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The University of North Carolina Symposia on Hemostasis began in 2002, with The First Symposium on Hemostasis with a Special Focus on FVIIa and Tissue Factor. They have occurred biannually since and have maintained the primary goal of establishing a forum for the sharing of outstanding advances made in the basic sciences of hemostasis. The 2024 11th Symposium on Hemostasis will bring together leading scientists from around the globe to present and discuss the latest research related to coagulation factors and platelet biology. In keeping with the tradition of the conference, we expect novel cross-disciplinary collaborations to result from bringing together fundamental scientists and physician-scientists from different backgrounds and perspectives. The aim of these collaborations is to springboard the next generation of important advances in the field . This year's program was designed to discuss Coagulation and Platelet Biology at the Intersection of Health and Disease. The goal is to develop a better understanding of the pathophysiologic mechanisms leading to hemostatic and thrombotic disorders as this understanding is critical for the continued development of safe and efficacious therapeutics. Included in this review article are illustrated capsules provided by our speakers that highlight the main conclusions of the invited talks., (© 2024 The Author(s).)

Background: Venous thromboembolism (VTE) is a complex disease with an incidence rate of about 1 in 1000 per year. Despite the availability of validated biomarkers for VTE, unprovoked events account for 50% of first events. Therefore, emerging high-throughput proteomics are promising methods for the expansion of VTE biomarkers. One such promising high-throughput platform is SomaScan, which uses a large library of synthetic oligonucleotide ligands known as aptamers to measure thousands of proteins., Objective: The aim of this study was to evaluate the viability of the aptamer-based SomaScan platform for VTE studies by examining its agreement with standard laboratory methods., Methods: We examined the agreement between eight established VTE biomarkers measured by SomaScan and standard laboratory immunoassay and viscosity-based instruments in 54 individuals (27 cases and 27 controls) from the Thrombophilia, Hypercoagulability and Environmental Risks in Venous Thromboembolism study. We performed the agreement analysis by using a regression model and predicting the estimates and the 95% prediction interval (PI) of the laboratory instrument values using SomaScan values., Results: SomaScan measurements exhibited overall poor agreement, particularly for D-dimer (average fit, 492.7 ng/mL; 95% PI, 110.0-1998.2) and fibrinogen (average fit, 3.3 g/L; 95% PI, 2.0-4.7)., Conclusion: Our results indicate that SomaScan measurement had poor agreement with the standard laboratory measurements. These results may explain why some genome-wide association studies with VTE proteins measured by SomaScan did not confirm previously identified loci. Therefore, SomaScan should be considered with caution in VTE studies., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Essentials Deep vein thrombosis (DVT) has a large unknown genetic component. We sequenced coding areas of 734 hemostasis-related genes in 899 DVT patients and 599 controls. Variants in F5, FGA-FGG, CYP4V2-KLKB1-F11, and ABO were associated with DVT risk. Associations in KLKB1 and F5 suggest a more complex genetic architecture than previously thought. SUMMARY: Background Although several genetic risk factors for deep vein thrombosis (DVT) are known, almost all related to hemostasis, a large genetic component remains unexplained. Objectives To identify novel genetic determinants by using targeted DNA sequencing. Patients/Methods We included 899 DVT patients and 599 controls from three case-control studies (DVT-Milan, Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis [MEGA], and the Thrombophilia, Hypercoagulability and Environmental Risks in Venous Thromboembolism [THE-VTE] study) for sequencing of the coding regions of 734 genes involved in hemostasis or related pathways. We performed single-variant association tests for common variants (minor allele frequency [MAF] ≥ 1%) and gene-based tests for rare variants (MAF ≤ 1%), accounting for multiple testing by use of the false discovery rate (FDR). Results Sixty-two of 3617 common variants were associated with DVT risk (FDR < 0.10). Most of these mapped to F5,ABO,FGA-FGG, and CYP4V2-KLKB1-F11. The lead variant at F5 was rs6672595 (odds ratio [OR] 1.58, 95% confidence interval [CI] 1.29-1.92), in moderate linkage with the known variant rs4524. Reciprocal conditional analyses suggested that intronic variation might drive this association. We also observed a secondary association at the F11 region: missense KLKB1 variant rs3733402 remained associated conditional on known variants rs2039614 and rs2289252 (OR 1.36, 95% CI 1.10-1.69). Two novel variant associations were observed, in CBS and MASP1, but these were not replicated in the meta-analysis data from the International Network against Thrombosis (INVENT) consortium. There was no support for a burden of rare variants contributing to DVT risk (FDR > 0.2). Conclusions We confirmed associations between DVT and common variants in F5,ABO,FGA-FGG, and CYP4V2-KLKB1-F11, and observed secondary signals in F5 and CYP4V2-KLKB1-F11 that warrant replication and fine-mapping in larger studies., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

It is uncertain whether antiphospholipid antibodies (APAs) increase the risk of recurrence after a first unprovoked venous thromboembolism (VTE). We tested for anticardiolipin antibodies, anti-β2 glycoprotein 1 antibodies, and lupus anticoagulant on 2 occasions ∼6 months apart in 307 patients with a first unprovoked VTE who were part of a prospective cohort study. We then determined if APAs were associated with recurrent thrombosis in the 290 patients who stopped anticoagulant therapy in response to negative D-dimer results. Compared with those without an APA, the hazard ratios for recurrent VTE were 1.8 (95% confidence interval [CI], 0.9-3.7; P = .09) in the 25.9% of patients with an APA on ≥1 occasions, 2.7 (95% CI, 1.1-.7; P = .03) in the 9.0% of patients with the same APA on 2 occasions, and 4.5 (95% CI, 1.5-13.0; P = .006) in the 3.8% of patients with 2 or 3 different APA types on either the same or different occasions. There was no association between having an APA and D-dimer levels. We conclude that having the same type of APA on 2 occasions or having >1 type of APA on the same or different occasions is associated with recurrent thrombosis in patients with a first unprovoked VTE who stop anticoagulant therapy in response to negative D-dimer tests. APA and D-dimer levels seem to be independent predictors of recurrence in patients with an unprovoked VTE. This trial was registered at www.clinicaltrials.gov as #NCT00720915., (© 2018 by The American Society of Hematology.)

