Background: Platelets are generated by megakaryocytes (MKs) in the bone marrow. A key step in this process involves the extension of MK protrusions across the sinusoidal vessel barrier through tiny endothelial pores. While the MK cell body remains in the marrow stroma, proplatelets elongate in the bloodstream. Transendothelial migration and elongation impose strong deformation and stretching on the plasma membrane. Objectives: Since MKs are known to sense and respond to mechanical cues, we investigated the contribution of stretch-activated mechanosensitive calcium channels to this process. Results: Absence of calcium prevented proplatelet formation from MK differentiated in a 3-dimensional medium but not in a 2-dimensional liquid medium. We found that the confined 3-dimensional environment promoted selective overexpression of both transient receptor potential vanilloid (TRPV) 4 and TRPV2 stretch-activated calcium channels. Using genetic and pharmacologic approaches, we showed that TRPV4 is essential for optimal proplatelet formation, independent of substrate adhesion. Mechanistically, the absence of TRPV4 maintained abnormally high active Ras homolog family member A levels, preventing optimal proplatelet formation. The TRPV2 receptor also contributes, as blocking both TRPV4 and TRPV2 produces an additive inhibitory effect. We confirmed these findings in human platelet formation. Inhibition of both TRPV4 and TRPV2 reduces platelet production from CD34 +-derived MKs in culture. Conclusions: These results highlight TRPV4 and TRPV2 as key mechanosensitive channels in platelet biogenesis, presenting potential targets to enhance platelet production.

Megakaryocytes, the progenitor cells of blood platelets, play a crucial role in hemostasis by residing in the bone marrow and ensuring continuous platelet production. Unlike other hematopoietic cells, megakaryocytes do not enter the blood circulation intact. They remain anchored within the bone marrow while extending cytoplasmic protrusions called proplatelets through the sinusoidal endothelial barrier. These proplatelets subsequently fragment into functional platelets. This unique process of intravasation facilitates efficient platelet production while maintaining the megakaryocyte cell body within the bone marrow niche, thus preventing potential thrombotic complications. How the extracellular matrix (ECM) influences the delicate balance between megakaryocyte retention and proplatelet extension remains largely unknown. Here, we investigate the spatial organization and functional role of ECM components in the megakaryocyte vascular niche of mice bone marrow. Our findings reveal that laminin and collagen IV form three-dimensional (3D) ECM cages encompassing megakaryocytes and anchor them to the sinusoidal basement membrane. Gene deletion shows the existence of laminin α4 in the ECM cage that is necessary to maintain megakaryocyte-sinusoid interactions. Notably, megakaryocytes actively contribute to the ECM cage assembly; β1/β3 integrin knockout weakens these structures, increasing intravasation and entire megakaryocyte entry into circulation. The retention of megakaryocytes by these 3D ECM cages depends on dynamic remodeling processes. Inhibition of ECM proteolysis results in denser cage formation, increasing the frequency of immature megakaryocytes with impaired demarcation membrane system (DMS) development. Thus, the ECM cage represents a novel concept of an active and dynamic 3D microenvironment that is continuously remodeled and essential for maintaining megakaryocyte perivascular positioning. This specific microarchitecture guides megakaryocyte maturation and intravasation, underscoring the critical role of ECM microarchitecture and dynamics in megakaryocyte function.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, with limited treatment options and a poor prognosis. Epithelial-to-mesenchymal transition (EMT) plays a critical role in promoting TNBC metastasis. The natural bioactive substance 3-hydroxy-β-ionone (3-HBI), which has been studied in other cancer types, has not yet been examined in TNBC. This study investigates its potential mechanisms in TNBC cells through a combination of computational and experimental approaches, focusing on apoptosis induction and EMT inhibition. Molecular docking and molecular dynamics simulations demonstrated strong and stable binding of 3-HBI to key apoptosis-related proteins (Bcl-2, Bax, caspase-3) and EMT regulators (Smad2, Smad3). In vitro, 3-HBI significantly reduced cell viability in MDA-MB-231, T47D, and MCF7 cells, with IC50 values of 388.40, 185.50, and 113.40 µg/mL, respectively. Moreover, 3-HBI suppressed clonogenic potential, induced apoptosis, and inhibited both migration and invasion. Molecular analysis showed increased expression of Bax, caspase-3, and E-cadherin, and reduced levels of Bcl-2, Smad2, Smad3, and N-cadherin. These findings provide the first mechanistic evidence that 3-HBI exerts anti-TNBC effects by promoting apoptosis and suppressing EMT, highlighting its potential as a promising therapeutic candidate for TNBC treatment.

