identify APOLD1 as an important regulator of vascular homeostasis and raise the need to consider testing of endothelial cell function in patients with inherited bleeding disorders without apparent platelet or coagulation defects

Oncogenic mutations in PIK3CA, encoding p110α-PI3K, are a common cause of venous and lymphatic malformations. Vessel type–specific disease pathogenesis is poorly understood, hampering development of efficient therapies. Here, we reveal a new immune-interacting subtype of Ptx3-positive dermal lymphatic capillary endothelial cells (iLECs) that recruit pro-lymphangiogenic macrophages to promote progressive lymphatic overgrowth. Mouse model of Pik3caH1047R-driven vascular malformations showed that proliferation was induced in both venous and lymphatic ECs but sustained selectively in LECs of advanced lesions. Single-cell transcriptomics identified the iLEC population, residing at lymphatic capillary terminals of normal vasculature, that was expanded in Pik3caH1047R mice. Expression of pro-inflammatory genes, including monocyte/macrophage chemokine Ccl2, in Pik3caH1047R-iLECs was associated with recruitment of VEGF-C–producing macrophages. Macrophage depletion, CCL2 blockade, or anti-inflammatory COX-2 inhibition limited Pik3caH1047R-driven lymphangiogenesis. Thus, targeting the paracrine crosstalk involving iLECs and macrophages provides a new therapeutic opportunity for lymphatic malformations. Identification of iLECs further indicates that peripheral lymphatic vessels not only respond to but also actively orchestrate inflammatory processes.

Vascular homeostasis is impaired in various diseases thereby contributing to the progression of their underlying pathologies. The endothelial immediate early gene Apolipoprotein L domain-containing 1 (APOLD1) helps to regulate endothelial function. However, its precise role in endothelial cell biology remains unclear. We have localized APOLD1 to endothelial cell contacts and to Weibel-Palade bodies (WPB) where it associates with von Willebrand factor (VWF) tubules. Silencing of APOLD1 in primary human endothelial cells disrupted the cell junction-cytoskeletal interface, thereby altering endothelial permeability accompanied by spontaneous release of WPB contents. This resulted in an increased presence of WPB cargoes, notably VWF and angiopoietin-2 in the extracellular medium. Autophagy flux, previously recognized as an essential mechanism for the regulated release of WPB, was impaired in the absence of APOLD1. In addition, we report APOLD1 as a candidate gene for a novel inherited bleeding disorder across three generations of a large family in which an atypical bleeding diathesis was associated with episodic impaired microcirculation. A dominant heterozygous nonsense APOLD1:p.R49* variant segregated to affected family members. Compromised vascular integrity resulting from an excess of plasma angiopoietin-2, and locally impaired availability of VWF may explain the unusual clinical profile of APOLD1:p.R49* patients. In summary, our findings identify APOLD1 as an important regulator of vascular homeostasis and raise the need to consider testing of endothelial cell function in patients with inherited bleeding disorders without apparent platelet or coagulation defects.

BACKGROUND: MAGT1 (magnesium transporter 1) is a subunit of the oligosaccharide protein complex with thiol-disulfide oxidoreductase activity, supporting the process of N-glycosylation. MAGT1 deficiency was detected in human patients with X-linked immunodeficiency with magnesium defect syndrome and congenital disorders of glycosylation, resulting in decreased cation responses in lymphocytes, thereby inhibiting the immune response against viral infections. Curative hematopoietic stem cell transplantation of patients with X-linked immunodeficiency with magnesium defect causes fatal bleeding and thrombotic complications. METHODS: We studied the role of MAGT1 deficiency in platelet function in relation to arterial thrombosis and hemostasis using several in vitro experimental settings and in vivo models of arterial thrombosis and transient middle cerebral artery occlusion model of ischemic stroke. RESULTS: MAGT1-deficient mice ( Magt1 −/y ) displayed accelerated occlusive arterial thrombus formation in vivo, a shortened bleeding time, and profound brain damage upon focal cerebral ischemia. These defects resulted in increased calcium influx and enhanced second wave mediator release, which further reinforced platelet reactivity and aggregation responses. Supplementation of MgCl 2 or pharmacological blockade of TRPC6 (transient receptor potential cation channel, subfamily C, member 6) channel, but not inhibition of store-operated calcium entry, normalized the aggregation responses of Magt1 −/y platelets to the control level. GP (glycoprotein) VI activation of Magt1 −/y platelets resulted in hyperphosphorylation of Syk (spleen tyrosine kinase), LAT (linker for activation of T cells), and PLC (phospholipase C) γ2, whereas the inhibitory loop regulated by PKC (protein kinase C) was impaired. A hyperaggregation response to the GPVI agonist was confirmed in human platelets isolated from a MAGT1-deficient (X-linked immunodeficiency with magnesium defect) patient. Haploinsufficiency of TRPC6 in Magt1 −/y mice could normalize GPVI signaling, platelet aggregation, and thrombus formation in vivo. CONCLUSIONS: These results suggest that MAGT1 and TRPC6 are functionally linked. Therefore, deficiency or impaired functionality of MAGT1 could be a potential risk factor for arterial thrombosis and stroke.