Essentials Size of pulmonary embolus (PE) at the time of recurrence compared with first event is unstudied. In a retrospective cohort of patients we compared PE size at recurrence versus the initial PE. Rates of massive PE at recurrence were low whether the first PE was massive or unmassive. Studies to determine PE size and severity at recurrence when anticoagulation is stopped are needed., Summary: Background The size of recurrent pulmonary embolus (PE) in relation to a prior event could be a factor in deciding whether to continue anticoagulation. There are no published data on this subject to help with counselling patients. Objectives To determine whether size of a first PE predicts the size at recurrence. Patients and methods This was a retrospective cohort study of consecutive patients with recurrent PE over 25 months at a single tertiary center. In confirmed cases the sizes of first and recurrent PEs were grouped into radiologically non-massive PE (RNMPE) or radiologically massive PE (RMPE) if there was bilateral main pulmonary artery thrombus, saddle PE or right ventricular strain on the computerized tomography pulmonary angiogram. Results Sixty-three patients were included in the study (37 exclusions). Thirty-seven patients were men and 26 women, with a median age of 72 years; 33.3% of PEs were unprovoked. Patients whose first PE was an RNMPE (46/63 or 73% of patients) had a 15.2% (95% confidence interval [CI], 7.6-28.2%) chance of RMPE at recurrence and a 32.6% (95% CI 20.9-47.0%) chance of having a larger PE at recurrence, whereas those who presented first with an RMPE (17/63 or 27% or patients) had a 17.6% (95% CI, 6.2-41.0%) chance of RMPE at recurrence (odds ratio, 1.19; CI, 0.27-5.27). Conclusions Risk of a massive PE at recurrence is low (and similar) irrespective of the size of the first PE in this single study. Further studies are warranted as this could help in decisions on long-term anticoagulation., (© 2017 International Society on Thrombosis and Haemostasis.)

ITP is an organ-specific autoimmune disorder characterised by a low platelet count whose cause is uncertain. A possible factor is food intolerance, although much of the information linking this with ITP is anecdotal. The role of food intolerance in ITP was studied by replacing a normal diet with an elemental diet (E028), but this did not increase platelet counts. Clear differences, however, were apparent between the volatile organic compounds (VOCs) in the urine headspace of patients with ITP and those present in healthy volunteers, which leads to speculation that abnormal metabolic activity of the intestinal microbiome may be a factor causing ITP. However, further work is needed to confirm this. There were also differences between the VOCs of patients on a normal diet and those on the elemental diet, and in this case, the VOCs involved are very likely to be of bacterial origin, as their production is affected by dietary manipulation. Many of these VOCs are known to be toxic., (Copyright © 2016. Published by Elsevier B.V.)

