RUNX1-deficient human megakaryocytes demonstrate thrombopoietic and platelet half-life and functional defects.

Heterozygous defects in runt-related transcription factor 1 (RUNX1) are causative of a familial platelet disorder with associated myeloid malignancy (FPDMM). Because RUNX1-deficient animal models do not mimic bleeding disorder or leukemic risk associated with FPDMM, development of a proper model system is critical to understanding the underlying mechanisms of the observed phenotype and to identifying therapeutic interventions. We previously reported an in vitro megakaryopoiesis system comprising human CD34+ hematopoietic stem and progenitor cells that recapitulated the FPDMM quantitative megakaryocyte defect through a decrease in RUNX1 expression via a lentiviral short hairpin RNA strategy. We now show that shRX-megakaryocytes have a marked reduction in agonist responsiveness. We then infused shRX-megakaryocytes into immunocompromised NOD scid gamma (NSG) mice and demonstrated that these megakaryocytes released fewer platelets than megakaryocytes transfected with a nontargeting shRNA, and these platelets had a diminished half-life. The platelets were also poorly responsive to agonists, unable to correct thrombus formation in NSG mice homozygous for a R1326H mutation in von Willebrand Factor (VWFR1326H), which switches the species-binding specificity of the VWF from mouse to human glycoprotein Ibα. A small-molecule inhibitor RepSox, which blocks the transforming growth factor ß1 (TGFß1) pathway and rescued defective megakaryopoiesis in vitro, corrected the thrombopoietic defect, defects in thrombus formation and platelet half-life, and agonist response in NSG/VWFR1326H mice. Thus, this model recapitulates the defects in FPDMM megakaryocytes and platelets, identifies previously unrecognized defects in thrombopoiesis and platelet half-life, and demonstrates for the first time, reversal of RUNX1 deficiency-induced hemostatic defects by a drug.

Defective RAB31-mediated megakaryocytic early endosomal trafficking of VWF, EGFR, and M6PR in RUNX1 deficiency.

Transcription factor RUNX1 is a master regulator of hematopoiesis and megakaryopoiesis. RUNX1 haplodeficiency (RHD) is associated with thrombocytopenia and platelet granule deficiencies and dysfunction. Platelet profiling of our study patient with RHD showed decreased expression of RAB31, a small GTPase whose cell biology in megakaryocytes (MKs)/platelets is unknown. Platelet RAB31 messenger RNA was decreased in the index patient and in 2 additional patients with RHD. Promoter-reporter studies using phorbol 12-myristate 13-acetate-treated megakaryocytic human erythroleukemia cells revealed that RUNX1 regulates RAB31 via binding to its promoter. We investigated RUNX1 and RAB31 roles in endosomal dynamics using immunofluorescence staining for markers of early endosomes (EEs; early endosomal autoantigen 1) and late endosomes (CD63)/multivesicular bodies. Downregulation of RUNX1 or RAB31 (by small interfering RNA or CRISPR/Cas9) showed a striking enlargement of EEs, partially reversed by RAB31 reconstitution. This EE defect was observed in MKs differentiated from a patient-derived induced pluripotent stem cell line (RHD-iMKs). Studies using immunofluorescence staining showed that trafficking of 3 proteins with distinct roles (von Willebrand factor [VWF], a protein trafficked to α-granules; epidermal growth factor receptor; and mannose-6-phosphate) was impaired at the level of EE on downregulation of RAB31 or RUNX1. There was loss of plasma membrane VWF in RUNX1- and RAB31-deficient megakaryocytic human erythroleukemia cells and RHD-iMKs. These studies provide evidence that RAB31 is downregulated in RHD and regulates megakaryocytic vesicle trafficking of 3 major proteins with diverse biological roles. EE defect and impaired vesicle trafficking is a potential mechanism for the α-granule defects observed in RUNX1 deficiency.

RUNX-1 haploinsufficiency causes a marked deficiency of megakaryocyte-biased hematopoietic progenitor cells.

Patients with familial platelet disorder with a predisposition to myeloid malignancy (FPDMM) harbor germline monoallelic mutations in a key hematopoietic transcription factor, RUNX-1. Previous studies of FPDMM have focused on megakaryocyte (Mk) differentiation and platelet production and signaling. However, the effects of RUNX-1 haploinsufficiency on hematopoietic progenitor cells (HPCs) and subsequent megakaryopoiesis remains incomplete. We studied induced pluripotent stem cell (iPSC)-derived HPCs (iHPCs) and Mks (iMks) from both patient-derived lines and a wild-type (WT) line modified to be RUNX-1 haploinsufficient (RUNX-1+/-), each compared with their isogenic WT control. All RUNX-1+/- lines showed decreased iMk yield and depletion of an Mk-biased iHPC subpopulation. To investigate global and local gene expression changes underlying this iHPC shift, single-cell RNA sequencing was performed on sorted FPDMM and control iHPCs. We defined several cell subpopulations in the Mk-biased iHPCs. Analyses of gene sets upregulated in FPDMM iHPCs indicated enrichment for response to stress, regulation of signal transduction, and immune signaling-related gene sets. Immunoblot analyses in FPDMM iMks were consistent with these findings, but also identified augmented baseline c-Jun N-terminal kinase (JNK) phosphorylation, known to be activated by transforming growth factor-ß1 (TGF-ß1) and cellular stressors. These findings were confirmed in adult human CD34+-derived stem and progenitor cells (HSPCs) transduced with lentiviral RUNX1 short hairpin RNA to mimic RUNX-1+/-. In both iHPCs and CD34+-derived HSPCs, targeted inhibitors of JNK and TGF-ß1 pathways corrected the megakaryopoietic defect. We propose that such intervention may correct the thrombocytopenia in patients with FPDMM.

RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival.

G protein-coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor-activating peptide, an increased maximum response to adenosine 5'-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10-/- platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18-/- mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18-/- and RGS10-/-18-/- mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.

New Concepts and Mechanisms of Platelet Activation Signaling.

Upon blood vessel injury, platelets are exposed to adhesive proteins in the vascular wall and soluble agonists, which initiate platelet activation, leading to formation of hemostatic thrombi. Pathological activation of platelets can induce occlusive thrombosis, resulting in ischemic events such as heart attack and stroke, which are leading causes of death globally. Platelet activation requires intracellular signal transduction initiated by platelet receptors for adhesion proteins and soluble agonists. Whereas many platelet activation signaling pathways have been established for many years, significant recent progress reveals much more complex and sophisticated signaling and amplification networks. With the discovery of new receptor signaling pathways and regulatory networks, some of the long-standing concepts of platelet signaling have been challenged. This review provides an overview of the new developments and concepts in platelet activation signaling.

Differential Roles of the NADPH-Oxidase 1 and 2 in Platelet Activation and Thrombosis.

OBJECTIVE: Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here, we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. APPROACH AND RESULTS: NOX1(-/Y) platelets showed selective defects in G-protein-coupled receptor-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide, a glycoprotein VI agonist. In contrast, NOX2(-/-) platelets showed potent inhibition of collagen-related peptide-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1(-/Y) platelets stimulated with thrombin, but not collagen-related peptide, whereas NOX2(-/-) platelets showed reduced ROS generation induced by collagen-related peptide or thrombin. Reduced ROS generation in NOX1/2-deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2, but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2(-/-) mice. Wild-type thrombocytopenic mice injected with NOX2(-/-) platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. CONCLUSIONS: NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/phospholipase Cγ2/calcium signaling pathway.

Signaling-mediated cooperativity between glycoprotein Ib-IX and protease-activated receptors in thrombin-induced platelet activation.

Thrombin-induced cellular response in platelets not only requires protease-activated receptors (PARs), but also involves another thrombin receptor, the glycoprotein Ib-IX complex (GPIb-IX). It remains controversial how thrombin binding to GPIb-IX stimulates platelet responses. It was proposed that GPIb-IX serves as a dock that facilitates thrombin cleavage of protease-activated receptors, but there are also reports suggesting that thrombin binding to GPIb-IX induces platelet activation independent of PARs. Here we show that GPIb is neither a passive thrombin dock nor a PAR-independent signaling receptor. We demonstrate a novel signaling-mediated cooperativity between PARs and GPIb-IX. Low-dose thrombin-induced PAR-dependent cell responses require the cooperativity of GPIb-IX signaling, and conversely, thrombin-induced GPIb-IX signaling requires cooperativity of PARs. This mutually dependent cooperativity requires a GPIb-IX-specific 14-3-3-Rac1-LIMK1 signaling pathway, and activation of this pathway also requires PAR signaling. The cooperativity between GPIb-IX signaling and PAR signaling thus drives platelet activation at low concentrations of thrombin, which are important for in vivo thrombosis.

The interaction of Gα13 with integrin ß1 mediates cell migration by dynamic regulation of RhoA.

Heterotrimeric G protein Gα13 is known to transmit G protein-coupled receptor (GPCR) signals leading to activation of RhoA and plays a role in cell migration. The mechanism underlying the role of Gα13 in cell migration, however, remains unclear. Recently we found that Gα13 interacts with the cytoplasmic domain of integrin ß3 subunits in platelets via a conserved ExE motif. Here we show that a similar direct interaction between Gα13 and the cytoplasmic domain of the integrin ß1 subunit plays a critical role in ß1-dependent cell migration. Point mutation of either glutamic acid in the Gα13-binding (767)EKE motif in ß1 or treatment with a peptide derived from the Gα13-binding sequence of ß1 abolished Gα13-ß1 interaction and inhibited ß1 integrin-dependent cell spreading and migration. We further show that the Gα13-ß1 interaction mediates ß1 integrin-dependent Src activation and transient RhoA inhibition during initial cell adhesion, which is in contrast to the role of Gα13 in mediating GPCR-dependent RhoA activation. These data indicate that Gα13 plays dynamic roles in both stimulating RhoA via a GPCR pathway and inhibiting RhoA via an integrin signaling pathway. This dynamic regulation of RhoA activity is critical for cell migration on ß1 integrin ligands.

Targeting integrin and integrin signaling in treating thrombosis.

