Clinical phenotype of severe and moderate haemophilia: who should receive prophylaxis and what is the target trough level?

Introduction One of the most often stated tenets of haemophilia care is that prophylaxis converts a person from a severe to a moderate phenotype. In this review, we argue that this is not an accurate assumption and that people on prophylaxis predominantly have factor VIII/IX levels in the mild range. Moderate haemophilia and prophylaxis People with moderate haemophilia, who are treating with on‐demand regimens, experience joint bleeds and often develop significant arthropathy. This is especially true for people with a baseline level of 1‒3 IU/dl, as first reported 55 years ago, and confirmed in more recent studies. Evidence is emerging suggesting that people with severe haemophilia who are using prophylaxis have better musculoskeletal outcomes than people with moderate haemophilia treated episodically. Trough levels The debate around the optimum trough level whilst on prophylaxis is ongoing. It is not appropriate to extrapolate information about baseline levels to recommendations about target trough levels on prophylaxis because these are different situations. Studies are emerging that support higher target trough levels than previously used, but in spite of this, the aim of achieving zero bleeds remains elusive with both factor replacement and non‐replacement therapies. Conclusions We recommend that people with moderate haemophilia, especially those with a baseline of 1–3 IU/dl, should be offered prophylaxis based on the same criteria as people with severe haemophilia. Trough levels should be maintained above 3 IU/dl or higher if a level of 3 IU/dl does not control breakthrough bleeding and prophylaxis should be tailored to the bleeding phenotype. This advice is in line with recently published guidelines from the World Federation of Haemophilia and the UK Haemophilia Centre Doctors’ Organisation

The procoagulant activity of tissue factor expressed on fibroblasts is increased by tissue factor-negative extracellular vesicles

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function. Fibroblasts expressed 6–9 x 104 TF molecules/cell but had low specific activity for FXa generation. We confirmed that this was associated with minimal externalized PS and PE and characterised for the first time the molecular species of PS/PE demonstrating that these differed from those found in platelets. Mechanical damage of fibroblasts, used to simulate vascular injury, increased externalized PS/PE and led to a 7-fold increase in FXa generation that was inhibited by annexin V and an anti-TF antibody. Platelet-derived extracellular vesicles (EVs), that did not express TF, supported minimal FVIIa-dependent FXa generation but substantially increased fibroblast TF activity. This enhancement in fibroblast TF activity could also be achieved using synthetic liposomes comprising 10% PS without TF. In conclusion, despite high levels of surface TF expression, healthy fibroblasts express low levels of external-facing PS and PE limiting their ability to generate FXa. Addition of platelet-derived TF-negative EVs or artificial liposomes enhanced fibroblast TF activity in a PS dependent manner. These findings contribute information about the mechanisms that control TF function in the fibroblast membrane

Expanded repertoire of RASGRP2 variants responsible for platelet dysfunction and severe bleeding.

Heritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterized. Pathogenic variants in RASGRP2, which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in 3 pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5422 controls. Eleven cases harbored 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included 5 high-impact variants predicted to prevent CalDAG-GEFI expression and 6 missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbβ3 signaling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical, and dental bleeding from childhood, requiring ≥1 blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to adenosine 5'-diphosphate and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a nonsyndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation

Mutations in tropomyosin 4 underlie a rare form of human macrothrombocytopenia.

Platelets are anuclear cells that are essential for blood clotting. They are produced by large polyploid precursor cells called megakaryocytes. Previous genome-wide association studies in nearly 70,000 individuals indicated that single nucleotide variants (SNVs) in the gene encoding the actin cytoskeletal regulator tropomyosin 4 (TPM4) exert an effect on the count and volume of platelets. Platelet number and volume are independent risk factors for heart attack and stroke. Here, we have identified 2 unrelated families in the BRIDGE Bleeding and Platelet Disorders (BPD) collection who carry a TPM4 variant that causes truncation of the TPM4 protein and segregates with macrothrombocytopenia, a disorder characterized by low platelet count. N-Ethyl-N-nitrosourea-induced (ENU-induced) missense mutations in Tpm4 or targeted inactivation of the Tpm4 locus led to gene dosage-dependent macrothrombocytopenia in mice. All other blood cell counts in Tpm4-deficient mice were normal. Insufficient TPM4 expression in human and mouse megakaryocytes resulted in a defect in the terminal stages of platelet production and had a mild effect on platelet function. Together, our findings demonstrate a nonredundant role for TPM4 in platelet biogenesis in humans and mice and reveal that truncating variants in TPM4 cause a previously undescribed dominant Mendelian platelet disorder.The research participants were enrolled in the Biomedical Research Centres/Units Inherited Diseases Genetic Evaluation (BRIDGE) Bleeding and Platelet Disorders (BPD) study (UK REC10/H0304/66). We are grateful to all the donors who allowed us to use their samples for this study. We thank Sofia Papadia from the NIHR BioResource for organizing the recalls of BRIDGE-BPD participants. The genome sequencing of the BRIDGE-BPD participants was supported by the NIHR BioResource–Rare Diseases (to ET, KD, and WHO). The NIHR BioResource–Rare Diseases is responsible for the delivery of the rare diseases pilot phase of the 100,000 Genomes Project and is funded by the National Institute for Health Research (NIHR; http://www.nihr.ac.uk). Research in the Ouwehand laboratory also receives funding support from the European Commission, NIHR, Wellcome Trust, Medical Research Council (MRC), and British Heart Foundation under numbers RP-PG-0310-1002 and RG/09/12/28096. SKW is supported by an MRC Clinical Training Fellowship (MR/K023489/1). ADM receives support from the Bristol NIHR Biomedical Research Unit for Cardiovascular Disease. This work was supported by a Project Grant (no. 575535), a Program Grant (no. 1016647), a Fellowship (1063008 to BTK and 1058344 to WSA), Project Grants (to PWG and ECH), and an Independent Research Institutes Infrastructure Support Scheme Grant (no. 361646) from the Australian National Health and Medical Research Council; a fellowship from the Sylvia and Charles Viertel Foundation (to BTK); a start-up grant, a fellowship, and a grant from the German Research Foundation (SFB 688, PL707/1-1 and PL707/2-1 to IP); the Kids’ Cancer Project (to PWG); a Fellowship from the European Hematology Association (to MRT) and the British Heart Foundation (PG/13/77/30375 to MRT); NHS Blood and Transplant (to WHO and MRT); the Australian Cancer Research Fund; and a Victorian State Government Operational Infrastructure Support Grant