ABSTRACT:Throughout thrombopoiesis megakaryocytes (MKs) form proplatelets within the bone marrow (BM) and release platelets into BM sinusoids. Casein kinase 1α (CK1α) is a major player and thus, an important therapeutic target in several hematological malignancies. This study aimed to define the role of CK1α for the essential steps of thrombopoiesis and to dissect potential mechanisms of thrombocytopenia. MK-specific CK1α-deficiency resulted in a macrothrombocytopenia. Ck1αPf4Δ/Pf4Δ mice displayed a substantial BM hyperplasia with pivotal changes in MK nuclear lobulation and reduced contact to BM sinusoids. Ck1αPf4Δ/Pf4Δ MKs displayed a defective cytoskeleton organization reflected by a decreased amount of polymerized filamentous actin and disturbed microtubule dynamics due to p21/p53 accumulation and impaired Rho-associated protein kinase (ROCK)/LIM domain kinase (LIMK)/cofilin signaling. Further, pronounced defects in DMS (demarcation membrane system) polarization and proplatelet formation of Ck1αPf4Δ/Pf4Δ MKs, unraveled CK1α as a prerequisite for thrombopoiesis. Our findings could be translated into a human approach, because a CRISPR/Cas9-mediated genetic deletion of CSNK1A1 in MKs derived from human CD34+ progenitor cells resulted in a substantial defect in human MK maturation and platelet production. The present observations elucidated CK1α as an important signaling molecule in MK cytoskeletal dynamics and polarization, proplatelet formation, and polyploidization, thus highlighting the crucial role of CK1α in platelet biogenesis.

Background: Hereditary xerocytosis (HX) is a dominant red blood cell membrane disorder characterized by dehydration and hemolysis. Most HX cases result from gain-of-function (GOF) mutations in the PIEZO1 gene, which encodes a mechanosensitive ion channel permeable to calcium when activated. Evolution of HX is characterized by frequent thromboembolic events after splenectomy. Objectives: Given the major role of calcium during platelet activation, we hypothesized that Piezo1 GOF may increase platelet activation and contribute to thrombosis. Methods: Human washed platelets were activated by various agonists in the presence of Yoda1 (a chemical Piezo1 activator). Aggregation was monitored in an aggregometer, secretion by flow cytometry and calpain activation by western blot. Mouse models were the R2482H knock-in, mimicking the human recurrent R2456H GOF mutation of PIEZO1, and the megakaryocyte lineage-specific Piezo1/2 knockout (KO). Ex vivo thrombus formation was performed under flow on a collagen surface. In vivo, tail bleeding time was measured. Results: Yoda1 alone had no effect on human platelets but potentiated agonistinduced platelet aggregation, secretion, and procoagulant activity, involving calpain activation. In ex vivo, GOF mouse platelets aggregated more than wild type, especially in flow conditions. Conversely, blood from KO mice formed smaller thrombi. In vivo, a shorter total tail bleeding time was found in Piezo1 GOF mice, while KO mice had a longer first bleeding time. Conclusion: These results therefore support a correlation between Piezo1 activated status and platelet activation and are consistent with the involvement of platelet Piezo1 GOF in thrombotic events reported in patients with HX.

Liver fibrosis, characterized by excessive extracellular matrix deposition, is driven by activated hepatic stellate cells (HSCs). Due to the limited availability of anti-fibrotic drugs, the research on therapeutic agents continues. Here we have investigated Moringa oleifera Lam. (MO), known for its various bioactive properties, for anti-fibrotic effects. This study has focused on 1-phenyl-2-pentanol (1-PHE), a compound derived from MO leaves, and its effects on LX-2 human hepatic stellate cell activation. TGF-β1-stimulated LX-2 cells were treated with MO extract or 1-PHE, and the changes in liver fibrosis markers were assessed at both gene and protein levels. Proteomic analysis and molecular docking were employed to identify potential protein targets and signaling pathways affected by 1-PHE. Treatment with 1-PHE downregulated fibrosis markers, including collagen type I alpha 1 chain (COL1A1), collagen type IV alpha 1 chain (COL4A1), mothers against decapentaplegic homologs 2 and 3 (SMAD2/3), and matrix metalloproteinase-2 (MMP2), and reduced the secretion of matrix metalloproteinase-9 (MMP-9). Proteomic analysis data showed that 1-PHE modulates the Wnt/β-catenin pathway, providing a possible mechanism for its effects. Our results suggest that 1-PHE inhibits the TGF-β1 and Wnt/β-catenin signaling pathways and HSC activation, indicating its potential as an anti-liver-fibrosis agent.

