Andrew D. Mumford

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.

Identification and characterization of a novel P2Y12 variant in a patient diagnosed with type 1 von Willebrand disease in the European MCMDM-1VWD study

We investigated whether defects in the P2Y(12) ADP receptor gene (P2RY12) contribute to the bleeding tendency in 92 index cases enrolled in the European MCMDM-1VWD study. A heterozygous mutation, predicting a lysine to glutamate (K174E) substitution in P2Y(12), was identified in one case with mild type 1 von Willebrand disease (VWD) and a VWF defect. Platelets from the index case and relatives carrying the K174E defect changed shape in response to ADP, but showed reduced and reversible aggregation in response to 10 muM ADP, unlike the maximal, sustained aggregation observed in controls. The reduced response was associated with an approximate 50% reduction in binding of [(3)H]2MeS-ADP to P2Y(12), whereas binding to the P2Y(1) receptor was normal. A hemagglutinin-tagged K174E P2Y(12) variant showed surface expression in CHO cells, markedly reduced binding to [(3)H]2MeS-ADP, and minimal ADP-mediated inhibition of forskolin-induced adenylyl cyclase activity. Our results provide further evidence for locus heterogeneity in type 1 VWD.

Evaluation of participants with suspected heritable platelet function disorders including recommendation and validation of a streamlined agonist panel

Light transmission aggregometry (LTA) is used worldwide for the investigation of heritable platelet function disorders (PFDs), but interpretation of results is complicated by the feedback effects of ADP and thromboxane A(2) (TxA(2)) and by the overlap with the response of healthy volunteers. Over 5 years, we have performed lumi-aggregometry on 9 platelet agonists in 111 unrelated research participants with suspected PFDs and in 70 healthy volunteers. Abnormal LTA or ATP secretion test results were identified in 58% of participants. In 84% of these, the patterns of response were consistent with defects in Gi receptor signaling, the TxA(2) pathway, and dense granule secretion. Participants with defects in signaling to Gq-coupled receptor agonists and to collagen were also identified. Targeted genotyping identified 3 participants with function-disrupting mutations in the P2Y(12) ADP and TxA(2) receptors. The results of the present study illustrate that detailed phenotypic analysis using LTA and ATP secretion is a powerful tool for the diagnosis of PFDs. Our data also enable subdivision at the level of platelet-signaling pathways and in some cases to individual receptors. We further demonstrate that most PFDs can be reliably diagnosed using a streamlined panel of key platelet agonists and specified concentrations suitable for testing in most clinical diagnostic laboratories.

An intact PDZ-motif is essential for correct P2Y12 purinoceptor traffic in human platelets

The platelet P2Y(12) purinoceptor (P2Y(12)R), which plays a crucial role in hemostasis, undergoes internalization and subsequent recycling to maintain receptor responsiveness, processes that are essential for normal platelet function. Here, we observe that P2Y(12)R function is compromised after deletion or mutation of the 4 amino acids at the extreme C-terminus of this receptor (ETPM), a putative postsynaptic density 95/disc large/zonula occludens-1 (PDZ)-binding motif. In cell line models, removal of this sequence or mutation of one of its core residues (P341A), attenuates receptor internalization and receptor recycling back to the membrane, thereby blocking receptor resensitization. The physiologic significance of these findings in the regulation of platelet function is shown by identification of a patient with a heterozygous mutation in the PDZ binding sequence of their P2Y(12)R (P341A) that is associated with reduced expression of the P2Y(12)R on the cell surface. Importantly, platelets from this subject showed significantly compromised P2Y(12)R recycling, emphasizing the importance of the extreme C-terminus of this receptor to ensure correct receptor traffic.

Factor V I359T: a novel mutation associated with thrombosis and resistance to activated protein C

Summary. We report a kindred in which two siblings suffered spontaneous venous thromboses in the second decade of life. Further investigation showed reduced coagulation factor V (FV) activity and activated protein C resistance (APCR) ratio but no other thrombophilic abnormalities. The reduction in APCR ratio persisted in a modified APCR assay in which FV activity was normalized between test and control plasmas. Analysis of the FV gene showed that the thrombotic individuals had a complex genotype that included two novel point mutations c.529G>T and c.1250T>C resulting in FV E119X and FV I359T substitutions inherited on different alleles. Individuals in the kindred with FV E119X or FV I359T substitutions alone were asymptomatic. We suggest that the FV I359T substitution confers pro‐thrombotic risk and APCR, but that this is only clinically manifest when co‐inherited with the FV E119X allele. The FV I359T substitution creates a new consensus sequence for N‐linked glycosylation within the FV heavy chain and we speculate that this abnormal glycosylation may disrupt activated protein C‐mediated proteolysis of the variant FV and FVa.

Src family kinases are essential for primary aggregation by G(i) -coupled receptors.

