Michael Laffan

Endothelial von Willebrand factor regulates angiogenesis

The regulation of blood vessel formation is of fundamental importance to many physiological processes, and angiogenesis is a major area for novel therapeutic approaches to diseases from ischemia to cancer. A poorly understood clinical manifestation of pathological angiogenesis is angiodysplasia, vascular malformations that cause severe gastrointestinal bleeding. Angiodysplasia can be associated with von Willebrand disease (VWD), the most common bleeding disorder in man. VWD is caused by a defect or deficiency in von Willebrand factor (VWF), a glycoprotein essential for normal hemostasis that is involved in inflammation. We hypothesized that VWF regulates angiogenesis. Inhibition of VWF expression by short interfering RNA (siRNA) in endothelial cells (ECs) caused increased in vitro angiogenesis and increased vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2)-dependent proliferation and migration, coupled to decreased integrin αvβ3 levels and increased angiopoietin (Ang)-2 release. ECs expanded from blood-derived endothelial progenitor cells of VWD patients confirmed these results. Finally, 2 different approaches, in situ and in vivo, showed increased vascularization in VWF-deficient mice. We therefore identify a new function of VWF in ECs, which confirms VWF as a protein with multiple vascular roles and defines a novel link between hemostasis and angiogenesis. These results may have important consequences for the management of VWD, with potential therapeutic implications for vascular diseases.

The relationship between ABO histo-blood group, factor VIII and von Willebrand factor

. ABO histo‐blood group is a major determinant of plasma levels of factor VIII (FVIII) and von Willebrand factor (vWF). Blood group O individuals have significantly (approximately 25%) lower plasma levels of both glycoproteins. This association is of clinical significance. Low plasma levels of either FVIII or vWF have long been established as causes of excess bleeding. Conversely, there is accumulating evidence that elevated FVIII–vWF levels may represent an important risk factor for ischaemic heart disease and venous thromboembolic disease. In spite of the well‐documented association between ABO blood group and FVIII–vWF levels, the underlying mechanism remains unknown. However, it has been established that the ABO effect is primarily mediated through a direct functional effect of the ABO locus on plasma vWF levels.

Bleeding symptoms and coagulation abnormalities in 337 patients with AL-amyloidosis.

Haemorrhage is a frequent manifestation of amyloidosis. We performed a retrospective clinical analysis of 337 patients with systemic immunoglobulin light‐chain (AL)‐amyloidosis, in whom whole‐body serum amyloid P component (SAP) scintigraphy and a clotting screen had been performed. Abnormal bleeding was noted in 94 cases (28%), and the coagulation screen was abnormal in 172 cases (51%). The most common abnormalities were prolongation of the thrombin time (TT; 108 cases, 32%) and the prothrombin time (PT; 82 cases, 24%). In multivariate analysis, a prolonged PT was the only coagulation abnormality associated with abnormal bleeding (P = 0·0012), but this was independent of the whole‐body amyloid load. Prolongation of the TT was associated with hepatic amyloid infiltration (P < 0·00001), with proteinuria (P < 0·001) and low serum albumin (P < 0·00001). In 154 patients who were studied further, subnormal factor X activity (FX:C) was found in 22 cases (14%). In cases with subnormal FX:C, the corresponding factor X antigen (FX:Ag) measurements were consistently higher (median FX:Ag/FX:C 2·5, range 0·81–9·25, n = 16) than cases with normal FX:C (median FX:Ag/FX:C 0·96, range 0·65–1·29, n = 28, P < 0·0001). No evidence was found of an FX inhibitor. Of the 48/154 (31%) cases with a prolonged TT, the reptilase time was also prolonged in 38/48 cases (79%). These data show that haemorrhage and abnormal coagulation are common in AL‐amyloidosis and are multifactorial in origin. We provide evidence suggesting that hepatic amyloid infiltration and nephrotic syndrome are determinants of the TT. In most patients, prolongation of the PT was explained by reduction in FX:C, but this was not wholly explained by a reduction in FX:Ag.

The use of fresh-frozen plasma in England: high levels of inappropriate use in adults and children

BACKGROUND: Fresh‐frozen plasma (FFP) is given to patients across a range of clinical settings, frequently in association with abnormalities of standard coagulation tests.

The diagnosis of von Willebrand disease: a guideline from the UK Haemophilia Centre Doctors' Organization.

