Diagnosis and laboratory monitoring of hemophilia A.

Acquired hemophilia A (AHA) is a rare disorder in which autoantibodies against factor VIII (FVIII) lead to a bleeding phenotype that varies from life-threatening to no bleeding at all. Prolonged activated partial thromboplastin times (APTT) in patients with a bleeding phenotype should be investigated to rule out AHA and should never be ignored in a preprocedure patient. Most inhibitors in AHA are heat and time dependent, so mixing studies performed only on an immediate mix are not useful: both lupus anticoagulants and treatment with direct oral anticoagulants can coexist with AHA and confound the diagnosis. Assays for intrinsic coagulation factors and von Willebrand factor should always be performed, regardless of the results of mixing studies. A Bethesda or modified Bethesda assay should be performed to quantify any inhibitor, and if susoctocog alfa (rpFVIII) is available, then an assay for cross-reacting antibodies should also be performed. At diagnosis and until complete remission, if the FVIII in the patient sample is >5 IU/dL, heat inactivation should be performed before the inhibitor assays are performed. While there are no conventional tests available to measure the effects of FVIII bypassing therapies, newer therapies may require monitoring, or their effects may need to be considered when choosing appropriate assays. Measurement of rpFVIII requires a 1-stage clotting assay, and measurement of patient FVIII while on emicizumab requires a chromogenic assay that does not contain human FX. Close communication is required between the treating clinicians and the laboratory to ensure that the correct tests are performed while patients are receiving treatments.

Increased bleeding and thrombosis in myeloproliferative neoplasms mediated through altered expression of inherited platelet disorder genes.

An altered thrombo-hemorrhagic profile has long been observed in patients with myeloproliferative neoplasms (MPNs). We hypothesized that this observed clinical phenotype may result from altered expression of genes known to harbor genetic variants in bleeding, thrombotic, or platelet disorders. Here, we identify 32 genes from a clinically validated gene panel that were also significantly differentially expressed in platelets from MPN patients as opposed to healthy donors. This work begins to unravel previously unclear mechanisms underlying an important clinical reality in MPNs. Knowledge of altered platelet gene expression in MPN thrombosis/bleeding diathesis opens opportunities to advance clinical care by: (1) enabling risk stratification, in particular, for patients undergoing invasive procedures, and (2) facilitating tailoring of treatment strategies for those at highest risk, for example, in the form of antifibrinolytics, desmopressin or platelet transfusions (not current routine practice). Marker genes identified in this work may also enable prioritization of candidates in future MPN mechanistic as well as outcome studies.

Identification of a homozygous recessive variant in PTGS1 resulting in a congenital aspirin-like defect in platelet function.

We have identified a rare missense variant on chromosome 9, position 125145990 (GRCh37), in exon 8 in PTGS1 (the gene encoding cyclo-oxygenase 1, COX-1, the target of anti-thrombotic aspirin therapy). We report that in the homozygous state within a large consanguineous family this variant is associated with a bleeding phenotype and alterations in platelet reactivity and eicosanoid production. Western blotting and confocal imaging demonstrated that COX-1 was absent in the platelets of three family members homozygous for the PTGS1 variant but present in their leukocytes. Platelet reactivity, as assessed by aggregometry, lumi-aggregometry and flow cytometry, was impaired in homozygous family members, as were platelet adhesion and spreading. The productions of COX-derived eicosanoids by stimulated platelets were greatly reduced but there were no changes in the levels of urinary metabolites of COX-derived eicosanoids. The proband exhibited additional defects in platelet aggregation and spreading which may explain why her bleeding phenotype was slightly more severe than those of other homozygous affected relatives. This is the first demonstration in humans of the specific loss of platelet COX-1 activity and provides insight into its consequences for platelet function and eicosanoid metabolism. Notably despite the absence of thromboxane A2 (TXA2) formation by platelets, urinary TXA2 metabolites were in the normal range indicating these cannot be assumed as markers of in vivo platelet function. Results from this study are important benchmarks for the effects of aspirin upon platelet COX-1, platelet function and eicosanoid production as they define selective platelet COX-1 ablation within humans.

An overview of thrombotic complications of old and new anticancer drugs.

