Publications
347 results found
Nowak AA, O'Brien HER, Henne P, et al., 2017, ADAMTS-13 glycans and conformation-dependent activity., Journal of Thrombosis and Haemostasis, Vol: 15, Pages: 1155-1166, ISSN: 1538-7933
Background:ADAMTS-13 activity can be regulated by its conformation, whereby interactions between the C-terminal CUB domains and the spacer domain maintain ADAMTS-13 in a closed conformation. ADAMTS-13 contains 10 N-linked glycans, with four sites present in theTSP2 through to CUB domains that may contribute to its conformation.Objectives/Methods:We hypothesized that glycosylation contributes to ADAMTS-13 conformation and function. The proteolytic activity of glycan-modified ADAMTS-13 was assessed under static and shear stress conditions.Results:Enzymatic removal of terminal silaic acid or entire N-linked glycan chains decreased activity against FRETS-VWF73 at pH 7.4 and against full-length von Willebrand factor (VWF) under shear stress. Using truncated ADAMTS-13, we demonstrated that this was attributable to loss of sialic acid from the glycans in the metalloprotease domain and an effect of N-linked glycosylation in the TSP2 through to CUB domains. Mutation of the N-linked glycan sites in the MDTCS domains reduced or abolished protein expression. However, the N707Q, N828Q, N1235Q and N1354Q (TSP2, TSP4, CUB1, and CUB2 domains, respectively) variants were expressed normally. Interestingly, the N707Q and N828Q variants showed reduced activity against FRETS-VWF73, but normal activity under flow conditions. In contrast, the N1235Q and N1354Q variants had enhanced activity against FRETS-VWF73 and VWF under shear stress. Immunoprecipitation experiments confirmed that loss of N-linked glycans in the CUB domains significantly reduced the interaction with the spacer domain and enhanced binding to the 6A6 anti-ADAMTS-13 antibody, which recognizes a cryptic epitope in the metalloprotease domain.Conclusions:Together, these data demonstrate that the N-linked glycans of ADAMTS-13 play a crucial role in regulating ADAMTS-13 activity.
Arachchillage DRJ, Laffan M, 2017, Pathogenesis and management of antiphospholipid syndrome, British Journal of Haematology, Vol: 178, Pages: 181-195, ISSN: 1365-2141
Antiphospholipid antibodies are a heterogeneous group of autoantibodies that have clear associations with thrombosis and pregnancy morbidity, and which together constitute the ‘antiphospholipid syndrome’ (APS). However, the pathophysiology of these complications is not well understood and their heterogeneity suggests that more than one pathogenic process may be involved. Diagnosis remains a combination of laboratory analysis and clinical observation but there have been significant advances in identifying specific pathogenic features, such as domain I-specific anti-β2-glycoprotein-I antibodies. This in turn has pointed to endothelial and complement activation as important factors in the pathogenesis of APS. Consequently, although anticoagulation remains the standard treatment for thrombotic APS and during pregnancy, the realisation that these additional pathways are involved in the pathogenesis of APS has significant implications for treatment: agents acting outside the coagulation system, such as hydroxychloroquine for pregnancy complications and sirolimus as an inhibitor of the mammalian target of rapamycin (mTOR) pathway, are now under evaluation and represent a radical change in thinking for haematologists. Conventional anticoagulation is also under challenge from new, direct acting anticoagulants. This review will provide a comprehensive overview of the evolving understanding of APS pathogenesis and how this and novel therapeutics will alter diagnosis and management.