The critical roles of integrins in thrombosis have enabled the successful development and clinical use of the first generation of integrin antagonists as represented by abciximab (Reopro), eptifibatide (Integrilin), and tirofiban (Aggrastat). These integrin αIIbß3 antagonists are not only potent antithrombotics but also have significant side effects. In particular, their induction of ligand-induced integrin conformational changes is associated with thrombocytopenia. Increased bleeding risk prevents integrin antagonists from being used at higher doses and in patients at risk for bleeding. To address the ligand-induced conformational changes caused by current integrin antagonists, compounds that minimally induce conformational changes in integrin αIIbß3 have been developed. Recent studies on the mechanisms of integrin signaling suggest that selectively targeting integrin outside-in signaling mechanisms allows for potent inhibition of thrombosis, while maintaining hemostasis in animal models.

Sex-dependent regulation of cytochrome P450 family members Cyp1a1, Cyp2e1, and Cyp7b1 by methylation of DNA.

Sexual differences are only partially attributable to hormones. Cultured male or female cells, even from embryos before sexual differentiation, differ in gene expression and sensitivity to toxins, and these differences persist in isolated primary cells. Male and female cells from Swiss Webster CWF mice manifest sex-distinct patterns of DNA methylation for X-ist and for cytochrome P450 (CYP; family members 1a1, 2e1m, and 7b1. Dnmt3l is differentially expressed but not differentially methylated, and Gapdh is neither differentially methylated nor expressed. CYP family genes differ in expression in whole tissue homogenates and cell cultures, with female Cyp expression 2- to 355-fold higher and Dnmt3l 12- to 32-fold higher in males. DNA methylation in the promoters of these genes is sex dimorphic; reducing methylation differences reduces to 1- to 6-fold differences in the expression of these genes. Stress or estradiol alters both methylation and gene expression. We conclude that different methylation patterns partially explain the sex-based differences in expression of CYP family members and X-ist, which potentially leads to inborn differences between males and females and their different responses to chronic and acute changes. Sex-differential methylation may have medical effects.

LIM kinase-1 selectively promotes glycoprotein Ib-IX-mediated TXA2 synthesis, platelet activation, and thrombosis.

Current antithrombotic drugs have an adverse effect on bleeding, highlighting the need for new molecular targets for developing antithrombotic drugs that minimally affect hemostasis. Here we show that LIMK1(-/-) mice have defective arterial thrombosis in vivo but do not differ from wild-type mice with respect to bleeding time. LIMK1(-/-) mice show a selective defect in platelet activation induced through the von Willebrand Factor (VWF) receptor, the glycoprotein Ib-IX-V complex (GPIb-IX), but not by GPIb-IX-independent platelet agonists. In fact, LIMK1(-/-) platelets show an enhanced reaction to certain GPIb-IX-independent agonists. The defect of LIMK1(-/-) platelets in GPIb-IX-mediated platelet activation is attributed to a selective inhibition in VWF/GPIb-IX-induced phosphorylation of cytosolic phospholipase A2 (cPLA2) and consequent thromboxane A2 (TXA2) production. Supplementing a TXA2 analog, U46619, corrected the defect of LIMK1(-/-) platelets in VWF-induced stable platelet adhesion. Although LIMK1(-/-) platelets also showed reduced actin polymerization after GPIb-IX-mediated platelet aggregation, actin polymerization inhibitors did not reduce TXA2 generation, but rather accelerated platelet aggregation, suggesting that the role of LIMK1 in GPIb-mediated platelet activation is independent of actin polymerization. Thus, LIMK1 plays a novel role in selectively mediating GPIb-IX-dependent TXA2 synthesis and thrombosis and represents a potential target for developing antithrombotic drugs with minimal bleeding side effect.

Sex of the cell dictates its response: differential gene expression and sensitivity to cell death inducing stress in male and female cells.

Sexual dimorphisms are typically attributed to the hormonal differences arising once sex differentiation has occurred. However, in some sexually dimorphic diseases that differ in frequency but not severity, the differences cannot be logically connected to the sex hormones. Therefore, we asked whether any aspect of sexual dimorphism could be attributed to chromosomal rather than hormonal differences. Cells taken from mice at d 10.5 postconception (PC) before sexual differentiation, at d 17.5 PC after the first embryonic assertion of sexual hormones, and at postnatal day 17 (puberty) were cultured and exposed to 400 microM ethanol or 20 microM camptothecin or to infection with influenza A virus (multiplicity of infection of 5). The results showed that untreated male and female cells of the same age grew at similar rates and manifested similar morphology. However, they responded differently to the applied stressors, even before the production of fetal sex hormones. Furthermore, microarray and qPCR analyses of the whole 10.5 PC embryos also revealed differences in gene expression between male and female tissues. Likewise, the exposure of cells isolated from fetuses and adolescent mice to the stressors and/or sex hormones yielded expression patterns that reflected chromosomal sex, with ethanol feminizing male cells and masculinizing female cells. We conclude that cells differ innately according to sex irrespective of their history of exposure to sex hormones. These differences may have consequences in the course of sexually dimorphic diseases and their therapy.