Humanized mouse models and mouse-adapted SARS-CoV-2 virus are increasingly used to study COVID-19 pathogenesis, so it is important to learn where the SARS-CoV-2 receptor ACE2 is expressed. Here we mapped ACE2 expression during mouse postnatal development and in adulthood. Pericytes in the CNS, heart, and pancreas express ACE2 strongly, as do perineurial and adrenal fibroblasts, whereas endothelial cells do not at any location analyzed. In a number of other organs, pericytes do not express ACE2, including in the lung where ACE2 instead is expressed in bronchial epithelium and alveolar type II cells. The onset of ACE2 expression is organ specific: in bronchial epithelium already at birth, in brain pericytes before, and in heart pericytes after postnatal day 10.5. Establishing the vascular localization of ACE2 expression is central to correctly interpret data from modeling COVID-19 in the mouse and may shed light on the cause of vascular COVID-19 complications.

The process of platelet production has so far been understood to be a 2-stage process: megakaryocyte maturation from hematopoietic stem cells followed by proplatelet formation, with each phase regulating the peripheral blood platelet count. Proplatelet formation releases into the bloodstream beads-on-a-string preplatelets, which undergo fission into mature platelets. For the first time, we show that preplatelet maturation is a third, tightly regulated, critical process akin to cytokinesis that regulates platelet count. We show that deficiency in cytokine receptor-like factor 3 (CRLF3) in mice leads to an isolated and sustained 25% to 48% reduction in the platelet count without any effect on other blood cell lineages. We show that Crlf3(-/-) preplatelets have increased microtubule stability, possibly because of increased microtubule glutamylation via the interaction of CRLF3 with key members of the Hippo pathway. Using a mouse model of JAK2 V617F essential thrombocythemia, we show that a lack of CRLF3 leads to long-term lineage-specific normalization of the platelet count. We thereby postulate that targeting CRLF3 has therapeutic potential for treatment of thrombocythemia.

Vascular malformations are congenital, chronically debilitating diseases. Somatic oncogenic mutations in PIK3CA , encoding p110α-PI3K, specifically cause venous and lymphatic malformations (LM), yet the basis of vessel type-restricted disease manifestation is unknown. Here we report endothelial subtype-specific responses to the common causative Pik3ca H1047R mutation, and reveal a new immunoregulatory subtype of dermal lymphatic capillary endothelial cells (iLECs) as a driver of LM pathology. Mouse model of Pik3ca H1047R -driven vascular malformations showed that cell proliferation was a common early response of venous and lymphatic ECs to oncogenic Pik3ca , but sustained selectively in LECs of advanced lesions. Lymphatic overgrowth was associated with increased pro-inflammatory cytokine levels and pro-lymphangiogenic myeloid cell infiltrate. Single-cell transcriptomics revealed a new LEC subtype at capillary terminals, characterized by the expression of immunoregulatory genes. Selective expansion and activation of iLECs in the Pik3ca H1047R mice was evidenced by proliferation and upregulation of pro-inflammatory genes. Importantly, macrophage depletion or anti-inflammatory COX-2 inhibition limited Pik3ca H1047R -driven lymphangiogenesis. This provides a therapeutic target for LM and suggests a paracrine crosstalk in which LEC-autonomous oncogenic Pik3ca signaling induces immune activation that in turn sustains pathological lymphangiogenesis. Identification of iLECs indicates that peripheral lymphatic vessels not only respond to inflammation but also actively orchestrate the immune response.