Randi AM, Laffan MA, 2017, Von Willebrand factor and angiogenesis: basic and applied issues, Journal of Thrombosis and Haemostasis, Vol: 15, Pages: 13-20, ISSN: 1538-7933
The recent discovery that von Willebrand factor (VWF) regulates blood vessel formation has opened a novel perspective on the function of this complex protein. VWF was discovered as a key component of haemostasis, capturing platelets at sites of endothelial damage and synthesized in megakaryocytes and endothelial cells (EC). In recent years, novel functions and binding partners have been identified for VWF. The finding that loss of VWF in EC results in enhanced, possibly dysfunctional angiogenesis is consistent with the clinical observations that in some patients with Von Willebrand disease (VWD), vascular malformations can cause severe gastrointestinal (GI) bleeding. In vitro and in vivo studies indicate that VWF can regulate angiogenesis through multiple pathways, both intracellular and extracellular, although their relative importance is still unclear. Investigation of these pathways has been greatly facilitated by the ability to isolate EC from progenitors circulating in the peripheral blood of normal controls and patients with VWD. In the next few years, these will yield further evidence on the molecular pathways controlled by VWF and shed light on this novel and fascinating area of vascular biology. In this article, we will review the evidence supporting a role for VWF in blood vessel formation, the link between VWF dysfunction and vascular malformations causing GI bleeding and how they may be causally related. Finally, we will discuss how these findings point to novel therapeutic approaches to bleeding refractory to VWF replacement therapy in VWD.
Sivapalaratnam S, Westbury SK, Stephens JC, et al., 2017, Rare variants in GP1BB are responsible for autosomal dominant macrothrombocytopenia, Blood, Vol: 129, Pages: 520-524, ISSN: 0006-4971
The von Willebrand receptor complex, which is composed of the glycoproteins Ibα, Ibβ, GPV, and GPIX, plays an essential role in the earliest steps in hemostasis. During the last 4 decades, it has become apparent that loss of function of any 1 of 3 of the genes encoding these glycoproteins (namely, GP1BA, GP1BB, and GP9) leads to autosomal recessive macrothrombocytopenia complicated by bleeding. A small number of variants in GP1BA have been reported to cause a milder and dominant form of macrothrombocytopenia, but only 2 tentative reports exist of such a variant in GP1BB. By analyzing data from a collection of more than 1000 genome-sequenced patients with a rare bleeding and/or platelet disorder, we have identified a significant association between rare monoallelic variants in GP1BB and macrothrombocytopenia. To strengthen our findings, we sought further cases in 2 additional collections in the United Kingdom and Japan. Across 18 families exhibiting phenotypes consistent with autosomal dominant inheritance of macrothrombocytopenia, we report on 27 affected cases carrying 1 of 9 rare variants in GP1BB.
Smith K, Starke R, Dufton N, et al., 2017, Von Willebrand Factor modulates blood vessel formation and function via Angiopoietin-2, 2nd Joint Meeting of the European-Society-for-Microcirculation (ESM) and European-Vascular-Biology-Organisation (EVBO), Publisher: KARGER, Pages: 38-38, ISSN: 1018-1172
Wahl S, Drong A, Lehne B, et al., 2016, Epigenome-wide association study of body mass index, and the adverse outcomes of adiposity, Nature, Vol: 541, Pages: 81-+, ISSN: 0028-0836
Approximately 1.5 billion people worldwide are overweight oraffected by obesity, and are at risk of developing type 2 diabetes,cardiovascular disease and related metabolic and inflammatorydisturbances1,2. Although the mechanisms linking adiposity toassociated clinical conditions are poorly understood, recent studiessuggest that adiposity may influence DNA methylation3–6, a keyregulator of gene expression and molecular phenotype7. Here weuse epigenome-wide association to show that body mass index(BMI; a key measure of adiposity) is associated with widespreadchanges in DNA methylation (187 genetic loci with P<1×10−7,range P=9.2×10−8 to 6.0×10−46; n=10,261 samples). Geneticassociation analyses demonstrate that the alterations in