Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.

Matrix stiffness controls megakaryocyte adhesion, fibronectin fibrillogenesis, and proplatelet formation through Itgß3.

Megakaryocytes (MKs) are the precursor cells of platelets, located in the bone marrow (BM). Once mature, they extend elongated projections named proplatelets through sinusoid vessels, emerging from the marrow stroma into the circulating blood. Not all signals from the microenvironment that regulate proplatelet formation are understood, particularly those from the BM biomechanics. We sought to investigate how MKs perceive and adapt to modifications of the stiffness of their environment. Although the BM is one of the softest tissue of the body, its rigidification results from excess fibronectin (FN), and other matrix protein deposition occur upon myelofibrosis. Here, we have shown that mouse MKs are able to detect the stiffness of a FN-coated substrate and adapt their morphology accordingly. Using a polydimethylsiloxane substrate with stiffness varying from physiological to pathological marrow, we found that a stiff matrix favors spreading, intracellular contractility, and FN fibrils assembly at the expense of proplatelet formation. Itgb3, but not Itgb1, is required for stiffness sensing, whereas both integrins are involved in fibrils assembly. In contrast, soft substrates promote proplatelet formation in an Itgb3-dependent manner, consistent with the ex vivo decrease in proplatelet formation and the in vivo decrease in platelet number in Itgb3-deficient mice. Our findings demonstrate the importance of environmental stiffness for MK functions with potential pathophysiological implications during pathologies that deregulate FN deposition and modulate stiffness in the marrow.

Combined deficiency of RAB32 and RAB38 in the mouse mimics Hermansky-Pudlak syndrome and critically impairs thrombosis.

The biogenesis of lysosome related organelles is defective in Hermansky-Pudlak syndrome (HPS), a disorder characterized by oculocutaneous albinism and platelet dense granule (DG) defects. The first animal model of HPS was the fawn-hooded rat, harboring a spontaneous mutation inactivating the small guanosine triphosphatase Rab38 This leads to coat color dilution associated with the absence of DGs and lung morphological defects. Another RAB38 mutant, the cht mouse, has normal DGs, which has raised controversy about the role of RAB38 in DG biogenesis. We show here that murine and human, but not rat, platelets also express the closely related RAB32. To elucidate the parts played by RAB32 and RAB38 in the biogenesis of DGs in vivo and their effects on platelet functions, we generated mice inactivated for Rab32, Rab38, and both genes. Single Rab38 inactivation mimicked cht mice, whereas single Rab32 inactivation had no effect in DGs, coat color, or lung morphology. By contrast, Rab32/38 double inactivation mimicked severe HPS, with strong coat and eye pigment dilution, some enlarged lung multilamellar bodies associated with a decrease in the number of DGs. These organelles were morphologically abnormal, decreased in number, and devoid of 5-hydroxytryptamine content. In line with the storage pool defect, platelet activation was affected, resulting in severely impaired thrombus growth and prolongation of the bleeding time. Overall, our study demonstrates the absence of impact of RAB38 or RAB32 single deficiency in platelet biogenesis and function resulting from full redundancy, and characterized a new mouse model mimicking HPS devoid of DG content.

Myosin IIA is critical for organelle distribution and F-actin organization in megakaryocytes and platelets.

During proplatelet formation, a relatively homogeneous content of organelles is transported from the megakaryocyte (MK) to the nascent platelets along microtubule tracks. We found that platelets from Myh9(-/-) mice and a MYH9-RD patient were heterogeneous in their organelle content (granules and mitochondria). In addition, Myh9(-/-) MKs have an abnormal cytoplasmic clustering of organelles, suggesting that the platelet defect originates in the MKs. Myosin is not involved in the latest stage of organelle traffic along microtubular tracks in the proplatelet shafts as shown by confocal observations of proplatelet buds. By contrast, it is required for the earlier distribution of organelles within the large MK preplatelet fragments shed into the sinusoid circulation before terminal proplatelet remodeling. We show here that F-actin is abnormally clustered in the cytoplasm of Myh9(-/-) MKs and actin polymerization is impaired in platelets. Myosin IIA is required for normal granule motility and positioning within MKs, mechanisms that may be dependent on organelle traveling and tethering onto F-actin cytoskeleton tracks. Altogether, our results indicate that the distribution of organelles within platelets critically depends on a homogeneous organelle distribution within MKs and preplatelet fragments, which requires myosin IIA.

Megakaryocyte-restricted MYH9 inactivation dramatically affects hemostasis while preserving platelet aggregation and secretion.

Mutations in the MYH9 gene encoding the nonmuscle myosin heavy chain IIA result in bleeding disorders characterized by a macrothrombocytopenia. To understand the role of myosin in normal platelet functions and in pathology, we generated mice with disruption of MYH9 in megakaryocytes. MYH9Delta mice displayed macrothrombocytopenia with a strong increase in bleeding time and absence of clot retraction. However, platelet aggregation and secretion in response to any agonist were near normal despite absence of initial platelet contraction. By contrast, integrin outside-in signaling was impaired, as observed by a decrease in integrin beta3 phosphorylation and PtdIns(3,4)P(2) accumulation following stimulation. Upon adhesion on a fibrinogen-coated surface, MYH9Delta platelets were still able to extend lamellipodia but without stress fiber-like formation. As a consequence, thrombus growth and organization, investigated under flow by perfusing whole blood over collagen, were strongly impaired. Thrombus stability was also decreased in vivo in a model of FeCl(3)-induced injury of carotid arteries. Overall, these results demonstrate that while myosin seems dispensable for aggregation and secretion in suspension, it plays a key role in platelet contractile phenomena and outside-in signaling. These roles of myosin in platelet functions, in addition to thrombocytopenia, account for the strong hemostatic defects observed in MYH9Delta mice.