Thrombosis is a common complication of cancer with a mean prevalence of 15%. Most commonly, this presents as venous thromboembolism; however, other manifestations such as arterial thrombosis or thrombotic microangiopathy may occur. Cancer itself is not only associated with risk factors for thrombotic complications, including intrinsic biological effect of malignant cells, accompanying operations, or the presence of indwellingvascular catheters, but there is also an additional risk caused by anticancer agents including chemotherapy and immunotherapy. In most cases the underlying pathogenetic factor that contributes to the thrombotic risk associated with chemotherapy is endothelial cell injury (or loss of protection of endothelial integrity, for example by vascular endothelial growth factor inhibition). In addition, individual anticancer agents may have specific prothrombotic effects. As in recent years more intense anticancer drugs are administered, such as in myeloablative conditioning regimens preceding stem cell transplantation, thrombosis and in particular thrombotic microangiopathy are a more frequent complication in anticancer treatment.

Novel manifestations of immune dysregulation and granule defects in gray platelet syndrome.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Coagulation and anticoagulation in the intraoperative setting.

Adequate function of the coagulation system is vital for an uncomplicated outcome of surgery. Clinically relevant perioperative bleeding complications may occur when surgical hemostasis is inadequate, but can also be caused by insufficient activity of the hemostatic system. Optimal surgical hemostasis and a satisfactory function of the coagulation system are complementary. In this article current insights on normal function and dysfunction of the coagulation system are reviewed as well as drugs that may affect a proper hemostatic response. We discuss coagulation disorders resulting in increased perioperative blood loss and conditions that may enhance the threat of postoperative thrombosis.

Publisher Correction: Protein-altering variants associated with body mass index implicate pathways that control energy intake and expenditure in obesity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Germline mutations in the transcription factor IKZF5 cause thrombocytopenia.

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of α granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD4+ T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans.

Diagnostic high-throughput sequencing of 2396 patients with bleeding, thrombotic, and platelet disorders.

A targeted high-throughput sequencing (HTS) panel test for clinical diagnostics requires careful consideration of the inclusion of appropriate diagnostic-grade genes, the ability to detect multiple types of genomic variation with high levels of analytic sensitivity and reproducibility, and variant interpretation by a multidisciplinary team (MDT) in the context of the clinical phenotype. We have sequenced 2396 index patients using the ThromboGenomics HTS panel test of diagnostic-grade genes known to harbor variants associated with rare bleeding, thrombotic, or platelet disorders (BTPDs). The molecular diagnostic rate was determined by the clinical phenotype, with an overall rate of 49.2% for all thrombotic, coagulation, platelet count, and function disorder patients and a rate of 3.2% for patients with unexplained bleeding disorders characterized by normal hemostasis test results. The MDT classified 745 unique variants, including copy number variants (CNVs) and intronic variants, as pathogenic, likely pathogenic, or variants of uncertain significance. Half of these variants (50.9%) are novel and 41 unique variants were identified in 7 genes recently found to be implicated in BTPDs. Inspection of canonical hemostasis pathways identified 29 patients with evidence of oligogenic inheritance. A molecular diagnosis has been reported for 894 index patients providing evidence that introducing an HTS genetic test is a valuable addition to laboratory diagnostics in patients with a high likelihood of having an inherited BTPD.

Protein-coding variants implicate novel genes related to lipid homeostasis contributing to body-fat distribution.

Body-fat distribution is a risk factor for adverse cardiovascular health consequences. We analyzed the association of body-fat distribution, assessed by waist-to-hip ratio adjusted for body mass index, with 228,985 predicted coding and splice site variants available on exome arrays in up to 344,369 individuals from five major ancestries (discovery) and 132,177 European-ancestry individuals (validation). We identified 15 common (minor allele frequency, MAF ≥5%) and nine low-frequency or rare (MAF <5%) coding novel variants. Pathway/gene set enrichment analyses identified lipid particle, adiponectin, abnormal white adipose tissue physiology and bone development and morphology as important contributors to fat distribution, while cross-trait associations highlight cardiometabolic traits. In functional follow-up analyses, specifically in Drosophila RNAi-knockdowns, we observed a significant increase in the total body triglyceride levels for two genes (DNAH10 and PLXND1). We implicate novel genes in fat distribution, stressing the importance of interrogating low-frequency and protein-coding variants.

High-throughput elucidation of thrombus formation reveals sources of platelet function variability.