Blood platelets are produced by megakaryocytes (MKs), their parent cells, which are in the bone marrow. Once mature, MK pierces through the sinusoid vessel, and the initial protrusion further elongates as proplatelet or buds to release platelets. The mechanisms controlling the decision to initiate proplatelet and platelet formation are unknown. Here, we show that the mechanical properties of the microenvironment prevent proplatelet and platelet release in the marrow stroma while allowing this process in the bloodstream. Loss of marrow confinement following myelosuppression led to inappropriate proplatelet and platelet release into the extravascular space. We further used an inert viscoelastic hydrogel to evaluate the impact of compressive stress. Transcriptional analysis showed that culture in three-dimensional gel induced upregulation of genes related to the Rho-GTPase pathway. We found higher Rho-GTPase activation, myosin light chain phosphorylation and F-actin under mechanical constraints while proplatelet formation was inhibited. The use of latrunculin-A to decrease F-actin promoted microtubule-dependent budding and proplatelet extension inside the gel. Additionally, ex vivo exposure of intact bone marrow to latrunculin-A triggered proplatelet extensions in the interstitial space. In vivo, this confinement-mediated high intracellular tension is responsible for the formation of the peripheral zone, a unique actin-rich structure. Cytoskeleton reorganization induces the disappearance of the peripheral zone upon reaching a liquid milieu to facilitate proplatelet and platelet formation. Hence, our data provide insight into the mechanisms preventing ectopic platelet release in the marrow stroma. Identifying such pathways is especially important for understanding pathologies altering marrow mechanics such as chemotherapy or 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 Jak2 V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2 V617F 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 JAK2 V617F 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.

Introduction: Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix (ECM) to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Primary Myelofibrosis (PMF) is a Chromosome Philadelphia-negative Myeloproliferative Neoplasm (MPN), characterized by altered stiffer ECM and megakaryocytosis, often driven by the gain-of-function mutation in JAK2 V617F. Here, we study a role for PIEZO1 in megakaryopoiesis and proplatelet formation under normal physiological conditions and in the context of PMF. Methods: Bone marrow Mks from C57BL/6J control and myelofibrotic mice carrying the human JAK2 V617F+ mutation, or Mks derived from stem cells of patients carrying the same mutation were analyzed at mRNA and protein levels for Piezo1 expression. Effects of Piezo1 activation or inhibition on Mk maturation and ploidy were assessed by flow cytometry, and proplatelet formation by fluorescent microscopy. Human peripheral blood samples from PMF patients, diagnosed according to established criteria, or healthy individuals were used to compare the two categories for Piezo1 relative expression on mRNA and protein levels and platelet biogenesis. Mk-specific double knockout mice of Piezo1/2 were generated to compare platelet counts under normal conditions or bone marrow ablation with 5-FU challenge. Culture of mouse and human Mks in different 3D microenvironments was used to demonstrate in vitro association of Piezo1 expression levels with different ECM stiffness substrates. Results: Mouse Mks express PIEZO1 and negligible levels of PIEZO2. Pharmacological activation of mouse PIEZO1 increases the number of immature CD41 + Mks, while the number of mature CD41 +CD42 + 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 pharmacological activation reduces Mk size, ploidy, maturation, and proplatelet development. Resulting effects of PIEZO1 activation on Mks resemble the profile in PMF. Intriguingly, Mks derived from PMF mice bearing the Jak2 V617F mutation show significantly elevated PIEZO1 expression, compared to wild-type controls. Pharmacological activation of PIEZO1 in these cells significantly augments the number of immature CD41 + Mks, and sharply reduces the number of mature CD41 +CD42 + cells and ploidy level. Accordingly, Mks isolated from bone marrow aspirates of JAK2 V617FPMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Additionally, PIEZO1 was overexpressed in 3D culture of human and mouse Mks in stiffer microenvironments as compared to softer ones and liquid medium. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2 V617FPMF patients are rescued upon PIEZO1 inhibition with GsMTx4. Discussion: Our data show that PIEZO1 might serve as a break of Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease. Our finding that inhibition of PIEZO1 activity partially rescues the aberrant Mk phenotype associated with PMF suggests that GsMTx4 holds potential as a therapeutic strategy to mitigate the pathological effects observed in Mk development in PMF patients.

