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Many common and rare variants associated with hematologic traits have been discovered through imputation on large-scale reference panels. However, the majority of genome-wide association studies (GWASs) have been conducted in Europeans, and determining causal variants has proved challenging. We performed a GWAS of total leukocyte, neutrophil, lymphocyte, monocyte, eosinophil, and basophil counts generated from 109,563,748 variants in the autosomes and the X chromosome in the Trans-Omics for Precision Medicine (TOPMed) program, which included data from 61,802 individuals of diverse ancestry. We discovered and replicated 7 leukocyte trait associations, including (1) the association between a chromosome X, pseudo-autosomal region (PAR), noncoding variant located between cytokine receptor genes (CSF2RA and CLRF2) and lower eosinophil count; and (2) associations between single variants found predominantly among African Americans at the S1PR3 (9q22.1) and HBB (11p15.4) loci and monocyte and lymphocyte counts, respectively. We further provide evidence indicating that the newly discovered eosinophil-lowering chromosome X PAR variant might be associated with reduced susceptibility to common allergic diseases such as atopic dermatitis and asthma. Additionally, we found a burden of very rare FLT3 (13q12.2) variants associated with monocyte counts. Together, these results emphasize the utility of whole-genome sequencing in diverse samples in identifying associations missed by European-ancestry-driven GWASs.

Background There is interest in deriving megakaryocytes (MKs) from pluripotent stem cells (iPSC) for biological studies. We previously found that genomic structural integrity and genotype concordance is maintained in iPSC-derived MKs. Objective To establish a comprehensive dataset of genes and proteins expressed in iPSC-derived MKs. Methods iPSCs were reprogrammed from peripheral blood mononuclear cells (MNCs) and MKs were derived from the iPSCs in 194 healthy European American and African American subjects. mRNA was isolated and gene expression measured by RNA sequencing. Protein expression was measured in 62 of the subjects using mass spectrometry. Results and conclusions MKs expressed genes and proteins known to be important in MK and platelet function and demonstrated good agreement with previous studies in human MKs derived from CD34+ progenitor cells. The percent of cells expressing the MK markers CD41 and CD42a was consistent in biological replicates, but variable across subjects, suggesting that unidentified subject-specific factors determine differentiation of MKs from iPSCs. Gene and protein sets important in platelet function were associated with increasing expression of CD41/42a, while those related to more basic cellular functions were associated with lower CD41/42a expression. There was differential gene expression by the sex and race (but not age) of the subject. Numerous genes and proteins were highly expressed in MKs but not known to play a role in MK or platelet function; these represent excellent candidates for future study of hematopoiesis, platelet formation, and/or platelet function.

Summary Whole-genome sequencing (WGS), a powerful tool for detecting novel coding and non-coding disease-causing variants, has largely been applied to clinical diagnosis of inherited disorders. Here we leveraged WGS data in up to 62,653 ethnically diverse participants from the NHLBI Trans-Omics for Precision Medicine (TOPMed) program and assessed statistical association of variants with seven red blood cell (RBC) quantitative traits. We discovered 14 single variant-RBC trait associations at 12 genomic loci, which have not been reported previously. Several of the RBC trait-variant associations (RPN1, ELL2, MIDN, HBB, HBA1, PIEZO1, and G6PD) were replicated in independent GWAS datasets imputed to the TOPMed reference panel. Most of these discovered variants are rare/low frequency, and several are observed disproportionately among non-European Ancestry (African, Hispanic/Latino, or East Asian) populations. We identified a 3 bp indel p.Lys2169del (g.88717175_88717177TCT[4]) (common only in the Ashkenazi Jewish population) of PIEZO1, a gene responsible for the Mendelian red cell disorder hereditary xerocytosis (MIM: 194380 ), associated with higher mean corpuscular hemoglobin concentration (MCHC). In stepwise conditional analysis and in gene-based rare variant aggregated association analysis, we identified several of the variants in HBB, HBA1, TMPRSS6, and G6PD that represent the carrier state for known coding, promoter, or splice site loss-of-function variants that cause inherited RBC disorders. Finally, we applied base and nuclease editing to demonstrate that the sentinel variant rs112097551 (nearest gene RPN1) acts through a cis-regulatory element that exerts long-range control of the gene RUVBL1 which is essential for hematopoiesis. Together, these results demonstrate the utility of WGS in ethnically diverse population-based samples and gene editing for expanding knowledge of the genetic architecture of quantitative hematologic traits and suggest a continuum between complex trait and Mendelian red cell disorders.