DNAmethylation are predominantly the consequence of adiposity,rather than the cause. We find that methylation loci are enrichedfor functional genomic features in multiple tissues (P<0.05), andshow that sentinel methylation markers identify gene expressionsignatures at 38 loci (P < 9.0 × 10−6, range P = 5.5 × 10−6 to6.1×10−35, n=1,785 samples). The methylation loci identify genesinvolved in lipid and lipoprotein metabolism, substrate transportand inflammatory pathways. Finally, we show that the disturbancesin DNA methylation predict future development of type 2 diabetes(relative risk per 1 standard deviation increase in methylation riskscore: 2.3 (2.07–2.56); P=1.1×10−54). Our results provide newinsights into the biologic pathways influenced by adiposity, and mayenable development of new strategies for prediction and preventionof type 2 diabetes and other adverse clinical consequences of obesity
Khan E, Ambrose N, Ahnstrom J, et al., 2016, A low balance between microparticles expressing tissue factor pathway inhibitor and tissue factor is associated with thrombosis in Behçet’s Syndrome, Clinical and Experimental Rheumatology, Vol: 6, ISSN: 1593-098X
Thrombosis is common in Behçet’s Syndrome (BS), and there is a need for better biomarkers for risk assessment. As microparticles expressing Tissue Factor (TF) can contribute to thrombosis in preclinical models, we investigated whether plasma microparticles expressing Tissue Factor (TF) are increased in BS. We compared blood plasma from 72 healthy controls with that from 88 BS patients (21 with a history of thrombosis (Th+) and 67 without (Th−). Using flow cytometry, we found that the total plasma MP numbers were increased in BS compared to HC, as were MPs expressing TF and Tissue Factor Pathway Inhibitor (TFPI) (all p < 0.0001). Amongst BS patients, the Th+ group had increased total and TF positive MP numbers (both p ≤ 0.0002) compared to the Th- group, but had a lower proportion of TFPI positive MPs (p < 0.05). Consequently, the ratio of TFPI positive to TF positive MP counts (TFPI/TF) was significantly lower in Th+ versus Th− BS patients (p = 0.0002), and no patient with a TFPI/TF MP ratio >0.7 had a history of clinical thrombosis. We conclude that TF-expressing MP are increased in BS and that an imbalance between microparticulate TF and TFPI may predispose to thrombosis.
Khan E, Ambrose N, Ahnstrom J, et al., 2016, A LOW BALANCE BETWEEN MICROPARTICLES EXPRESSING TISSUE FACTOR PATHWAY INHIBITOR AND TISSUE FACTOR IS ASSOCIATED WITH THROMBOSIS IN BEHCET'S SYNDROME, Publisher: CLINICAL & EXPER RHEUMATOLOGY, Pages: S143-S143, ISSN: 0392-856X
Arachchillage DJ, Deplano S, Dunnett E, et al., 2016, Efficacy and Safety of Prothrombin Complex Concentrate in Patients Undergoing Major Cardiac Surgery, 58th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH), Publisher: AMER SOC HEMATOLOGY, ISSN: 0006-4971
- Author Web Link
- Cite
- Citations: 2
Danaee A, Gomez K, Laffan M, et al., 2016, Patients with Splanchnic Vein Thrombosis Demonstrate Significantly Increased Platelet Activity, 58th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH), Publisher: AMER SOC HEMATOLOGY, ISSN: 0006-4971
Wong CL, Innes AJ, Ma B, et al., 2016, Differential Expression of Genes Associated with Oncogene-Induced Senescence and Senescence Associated Secretory Phenotype in the Absence of Differential Expression of High Molecular Risk Genes and Genes Associated with JAK-STAT Pathway in Sorted Cells of Patients with Polycythemia Vera and Primary Myelofibrosis, 59th Annual Meeting and Exposition of the American-Society-of-Hematology (ASH)/Symposium on the Basic Science of Hemostasis and Thrombosis, Publisher: American Society of Hematology, Pages: 4283-4283, ISSN: 0006-4971
Chion A, O'Sullivan JM, Drakeford C, et al., 2016, N-linked glycan sites within the A2 domain of von Willebrand factor modulate macrophage-mediated clearance, Blood, Vol: 128, Pages: 1959-1968, ISSN: 0006-4971