Unlabelled: ESSENTIALS: An IgA paraprotein with anti-thrombin activity was not associated with a severe bleeding phenotype. This observation challenges the paradigm that anticoagulant therapy necessarily increases bleeding risk. Characterization of the antibody showed that it specifically binds to thrombin exosite I. A therapeutic drug with the properties of this antibody might be an antithrombotic that doesn't cause bleeding., Background: We report the case of a 54-year-old female who presented with a traumatic subdural hemorrhage. Coagulation tests were markedly prolonged due to the presence of an anti-thrombin IgA paraprotein at 3 g L(-1) . The patient made a complete recovery and has had no abnormal bleeding during a 7-year follow-up, despite the persistence of the paraprotein., Objectives: To determine how the paraprotein prolonged clotting tests by defining its target and its epitope., Methods: The paraprotein was purified and added to normal pooled plasma for in vitro clotting assays. Binding studies were conducted to determine the affinity of the IgA for thrombin. The Fab was isolated and crystallized with thrombin., Results: The purified IgA was sufficient to confer the patient's in vitro coagulation profile in normal pooled plasma, and was found to bind specifically and with high affinity to thrombin. A crystal structure of the Fab fragment in complex with thrombin revealed an exosite I interaction involving CDRH3 of the antibody., Conclusions: Although the patient originally presented with a subdural bleed, the hematoma resolved without intervention, and no other bleeding event occurred during the subsequent 7 years. During this period, the patient's IgA paraprotein levels have remained constant at 3 g L(-1) , suggesting that the presence of a high-affinity, exosite I-directed antibody is consistent with normal hemostasis. A therapeutic derivative of this antibody might therefore permit antithrombotic dose escalation without an associated increase in the risk of bleeding., (© 2015 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Background: Previous analyses reported a higher risk of recurrent venous thrombosis in men than women., Objectives: We aimed to assess the risk of recurrence in men compared with women whilst taking female reproductive risk factors (oral contraception, postmenopausal hormone therapy and pregnancy) into account. In addition, we hypothesized that the sex difference in venous thrombosis was related to F9 Malmö, an X-linked prothrombotic factor., Methods: In four pooled European cohorts (CARROT study, Glasgow, UK; CVTE study, Cambridge, UK; AUREC study, Vienna, Austria; and LETS follow-up study, Leiden, the Netherlands), the risk of recurrent venous thrombosis was calculated in men, women with reproductive risk factors and women without reproductive risk factors at the time of their first venous thrombosis. F9 Malmö was genotyped and carriers and non-carriers contrasted., Results: In total, 2185 patients with a first venous thrombosis, 1043 men and 1142 women, were included. Overall, men had a 2.8-fold (95% confidence interval [CI], 2.2-3.4) higher risk of recurrent venous thrombosis than women. This risk was 5.2-fold (95% CI, 3.5-7.7) higher in men than in women with reproductive risk factors, and 2.3-fold (95% CI, 1.7-3.2) higher in men than in women without reproductive risk factors. No difference in risk of recurrence was found for carriers vs. non-carriers of F9 Malmö., Conclusion: Men experienced a recurrent venous thrombosis twice as often as women without reproductive risk factors. These findings indicate that men have a higher intrinsic risk of venous thrombosis than women, which is partly masked by female reproductive risk factors. The sex difference cannot be explained by F9 Malmö., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: D-dimer and thrombin generation have been associated with the risk of recurrent venous thrombosis. However, for both measurements, different assays are available, and in vitro thrombin generation may be affected by the problem of contact activation during blood sampling., Objectives: To determine the association between hypercoagulability and first and recurrent thrombosis by the use of different D-dimer and thrombin generation assays, to assess whether the addition of corn trypsin inhibitor (CTI) prior to blood sampling to inhibit contact activation improved the association between thrombin generation and thrombosis risk, and to calculate the DASH score with two different D-dimer assays., Methods: A case-control study (626 patients and 361 controls) with subsequent follow-up of the cases was performed (2987 patient-years after stopping of anticoagulant therapy). Blood was drawn 2-3 months after discontinuation of anticoagulation for the first event in citrate tubes with and without CTI., Results/conclusions: An elevated D-dimer level and elevated thrombin generation were associated with an increased risk of a first event regardless of the assay used (odds ratios: 1.8-3.4). An elevated D-dimer level but not elevated thrombin generation was associated with the risk of recurrence. Patients with elevated D-dimer levels had a more than two-fold increased recurrence rate (Vidas - hazard ratio [HR] 2.3, 95% confidence interval [CI] 1.4-3.8; HemosIL - HR 2.4, 95% CI 1.5-3.9; Thrombinoscope and Technoclone assay - HR 1.3). Elimination of contact factor activation did not improve the predictive value of thrombin generation. In patients with unprovoked first events, the DASH score had a similar predictive value for deep vein thrombosis and pulmonary embolism, both when calculated with Vidas D-dimer and when calculated with HemosIL D-dimer., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: In order to stratify patients with a first unprovoked venous thromboembolism (VTE) according to their recurrence risk and to identify those who would actually benefit from indefinite anticoagulation, three prediction models have been developed so far; none of them has been yet externally validated., Objective: To externally validate the Vienna Prediction Model (VPM), a prediction guide for estimating the recurrence risk after a first unprovoked VTE developed through Cox modeling and including sex, D-dimer and index VTE site as predictors., Patients/methods: Nine hundred and four patients pooled from five prospective studies evaluating the prognostic value of D-dimer for VTE recurrence served as the validation cohort. The validity of the VPM in stratifying patients according to their relative recurrence risk (discrimination) and in predicting the absolute recurrence risk (calibration) was tested with survival analysis methods., Results: The ability of the VPM to distinguish patients' risk for recurrent VTE in the validation cohort was at least as good as in the original cohort, with a calibration slope of 1.17 (95% confidence interval 0.71-1.64; P = 0.456 for the hypothesis of a significant difference from 1), and a c-statistic of 0.626 (vs. 0.651 in the original derivation cohort). The VPM absolute predictions in terms of cumulative rates tended to underestimate the observed recurrence rates at 12 months., Conclusions: By using a pooled individual patient database as a validation cohort, we confirmed the ability of the VPM to stratify patients with a first unprovoked VTE according to their risk of recurrence., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: There is currently little evidence defining the clinical importance of detecting and treating isolated distal DVT (IDDVT). International guidelines vary regarding diagnostic and therapeutic advice. The potential benefits of anticoagulation are unquantified. We sought to evaluate the feasibility of a randomized controlled study within a modern framework and provide a primary outcome point estimate., Methods: In this open-label, external pilot randomized controlled trial, consecutive, symptomatic, ambulatory patients with IDDVT were approached for inclusion. Participants were allocated to receive either therapeutic anticoagulation or conservative management. Patients underwent blinded color-duplex imaging at 7 and 21 days and follow-up at 3 months. Principal feasibility outcomes included recruitment rate and attrition. The principal clinical outcome was a composite including proximal propagation, pulmonary embolism, death attributable to VTE disease, or major bleeding. Analysis was by intention to treat., Results: In total, 93 patients with IDDVT were screened, and 70 of those eligible (88.6%) were recruited. All patients but one were followed-up by direct contact after 90 days. Allocation crossover occurred in 15 patients (21.4%). The principal clinical outcome occurred in four of 35 of those conservatively treated (11.4%) and zero of 35 in the anticoagulated group (absolute risk reduction, 11.4%; 95% CI, -1.5 to 26.7, P = .11, number needed to treat of nine). There were no major bleeding episodes., Conclusions: We have established the feasibility of definitive study regarding the value of therapeutic anticoagulation in IDDVT and provide an approximate point estimate for serious complications with a contemporary conservative strategy., Trial Registry: Current Controlled Trials; No.: ISRCTN75175695; URL: www.controlled-trials.com.

Background: Traditionally, a fibrinogen level > 1 g L(-1) has been viewed as the critical plasma concentration required for hemostasis. No definitive trial has investigated the plasma fibrinogen hemostatic threshold and fibrinogen replacement in complex surgical patients with acquired bleeding., Objectives: To explore the plasma fibrinogen level required for hemostasis in cardiothoracic surgery patients and assess the association of fibrinogen replacement therapy (using cryoprecipitate or fibrinogen concentrate) with reducing postoperative bleeding rate., Patients/methods: Data from a prospectively collated database were used to examine the relationship between postoperative plasma fibrinogen level and the postoperative rate of bleeding within the hour of plasma fibrinogen measurement (n = 430) and to explore the effect of cryoprecipitate infusion (n = 76) or fibrinogen concentrate administration (n = 8) on postoperative bleeding rate., Results: A low plasma fibrinogen level was significantly associated with bleeding, with an odds ratio of 3.06 for every 1 g L(-1) decrease in fibrinogen (95% confidence interval 1.05-8.90) with adjustment for confounders. A fibrinogen threshold associated with excess bleeding was not identified, but this relationship was a continuum. There was no reduction in bleeding following administration of cryoprecipitate or fibrinogen concentrate to raise the post-infusion fibrinogen level to a median of 2.00 and 1.70 g L(-1) , respectively., Conclusions: There is a continuum of bleeding severity with reducing fibrinogen concentration. Fibrinogen concentrate or cryoprecipitate infusion did not significantly reduce bleeding rate; however, confirmation by a randomized controlled trial is required. It remains uncertain whether low postoperative fibrinogen levels are causally associated with postoperative bleeding., (© 2013 International Society on Thrombosis and Haemostasis.)

