Multiomic profiling reveals metabolic alterations mediating aberrant platelet activity and inflammation in myeloproliferative neoplasms.

Platelets from patients with myeloproliferative neoplasms (MPNs) exhibit a hyperreactive phenotype. Here, we found elevated P-selectin exposure and platelet-leukocyte aggregates indicating activation of platelets from essential thrombocythemia (ET) patients. Single-cell RNA-seq analysis of primary samples revealed significant enrichment of transcripts related to platelet activation, mTOR, and oxidative phosphorylation in ET patient platelets. These observations were validated via proteomic profiling. Platelet metabolomics revealed distinct metabolic phenotypes consisting of elevated ATP generation accompanied by increases in the levels of multiple intermediates of the tricarboxylic acid cycle, but lower α-ketoglutarate (α-KG) in MPN patients. Inhibition of PI3K/AKT/mTOR signaling significantly reduced metabolic responses and hyperreactivity in MPN patient platelets, while α-KG supplementation markedly reduced oxygen consumption and ATP generation. Ex vivo incubation of platelets from both MPN patients and Jak2 V617F-knockin mice with α-KG supplementation significantly reduced platelet activation responses. Oral α-KG supplementation of Jak2 V617F mice decreased splenomegaly and reduced hematocrit, monocyte, and platelet counts. Finally, α-KG treatment significantly decreased proinflammatory cytokine secretion from MPN CD14+ monocytes. Our results reveal a previously unrecognized metabolic disorder in conjunction with aberrant PI3K/AKT/mTOR signaling that contributes to platelet hyperreactivity in MPN patients.

N-Acetyl Cysteine Prevents Arterial Thrombosis in a Dose-Dependent Manner In Vitro and in Mice.

BACKGROUND: Platelet-rich thrombi occlude arteries causing fatal infarcts like heart attacks and strokes. Prevention of thrombi by current antiplatelet agents can cause major bleeding. Instead, we propose using N-acetyl cysteine (NAC) to act against the protein VWF (von Willebrand factor), and not platelets, to prevent arterial thrombi from forming. METHODS: NAC was assessed for its ability to prevent arterial thrombosis by measuring platelet accumulation rate and occlusion time using a microfluidic model of arterial thrombosis with human blood. Acute clot formation, clot stability, and tail bleeding were measured in vivo with the murine modified Folts model. The effect of NAC in the murine model after 6 hours was also measured to determine any persistent effects of NAC after it has been cleared from the blood. RESULTS: We demonstrate reduction of thrombi formation following treatment with NAC in vitro and in vivo. Human whole blood treated with 3 or 5 mmol/L NAC showed delayed thrombus formation 2.0× and 3.7× longer than control, respectively (P<0.001). Blood treated with 10 mmol/L NAC did not form an occlusive clot, and no macroscopic platelet aggregation was visible (P<0.001). In vivo, a 400-mg/kg dose of NAC prevented occlusive clots from forming in mice without significantly affecting tail bleeding times. A lower dose of NAC significantly reduced clot stability. Mice given multiple injections showed that NAC has a lasting and cumulative effect on clot stability, even after being cleared from the blood (P<0.001). CONCLUSIONS: Both preclinical models demonstrate that NAC prevents thrombus formation in a dose-dependent manner without significantly affecting bleeding time. This work highlights a new pathway for preventing arterial thrombosis, different from antiplatelet agents, using an amino acid derivative as an antithrombotic therapeutic.

A single F153Sß3 mutation causes constitutive integrin αIIbß3 activation in a variant form of Glanzmann thrombasthenia.