Introduction Genetic differences between races have been hypothesized to contribute to differences in outcome from acute coronary syndromes (ACS). Our objective was to assess racial differences in genetic variations in the platelet serotonin transporter (5HTT) and receptor in patients with ACS. Materials and methods 127 consecutive patients, African Americans (AA) = 27; Caucasian (C) =100, admitted with ACS were evaluated for platelet function by serotonin (5HT) induced platelet activation. All patients were genotyped for two polymorphisms in the serotonin-transporter-linked polymorphic region (5-HTTLPR) S/L and LG/LA and one polymorphism of the serotonin 2A receptor (5-HT2A, T102C) gene. All patients were followed for major and minor adverse cardiac events at 12 months. Results AA when compared to C had a lower prevalence of the HTTLPR S allele (21% vs 45%, p = 0.0003) and a higher prevalence of the LG allele (24% vs 4.5%, p = 0.0001). Allelic frequency of the 5-HT2A T102C allele was not significantly different between the races. Platelet activation was lower in AA compared to C, median EC50 5HT was 12.08 μg vs 2.14 μg (p = 0.001). The 5-HTTLPR and the 5-HT2A polymorphisms were not associated with platelet functional responses to serotonin. There were no significant differences in major or minor adverse cardiac events in patients with serotonin transporter or receptor polymorphisms. Conclusion We found a lower prevalence of the S allele and a higher prevalence of the G allele in AA with ACS. We also found decreased platelet activation in AA which did not correlate with serotonin-related platelet polymorphisms. It is unclear if other contributing factors may explain these platelet functional differences.

Objective High-quality prospective trials of hemostatic “rescue” therapy to control massive bleeding in cardiac surgery are lacking. Wide variability in the care of patients with severe bleeding following cardiopulmonary bypass has precluded accurate comparison of treatment groups in previous studies. This study identified the use of a management protocol for early identification and uniform treatment of patients with massive bleeding for application in future trials of hemostatic rescue agents. Design A prospective, nonblinded, interventional feasibility study. Setting A university teaching hospital. Participants Forty-three adult patients undergoing complex cardiac surgery. Interventions Study participants undergoing high-risk cardiac surgery received standardized treatment in accordance with a bleeding management protocol. Measurements and Main Results Twenty-seven patients (63%) had severe bleeding following heparin reversal and received conventional hemostatic resuscitation per protocol. Six patients had massive refractory bleeding. Compliance with protocol tasks was≥90% in 4 of 5 categories (anticoagulation, hemostasis scoring, recording blood loss, protocol transfusion) with the exception being submission of laboratory samples (76%). Measured bleeding rates (mL/h) following heparin reversal were clearly differentiated in those with hemostasis scores≥3 compared to those with scores≤2 (1,420±957 v 147±96; p Conclusions Adherence to a management protocol for massive bleeding is feasible and allows for homogenous treatment of patients before study arm randomization in future “rescue” therapy trials. The authors’ protocol allowed for prompt and accurate identification of patients with severe bleeding refractory to conventional therapy. This review resolved several key barriers in the design of severe bleeding management trials.

Most ischemic strokes in humans are caused by ruptured arterial atheroma, which activate platelets and produce thrombi that occlude cerebral vessels.To simulate these events, we threaded a catheter through the internal carotid artery toward the middle cerebral artery (MCA) orifice and injected collagen directly into the cerebral circulation of male C57Bl/6 mice and Wistar rats.Laser-Doppler flowmetry demonstrated reductions in cerebral blood flow (CBF) of ∼80% in mice and ∼60% in rats. CBF spontaneously increased but remained depressed after catheter withdrawal. Magnetic resonance imaging showed that ipsilateral CBF was reduced at 3h after collagen injection and markedly improved at 48 h. Micro-computed tomography revealed reduced blood vessel density in the ipsilateral MCA territory at 3 h. Gross examination of excised brains revealed thrombi within ipsilateral cerebral arteries at 3 h, but not 24 h, after collagen injection. Immunofluorescence microscopy confirmed that platelets and fibrinogen/fibrin were major components of these thrombi at both macrovascular and microvascular levels. Cerebral infarcts comprising ∼30% of hemispheric volume and neurobehavioral deficits were observed 48 h after ischemic injury in both mice and rats.Collagen injection caused brain injury that was similar in magnitude and variability to mechanical MCA occlusion or injection of a pre-formed clot; however, alterations in CBF and the mechanism of vascular occlusion were more consistent with clinical ischemic stroke.This novel rodent model of ischemic stroke has pathophysiologic characteristics consistent with clinical atherothrombotic stroke, is technically feasible, and creates reproducible brain injury.