Orally active small molecules that selectively and specifically inhibit coagulation serine proteases have been developed for clinical use. Dabigatran etexilate, rivaroxaban and apixaban are given at fixed doses and do not require monitoring. In most circumstances, these drugs have predictable bioavailability, pharmacokinetic effects, and pharmacodynamic effects. However, there will be clinical circumstances when assessment of the anticoagulant effect of these drugs will be required. The effect of these drugs on laboratory tests has been determined in vitro by spiking normal samples with a known concentration of active compound, or ex vivo by using plasma samples from volunteers and patients. Data on the sensitivity of different reagents are now available, and so guidance as to the effect and interpretation of a test result is now possible. Laboratories should be aware of the sensitivity of their own assays to each drug. This may be achieved by using appropriate calibrated plasma samples., (© 2013 International Society on Thrombosis and Haemostasis.)

Objective: We compared the performance of aggregate data (AD)--based and individual patient data (IPD)-based meta-analyses to synthesize evidence on the ability of D-dimer to distinguish recurrence risk in patients with unprovoked venous thromboembolism (VTE) who stopped anticoagulation., Study Design and Setting: We compared the results of the published AD-based rate ratio of VTE recurrence for positive vs. negative D-dimer, estimated by a mixed-effect Poisson model, with those of the IPD-based hazard ratio obtained by a Cox regression stratified by trial. We performed three additional analyses to investigate the methodological reasons for differences between the two approaches, comparing the IPD Cox regression with AD generated from IPD Poisson regression (to control for differences in population on study), AD time-to-event meta-analysis, and AD generated from IPD meta-regression., Results: Published analyses agreed in direction and statistical significance when estimating the prognostic value of D-dimer even if IPD estimates suggested a stronger effect. The additional analyses suggested that differences in study populations might explain this slight difference. Poor reporting in published studies precluded a true comparison of AD- and IPD-based assessments of heterogeneity sources., Conclusion: AD and IPD meta-analyses yielded similar estimates of D-dimer effect to distinguish risk for recurrent VTE. The IPD approach was justified by the need to investigate sources of heterogeneity., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Oral direct inhibitors (ODIs) of thrombin and factor Xa are now approved as anticoagulant drugs. The first two drugs to complete phase III clinical trials for thromboprophylaxis in orthopaedic surgery and treatment of patients with atrial fibrillation or venous thromboembolism were dabigatran and rivaroxaban. These small molecules are given at fixed dose with no requirement for monitoring as pharmacokinetic and pharmacodynamic responses are reliably predicted in patients with adequate renal function who are not taking other interacting drugs. However, there will be clinical circumstances in specific patients when measurement of the anticoagulant effect of an ODI will be required. © 2013 International Society on Thrombosis and Haemostasis., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: In patients with unprovoked venous thromboembolism (VTE), the optimal duration of anticoagulation is anchored on estimating the risk of disease recurrence., Objectives: We aimed to develop a score that could predict the recurrence risk following a first episode of unprovoked VTE, pooling individual patient data from seven prospective studies., Methods: One thousand eight hundred and eighteen cases with unprovoked VTE treated for at least 3 months with a vitamin K antagonist were available for analysis. Optimism-corrected Cox regression coefficients were used to develop a recurrence score that was subsequently internally validated by bootstrap analysis., Results: Abnormal D-dimer after stopping anticoagulation, age <50 years, male sex and VTE not associated with hormonal therapy (in women) were the main predictors of recurrence and were used to derive a prognostic recurrence score (DASH, D-dimer, Age, Sex, Hormonal therapy) showing a satisfactory predictive capability (ROC area =0.71). The annualized recurrence risk was 3.1% (95% confidence interval [CI], 2.3-3.9) for a score ≤ 1, 6.4% (95% CI, 4.8-7.9) for a score=2 and 12.3% (95% CI, 9.9-14.7) for a score ≥ 3. By considering at low recurrence risk those patients with a score ≤ 1, life-long anticoagulation might be avoided in about half of patients with unprovoked VTE., Conclusions: The DASH prediction rule appears to predict recurrence risk in patients with a first unprovoked VTE and may be useful to decide whether anticoagulant therapy should be continued indefinitely or stopped after an initial treatment period of at least 3 months., (© 2012 International Society on Thrombosis and Haemostasis.)