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.

Bone marrow megakaryocytes (MKs) undergo a maturation involving contacts with the microenvironment before extending proplatelets through sinusoids to deliver platelets in the bloodstream. We demonstrated that MKs assemble linear F-actin-enriched podosomes on collagen I fibers. Microscopy analysis evidenced an inverse correlation between the number of dot-like versus linear podosomes over time. Confocal videomicroscopy confirmed that they derived from each-other. This dynamics was dependent on myosin IIA. Importantly, MKs progenitors expressed the Tks4/5 adaptors, displayed a strong gelatinolytic ability and did not form linear podosomes. While maturing, MKs lost Tks expression together with digestive ability. However, those MKs were still able to remodel the matrix by exerting traction on collagen I fibers through a collaboration between GPVI, ß1 integrin and linear podosomes. Our data demonstrated that a change in structure and composition of podosomes accounted for the shift of function during megakaryopoiesis. These data highlight the fact that members of the invadosome family could correspond to different maturation status of the same entity, to adapt to functional responses required by differentiation stages of the cell that bears them.

Platelets are produced by megakaryocytes, specialized cells located in the bone marrow. The possibility to image megakaryocytes in real time and their native environment was described more than 10 years ago and sheds new light on the process of platelet formation. Megakaryocytes extend elongated protrusions, called proplatelets, through the endothelial lining of sinusoid vessels. This paper presents a protocol to simultaneously image in real time fluorescently labeled megakaryocytes in the skull bone marrow and sinusoid vessels. This technique relies on a minor surgery that keeps the skull intact to limit inflammatory reactions. The mouse head is immobilized with a ring glued to the skull to prevent movements from breathing. Using two-photon microscopy, megakaryocytes can be visualized for up to a few hours, enabling the observation of cell protrusions and proplatelets in the process of elongation inside sinusoid vessels. This allows the quantification of several parameters related to the morphology of the protrusions (width, length, presence of constriction areas) and their elongation behavior (velocity, regularity, or presence of pauses or retraction phases). This technique also allows simultaneous recording of circulating platelets in sinusoid vessels to determine platelet velocity and blood flow direction. This method is particularly useful to study the role of genes of interest in platelet formation using genetically modified mice and is also amenable to pharmacological testing (study the mechanisms, evaluating drugs in the treatment of platelet production disorders). It has become an invaluable tool, especially to complement in vitro studies as it is now known that in vivo and in vitro proplatelet formation rely on different mechanisms. It has been shown, for example, that in vitro microtubules are required for proplatelet elongation per se. However, in vivo, they rather serve as a scaffold, elongation being mainly promoted by blood flow forces.

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D culture has thus been developed to better approach the in vivo situation. Megakaryocytes differentiate from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). The BM is one of the softest tissues of the body, confined inside the bone. The bone being poorly extensible at the cell scale, megakaryocytes are concomitantly subjected to a weak stiffness and high confinement. This protocol presents a method for the recovery of mouse lineage negative (Lin-) HSPCs by immuno-magnetic sorting and their differentiation into mature megakaryocytes in a 3D medium composed of methylcellulose. Methylcellulose is non-reactive towards megakaryocytes and its stiffness may be adjusted to that of normal bone marrow or increased to mimic a pathological fibrotic marrow. The process to recover the megakaryocytes for further cell analyses is also detailed in the protocol. Although proplatelet extension is prevented within the 3D milieu, it is described below how to resuspend the megakaryocytes in liquid medium and to quantify their capacity to extend proplatelets. Megakaryocytes grown in 3D hydrogel have a higher capacity to form proplatelets compared to those grown in a liquid milieu. This 3D culture allows i) to differentiate progenitors towards megakaryocytes reaching a higher maturation state, ii) to recapitulate phenotypes that may be observed in vivo but go unnoticed in classical liquid cultures, and iii) to study transduction pathways induced by the mechanical cues provided by a 3D environment.