This report identifies a novel variant form of the inherited bleeding disorder Glanzmann thrombasthenia, exhibiting only mild bleeding in a physically active individual. The platelets cannot aggregate ex vivo with physiologic agonists of activation, although microfluidic analysis with whole blood displays moderate ex vivo platelet adhesion and aggregation consistent with mild bleeding. Immunocytometry shows reduced expression of αIIbß3 on quiescent platelets that spontaneously bind/store fibrinogen, and activation-dependent antibodies (ligand-induced binding site-319.4 and PAC-1) report ß3 extension suggesting an intrinsic activation phenotype. Genetic analysis reveals a single F153Sß3 substitution within the ßI-domain from a heterozygous T556C nucleotide substitution of ITGB3 exon 4 in conjunction with a previously reported IVS5(+1)G>A splice site mutation with undetectable platelet messenger RNA accounting for hemizygous expression of S153ß3. F153 is completely conserved among ß3 of several species and all human ß-integrin subunits suggesting that it may play a vital role in integrin structure/function. Mutagenesis of αIIb-F153Sß3 also displays reduced levels of a constitutively activated αIIb-S153ß3 on HEK293T cells. The overall structural analysis suggests that a bulky aromatic, nonpolar amino acid (F,W)153ß3 is critical for maintaining the resting conformation of α2- and α1-helices of the ßI-domain because small amino acid substitutions (S,A) facilitate an unhindered inward movement of the α2- and α1-helices of the ßI-domain toward the constitutively active αIIbß3 conformation, while a bulky aromatic, polar amino acid (Y) hinders such movements and restrains αIIbß3 activation. The data collectively demonstrate that disruption of F153ß3 can significantly alter normal integrin/platelet function, although reduced expression of αIIb-S153ß3 may be compensated by a hyperactive conformation that promotes viable hemostasis.

SVEP1 is an endogenous ligand for the orphan receptor PEAR1.

Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein that causally promotes vascular disease and associates with platelet reactivity in humans. Here, using a human genomic and proteomic approach, we identify a high affinity, disease-relevant, and potentially targetable interaction between SVEP1 and the orphan receptor Platelet and Endothelial Aggregation Receptor 1 (PEAR1). This interaction promotes PEAR1 phosphorylation and disease associated AKT/mTOR signaling in vascular cells and platelets. Mice lacking SVEP1 have reduced platelet activation, and exogenous SVEP1 induces PEAR1-dependent activation of platelets. SVEP1 and PEAR1 causally and concordantly relate to platelet phenotypes and cardiovascular disease in humans, as determined by Mendelian Randomization. Targeting this receptor-ligand interaction may be a viable therapeutic strategy to treat or prevent cardiovascular and thrombotic disease.

Single-cell transcriptional analysis of human endothelial colony-forming cells from patients with low VWF levels.

von Willebrand factor (VWF) plays a key role in normal hemostasis, and deficiencies of VWF lead to clinically significant bleeding. We sought to identify novel modifiers of VWF levels in endothelial colony-forming cells (ECFCs) using single-cell RNA sequencing (scRNA-seq). ECFCs were isolated from patients with low VWF levels (plasma VWF antigen levels between 30 and 50 IU/dL) and from healthy controls. Human umbilical vein endothelial cells were used as an additional control cell line. Cells were characterized for their Weibel Palade body (WPB) content and VWF release. scRNA-seq of all cell lines was performed to evaluate for gene expression heterogeneity and for candidate modifiers of VWF regulation. Candidate modifiers identified by scRNA-seq were further characterized with small-interfering RNA (siRNA) experiments to evaluate for effects on VWF. We observed that ECFCs derived from patients with low VWF demonstrated alterations in baseline WPB metrics and exhibit impaired VWF release. scRNA-seq analyses of these endothelial cells revealed overall decreased VWF transcription, mosaicism of VWF expression, and genes that are differentially expressed in low VWF ECFCs and control endothelial cells (control ECs). An siRNA screen of potential VWF modifiers provided further evidence of regulatory candidates, and 1 such candidate, FLI1, alters the transcriptional activity of VWF. In conclusion, ECFCs from individuals with low VWF demonstrate alterations in their baseline VWF packaging and release compared with control ECs. scRNA-seq revealed alterations in VWF transcription, and siRNA screening identified multiple candidate regulators of VWF.