Venous thromboembolism (VTE) is a frequent cause of morbidity and mortality in patients with cancer and those having chemotherapy. However, the incidence of VTE during radical treatment for patients with oesophago-gastric cancer is poorly documented. The incidence of VTE was assessed in 200 consecutive patients with oesophago-gastric cancer having surgery with curative intent; 132 (66%) had neo-adjuvant chemotherapy, 37 (18.5%) had adjuvant chemotherapy and 64 (32%) had no chemotherapy. Patients received 40 mg of Enoxaparin subcutaneously daily during the peri-operative hospital stay. Asymptomatic VTE were detected by routine chest computed tomography (CT) pre and post surgery. Symptomatic patients with suspected VTE were investigated and treated as clinically appropriate. Twenty six patients (13%) developed VTE of which 14 (54%) were symptomatic; 12/26 (46%) VTE were detected pre-operatively, all during or after neo-adjuvant chemotherapy, and 14/26 (54%) post-operatively. There were two post-operative deaths caused by pulmonary emboli occurring at days 24 and 56 respectively despite peri-operative VTE prophylaxis. Multivariate analysis demonstrated that neo-adjuvant chemotherapy was the only factor that predicted pre-operative VTE (p = 0.073) and any VTE (p = 0.045). This study found a 13% incidence of VTE in patients undergoing therapy with curative intent for oesophago-gastric cancer and a statistically significant association between neo-adjuvant chemotherapy and VTE. Half of the patients with VTE were asymptomatic but two had fatal PE's. Current VTE prophylaxis regimens for this patient group may be inadequate., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Aim: To determine if the mode of presentation of venous thromboembolism (VTE), as deep vein thrombosis (DVT) or pulmonary embolism (PE), predicts the likelihood and type of recurrence., Methods: We carried out a patient-level meta-analysis of seven prospective studies in patients with a first VTE who were followed after anticoagulation was stopped. We used Kaplan-Meier analysis to determine the cumulative incidence of recurrent VTE according to mode of presentation, and multivariable Cox regression to calculate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs) for mode of and extent of DVT as potential risk factors for recurrence., Results: The 5-year cumulative rate of recurrent VTE in 2554 patients was 22.6%. In 869 (36.1%) patients with PE, the 5-year rate of any recurrence (DVT or PE) was 22.0%, and recurrence as PE was 10.6%. In 1365 patients with proximal DVT, the 5-year recurrence rate was 26.4%, and recurrence with PE was 3.6%. The risk of recurrence as PE was 3.1-fold greater in patients presenting with symptomatic PE than in patients with proximal DVT (HR, 3.1; 95% CI, 1.9-5.1). Patients with proximal DVT had a 4.8-fold higher cumulative recurrence rate than those with distal DVT (HR, 4.8; 95% CI, 2.1-11.0)., Conclusion: Whilst DVT and PE are manifestations of the same disease, the phenotypic expression is predetermined. Patients presenting with PE are three times more likely to suffer recurrence as PE than patients presenting with DVT. Patients presenting with calf DVT are at low risk of recurrence and at low risk of recurrence as PE., (© 2010 International Society on Thrombosis and Haemostasis.)

The thrombophilia paradox whereby thrombophilia testing identifies defects associated with an increased risk of a first venous thrombosis but not of a particularly high risk of recurrence is likely the result of limitations imposed by a limited dichotomous testing strategy compounded by test inaccuracy and imprecision. Consequently, the observed intermediate phenotype (defined by limited laboratory test results) is not fully concordant with the heritable genotype. The next generation of thrombophilia tests, which utilize either individual genomic analysis or global measurement of the composite plasma intermediate phenotype, may more accurately quantify the thrombophilic risk. In conjunction with clinical risk assessment a more quantitative measurement of hypercoagulability and definition of the prothrombotic state should facilitate transition of clinical management from a disease-focused to a more patient-focused strategy.

Venous thromboembolism (VTE) comprises deep vein thrombosis (DVT) with or without symptomatic pulmonary embolus (PE). The incidence of a first episode of VTE is 1.5 per 1000 person-years [1] (J Thromb Haemost, 2007;5:692-9) with a per-person lifetime incidence of 5% [2] (Arch Intern Med 1998;158:585-93). The risk of recurrence after DVT and PE is similar but the pattern of recurrence tends to reflect the initial event, for example recurrence with PE is more common in patients with previous PE [3] (Circulation 2003;107:122-30). At least 50% of patients, who present with symptomatic DVT, have asymptomatic PE and conversely, a majority presenting with symptomatic PE have asymptomatic DVT [3] (Circulation 2003;107:122-30). This suggests that whilst DVT and PE are manifestations of the same pathology, the phenotypic expression of the disease is predetermined. This may be an important consideration for long-term anticoagulant therapy as the risk of fatal PE is the greatest in patients with previous PE [4] (Ann Intern Med 2007;147:766-74). At present, the only factor reported to be associated with the pattern of VTE is the factor V Leiden mutation [5] (Thromb Haemost 1999;81:345-8). This suggests that the kinetics of thrombin generation and the resulting fibrinolytic response may influence clot structure and likelihood of embolization.

Objective: To determine the predictive value of measurement of parameters of thrombin generation for unprovoked recurrent venous thrombosis., Methods: Measurements were made of thrombin generation in a prospective cohort study of 188 patients with a first episode of venous thrombosis that was unprovoked, or provoked by a non-surgical trigger., Results: The endogenous thrombin potential (ETP) was the only parameter associated with unprovoked recurrent thrombosis in a multivariate model [hazard ratio (HR) 1.3 per 100 nmol L min(-1) increase, 95% confidence interval (CI) 1.0-1.6]. Patients with a high ETP had a significantly higher rate of unprovoked recurrence than those with a low ETP (HR 2.9, 95% CI 1.3-6.6, cumulative recurrence at 4 years 27% vs. 11%). Patients with an unprovoked first event had a significantly higher rate of unprovoked recurrence than those with a provoking factor (HR 2.7, 95% CI 1.2-6.1), and in these patients there was a significantly higher rate of unprovoked recurrence in association with a high ETP (HR 4.0, 95% CI 1.3-11.8). After adjustment for D-dimer, thrombophilia, sex, and whether or not the first event was unprovoked, a high ETP remained a significant predictor of recurrence (HR 2.6, 95% CI 1.2-6.0)., Conclusions: This study demonstrates a high rate of unprovoked recurrent venous thrombosis in patients presenting with a first episode of venous thrombosis and a high ETP.