The last stage of megakaryopoiesis leads to cytoplasmic extensions from mature megakaryocytes, the so-called proplatelets. Much has been learned about the proplatelet formation using in vitro-differentiated megakaryocytes; however, there is an increasing evidence that conventional culture systems do not faithfully recapitulate the differentiation/maturation process that takes places inside the bone marrow. In this manuscript, we present an explant method initially described in 1956 by Thiéry and Bessis to visualize megakaryocytes which have matured in their native environment, thus circumventing potential artifacts and misinterpretations. Fresh bone marrows are collected by flushing the femurs of mice, sliced into 0.5 mm cross sections, and placed in an incubation chamber at 37 °C containing a physiological buffer. Megakaryocytes become gradually visible at the explant periphery and are observed up to 6 hours under an inverted microscope coupled to a video camera. Over time, megakaryocytes change their shape, with some cells having a spherical form and others developing thick extensions or extending many thin proplatelets with extensive branching. Both qualitative and quantitative investigations are carried out. This method has the advantage of being simple, reproducible, and fast as numerous megakaryocytes are present, and classically half of them form proplatelets in 6 hours compared to 4 days for cultured mouse megakaryocytes. In addition to the study of mutant mice, an interesting application of this method is the straightforward evaluation of the pharmacological agents on the proplatelet extension process, without interfering with the differentiation process that may occur in cultures.

The main function of blood platelets is to ensure hemostasis and prevent hemorrhages. The 1011 platelets needed daily are produced in a well-orchestrated process. However, this process is not yet fully understood and in vitro platelet production is still inefficient. Platelets are produced in the bone marrow by megakaryocytes, highly specialized precursor cells that extend cytoplasmic projections called proplatelets (PPTs) through the endothelial barrier of sinusoid vessels. In this Cell Science at a Glance article and the accompanying poster we discuss the mechanisms and pathways involved in megakaryopoiesis and platelet formation processes. We especially address the - still underestimated - role of the microenvironment of the bone marrow, and present recent findings on how PPT extension in vivo differs from that in vitro and entails different mechanisms. Finally, we recapitulate old but recently revisited evidence that - although bone marrow does produce megakaryocytes and PPTs - remodeling and the release of bona fide platelets, mainly occur in the downstream microcirculation.

Platelets are produced by bone marrow megakaryocytes through cytoplasmic protrusions, named native proplatelets (nPPT), into blood vessels. Proplatelets also refer to protrusions observed in megakaryocyte culture (cPPT) that are morphologically different. Contrary to cPPT, the mechanisms of nPPT formation are poorly understood. We show here in living mice that nPPT elongation is in equilibrium between protrusive and retraction forces mediated by myosin-IIA. We also found, using WT and β1-tubulin-deficient mice, that microtubule behavior differs between cPPT and nPPT, being absolutely required in vitro, while less critical in vivo. Remarkably, microtubule depolymerization in myosin-deficient mice did not affect nPPT elongation. We then calculated that blood Stokes'forces may be sufficient to promote nPPT extension, independently of myosin and microtubules. Together, we propose a new mechanism for nPPT extension that might explain contradictions between severely affected cPPT production and moderate platelet count defects in some patients and animal models.

BACKGROUND:Blood platelets are anucleate cell fragments that prevent bleeding and minimize blood vessel injury. They are formed from the cytoplasm of megakaryocytes located in the bone marrow. For successful platelet production, megakaryocyte fragments must pass through the sinusoid endothelial barrier by a cell biology process unique to these giant cells as compared with erythrocytes and leukocytes. Currently, the mechanisms by which megakaryocytes interact and progress through the endothelial cells are not understood, resulting in a significant gap in our knowledge of platelet production.OBJECTIVE:The aim of this study was to investigate how megakaryocytes interact and progress through the endothelial cells of mouse bone marrow sinusoids.METHODS:We used a combination of fluorescence, electron, and three-dimensional microscopy to characterize the cellular events between megakaryocytes and endothelial cells.RESULTS:We identified protrusive, F-actin-based podosome-like structures, called in vivo-MK podosomes, which initiate the formation of pores through endothelial cells. These structures present a collective and spatial organization through their interconnection via a contractile network of actomyosin, essential to regulate the endothelial openings. This ensures proper passage of megakaryocyte-derived processes into the blood circulation to promote thrombopoiesis.CONCLUSION:This study provides novel insight into the in vivo function of podosomes of megakaryocytes with critical importance to platelet production.

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.