Enhanced von Willebrand factor (VWF) clearance is important in the etiology of von Willebrand disease. However the molecular mechanisms underlying VWF clearance remain poorly understood. In this study, we have investigated the role of VWF domains and specific glycan moieties in regulating in vivo clearance. Our findings demonstrate that the A1 domain of VWF contains a receptor-recognition site that plays a key role in regulating the interaction of VWF with macrophages. In A1-A2-A3 and full-length VWF, this macrophage-binding site is cryptic but becomes exposed following exposure to shear or ristocetin. Previous studies have demonstrated that the N-linked glycans within the A2 domain play an important role in modulating susceptibility to ADAMTS13 proteolysis. We further demonstrate that these glycans presented at N1515 and N1574 also play a critical role in protecting VWF against macrophage-binding and clearance. Indeed, loss of the N-glycan at N1515 resulted in markedly enhanced VWF clearance that was significantly faster than that observed with any previously described VWF mutations. In addition, A1-A2-A3 fragments containing the N1515Q or N1574Q substitutions also demonstrated significantly enhanced clearance. Importantly, clodronate-induced macrophage depletion significantly attenuated the increased clearance observed with N1515Q and N1574Q in both full-length VWF and in A1-A2-A3. Finally, we further demonstrate that loss of these N-linked glycans does not enhance clearance in VWF in the presence of a structurally constrained A2 domain. Collectively, these novel findings support the hypothesis that conformation of the VWF A domains plays a critical role in modulating macrophage-mediated clearance of VWF in vivo.
Griffiths S, Woo C, Mansoubi V, et al., 2016, Thromboelastography (TEG (R)) demonstrates that tinzaparin 4500 international units has no detectable anticoagulant activity after caesarean section, International Journal of Obstetric Anesthesia, Vol: 29, Pages: 50-56, ISSN: 0959-289X
BackgroundLow molecular weight heparin is routinely used for thromboprophylaxis in pregnancy and the puerperium. Consensus guidelines recommend waiting 10–12 h after administration of a thromboprophylactic dose of low molecular weight heparin before performing a neuraxial block or removing an epidural catheter. Thromboelastography (TEG®) has been reported to be sensitive to the effects of enoxaparin 4 h after administration. The purpose of this study was to use TEG to examine coagulation changes in the first 10 h after a thromboprophylactic dose of tinzaparin in an attempt to ratify the current consensus guidelines about timing of neuraxial blockade and epidural catheter removal.MethodsTwenty-four women who had undergone caesarean delivery and were classified as low or intermediate risk of thrombosis were recruited. Blood samples were taken before subcutaneous administration of tinzaparin 4500 IU, and at 4, 8 and 10 h post-dose. Standard TEG analyses were performed using plain and heparinase cuvettes and samples were also sent for laboratory anti-Xa assay. Thromboelastograph profiles were analysed for a low molecular weight heparin effect.ResultsAnalysis revealed no significant differences in R time, K time, alpha angle or maximum amplitude between plain and heparinase samples at any time point. Apart from a small statistically significant (P=0.033) decrease in maximum amplitude of 2.8% (95% CI 0.3 to 5.4%) at 4 h, there were no significant changes in coagulation for any TEG parameter. Anti-Xa levels were virtually undetectable in all patients over the 10 h period (median 0.00 U/mL; range 0.00–0.13 U/mL).ConclusionA thromboprophylactic dose of tinzaparin 4500 IU had little detectable effect on coagulation as assessed by TEG and anti-Xa assay. These findings support consensus guidelines which state that it is acceptable to perform neuraxial blockade or remove an epidural catheter 10–12 h after a thromboprophylactic dose of tinzaparin. Rather tha
Williams H, Pollard D, Maposa W, et al., 2016, Developing a regional Dental Services Pathway for inherited bleeding disorders in North London, UK, Haemophilia, Vol: 22, Pages: 83-83, ISSN: 1365-2516
Austin S, Chowdary P, Kadam A, et al., 2016, Use of long term prophylaxis in London haemophilia centres, Haemophilia, Vol: 22, Pages: 110-110, ISSN: 1365-2516
Simoni I, Sivapalaratnam S, Greene D, et al., 2016, A comprehensive next generation sequencing test for the diagnosis of inherited bleeding, thrombotic and platelet disorders, 21st Congress of the European Hematology Association, Publisher: Ferrata Storti Foundation, Pages: 307-307, ISSN: 0390-6078