Summary.u2002 Introduction and Background: Adrenaline stimulates biphasic aggregation in plasma through the Gi‐coupled α2A‐adrenoreceptor. In the present study, we demonstrate that both primary and secondary wave aggregation induced by adrenaline in plasma is blocked by two structurally distinct inhibitors of Src family kinases, dasatinib and PD0173952. Methods and Results: In contrast, primary aggregation is partially inhibited or unaffected in the presence of inhibitors of cyclo‐oxygenase, phosphoinositide (PI) 3‐kinases, and P2Y1 and P2Y12 ADP receptors, although secondary aggregation is abolished. The ability of adrenaline to inhibit adenylyl cyclase and to synergize with platelet agonists in mediating platelet activation in plasma is retained in the presence of Src family kinase inhibition. Moreover, adrenaline does not activate Src family kinases, as determined by western blotting of their regulatory tyrosines, suggesting that constitutive signaling from Src family kinases may underlie their role in activation. Adrenaline is widely used in clinical laboratories for investigation of patients with suspected bleeding disorders. In a group of 90 unrelated patients with a clinically diagnosed platelet bleeding disorder, we identified four who did not exhibit primary wave aggregation in response to adrenaline, although the catecholamine potentiated the response to other agonists, and five who failed to undergo secondary wave aggregation. In contrast, adrenaline stimulated biphasic aggregation in 60 controls. All of the patients with a defective response to adrenaline had impaired ADP‐induced platelet activation. Conclusions: The present results indicate a previously unappreciated role for Src family kinases in mediating Gi signaling in plasma, and demonstrate heterogeneity in response to adrenaline in patients with a clinically diagnosed platelet disorder.

Cerebral palsy in siblings caused by compound heterozygous mutations in the gene encoding protein C

We report two sisters with extensive bilateral periventricular haemorrhagic infarction (PVHI) causing cerebral palsy (CP). The older sister presented at 20u2003months with cortical visual blindness, spastic diplegia, and purpura fulminans. The younger sister presented aged 3u2003days old with apnoeas and multifocal seizures. She subsequently had global developmental delay, cortical visual blindness, spastic quadriplegia, epilepsy, and purpura fulminans at age 2u2003years. Neuroimaging of both siblings showed bilateral PVHI consistent with bilateral cerebral intramedullary venous thrombosis occurring at under 28u2003weeks’ gestation for the older sister and around time of birth for the younger sister. At latest follow‐up, the older sister (13y) has spastic diplegia at Gross Motor Function Classification System (GMFCS) level II, and the younger sister (10y) has spastic quadriplegia at GMFCS level IV. Both sisters showed partial quantitative reduction in plasma protein C antigen and severe qualitative reduction in plasma protein C anticoagulant activity. They were heterozygous for two independent mutations in the protein C gene (PROC). There was no other risk factor for CP. To our knowledge, this is the first family reported with compound heterozygous PROC mutations as the likely genetic cause of familial CP. This report adds to the list of known monogenic causes of CP.

Severe prekallikrein deficiency associated with homozygosity for an Arg94Stop nonsense mutation

An elderly patient with no abnormal bleeding presented with prolongation of the activated partial thromboplastin time (aPTT). Preincubation of plasma with aPTT reagent caused shortening of the abnormal clotting time. Plasma prekallikrein (PK) activity and antigen were <5u2003u/dL. Molecular analysis showed a homozygous Arg94Stop substitution in the PK gene, predicted to prevent expression of the mutant allele. The five heterozygous offspring of the proband each showed a normal aPTT but reduced PK activity and antigen. This is the first description of a kindred in which absence of expression of one or both PK alleles has been confirmed by genotype.

The protein C ω‐loop substitution Asn2Ile is associated with reduced protein C anticoagulant activity

We report a kindred with heritable protein C (PC) deficiency in which two siblings with severe thrombosis showed a composite type I and IIb PC deficiency phenotype, identified using commercial PC assays (proband: PC antigen 42u2003u/dl, amidolytic activity 40u2003u/dl, anticoagulant activity 9u2003u/dl). The independent PROC nucleotide variations c.669C>A (predictive of Ser181Arg) and c.131C>T (predictive of Asn2Ile) segregated with the type I and type IIb PC deficiency phenotypes respectively, but co‐segregated in the siblings with severe thrombosis. Soluble thrombomodulin (sTM)‐mediated inhibition of plasma thrombin generation from an individual with PC‐Asn2Ile was lower (endogenous thrombin potential (ETP) 56u2003±u20031% that of ETP determined without sTM) than control plasma (ETP 15u2003±u20032%) indicating reduced PC anticoagulant activity. Recombinant APC‐Asn2Ile exhibited normal amidolytic activity but impaired anticoagulant activity. Protein S (PS)‐dependent anticoagulant activity of recombinant APC‐Asn2Ile and binding of recombinant APC‐Asn2Ile to endothelial protein C receptor (EPCR) were reduced compared to recombinant wild‐type APC. Asn2 lies within the ω‐loop of the PC/APC Gla domain and this region is critical for calcium‐induced folding and subsequent interactions with anionic phospholipids, EPCR and PS. The disruption of these interactions in this naturally‐occurring PC variant highlights their collective importance in mediating APC anticoagulant activity in vivo.

A vitamin K epoxide reductase complex subunit 1 mutation in an Irish patient with warfarin resistance

BackgroundOral anticoagulant use continues to increase rapidly. In this setting, it is important to recognise that some normal individuals can demonstrate resistance to anticoagulation with warfarin. Such patients require high daily doses of warfarin (>20xa0mg) in order to maintain an international normalised ratio (INR) within the target therapeutic range (2–3). We describe the case of an elderly gentleman with atrial fibrillation who demonstrated true warfarin resistance.MethodsWe performed vitamin K epoxide reductase subunit 1 (VKORC1) gene coding sequence analysis using polymerase chain reaction primers.ResultsWe demonstrated that our patient was heterozygous for a 383 T→G transition in exon 2 of the VKORC1 gene.ConclusionsThis is the first documented Irish case of true warfarin resistance as a result of a mutation in VKORC1, a novel gene encoding a component of the epoxide reductase enzyme complex which is an essential component in the recycling pathway of vitamin K and is postulated to be one of the sites of action of warfarin.