Vascular endothelial growth factor C (VEGF-C) induces lymphangiogenesis via VEGF receptor 3 (VEGFR3), which is encoded by the most frequently mutated gene in human primary lymphedema. Angiopoietins (Angs) and their Tie receptors regulate lymphatic vessel development, and mutations of the ANGPT2 gene were recently found in human primary lymphedema. However, the mechanistic basis of Ang2 activity in lymphangiogenesis is not fully understood. Here, we used gene deletion, blocking Abs, transgene induction, and gene transfer to study how Ang2, its Tie2 receptor, and Tie1 regulate lymphatic vessels. We discovered that VEGF-C–induced Ang2 secretion from lymphatic endothelial cells (LECs) was involved in full Akt activation downstream of phosphoinositide 3 kinase (PI3K). Neonatal deletion of genes encoding the Tie receptors or Ang2 in LECs, or administration of an Ang2-blocking Ab decreased VEGFR3 presentation on LECs and inhibited lymphangiogenesis. A similar effect was observed in LECs upon deletion of the PI3K catalytic p110α subunit or with small-molecule inhibition of a constitutively active PI3K located downstream of Ang2. Deletion of Tie receptors or blockade of Ang2 decreased VEGF-C–induced lymphangiogenesis also in adult mice. Our results reveal an important crosstalk between the VEGF-C and Ang signaling pathways and suggest new avenues for therapeutic manipulation of lymphangiogenesis by targeting Ang2/Tie/PI3K signaling.

Inherited platelet disorders resulting from platelet function defects and a normal platelet count cause a moderate or severe bleeding diathesis. Since the description of Glanzmann thrombasthenia resulting from defects of ITGA2B and ITGB3, new inherited platelet disorders have been discovered, facilitated by the use of high throughput sequencing and genomic analyses. Defects of RASGRP2 and FERMT3 responsible for severe bleeding syndromes and integrin activation have illustrated the critical role of signaling molecules. Important are mutations of P2RY12 encoding the major ADP receptor causal for an inherited platelet disorder with inheritance characteristics that depend on the variant identified. Interestingly, variants of GP6 encoding the major subunit of the collagen receptor GPVI/FcRγ associate only with mild bleeding. The numbers of genes involved in dense granule defects including Hermansky-Pudlak and Chediak Higashi syndromes continue to progress and are updated. The ANO6 gene encoding a Ca2+-activated ion channel required for phospholipid scrambling is responsible for the rare Scott syndrome and decreased procoagulant activity. A novel EPHB2 defect in a familial bleeding syndrome demonstrates a role for this tyrosine kinase receptor independent of the classical model of its interaction with ephrins. Such advances highlight the large diversity of variants affecting platelet function but not their production, despite the difficulties in establishing a clear phenotype when few families are affected. They have provided insights into essential pathways of platelet function and have been at the origin of new and improved therapies for ischemic disease. Nevertheless, many patients remain without a diagnosis and requiring new strategies that are now discussed.

The lymphatic vasculature is emerging as a multifaceted regulator of tissue homeostasis and regeneration. Lymphatic vessels drain fluid, macromolecules, and immune cells from peripheral tissues to lymph nodes (LNs) and the systemic circulation. Their recently uncovered functions extend beyond drainage and include direct modulation of adaptive immunity and paracrine regulation of organ growth. The developmental mechanisms controlling lymphatic vessel growth have been described with increasing precision. It is less clear how the essential functional features of lymphatic vessels are established and maintained. We discuss the mechanisms that maintain lymphatic vessel integrity in adult tissues and control vessel repair and regeneration. This knowledge is crucial for understanding the pathological vessel changes that contribute to disease, and provides an opportunity for therapy development.

Endothelial integrity is vital for homeostasis and adjusted to tissue demands. Although fluid uptake by lymphatic capillaries is a critical attribute of the lymphatic vasculature, the barrier function of collecting lymphatic vessels is also important by ensuring efficient fluid drainage as well as lymph node delivery of antigens and immune cells. Here, we identified the transmembrane ligand EphrinB2 and its receptor EphB4 as critical homeostatic regulators of collecting lymphatic vessel integrity. Conditional gene deletion in mice revealed that EphrinB2/EphB4 signalling is dispensable for blood endothelial barrier function, but required for stabilization of lymphatic endothelial cell (LEC) junctions in different organs of juvenile and adult mice. Studies in primary human LECs further showed that basal EphrinB2/EphB4 signalling controls junctional localisation of the tight junction protein CLDN5 and junction stability via Rac1/Rho-mediated regulation of cytoskeletal contractility. EphrinB2/EphB4 signalling therefore provides a potential therapeutic target to selectively modulate lymphatic vessel permeability and function.