Stritt S, Nurden P, Turro E, et al., 2016, A gain-of-function variant in DIAPH1 causes dominant macrothrombocytopenia and hearing loss, Blood, Vol: 127, Pages: 2903-2914, ISSN: 0006-4971
Macrothrombocytopenia (MTP) is a heterogeneous group of disorders characterized by enlarged and reduced numbers of circulating platelets, sometimes resulting in abnormal bleeding. In most MTP, this phenotype arises because of altered regulation of platelet formation from megakaryocytes (MKs). We report the identification of DIAPH1, which encodes the Rho-effector diaphanous-related formin 1 (DIAPH1), as a candidate gene for MTP using exome sequencing, ontological phenotyping, and similarity regression. We describe 2 unrelated pedigrees with MTP and sensorineural hearing loss that segregate with a DIAPH1 R1213* variant predicting partial truncation of the DIAPH1 diaphanous autoregulatory domain. The R1213* variant was linked to reduced proplatelet formation from cultured MKs, cell clustering, and abnormal cortical filamentous actin. Similarly, in platelets, there was increased filamentous actin and stable microtubules, indicating constitutive activation of DIAPH1. Overexpression of DIAPH1 R1213* in cells reproduced the cytoskeletal alterations found in platelets. Our description of a novel disorder of platelet formation and hearing loss extends the repertoire of DIAPH1-related disease and provides new insight into the autoregulation of DIAPH1 activity.
Simeoni I, Stephens JC, Hu F, et al., 2016, A high-throughput sequencing test for diagnosing inherited bleeding, thrombotic, and platelet disorders, Blood, Vol: 127, Pages: 2791-2803, ISSN: 0006-4971
Inherited bleeding, thrombotic and platelet disorders (BPDs) are diseases affecting approximately 300 individuals per million births. With the exception of haemophilia and von Willebrand disease patients, a molecular analysis for patients with a BPD is often unavailable. Many specialised tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing (HTS) platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants (SNVs), short insertions/deletions (indels) and large copy number variants (CNVs), though not inversions, which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples respectively from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology while the remainder had ana priorihighly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only eight of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.
Lentaigne C, Freson K, Laffan MA, et al., 2016, Inherited platelet disorders: toward DNA-based diagnosis, Blood, Vol: 127, Pages: 2814-2823, ISSN: 0006-4971
Variations in platelet number, volume, and function are largely genetically controlled, and many loci associated with platelet traits have been identified by genome-wide association studies (GWASs).(1) The genome also contains a large number of rare variants, of which a tiny fraction underlies the inherited diseases of humans. Research over the last 3 decades has led to the discovery of 51 genes harboring variants responsible for inherited platelet disorders (IPDs). However, the majority of patients with an IPD still do not receive a molecular diagnosis. Alongside the scientific interest, molecular or genetic diagnosis is important for patients. There is increasing recognition that a number of IPDs are associated with severe pathologies, including an increased risk of malignancy, and a definitive diagnosis can inform prognosis and care. In this review, we give an overview of these disorders grouped according to their effect on platelet biology and their clinical characteristics. We also discuss the challenge of identifying candidate genes and causal variants therein, how IPDs have been historically diagnosed, and how this is changing with the introduction of high-throughput sequencing. Finally, we describe how integration of large genomic, epigenomic, and phenotypic datasets, including whole genome sequencing data, GWASs, epigenomic profiling, protein-protein interaction networks, and standardized clinical phenotype coding, will drive the discovery of novel mechanisms of disease in the near future to improve patient diagnosis and management.
