS) in megakaryocytes forms the plasma membrane (PM) of future platelets. Using confocal microscopy, electron tomography, and large volume focused ion beam/scanning electron microscopy (FIB/SEM), we determined the sequential steps of DMS formation. We identified a pre-DMS that initiated at the cell periphery and was precisely located between the nuclear lobes. At all developmental stages, the DMS remained continuous with the cell surface. The number of these connections correlated well with the nuclear lobulation, suggesting a relationship with cleavage furrow formation and abortive cytokinesis. On DMS expansion, Golgi complexes assembled around the pre-DMS, and fusion profiles between trans-golgi network-derived vesicles and the DMS were observed. Brefeldin-A reduced DMS expansion, indicating that the exocytic pathway is essential for DMS biogenesis. Close contacts between the endoplasmic reticulum (ER) and the DMS were detected, suggesting physical interaction between the 2 membrane systems. FIB/SEM revealed that the DMS forms an intertwined tubular membrane network resembling the platelet open canalicular system. We thus propose the following steps in DMS biogenesis:(1) focal membrane assembly at the cell periphery; (2) PM invagination and formation of a perinuclear pre-DMS; (3) expansion through membrane delivery from Golgi complexes; and (4) ER-mediated lipid transfer. (Blood. 2014;123(6):921-930) IntroductionThe maturation of megakaryocytes (MKs) includes the development of a unique and extensive membrane system known as the demarcation membrane system (DMS), which divides the cytoplasm into small platelet territories. One MK is thought to produce an average of 4000 platelets. Although it has been known for many years that the DMS ultimately forms the cell membrane of the future platelets, 1,2 the exact mechanism of the formation of this unique membrane system remains unclear. Early electron microscopy (EM) examination of late stage mature MKs led to the proposal that the DMS demarcates already preformed platelets. 3 Observations by De Bruyn of long MK extensions protruding into the sinusoidal lumen modified this idea and suggested that the DMS divides the MK cytoplasm into intertwined and compacted cylindrical regions. 4 Such a model had already been proposed earlier by Thiery and Bessis, who observed that MKs from bone marrow (BM) explants extended elongated projections they called proplatelets. 5 Studies using cultured MKs led to a more refined model, where the DMS appears to function as a membrane reservoir for the extension of proplatelets, which would then fragment into platelets along their length 6 or at their tips. [7][8][9] More recently, using intravital microscopy...

“…28 Indeed, the large cytoplasmic fragments and the isolated platelet-sized fragments that we observed in real time to form on the coverslip during the flow assay resembled those seen downstream of the pulmonary circulation in vivo. 33 In our conditions, high shear rates were essential to proplatelet and platelet formation during an exposure time of 20 minutes. In contrast, no proplatelet or platelet was generated in static conditions during this short period.…”

mentioning

confidence: 99%

“…21 Thus MK fragmentation leading to platelets may occur intravascularly and be finely regulated depending on the release of VWF from Weibel-Palade bodies, as well as on the VWF content of endothelial cells that is known to be heterogeneous along the vascular bed, and particularly high in the pulmonary artery. 44,45 Indeed, there is evidence that platelet counts are higher in pulmonary venous samples than in pulmonary arterial blood, 28,33 suggesting that circulating MK fragments and proplatelets may be processed into platelets when passing through the lung capillaries, where they are exposed to high shear rates. Shear rates conditions used in this study appear relevant to the pulmonary microcirculation, which has been suggested to be a site of platelet production.…”

mentioning

confidence: 99%

Exposure of human megakaryocytes to high shear rates accelerates platelet production

Dunois-Lardé

Capron

Fichelson

et al. 2009

120

139

Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation. (Blood. 2009;114:1875-1883) IntroductionMegakaryocyte (MK) differentiation is a continuous process characterized by sequential steps. MK ploidy increases through endomitosis, with parallel increase in cell size. Synthesis of storage organelles is enhanced and plasma membrane invaginates, resulting in the formation of demarcation membranes. Finally, mature MKs undergo complete cytoskeleton reorganization with microtubule involvement, to induce pseudopodial elongations called proplatelets. 1,2 Platelets are released from the tips of proplatelets that contain the organelles. 3 In static conditions, platelets are formed from mature MKs cultured in the presence of thrombopoietin (TPO). 3 MK fragmentation into platelets does not occur in the extravascular, hematopoietic space in normal bone marrow. To achieve platelet formation, MKs need to migrate into the sinusoid capillaries and mature MKs are generally located in the vicinity of sinusoids, more precisely on their abluminal side. 4 MK fragmentation occurs only in the intravascular space, and mature MKs have been identified in the blood circulation where they may interact via their surface receptors with proteins expressed on endothelial cells. [4][5][6] Adhesion of MKs to endothelial cells is essential for robust platelet production in vitro, a process that is believed to mimic what occurs in vivo when MKs interact with bone marrow endothelial cells within the vascular niche. 7 Recently, the process of proplatelet formation was examined in mouse skull bone marrow in vivo: the authors observed large MK fragments resembling imma...