Fibrinolysis in trauma patients: wide variability demonstrated by the Lysis Timer.

Hyperfibrinolysis contributes to the pathophysiology of trauma-induced coagulopathy. At present, systematic administration of tranexamic acid (TXA) is recommended in all patients in the early phase of trauma. However, there is some debate regarding whether TXA is beneficial in all trauma patients. A rapid and accurate tool to diagnose hyperfibrinolysis may be useful for tailoring TXA treatment. We conducted a proof-of-concept study of consecutive adult trauma patients. A first blood sample was obtained at the time of pre-hospital care (T1). Patients received 1 g of TXA after T1. A second sample was obtained on arrival at the emergency unit (T2). We examined coagulation, fibrin and fibrinogen formation and degradation. Fibrinolysis was assessed by determining tissue plasminogen activator (t-PA) antigen and plasminogen activator inhibitor 1 (PAI-1) activity and global fibrinolysis capacity assay using a device developed by Hyphen BioMed: the Lysis Timer (GFC/LT). The study population consisted of 20 patients (42 ± 21 years, index of severity score 32 ± 21). Both coagulation and fibrinolysis were altered at T1. GFC/LT values exhibited hyperfibrinolysis only in five patients. Principal component analysis carried out at T1 showed two main axes of alteration. The major axis was related to coagulation, altered in all patients, while the second axis was related to fibrinolysis. GFC/LT was mainly influenced by PAI-1 activity while fibrin monomers were related to the severity of trauma. At T2, GFC/LT exhibited the marked effect of TXA on clot lysis time. In conclusion, GFC/LT demonstrated huge variation in the fibrinolytic response to trauma.

Platelet glycoprotein VI binds to polymerized fibrin and promotes thrombin generation.

Fibrin, the coagulation end product, consolidates the platelet plug at sites of vascular injury and supports the recruitment of circulating platelets. In addition to integrin αIIbß3, another as-yet-unidentified receptor is thought to mediate platelet interaction with fibrin. Platelet glycoprotein VI (GPVI) interacts with collagen and several other adhesive macromolecules. We evaluated the hypothesis that GPVI could be a functional platelet receptor for fibrin. Calibrated thrombin assays using platelet-rich plasma (PRP) showed that tissue factor-triggered thrombin generation was impaired in GPVI-deficient patients and reduced by the anti-GPVI Fab 9O12. Assays on reconstituted PRP and PRP from fibrinogen-deficient patients revealed a fibrinogen-dependent enhancement of thrombin generation, which relied on functional GPVI. The effect of GPVI was found to depend on fibrin polymerization. A binding assay showed a specific interaction between GPVI-Fc and fibrin, inhibited by the Fab 9O12. This Fab also reduced platelet adhesion to fibrin at low (300 s(-1)) and high (1500 s(-1)) wall shear rates. Platelets adherent to fibrin displayed shape change, exposure of procoagulant phospholipids, and the formation of small clots. When hirudinated blood was perfused at 1500 s(-1) over preformed fibrin-rich clots, the Fab 9O12 decreased the recruitment of platelets by up to 85%. This study identifies GPVI as a platelet receptor for polymerized fibrin with 2 major functions: (1) amplification of thrombin generation and (2) recruitment of circulating platelets to clots. These so-far-unrecognized properties of GPVI confer on it a key role in thrombus growth and stabilization.

Management of bleeding and transfusion during liver transplantation before and after the introduction of a rotational thromboelastometry-based algorithm.

Orthotopic liver transplantation (OLT) remains a potentially hemorrhagic procedure. Rotational thromboelastometry (ROTEM) is a point-of-care device used to monitor coagulation during OLT. Whether it allows blood loss and transfusions to be reduced during OLT remains controversial. Excellent correlations and predictive values have been found between ROTEM parameters and fibrinogen. We hypothesized that the use of a ROTEM-based transfusion algorithm during OLT would lead to more fibrinogen transfusion and decreased bleeding and blood transfusion. Sixty adult patients were consecutively included in a prospective, without-versus-with study: 30 in the group without ROTEM results and 30 in the group with the ROTEM-based algorithm. A small and nonsignificant increase in median fibrinogen transfusions was found for the with group (6.0 g versus 4.5 g, P = 0.50). It was not associated with a decrease in blood transfusions or in the number of patients exposed to blood products.

Interest of Antiplatelet Drug Testing after an Acute Ischemic Stroke.