GWAS meta-analysis of intrahepatic cholestasis of pregnancy implicates multiple hepatic genes and regulatory elements

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility

目次 (泌尿器科紀要 第55巻第7号)

Blood coagulation functions as part of the innate immune system by preventing bacterial invasion and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca2+- and phosphatidylserine (PS)- dependent manner. In wild-type mice, hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) enhanced coagulation and restored normal hemostasis in clotting-deficient animals genetically lacking p12-LOX or 12/15-LOX activity. Murine platelets generated 22 eoxPL species, all of which were missing in the absence of p12-LOX. Humans with the thrombotic disorder antiphospholipid syndrome (APS) had statistically significantly increased HETE-PLs in platelets and leukocytes, as well as greater HETE-PL immunoreactivity, than healthy controls. HETE-PLs enhanced membrane binding of the serum protein β2GPI (β2-glycoprotein I), an event considered central to the autoimmune reactivity responsible for APS symptoms. Correlation network analysis of 47 platelet eoxPL species in platelets from APS and control subjects identified their enzymatic origin and revealed a complex network of regulation, with the abundance of 31 p12-LOX-derived eoxPL molecules substantially increased in APS. In summary, circulating blood cells generate networks of eoxPL molecules, including HETE-PLs, which change membrane properties to enhance blood coagulation and contribute to the excessive clotting and immunoreactivity of patients with APS

Phenotype description and response to thrombopoietin receptor agonist in DIAPH1-related disorder

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Phenotype description and response to thrombopoietin receptor agonist in DIAPH1-related disorder.

DIAPH1-related disorder has a bilineage hematological phenotype of macrothrombocytopenia and neutropenia associated with hearing loss. Eltrombopag increased proplatelet formation from cultured DIAPH1-related disorder megakaryocytes and improved platelet counts in vivo

Expanded repertoire of RASGRP2 variants responsible for platelet dysfunction and severe bleeding

Heritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterised. Pathogenic variants in RASGRP2, which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in three pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2,042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5,422 controls. Eleven cases harboured 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included five high-impact variants predicted to prevent CalDAG-GEFI expression and six missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbβ3 signalling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical and dental bleeding from childhood, requiring at least one blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to ADP and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a non-syndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation.status: publishe

Expanded repertoire of RASGRP2 variants responsible for platelet dysfunction and severe bleeding.

Heritable platelet function disorders (PFDs) are genetically heterogeneous and poorly characterized. Pathogenic variants in RASGRP2, which encodes calcium and diacylglycerol-regulated guanine exchange factor I (CalDAG-GEFI), have been reported previously in 3 pedigrees with bleeding and reduced platelet aggregation responses. To better define the phenotype associated with pathogenic RASGRP2 variants, we compared high-throughput sequencing and phenotype data from 2042 cases in pedigrees with unexplained bleeding or platelet disorders to data from 5422 controls. Eleven cases harbored 11 different, previously unreported RASGRP2 variants that were biallelic and likely pathogenic. The variants included 5 high-impact variants predicted to prevent CalDAG-GEFI expression and 6 missense variants affecting the CalDAG-GEFI CDC25 domain, which mediates Rap1 activation during platelet inside-out αIIbβ3 signaling. Cases with biallelic RASGRP2 variants had abnormal mucocutaneous, surgical, and dental bleeding from childhood, requiring ≥1 blood or platelet transfusion in 78% of cases. Platelets displayed reduced aggregation in response to adenosine 5'-diphosphate and epinephrine, but variable aggregation defects with other agonists. There were no other consistent clinical or laboratory features. These data enable definition of human CalDAG-GEFI deficiency as a nonsyndromic, recessive PFD associated with a moderate or severe bleeding phenotype and complex defects in platelet aggregation