Platelet α-granules are required for occlusive high-shear-rate thrombosis.

von Willebrand factor (VWF) is essential for the induction of arterial thrombosis. In this study, we investigated the critical role of platelet VWF in occlusive thrombosis formation at high shear in mice that do not express platelet VWF (Nbeal2-/-). Using in silico modeling, in vitro high-shear microfluidics, and an in vivo Folts model of arterial thrombosis we reproduced the platelet dynamics that occur under pathological flow in a stenosed vessel. Computational fluid dynamics (CFDs) simulated local hemodynamics in a stenosis based on arterial geometries. The model predicted shear rates, time course of platelet adhesion, and time to occlusion. These predictions were validated in vitro and in vivo. Occlusive thrombosis developed in wild-type control mice that had normal levels of plasma VWF and platelet VWF in vitro and in vivo. Occlusive thrombosis did not form in the Nbeal2-/- mice that had normal plasma VWF and an absence of platelet VWF. Occlusive thrombosis was corrected in Nbeal2-/- microfluidic assays by the addition of exogenous normal platelets with VWF. Combining model and experimental data, we demonstrated the necessary requirement of platelet VWF in α-granules in forming an occlusive thrombus under high shear. These results could inspire new pharmacological targets specific to pathological conditions and prevent arterial thrombosis.

The CXCR1/2 ligand NAP-2 promotes directed intravascular leukocyte migration through platelet thrombi.

Thrombosis promotes leukocyte infiltration into inflamed tissues, leading to organ injury in a broad range of diseases; however, the mechanisms by which thrombi guide leukocytes to sites of vascular injury remain ill-defined. Using mouse models of endothelial injury (traumatic or ischemia reperfusion), we demonstrate a distinct process of leukocyte recruitment, termed "directed intravascular migration," specifically mediated by platelet thrombi. Single adherent platelets and platelet aggregates stimulated leukocyte shape change at sites of endothelial injury; however, only thrombi were capable of inducing directed intravascular leukocyte migration. Leukocyte recruitment and migration induced by platelet thrombi occurred most prominently in veins but could also occur in arteries following ischemia-reperfusion injury. In vitro studies demonstrated a major role for platelet-derived NAP-2 (CXCL-7) and its CXCR1/2 receptor in regulating leukocyte polarization and motility. In vivo studies demonstrated the presence of an NAP-2 chemotactic gradient within the thrombus body. Pharmacologic blockade of CXCR1/2 as well as genetic deletion of NAP-2 markedly reduced leukocyte shape change and intrathrombus migration. These studies define a distinct process of leukocyte migration that is initiated by homotypic adhesive interactions between platelets, leading to the development of an NAP-2 chemotactic gradient within the thrombus body that guides leukocytes to sites of vascular injury.

High shear-dependent loss of membrane integrity and defective platelet adhesion following disruption of the GPIbα-filamin interaction.

Platelets have evolved a highly specialized membrane skeleton that provides stability to the plasma membrane and facilitates adhesion under high shear stress. The cytoskeletal anchorage of glycoprotein (GP) Ibα plays an important role in regulating the membrane skeleton. However, its role in regulating membrane stability remains unknown. To investigate this role, we have developed a new mouse model that expresses wild-type human GPIbα (hGPIbα(WT)), or a mutant form of human GPIbα that has a selective defect in its ability to bind filamin A and anchor to the membrane skeleton (hGPIbα(FW)-Phe568Ala and Trp570Ala substitutions). Our study demonstrates that the link between platelet GPIb and the cytoskeleton does not alter the intrinsic ligand binding function of GPIbα or the ability of the receptor to stimulate integrin α(IIb)ß(3)-dependent spreading. However, exposure of hGPIbα(FW) platelets to pathologic shear rate levels (5000 to 40,000 s(-1)) leads to the development of unstable membrane tethers, defective platelet adhesion, and loss of membrane integrity, leading to complete disintegration of the platelet cell body. These outcomes suggest that the GPIbα-filamin A interaction not only regulates the architecture of the membrane skeleton, but also maintains the mechanical stability of the plasma membrane under conditions of high shear.