BACKGROUND: Stroke occurrence despite chronic antiplatelet drug (APD) treatment is frequent. We aimed at evaluating the relevance of platelet aggregation testing in the identification of stroke etiology in this context. METHODS: Patients admitted for a suspected acute ischemic stroke, while under APD (aspirin and/or clopidogrel), were prospectively included. The efficacy of the APD was evaluated using a Multiplate™ assay. Resistance was confirmed using light transmission aggregometry. A standardized diagnostic work-up was performed to identify stroke mechanism according to the TOAST and the ASCO classifications. We evaluated the influence of APD functional status on stroke severity and identified potential determinants of resistance. RESULTS: APD resistance was observed in 53 of the 287 patients (18.5%). No difference in stroke mechanism depending on APD efficacy was observed. Patients sensitive to APD had less severe initial stroke severity (mean National Institutes of Health Stroke Scale 3.9 ± 5.6 vs. 7.2 ± 6.8; p < 0.01). Main determinants for APD resistance were a worse control of the diabetes and higher baseline levels of inflammation (mean CRP 26.4 ± 56.0 vs. 9.3 ± 21.0; p < 0.01). CONCLUSIONS: Platelet function testing does not provide orientation concerning stroke mechanism in patients who were previously on APDs. However, the high frequency of APD resistance and its association with inflammation and stroke severity are confirmed.

Subclavian central venous catheter-related thrombosis in trauma patients: incidence, risk factors and influence of polyurethane type.

INTRODUCTION: The incidence of deep venous thrombosis (DVT) related to a central venous catheter varies considerably in ICUs depending on the population included. The aim of this study was to determine subclavian central venous catheter (SCVC)-related DVT risk factors in severely traumatized patients with regard to two kinds of polyurethane catheters. METHODS: Critically ill trauma patients needing a SCVC for their usual care were prospectively included in an observational study. Depending on the month of inclusion, patients received one of the two available products in the emergency unit: either an aromatic polyurethane SCVC or an aliphatic polyurethane SCVC. Patients were screened weekly by ultrasound for SCVC-related DVT. Potential risk factors were collected, including history-related, trauma-related and SCVC-related characteristics. RESULTS: A total of 186 patients were included with a median Injury Severity Sore of 30 and a high rate of severe brain injuries (21% of high intracranial pressure). Incidence of SCVC-related DVT was 37% (95% confidence interval: 26 to 40) in patients or 20/1,000 catheter-days. SCVC-related DVT occurred within 8 days in 65% of cases. There was no significant difference in DVT rates between the aromatic polyurethane and aliphatic polyurethane SCVC groups (38% vs. 36%). SCVC-related DVT independent risk factors were age>30 years, intracranial hypertension, massive transfusion (>10 packed red blood cell units), SCVC tip position in the internal jugular or in the innominate vein, and ipsilateral jugular catheter. CONCLUSION: SCVC-related DVT concerned one-third of these severely traumatized patients and was mostly clinically silent. Incidence did not depend on the type of polyurethane but was related to age>30 years, intracranial hypertension or misplacement of the SCVC. Further studies are needed to assess the cost-effectiveness of routine screening in these patients in whom thromboprophylaxis may be hazardous.

C-reactive protein and D-dimer in cerebral vein thrombosis: Relation to clinical and imaging characteristics as well as outcomes in a French cohort study.

Introduction: Cerebral venous sinus thrombosis (CVST) is a rare disease with highly variable clinical presentation and outcomes. Clinical studies suggest a role of inflammation and coagulation in CVST outcomes. The aim of this study was to investigate the association of inflammation and hypercoagulability biomarkers with CVST clinical manifestations and prognosis. Methods: This prospective multicenter study was conducted from July 2011 to September 2016. Consecutive patients referred to 21 French stroke units and who had a diagnosis of symptomatic CVST were included. High-sensitivity C-reactive protein (hs-CRP), neutrophil-to-lymphocyte ratio (NLR), D-dimer, and thrombin generation using calibrated automated thrombogram system were measured at different time points until 1 month after anticoagulant therapy discontinuation. Results: Two hundred thirty-one patients were included. Eight patients died, of whom 5 during hospitalization. The day 0 hs-CRP levels, NLR, and D-dimer were higher in patients with initial consciousness disturbance than in those without (hs-CRP: 10.2 mg/L [3.6-25.5] vs 23.7 mg/L [4.8-60.0], respectively; NLR: 3.51 [2.15-5.88] vs 4.78 [3.10-9.59], respectively; D-dimer: 950 µg/L [520-2075] vs 1220 µg/L [950-2445], respectively). Patients with ischemic parenchymal lesions (n = 31) had a higher endogenous thrombin potential5pM than those with hemorrhagic parenchymal lesions (n = 31): 2025 nM min (1646-2441) vs 1629 nM min (1371-2090), respectively (P = .0082). Using unadjusted logistic regression with values >75th percentile, day 0 hs-CRP levels of >29.7 mg/L (odds ratio, 10.76 [1.55-140.4]; P = .037) and day 5 D-dimer levels of >1060 mg/L (odds ratio, 14.63 [2.28-179.9]; P = .010) were associated with death occurrence. Conclusion: Two widely available biomarkers measured upon admission, especially hs-CRP, could help predict bad prognosis in CVST in addition to patient characteristics. These results need to be validated in other cohorts.