Background: Individual single nucleotide polymorphisms (SNPs) associated with an increased risk of a first venous thrombosis do not predict risk of recurrent thrombosis., Objective: To assess the risk of recurrent venous thrombosis associated with multiple SNPs., Patients/methods: Fifteen nucleotide polymorphisms (SNPs), either established or putative risk factors for venous thrombosis, were measured in 817 unselected patients presenting with a first episode of venous thrombosis. Data from patients enrolled in the Leiden Thrombophilia Study (LETS) (n = 443) and the first Cambridge Prospective Cohort Study (n = 374) were combined. Hazard ratios for recurrence of thrombosis were calculated for individual SNPs., Results: Of the total study population, 117 patients had a recurrent event after a mean follow-up of 4.6 years. The overall incidence rate was 30.8/1000 person years, corresponding with an annual risk of 3.1%. None of the individual SNPs was more than weakly associated with the risk of recurrent venous thrombosis. With addition of sequential SNPs, added in rank order of risk, the hazard ratios for recurrence increased by 1.7-fold for carriers (3.8% of all patients) of the first two SNPs, 2.7-fold for carriers of three (2.3%) and 5.1-fold for carriers of four (0.4%). With addition of each SNP the number of carriers rapidly reduced., Conclusions: Although there is a substantially increased risk of recurrent thrombosis for carriers of several genetic variants, the clinical utility of multiple SNP analysis at present would be limited to a small proportion of patients.

Background: The aim of the study was to determine the predictive value of D-dimer measurement for unprovoked recurrent venous thrombosis and the influence of sex, age and type of first event (unprovoked or provoked)., Methods: Prospective cohort study of 272 patients with a first episode of venous thrombosis that was unprovoked or provoked by a non-surgical trigger., Findings: The cumulative rate of unprovoked recurrence in patients with a positive D-dimer was 20% at 5 years [5.5/100 patient-years, 95% confidence interval (CI) 3.7-7.8] and in patients with a negative D-dimer 17% (4.1/100 patient-years, 95% CI 2.3-6.9). The rates are not different (hazard ratio 1.3, 95% CI 0.7-2.5). After adjustment for clinical risk factors a positive D-dimer result was significantly associated with an increased risk of unprovoked recurrent thrombosis (hazard ratio 2.0, 95% CI 1.01-3.9). The strongest indicator of risk of recurrence was male sex (hazard ratio 3.3 unadjusted and 2.9 after adjustment). The only determinant of D-dimer in a linear regression model was age (P < 0.001)., Conclusions: The analysis indicates that clinical risk factors confound the association between D-dimer and risk of recurrence and when adjusted for these confounders a positive D-dimer result is significantly associated with unprovoked recurrence. The clinical utility of D-dimer measurement in individual patients should be interpreted in conjunction with clinical risk factors.

Background: Despite increasing use of recombinant factor VIIa (rFVIIa) in non-haemophiliac patients, it is unclear when rFVIIa might be effective., Methods: A single centre review of consecutive non-haemophiliac patients receiving rFVIIa for the management of severe haemorrhage. Treatments with rFVIIa were at a dose of 90 mug kg(-1) repeated at three hourly intervals at the clinicians' discretion., Results: Eighteen patients received rFVIIa. Six patients survived to discharge and 12 patients died in hospital. The median (range) Sequential Organ Failure Assessment (SOFA) score at the time of administration of rFVIIa for the group that survived was 8.0 (5-12) compared with the group that died 12.0 (7.0-14.0) (P=0.03). One of the patients who survived (17%) had organ failure at the time of rFVIIa administration compared with 11 of those who died (92%) (P=0.004). Fifteen patients survived long enough to consider a second dose of rFVIIa, one patient who survived to discharge needed more than one dose (1/6, 17%), compared with seven of those who later died in hospital (7/9, 78%) (P=0.04). The survivors had a significant reduction in blood product requirements after rFVIIa, while patients who died did not. Neither the prothrombin time nor the activated partial thromboplastin time before or after rFVIIa predicted survival., Conclusions: High SOFA score and failure to respond to one adequate dose of rFVIIa appear to identify patients with poor prognosis. These observations may help in determining when rFVIIa treatment is likely to be futile.

Background: The development of inhibitors in hemophiliacs is a severe complication of factor VIII (FVIII) replacement therapy and is a process driven by FVIII specific T helper cells., Objectives: To finely map T cell epitopes within the whole FVIII protein in order to investigate the possibility of engineering FVIII variants with reduced propensity for inhibitor development., Patients and Methods: T cell lines were generated from five patients with severe hemophilia who had developed inhibitors, and were screened for T cell proliferation against pools of overlapping peptides spanning the entire B domain deleted (BDD) FVIII sequence. Positive peptide pools were decoded by screening individual peptides against the T cell lines. Positive peptides, and mutants thereof, were tested for their ability to bind major histocompatibility complex (MHC) Class II and stimulate T cell proliferation in a panel of healthy donors. The activities of the corresponding mutant proteins were assessed via chromogenic assay., Results: One peptide, spanning FVIII amino acids 2098-2112, elicited a vigorous response from one hemophiliac donor, induced strong T cell responses in the panel of healthy donors and bound to a number of HLA-DR alleles. Mutations were made in this peptide that removed its ability to stimulate T cells of healthy donors and to bind to MHC Class II while retaining full activity when incorporated into a mutant BDD-FVIII protein., Conclusions: Fine T cell epitope mapping of the entire FVIII protein is feasible, although challenging, and this knowledge may be used to create FVIII variants which potentially have reduced immunogenicity.