Laffan M, Douglas S, Littlewood R, 2016, Big data analytics & pathology services in NHS: achieving step changes in capacity, 21st Congress of the European Hematology Association, Publisher: Ferrata Storti Foundation, Pages: 602-602, ISSN: 0390-6078
Laffan M, 2016, Identifying the genetic basis of rare bleeding and platelet phenotypes using systematic phenotyping and genome sequencing, Journal of Thrombosis and Haemostasis, Vol: 14, Pages: 72-72, ISSN: 1538-7933
Stritt S, Nurden P, Favier R, et al., 2016, Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg<SUP>2+</SUP> homeostasis and cytoskeletal architecture, Publisher: WILEY-BLACKWELL, Pages: 107-107, ISSN: 1538-7933
Chamali B, Finnamore H, Manning R, et al., 2016, Dietary supplement use and nosebleeds in hereditary haemorrhagic telangiectasia - an observational study., Intractable & rare diseases research, Vol: 5, Pages: 109-113, ISSN: 2186-3644
Understanding potential provocations of haemorrhage is important in a range of clinical settings, and particularly for people with abnormal vasculature. Patients with hereditary haemorrhagic telangiectasia (HHT) can report haemorrhage from nasal telangiectasia in real time, and suggested dietary factors may precipitate nosebleeds. To examine further, nosebleed severity, dietary supplement use, and blood indices were evaluated in an unselected group of 50 HHT patients recruited from a specialist UK service. Using the validated Epistaxis Severity Score, nosebleed severity ranged from 0 to 9.1 out of 10 (median 3.9). Using a Food Frequency Questionnaire, 24/50 (48%) participants reported use of dietary supplements in the previous year. A third (18/50; 36%) had used self prescribed, non-iron containing dietary supplements, ingesting between 1 and 3 different supplements each day. Eight (16%) used fish oils. Despite having more severe epistaxis (p = 0.012), the 12 iron supplement users had higher serum iron concentrations, and were able to maintain their red blood cell indices. In contrast, there was no evident benefit for the participants using non iron supplements. Furthermore, platelet counts and serum fibrinogen tended to be lower in fish oil/supplement users, and one fish oil user demonstrated reduced in vitro platelet aggregation. In conclusion, in this small study, a third of HHT patients used non-iron dietary supplements, and one in six ingested fish oils, unaware of their known anti-platelet activity. The scale of use, and potential of these "natural health supplements" to exacerbate nosebleeds has not been appreciated previously in HHT.
Wong CL, Gerrard G, Norziha ZA, et al., 2016, A prospective multicentre study of the demographic, molecular and clinical landscape of myeloproliferative neoplasm in a developing country, 36th World Congress of the International-Society-of-Hematology, Publisher: Wiley, Pages: 135-135, ISSN: 1365-2141
Patel A, Jhanji S, Pavlu J, et al., 2016, Mesenchymal stromal stem cell (MSC) immunotherapy for experimental septic shock: systematic review and meta-analysis with trial sequential analysis of mortality, 42nd Annual Meeting of the European Society for Blood and Marrow Transplantation, Publisher: Nature Publishing Group, Pages: S129-S129, ISSN: 1476-5365
Stritt S, Nurden P, Favier R, et al., 2016, Defects in TRPM7 channel function deregulate thrombopoiesis through altered cellular Mg2+ homeostasis and cytoskeletal architecture, Nature Communications, Vol: 7, ISSN: 2041-1723
Mg2+ plays a vital role in platelet function, but despite implications for life-threatening conditions such as stroke or myocardial infarction, the mechanisms controlling [Mg2+]i in megakaryocytes (MKs) and platelets are largely unknown. Transient receptor potential melastatin-like 7 channel (TRPM7) is a ubiquitous, constitutively active cation channel with a cytosolic α-kinase domain that is critical for embryonic development and cell survival. Here we report that impaired channel function of TRPM7 in MKs causes macrothrombocytopenia in mice (Trpm7fl/fl-Pf4Cre) and likely in several members of a human pedigree that, in addition, suffer from atrial fibrillation. The defect in platelet biogenesis is mainly caused by cytoskeletal alterations resulting in impaired proplatelet formation by Trpm7fl/fl-Pf4Cre MKs, which is rescued by Mg2+ supplementation or chemical inhibition of non-muscle myosin IIA heavy chain activity. Collectively, our findings reveal that TRPM7 dysfunction may cause macrothrombocytopenia in humans and mice.