Accumulating clinical observations implicate vascular inflammation as an underlying cause of coagulopathy in severely ill COVID-19 patients and it was recently suggested that SARS-CoV-2 virus particles infect endothelial cells. Here, we show that endothelial cells do not express angiotensin-converting enzyme-2 (ACE2), the SARS-CoV-2 receptor. Instead, pericytes and microvascular smooth muscle cells express ACE2 in an organotypic manner. Pericyte deficiency leads to increased endothelial expression and release of Von Willebrand factor and intravascular platelet and fibrin aggregation, suggesting that pericytes limit endothelial pro-thrombotic responses. That pericytes and not endothelial cells express ACE2 may provide important clues to the pathology of COVID-19, as pericytes are normally shielded behind an endothelial barrier and may get infected only when this barrier is compromised by COVID-19 risk factors.

Platelet aggregate formation is a multistep process involving receptor-mediated, as well as biomechanical, signaling cascades, which are highly dependent on actin dynamics. We have previously shown that actin depolymerizing factor (ADF)/n-cofilin and Twinfilin 2a, members of the ADF homology (ADF-H) protein family, have distinct roles in platelet formation and function. Coactosin-like 1 (Cotl1) is another ADF-H protein that binds actin and was also shown to enhance biosynthesis of pro-inflammatory leukotrienes (LT) in granulocytes. Here, we generated mice lacking Cotl1 in the megakaryocyte lineage (Cotl1−/−) to investigate its role in platelet production and function. Absence of Cotl1 had no impact on platelet counts, platelet activation or cytoskeletal reorganization under static conditions in vitro. In contrast, Cotl1 deficiency markedly affected platelet aggregate formation on collagen and adhesion to immobilized von Willebrand factor at high shear rates in vitro, pointing to an impaired function of the platelet mechanoreceptor glycoprotein (GP) Ib. Furthermore, Cotl1−/−platelets exhibited increased deformability at high shear rates, indicating that the GPIb defect may be linked to altered biomechanical properties of the deficient cells. In addition, we found that Cotl1 deficiency markedly affected platelet LT biosynthesis. Strikingly, exogenous LT addition restored defective aggregate formation of Cotl1−/− platelets at high shear in vitro, indicating a critical role of platelet-derived LT in thrombus formation. In vivo, Cotl1 deficiency translated into prolonged tail bleeding times and protection from occlusive arterial thrombus formation. Together, our results show that Cotl1 in platelets is an integrator of biomechanical and LT signaling in hemostasis and thrombosis.

Maintenance of blood vessel integrity is crucial for vascular homeostasis and is mainly controlled at the level of endothelial cell (EC) junctions. Regulation of endothelial integrity has largely been investigated in the mature quiescent vasculature. Less is known about how integrity is maintained during vascular growth and remodeling involving extensive junctional reorganization. Here, we show that embryonic mesenteric blood vascular remodeling is associated with a transient loss of venous integrity and concomitant extravasation of red blood cells (RBCs), followed by their clearance by the developing lymphatic vessels. In wild-type mouse embryos, we observed activated platelets extending filopodia at sites of inter-EC gaps. In contrast, embryos lacking the activatory C-type lectin domain family 1, member b (CLEC1B) showed extravascular platelets and an excessive number of RBCs associated with and engulfed by the first lymphatic EC clusters that subsequently form lumenized blood-filled vessels connecting to the lymphatic system. These results uncover novel functions of platelets in maintaining venous integrity and lymphatic vessels in clearing extravascular RBCs during developmental remodeling of the mesenteric vasculature. They further provide insight into how vascular abnormalities characterized by blood-filled lymphatic vessels arise.

Objective— TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg 2+ and Ca 2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown. Approach and Results— Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice ( Trpm7 R/R ) causes a marked signaling defect in platelets. Trpm7 R/R platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca 2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C γ2 and β3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca 2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo. Conclusions— Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca 2+ entry in platelets. The protection of Trpm7 R/R mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target.

Key Points Profilin 1–mediated cytoskeletal dynamics regulate platelet β1- and β3-integrin function and turnover. Profilin 1 deficiency in platelets impairs hemostasis and results in a marked protection from arterial thrombosis.

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V.

Objective— TRPM7 (transient receptor potential melastatin-like 7 channel) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg 2+ and Ca 2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown. Approach and Results— Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice ( Trpm7 R/R ) causes a marked signaling defect in platelets. Trpm7 R/R platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca 2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of spleen tyrosine kinase and phospholipase C γ2 and β3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca 2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo. Conclusions— Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca 2+ entry in platelets. The protection of Trpm7 R/R mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target.