Summary.  von Willebrand disease (VWD) is the commonest inherited bleeding disorder. However, despite an increasing understanding of the pathophysiology of VWD, the diagnosis of VWD is frequently difficult because of uncertainty regarding the relationship between laboratory assays and function in vivo. The objective of this guideline is to provide contemporary advice on a rational approach to the diagnosis of VWD. This is the second edition of this UK Haemophilia Centre Doctors’ Organisation (UKHCDO) guideline and supersedes the previous edition which was published in 1997.

Recombinant FVIIa in the management of uncontrolled hemorrhage

BACKGROUND: Recombinant FVIIa (rFVIIa/NovoSeven) is a novel hemostatic agent originally developed to treat patients with hemophilia who had developed inhibitors. Several case reports have suggested that rFVIIa may be effective in treating patients without a pre‐existing bleeding disorder who have uncontrolled bleeding.

Transcriptional diversity during lineage commitment of human blood progenitors

Introduction Blood production in humans culminates in the daily release of around 1011 cells into the circulation, mainly platelets and red blood cells. All blood cells originate from a minute population of hematopoietic stem cells (HSCs) that expands and differentiates into progenitor cells with increasingly restricted lineage choice. Characterizing alternative splicing events involved in hematopoiesis is critical for interpreting the effects of mutations leading to inherited disorders and blood cancers and for the rational design of strategies to advance transplantation and regenerative medicine. Overview of methodology. RNA-sequencing reads from human blood progenitors [opaque cells in (A)] were mapped to the transcriptome to quantify gene and transcript expression. Reads were also mapped to the genome to identify novel splice junctions and characterize alternative splicing events (B). Rationale To address this, we explored the transcriptional diversity of human blood progenitors by sequencing RNA from six progenitor and two precursor populations representing the classical myeloid commitment stages of hematopoiesis and the main lymphoid stage. Data were aligned to the human reference transcriptome and genome to quantify known transcript isoforms and to identify novel splicing events, respectively. We used Bayesian polytomous model selection to classify transcripts into distinct expression patterns across the three cell types that comprise each differentiation step. Results We identified extensive transcriptional changes involving 6711 genes and 10,724 transcripts and validated a number of these. Many of the changes at the transcript isoform level did not result in significant changes at the gene expression level. Moreover, we identified transcripts unique to each of the progenitor populations, observing enrichment in non–protein-coding elements at the early stages of differentiation. We discovered 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes and often resulting in the gain or loss of functional domains. Of the alternative splice sites displaying differential usage, 73% resulted in exon-skipping events involving at least one protein domain (38.5%) or introducing a premature stop codon (26%). Enrichment analysis of RNA-binding motifs provided insights into the regulation of cell type–specific splicing events. To demonstrate the importance of specific isoforms in driving lineage fating events, we investigated the role of a transcription factor highlighted by our analyses. Our data show that nuclear factor I/B (NFIB) is highly expressed in megakaryocytes and that it is transcribed from an unannotated transcription start site preceding a novel exon. The novel NFIB isoform lacks the DNA binding/dimerization domain and therefore is unable to interact with its binding partner, NFIC. We further show that NFIB and NFIC are important in megakaryocyte differentiation. Conclusion We produced a quantitative catalog of transcriptional changes and splicing events representing the early progenitors of human blood. Our analyses unveil a previously undetected layer of regulation affecting cell fating, which involves transcriptional isoforms switching without noticeable changes at the gene level and resulting in the gain or loss of protein functions. A BLUEPRINT of immune cell development To determine the epigenetic mechanisms that direct blood cells to develop into the many components of our immune system, the BLUEPRINT consortium examined the regulation of DNA and RNA transcription to dissect the molecular traits that govern blood cell differentiation. By inducing immune responses, Saeed et al. document the epigenetic changes in the genome that underlie immune cell differentiation. Cheng et al. demonstrate that trained monocytes are highly dependent on the breakdown of sugars in the presence of oxygen, which allows cells to produce the energy needed to mount an immune response. Chen et al. examine RNA transcripts and find that specific cell lineages use RNA transcripts of different length and composition (isoforms) to form proteins. Together, the studies reveal how epigenetic effects can drive the development of blood cells involved in the immune system. Science, this issue 10.1126/science.1251086, 10.1126/science.1250684, 10.1126/science.1251033 RNA sequencing identifies how different cell fate decisions are made during blood cell differentiation. Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type–specific expression changes: 6711 genes and 10,724 transcripts, enriched in non–protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation—the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine.