Background: We have analyzed the influence of gender on risk of recurrence after a first episode of venous thromboembolism (VTE)., Methods: The Cambridge Venous Thromboembolism Study (CVTE) is a single-center study of a cohort of unselected patients with a first episode of objectively proven VTE., Results: Recurrence rates were significantly higher in men compared with women [log rank chi2=11.82; hazard ratio (HR) 2.66; 95% confidence interval (CI) 1.49, 4,77; P=0.0006]. The cumulative recurrence rate at 2 years was 19.2% in men and 7.7% in women. There was no evidence of a difference in recurrence between men with or without thrombophilia (log rank chi2=0.03; HR 1.08; 95% CI 0.49, 2.37; P=0.855). The high recurrence rate in men compared with women was still observed when only patients with idiopathic VTE were analyzed (log rank chi2=4.38; HR 2.31; 95% CI 1.027, 5.20; P=0.0363). The recurrence risk was highest in men with a first idiopathic event at 25.7% compared with 11.7% for women in the same category., Conclusion: The risk of recurrent VTE is higher in men than in women.

Background: Measurement of thrombin generation by calibrated automated thrombography (CAT) could fulfill the requirements of a global test of coagulability and is potentially applicable to routine clinical laboratory practice. The purpose of this study was to determine if corn trypsin inhibitor (CTI) could be used to abolish contact factor activation in this assay, thus allowing accurate measurement of low tissue factor (TF) concentration-triggered thrombin generation on samples taken in a routine clinical setting., Methods: The endogenous thrombin potential (ETP) was measured by CAT., Results: The study demonstrated that addition of CTI after plasma separation is not sufficient and blood must be drawn into tubes containing CTI if in-vitro contact factor-activated thrombin generation is to be abolished. Contact factor-activated thrombin generation is completely inhibited at a CTI concentration of 18.3 microg mL(-1) whole blood. Increasing the CTI concentration above this level does not lead to suppression of the TF-triggered ETP. At a TF concentration of 2 pmol, ETPs were significantly lower in the presence of CTI (P < 0.001). The difference (no CTI minus CTI) between results ranged from - 1 to 2159 nM min(-1) (median - 754). Whilst the low concentration TF-ETP assay was not optimized to distinguish degrees of coagulability between patient samples, there was a significant difference in ETP between normal and hemophilia samples and samples from patients with a clinical prothrombotic tendency., Conclusions: CTI can be applied to ETP measurement by CAT. This permits the use of CAT in a low TF-triggered thrombin generation assay without concern for the effect of interference from in-vitro contact factor activation and the optimum reagent conditions for using CAT as a global test of coagulability in clinical practice can now be defined.

Background: Stratification for risk of recurrence after a first episode of venous thromboembolism (VTE) would affect the duration of anticoagulant therapy. We aimed to determine the incidence of recurrence of VTE in relation to clinical risk factors and standard laboratory testing for heritable thrombophilic defects., Methods: We established a database to prospectively follow-up a cohort of unselected patients who had had a first episode of objectively proven VTE. We excluded patients with malignant disease and antiphospholipid syndrome. All patients were offered testing for heritable thrombophilia., Findings: At 2 years, the cumulative recurrence rate in 570 patients was 11%. Incidence was lowest after surgery-related VTE (0%) and highest after unprecipitated VTE (19.4%) (p<0.001). 85% of patients were tested for heritable thrombophilic defects. Recurrence rates were not related to presence or absence of laboratory evidence of heritable thrombophilia (hazard ratio 1.50 [95% CI 0.82-2.77]; p=0.187). In patients with a first event that was unprecipitated or was associated with a non-surgical trigger, recurrence rates did not differ in patients with or without thrombophilia (1.34 [0.73-2.46]; p=0.351)., Interpretation: In unselected patients who have had a first episode of VTE, testing for heritable thrombophilia does not allow prediction of recurrent VTE in the first 2 years after anticoagulant therapy is stopped. However, assessment of clinical risk factors associated with the first episode of VTE does predict risk of recurrence. Patients with postoperative VTE have a very low rate of recurrence.

Cytochrome P-450 2C9 is the principle enzyme that terminates the anticoagulant effect of warfarin. Genetic polymorphisms in CYP2C9 producing variants with altered catalytic properties have been identified. Patients (n = 561) with a target international normalized ratio (INR) of 2.5 who had been treated with warfarin for more than 2 months were anonymously genotyped for the wild-type CYP2C9*1 allele and the 2C9*2 and 2C9*3 variants. The mean maintenance dose of warfarin in patients who were wild-type for both alleles was 5.01 mg. The maintenance dose of warfarin was significantly related to genotype (Kruskall-Wallis, chi(2) = 17.985, P =.001) with mean maintenance doses in patients with variant alleles between 61% and 86% of that in wild-type patients. The odds ratio for the 2C9*2 allele in patients with a maintenance dose of 1. 5 mg or less was 5.42 (95% CI 1.68-17.4). The odds ratio for one or more variant alleles in patients developing an INR of 8.0 or greater was 1.52 (95% CI 0.64-3.58). The SD of the mean INR, percentage of high INRs, and person-time spent in range were determined as parameters of stability. There was no difference between patients grouped according to genotype for any parameter of stability. This study confirmed an association between CYP2C9 genotype and warfarin sensitivity. However, the possession of a variant allele does not increase the likelihood of severe over-anticoagulation or stability of anticoagulation during long-term therapy. (Blood. 2000;96:1816-1819)

Plasma and platelet factor Va represent different substrates for activated protein C (APC). In this study, we have measured platelet-dependent APC resistance and the effect of aspirin and a platelet glycoprotein IIbIIIa antagonist (GR144053F) on this phenomenon. In platelet rich plasma (PRP), progressive APC resistance was observed with increasing platelet activation. APC sensitivity ratios of 1.8, 1.7, and 1.4 were observed after platelet activation with thrombin receptor activating peptide (TRAP), collagen, and A23187, respectively. Ultracentrifugation at 77,000g for 1 hour abolished APC resistance indicating that the phenotype is associated exclusively with the platelet membrane. APC resistance was not observed in the presence of phosphatidylcholine-phosphatidylserine (PCPS) vesicles or purified human plasma lipoproteins. APC resistance was observed in the presence of platelet-derived microparticles, but to a lesser degree than that in the presence of activated platelets. The platelet-dependent APC resistance phenotype was also observed when endogenous APC was generated by Protac (American Diagnostica, Inc, Greenwich, CT). In vitro inhibition of platelet activation with aspirin had no effect, but the fibrinogen receptor antagonist, GR144053F, inhibited platelet-dependent APC resistance. These results indicate that platelet activation results in an APC-resistant phenotype comparable to that observed in the plasma of patients with factor V gene mutations affecting critical APC cleavage sites. This suggests that platelet activation at the site of endothelial damage downregulates a critical natural anticoagulant mechanism. The antithrombotic effect of aspirin may be due to an indirect effect on platelet-dependent APC resistance with reduced platelet retention within a developing thrombus. The more potent antithrombotic effect of glycoprotein IIbIIIa antagonists may in addition be the result of reduced platelet factor Va expression and modulation of the platelet-dependent APC resistance phenotype.