Turro E, Greene D, Wijgaerts A, et al., 2016, A dominant gain-of-function mutation in universal tyrosine kinase SRC causes thrombocytopenia, myelofibrosis, bleeding, and bone pathologies, Science Translational Medicine, Vol: 8, ISSN: 1946-6234
The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC’s self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr419 phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients’ platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of α-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC–positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients’ blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.
Solecka BA, Weise C, Laffan MA, et al., 2016, Site-specific analysis of von Willebrand factor O-glycosylation, Journal of Thrombosis and Haemostasis, Vol: 14, Pages: 733-746, ISSN: 1538-7933
BackgroundO-glycosylation of von Willebrand factor (VWF) affects many of its functions; however, there is currently no information on the occupancy of the 10 putative O-glycosylation sites.ObjectivesThe aim of this study was the site-specific analysis of VWF O-glycosylation.MethodsTryptic VWF-O-glycopeptides were isolated by lectin affinity chromatography and/or by reverse-phase high-performance liquid chromatography. Subsequently, the purified glycopeptides were analyzed by glycosidase digestion and mass spectrometry.ResultsWe found that all 10 predicted O-glycosylation sites in VWF are occupied. The majority of the glycan structures on all glycosylation sites is represented by disialyl core 1 O-glycan. The presence of core 2 O-glycan was also confirmed; interestingly, this structure was not evenly distributed among all 10 glycosylation sites. Analysis of the glycopeptides flanking the A1 domain revealed that generally more core-2-type O-glycan was present on the C-terminal Cluster 2 glycopeptide (encompassing T1468, T1477, S1486 and T1487) compared with the N-terminal Cluster 1 glycopeptide (encompassing T1248, T1255, T1256 and S1263). Disialosyl motifs were present on both glycopeptides flanking the A1 domain and on the glycosylation site T2298 in the C1 domain. In addition, we identify sulfation of core 2 O-glycans and the presence of the rare Tn antigen.ConclusionsThis is the first study to describe the qualitative and semi-quantitative distribution of O-glycan structures on all 10 O-glycosylation sites, which will provide a valuable starting point for further studies exploring the functional and structural implications of O-glycosylation in VWF.
Laffan M, 2016, A whole genome approach to platelet and bleeding disorders, Hamostaseologie, Vol: 36, ISSN: 0720-9355
The sequencing of hundreds of thousands of human exomes and hundreds of thousands of whole genomes is providing a progressively accurate and complete catalogue of human genetic variation. The initial studies to use genome wide data to help understand platelet disorders performed genome wide association studies to identify loci linked to variations in blood cell parameters. These studies used normal variation to find corresponding genetic variation. We next wished to investigate the genetic basis of bleeding disorders which may also provide a key to novel genes regulating platelet and haemostatic functions. The BRIDGE consortium (www.bridgestudy.org) is funded by the NIHR and brings together 13 rare disease gene discovery projects. The aim of these projects is to investigate as yet undiagnosed rare inherited diseases and identify the underlying mutational basis. We have used a cluster analysis based on the Human Phenotype Ontology in combination with next generation sequencing techniques to help identify patients with similar phenotypes which we hypothesise will arise from defects in the same gene. Preliminary results validate the clustering approach and have also resulted in a number of novel genes important for normal and pathogenic platelet physiology.
This data is extracted from the Web of Science and reproduced under a licence from Thomson Reuters. You may not copy or re-distribute this data in whole or in part without the written consent of the Science business of Thomson Reuters.