In combination with microspotting, whole-blood microfluidics can provide high-throughput information on multiple platelet functions in thrombus formation. Based on assessment of the inter- and intra-subject variability in parameters of microspot-based thrombus formation, we aimed to determine the platelet factors contributing to this variation. Blood samples from 94 genotyped healthy subjects were analyzed for conventional platelet phenotyping: i.e. hematologic parameters, platelet glycoprotein (GP) expression levels and activation markers (24 parameters). Furthermore, platelets were activated by ADP, CRP-XL or TRAP. Parallel samples were investigated for whole-blood thrombus formation (6 microspots, providing 48 parameters of adhesion, aggregation and activation). Microspots triggered platelet activation through GP Ib-V-IX, GPVI, CLEC-2 and integrins. For most thrombus parameters, inter-subject variation was 2-4 times higher than the intra-subject variation. Principal component analyses indicated coherence between the majority of parameters for the GPVI-dependent microspots, partly linked to hematologic parameters, and glycoprotein expression levels. Prediction models identified parameters per microspot that were linked to variation in agonist-induced αIIbß3 activation and secretion. Common sequence variation of GP6 and FCER1G, associated with GPVI-induced αIIbß3 activation and secretion, affected parameters of GPVI-and CLEC-2-dependent thrombus formation. Subsequent analysis of blood samples from patients with Glanzmann thrombasthenia or storage pool disease revealed thrombus signatures of aggregation-dependent parameters that were subject-dependent, but not linked to GPVI activity. Taken together, this high-throughput elucidation of thrombus formation revealed patterns of inter-subject differences in platelet function, which were partly related to GPVI-induced activation and common genetic variance linked to GPVI, but also included a distinct platelet aggregation component.

Disseminated intravascular coagulation: an update on pathogenesis and diagnosis.

INTRODUCTION: Activation of the hemostatic system can occur in many clinical conditions. However, a systemic and strong activation of coagulation complicating clinical settings such as sepsis, trauma or malignant disease may result in the occurrence disseminated intravascular coagulation (DIC). Areas covered: This article reviews the clinical manifestation and relevance of DIC, the various conditions that may precipitate DIC and the pathogenetic pathways underlying the derangement of the hemostatic system, based on clinical and experimental studies. In addition, the (differential) diagnostic approach to DIC is discussed. Expert commentary: In recent years a lot of precise insights in the pathophysiology of DIC have been uncovered, leading to a better understanding of pathways leading to the hemostatic derangement and providing points of impact for better adjunctive treatment strategies. In addition, simple diagnostic algorithms have been developed and validated to establish a diagnosis of DIC in clinical practice.

Publisher Correction: Protein-altering variants associated with body mass index implicate pathways that control energy intake and expenditure in obesity.

In the published version of this paper, the name of author Emanuele Di Angelantonio was misspelled. This error has now been corrected in the HTML and PDF versions of the article.

Publisher Correction: Protein-altering variants associated with body mass index implicate pathways that control energy intake and expenditure in obesity.

In the version of this article originally published, one of the two authors with the name Wei Zhao was omitted from the author list and the affiliations for both authors were assigned to the single Wei Zhao in the author list. In addition, the ORCID for Wei Zhao (Department of Biostatistics and Epidemiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA) was incorrectly assigned to author Wei Zhou. The errors have been corrected in the HTML and PDF versions of the article.

Protein-altering variants associated with body mass index implicate pathways that control energy intake and expenditure in obesity.

Genome-wide association studies (GWAS) have identified >250 loci for body mass index (BMI), implicating pathways related to neuronal biology. Most GWAS loci represent clusters of common, noncoding variants from which pinpointing causal genes remains challenging. Here we combined data from 718,734 individuals to discover rare and low-frequency (minor allele frequency (MAF) < 5%) coding variants associated with BMI. We identified 14 coding variants in 13 genes, of which 8 variants were in genes (ZBTB7B, ACHE, RAPGEF3, RAB21, ZFHX3, ENTPD6, ZFR2 and ZNF169) newly implicated in human obesity, 2 variants were in genes (MC4R and KSR2) previously observed to be mutated in extreme obesity and 2 variants were in GIPR. The effect sizes of rare variants are ~10 times larger than those of common variants, with the largest effect observed in carriers of an MC4R mutation introducing a stop codon (p.Tyr35Ter, MAF = 0.01%), who weighed ~7 kg more than non-carriers. Pathway analyses based on the variants associated with BMI confirm enrichment of neuronal genes and provide new evidence for adipocyte and energy expenditure biology, widening the potential of genetically supported therapeutic targets in obesity.