The diagnosis and management of von Willebrand disease: a United Kingdom Haemophilia Centre Doctors Organization guideline approved by the British Committee for Standards in Haematology

The guideline group was selected to be representative of UKbased medical experts. MEDLINE and EMBASE were searched systematically for publications in English from 2002 using the key word Willebrand. The writing group produced the draft guideline, which was subsequently reviewed by the A United Kingdom Haemophilia Centre Doctors Organization (UKHCDO) advisory committee, a British Committee for Standards in Haematology (BCSH) sounding board of approximately 50 UK haematologists, and the BCSH executive; comments were incorporated where appropriate. The ‘GRADE’ system was used to quote levels and grades of evidence, details of which can be found in at http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_ GRADES_OF_RECOMMENDATION/43_GRADE.html. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of patients with von Willebrand disease.

Guideline on the management of bleeding in patients on antithrombotic agents

The guideline writing group was selected to be representativeof UK-based medical experts. The MEDLINE and EMBASEdatabases were searched systematically for publications in Eng-lish from 1966 to June 2011 and 1980 to June 2011 respec-tively, using the following strategy: Approved and proprietarynames of the antithrombotic agents described in the guidelinewere combined with terms relating to antidote, reversal, haem-orrhage, (activated) prothrombin complex concentrate, factorVIII inhibitor bypass activity (FEIBA), Beriplex, Octaplex,recombinant activated factor VII (rFVIIa), Novoseven, freshfrozen plasma, tranexamic acid, antifibrinolytic, platelet trans-fusion, and desmopressin. Identified papers were also searchedfor additional references. The writing group produced the draftguideline which was subsequently revised by consensus bymembers of the Haemostasis and Thrombosis task Force of theBritish Committee for Standards in Haematology (BCSH).The guideline was then reviewed by a sounding board ofapproximately 50 UK haematologists, the BCSH and theBritish Society for Haematology Committee and commentsincorporated where appropriate. Criteria used to quote levelsand grades of evidence are as outlined in: http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMENDATION/43_GRADE.html.The objective of this document is to guide healthcareprofessionals on the management of patients receivingantithrombotic drugs who experience significant bleeding orwho require emergency surgery or an invasive procedure.Guidance on reversal of vitamin K antagonists (VKAs;warfarin, phenprocoumon, acenocoumarol (sinthrome) andphenindione has been described previously (Keeling et al,2011).Antithrombotic drugs are used increasingly in patientgroups at greater bleeding risk. Although major haemorrhageis infrequent, management can be difficult especially withantithrombotics for which there are no specific reversalagents. Bleeding during antithrombotic therapy is associatedwith high morbidity and mortality (Linkins et al, 2003; Eikel-boom et al, 2006; Mannucci & Levi, 2007). Before any anti-thrombotic treatment is started, the risks and benefits shouldbe carefully considered. In this guideline we consider themanagement of bleeding in patients on the more widely usedantithrombotic agents including heparin, anti-IIa and anti-Xainhibitors, oral VKAs, anti-platelet drugs as well as the fibri-nolytic agents.

Thrombin generation and phenotypic correlation in haemophilia A

Summary.  The clinical phenotype of patients with haemophilia A (HA) often differs between individuals with the same factor VIII (FVIII) gene defect (e.g. within the same family) or the same coagulant activity of FVIII (FVIII:C). We proposed that because the thrombin generation assay in platelet‐poor plasma of HA patients provides more information [peak thrombin concentration, endogenous thrombin potential (ETP), rate of thrombin generation and lag‐time] than a clot‐based FVIII assay it might provide insight into these differences. We therefore investigated the relation between the results of the thrombin generation assay and the clinical severity in nine families with HA (23 patients with different phenotypes). We also examined the contribution of prothrombotic risk factors: (FV Leiden G1691A and prothrombin G20210A), the coagulant activity of FVIII and tissue factor (5′UTR) polymorphisms. Our data detect marked differences between individuals but these did not correlate with the reported clinical phenotype. These differences were also reflected in a marked difference in response to the therapeutic amounts of FVIII. This might account for differences in amounts of treatment consumption. Reduced peak and possibly rate of thrombin generation, rather than FVIII:C or ETP appear to represent the critical defects in FVIII‐deficient plasma. We suggest that the analysis of parameters in thrombin generation is a useful tool to detect bleeding tendency in HA but not to predict the modulation of the haemorrhagic tendency in patients within families. However the presence of the other factors such as vessel wall components, protein C and platelets might need to be incorporated into this system.