Markers of systemic inflammation, including blood leukocyte count, are associated with increased cardiovascular risk, but the mechanisms underlying this association are unclear. Leukocytes may promote platelet reactivity and thrombus formation, providing a basis for increased risk, but a relation between leukocyte count and platelet function has not been studied.We evaluated the relation of blood leukocyte count, C-reactive protein (CRP), and interleukin-6 (IL-6) to platelet aggregation to collagen, ADP and arachidonic acid, and to urinary excretion of 11-dehydro thromboxane B2. Studies were conducted in 1600 individuals (45.0+/-12.9 years, 42.7% male) at risk for coronary artery disease (CAD) before and after low dose aspirin.At baseline, platelet reactivity increased with increasing quartile of leukocyte count (median counts for each quartile were normal) for all measures of platelet function (P0.0001). These relations were unchanged by aspirin. The relation between leukocyte count and each measure of platelet reactivity remained significant (P0.05) after multivariable adjustment for CRP, IL-6, cardiac risk factors, hematologic variables, and platelet thromboxane production. CRP and IL-6 were independently associated with few measures of platelet reactivity.Increasing quartile of leukocyte count, even within the normal range, is associated with increasing platelet reactivity in individuals at risk for CAD. This relationship is not altered by aspirin and is independent of inflammatory markers and platelet thromboxane production. Additional studies are needed to determine the mechanism(s) for this association and therapies to reduce cardiovascular risk in patients with elevated leukocyte counts.

Compared to men, women have greater platelet aggregation before and after low-dose aspirin. It is not known whether high-dose aspirin therapy brings residual platelet aggregation in women closer to men. Our objective was to compare the inhibition of platelet aggregation in women and men after low and high-dose aspirin. We enrolled healthy subjects (N=106) in a trial of 14 days of aspirin 81 mg/day followed by 14 days of 325 mg/day. Platelet function was measured at baseline and following both aspirin doses. Women had greater baseline platelet activation measures. After both aspirin doses, both sexes had near complete suppression of platelet aggregation to arachidonic acid in whole blood and in platelet-rich plasma (PRP), the direct cyclooxygenase-1 (COX-1) pathway affected by aspirin. For indirect pathways, women had significantly greater residual platelet activation to collagen and adenosine diphosphate (ADP) in whole blood after both aspirin doses and in response to collagen and ADP in PRP after aspirin 325 mg/day only. After aspirin 325 mg/day, women continued to have greater residual platelet aggregation compared to men after aspirin 81 mg/day in response to collagen (p=0.016 in whole blood and p=0.037 in PRP), ADP (p

Summary. Background: Variations in platelet function among individuals may be related to differences in platelet-related genes. The major goal of our study was to estimate the contribution of inheritance to the variability in platelet function in unaffected individuals from white and African American families with premature coronary artery disease.Methods: Platelet reactivity, in the absence of antiplatelet agents, was assessed by in vitro aggregation and the platelet function analyzer closure time. Heritability was estimated using a variance components model.Results: Both white (n = 687) and African American (n = 321) subjects exhibited moderate to strong heritability (h2) for epinephrine- and adenosine diphosphate-induced aggregation (0.36–0.42 for white and >0.71 for African American subjects), but heritability for collagen-induced platelet aggregation in platelet-rich plasma was prominent only in African American subjects. Platelet lag phase after collagen stimulation was heritable in both groups (0.47–0.50). A limited genotype analysis demonstrated that the C825T polymorphism of GNB3 was associated with the platelet aggregation response to 2 μM epinephrine, but the effect differed by race.Conclusions: Considering the few and modest genetic effects reported to affect platelet function, our findings suggest the likely existence of undiscovered important genes that modify platelet reactivity, some of which affect multiple aspects of platelet biology.

Resistance to inhibition of platelet function by aspirin may contribute to future myocardial infarction and stroke. Adverse cardiovascular outcomes have been associated with aspirin resistance on several different platelet function assays, including the level of urinary 11-dehydro thromboxane B2 (Tx-M), platelet aggregation to arachidonic acid and adenosine diphosphate, and closure time on the platelet function analyzer-100. We examined the concordance of these aspirin-resistance assays and their relation to cardiovascular risk factors in a primary prevention population. Asymptomatic patients (n = 1,311) at increased risk for coronary heart disease were evaluated before and after 2 weeks of aspirin (81 mg/day). Aspirin resistance was defined according to published criteria for these 3 assays of platelet function. Subjects were characterized for the presence of atherosclerosis risk factors. Agreement among the 3 assays was poor. Only 5 patients met aggregation criteria for aspirin resistance. Attenuated suppression of urinary Tx-M by aspirin was associated with a greater atherosclerotic risk profile and Framingham risk score in multivariable regression analysis. Aspirin resistance by platelet function analyzer-100 was associated only with increased von Willebrand factor levels and not with atherosclerotic risk profile. In conclusion, in a primary prevention population, different published criteria for aspirin resistance classify distinct groups of patients as aspirin resistant with very little overlap. Higher Tx-M, which reflects decreased suppression of thromboxane production in vivo, is the only criterion associated with atherosclerosis risk factors, suggesting that this measurement may represent the most relevant approach for identifying asymptomatic subjects whose aspirin treatment will "fail."