Regulated reorganization of the actin cytoskeleton is a prerequisite for proper platelet production and function. Consequently, defects in proteins controlling actin dynamics have been associated with platelet disorders in humans and mice. Twinfilin 2a (Twf2a) is a small actin-binding protein that inhibits actin filament assembly by sequestering actin monomers and capping filament barbed ends. Moreover, Twf2a binds heterodimeric capping proteins, but the role of this interaction in cytoskeletal dynamics has remained elusive. Even though Twf2a has pronounced effects on actin dynamics in vitro, only little is known about its function in vivo. Here, we report that constitutive Twf2a-deficient mice (Twf2a-/-) display mild macrothrombocytopenia due to a markedly accelerated platelet clearance in the spleen. Twf2a-/- platelets showed enhanced integrin activation and α-granule release in response to stimulation of (hem) immunoreceptor tyrosine-based activation motif (ITAM) and G-protein-coupled receptors, increased adhesion and aggregate formation on collagen I under flow, and accelerated clot retraction and spreading on fibrinogen. In vivo, Twf2a deficiency resulted in shortened tail bleeding times and faster occlusive arterial thrombus formation. The hyperreactivity of Twf2a-/- platelets was attributed to enhanced actin dynamics, characterized by an increased activity of n-cofilin and profilin 1, leading to a thickened cortical cytoskeleton and hence sustained integrin activation by limiting calpain-mediated integrin inactivation. In summary, our results reveal the first in vivo functions of mammalian Twf2a and demonstrate that Twf2a-controlled actin rearrangements dampen platelet activation responses in a n-cofilin- and profilin 1-dependent manner, thereby indirectly regulating platelet reactivity and half-life in mice.

Platelets are continuously produced from megakaryocytes (MK) in the bone marrow by a cytoskeleton-driven process of which the molecular regulation is not fully understood. As revealed in this thesis, MK/ platelet-specific Profilin1 (Pfn1) deficiency results in micro- thrombocytopenia, a hallmark of the Wiskott-Aldrich syndrome (WAS) in humans, due to accelerated platelet turnover and premature platelet release into the bone marrow. Both Pfn1-deficient mouse platelets and platelets isolated from WAS patients contained abnormally organized and hyper-stable microtubules. These results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients. In contrast, Twinfilin2a (Twf2a) was established as a central regulator of platelet reactivity and turnover. Twf2a-deficient mice revealed an age-dependent macrothrombocytopenia that could be explained by a markedly decreased platelet half-life, likely due to the pronounced hyper-reactivity of \(Twf2a^{-/-}\) platelets. The latter was characterized by sustained integrin acti- vation and thrombin generation in vitro that translated into accelerated thrombus formation in vivo. To further elucidate mechanisms of integrin activation, Rap1-GTP-interacting adaptor molecule (RIAM)-null mice were generated. Despite the proposed critical role of RIAM for platelet integrin activation, no alterations in this process could be found and it was concluded that RIAM is dispensable for the activation of β1 and β3 integrins, at least in platelets. These findings change the current mechanistic understanding of platelet integrin activation. Outside-in signaling by integrins and other surface receptors was supposed to regulate MK migration, but also the temporal and spatial formation of proplatelet protrusions. In this the- sis, phospholipase D (PLD) was revealed as critical regulator of actin dynamics and podo- some formation in MKs. Hence, the unaltered platelet counts and production in \(Pld1/2^{-/-}\) mice and the absence of a premature platelet release in the bone marrow of \(Itga2^{-/-}\) mice question the role of podosomes in platelet production and raise the need to reconsider the proposed inhibitory signaling by α2β1 integrins on proplatelet formation. Non-muscle myosin IIA (NMMIIA) has been implicated as a downstream effector of the in- hibitory signals transmitted via α2β1 integrins. Besides Rho-GTPase signaling, also \(Mg^{2+}\) and transient receptor potential melastatin-like 7 (TRPM7) channel α-kinase are known regulators of NMMIIA activity. In this thesis, TRPM7 was identified as major regulator of \(Mg^{2+}\) homeostasis in MKs and platelets. Furthermore, decreased \([Mg^{2+}]_i\) led to deregulated NMMIIA activity and altered cytoskeletal dynamics that impaired thrombopoiesis and resulted in macrothrombocytopenia in humans and mice.