Treatment with coumarin oral anticoagulants, such as warfarin, is effective antithrombotic therapy, but patients treated with these drugs are at significant risk of bleeding. The risk of haemorrhage increases with increasing intensity of anticoagulation and overanticoagulation is common. Reversal can be achieved by stopping the coumarin drug or administration of vitamin K, fresh frozen plasma or coagulation factor concentrates. However, there are surprisingly few studies defining the optimum dose of these products and there are no randomized studies comparing the relative benefit and risk of coagulation factor concentrates versus fresh frozen plasma. Guidelines for the management of overdose are based on level III and IV evidence and are, therefore, only grade B recommendations at best. Further studies are required to determine the most effective use of products and the dose required for safe reversal of overanticoagulation.

A new factor V mutation associated with resistance to activated protein C and thrombosis (factor V Cambridge, Arg306-->Thr) was found in one patient from a carefully selected group of 17 patients with venous thrombosis and confirmed APC resistance in the absence of the common Gln506 mutation. The Arg306 mutation was also present in a first degree relative who also had APC resistance. Other potential causes of APC resistance, such as a mutation at the Arg679 site and the factor V HR2 haplotype, were excluded. Subsequent screening of 585 patients with venous thromboembolism and 226 blood donors did not show any other individual with this mutation. Factor VThr306 is the first description of a mutation affecting the Arg306 APC cleavage site and is the only mutation, other than factor V Leiden (Arg506-->Gln), that has been found in association with APC resistance. This finding confirms the physiologic importance of the Arg306 APC-cleavage site in the regulation of the prothrombinase complex. It also supports the concept that APC resistance and venous thrombosis can result from a variety of genetic mutations affecting critical sites in the factor V cofactor.

The importance of lipoproteins in the etiology of atherosclerosis is well established. Evidence is now accumulating to implicate thrombin in the pathogenesis of atherosclerosis. We have investigated whether atherogenic lipoproteins can support thrombin generation. In the absence of platelets or endothelial cells, both very low-density lipoprotein (VLDL) and oxidized low-density lipoprotein (LDL) support assembly of the prothrombinase complex and generation of thrombin. Thrombin generation (per microgram of apolipoprotein) supported by VLDL was 19.4-fold greater than that supported by high-density lipoprotein (HDL), P < .00001, and 11.7-fold greater than that supported by LDL, P < .00001. Oxidation of LDL increased lipoprotein-supported thrombin generation 12-fold compared to unmodified LDL, P < .0001. We have shown that the phenomenon of lipoprotein-supported thrombin generation is mediated predominantly by specific phospholipids and is enhanced by oxidation of these phospholipids. The addition of vitamin E (alpha-tocopherol) markedly reduced the increase in thrombin generation observed after oxidation of LDL (822 +/- 57 v 138 +/- 47 nmol/L; P < .0001). These effects suggest that lipoproteins are important in the production of thrombin and that vitamin E may confer protection from the detrimental effects of lipoprotein oxidation by limiting thrombin formation. These results suggest that atherogenic lipoproteins are linked to the development of atherosclerosis in part by their capacity to support thrombin generation.

Thrombin activation requires assembly of a prothrombinase complex of activated coagulation factors on an anionic phospholipid surface, classically provided by activated platelets. We have previously shown that anionic phosphatidylserine is exposed by rat vascular smooth muscle cells (VSMCs) undergoing apoptosis after serum withdrawal. In this study, using a chromogenic assay, we have shown thrombin generation by apoptotic VSMCs expressing c-myc (VSMC-myc) with an area under the thrombin-generation curve (AUC) of 305 +/- 17 nmol x min/L and a peak thrombin (PT) of 154 +/- 9 nmol/L. The thrombin-generating potential of the apoptotic VSMC-myc cells was greater than that of unactivated platelets (P = .003 for AUC; P = .0002 for PT) and similar to calcium-ionophore activated platelets (AUC of 332 +/- 15 nmol x min/L, P = .3; PT of 172 +/- 8 nmol/L, P = .2). Thrombin activation was also seen with apoptotic human VSMCs (AUC of 211 +/- 8 nmol x min/L; PT of 103 +/- 4 nmol/L) and was inhibited by annexin V (P < .0001 for AUC and PT). VSMC-myc cells maintained in serum generated less thrombin than after serum withdrawal (P = .0002 for AUC and PT). VSMCs derived from human coronary atherosclerotic plaques that apoptose even in serum also generated thrombin (AUC of 260 +/- 2 nmol x min/L; PT of 128 +/- 4 nmol/L). We conclude that apoptotic VSMCs possess a significant thrombin-generating capacity secondary to phosphatidylserine exposure. Apoptotic cells within atherosclerotic plaques may allow local thrombin activation, thereby contributing to disease progression.

Perianal sepsis in patients with acute leukaemia is a life threatening complication which carries an extremely poor prognosis. The usual approach to this problem is simple incision and parenteral antibiotics but we have recently treated two patients with wide excision combined with a defunctioning colostomy which resulted in rapid and complete eradication of sepsis.