Cerebral Venous Thrombosis: Clinical, Radiological, Biological, and Etiological Characteristics of a French Prospective Cohort (FPCCVT)-Comparison With ISCVT Cohort.

Introduction: Cerebral venous thrombosis (CVT) is a rare disease with highly variable clinical presentation and outcome. Etiological assessment may be negative. The clinical and radiological presentation and evolution can be highly variable. The mechanisms involved in this variability remain unknown. Objective: The aim of this multicenter French study registered on ClinicalTrials.gov (NCT02013635) was therefore to prospectively recruit a cohort of patients with cerebral venous thrombosis (FPCCVT) in order to study thrombin generation and clot degradation, and to evaluate their influence on clinical radiological characteristics. The first part of the study was to compare our cohort with a reference cohort. Methods: This prospective, multicenter, French study was conducted from July 2011 to September 2016. Consecutive patients (aged >15 years) referred to the stroke units of 21 French centers and who had a diagnosis of symptomatic CVT were included. All patients gave their written informed consent. The diagnosis of CVT had to be confirmed by imaging. Clinical, radiological, biological, and etiological characteristics were recorded at baseline, at acute phase, at 3 months and at last follow-up visit. Thrombophilia screening and the choice of treatment were performed by the attending physician. All data were compared with data from the International Study on CVT published by Ferro et al. Results: Two hundred thirty-one patients were included: 117 (50.6%) had isolated intracranial hypertension, 96 (41.5%) had focal syndrome. During hospitalization, 229 (99.1%) patients received anticoagulant treatment. Median length of hospital stay was 10 days. Five patients died during hospitalization (2.2%). At 3 months, 216 patients (97.0%) had follow-up with neurological data based on an outpatient visit. The mean duration of antithrombotic treatment was 9 months, and the mean time to last follow-up was 10.5 months. At the end of follow-up, eight patients had died, and 26 patients were lost to follow-up. At least one risk factor was identified in 200 patients. Conclusions: We demonstrated that the FPCCVT cohort had radiological, biological, and etiological characteristics similar to the historical ISCVT cohort. Nevertheless, the initial clinical presentation was less severe in our study probably due to an improvement in diagnostic methods between the two studies.

Comparison of Two Thrombin Generation Methods, CAT and ST-Genesia, in Liver Transplant Patients.

BACKGROUND: During liver transplantation (LT), thrombin generation (TG) is altered. The most frequently used assay for TG is the Calibrated Automated Thrombogram (CAT). It is designed for series of plasmas and is semi-automated. Complete automation has led to a new device, the ST-Genesia, enabling quantitative standardized TG evaluation. OBJECTIVE: The aim of this observational study was to compare the TG results of the CAT and the ST-Genesia on frozen-thawed plasma samples prepared from the blood of LT patients. PATIENTS AND METHODS: Poor platelet plasma aliquots were prepared from blood samples from six LT patients selected to get the whole range of TG and were assessed with CAT (recombinant human tissue factor [TF] concentration 5 pm) and with ST-Genesia Bleedscreen assay (BS, using 'low' recombinant human TF concentration) and Thromboscreen assay (TS, using 'medium' recombinant human TF concentration). The TG parameters studied were: lag time, peak, time to peak, endogenous thrombin potential, velocity index and start tail. RESULTS: BS and TS did not differ significantly from each other whatever the parameter studied, whereas most of the CAT parameters were significantly different from those obtained with BS and TS. Hierarchical clustering analysis of the different parameters of TG showed three homogeneous groups. One cluster gathered TG quantitative parameters from ST-Genesia. A second cluster gathered all the kinetic parameters. The last cluster isolated the quantitative parameters of CAT. CONCLUSION: In patients undergoing LT, TG performed with CAT and with ST-Genesia provided different results, for unknown reasons.

Fibrinolysis during liver transplantation: analysis by the Thrombodynamics method.

An issue in orthotopic liver transplantation (OLT) is the diagnosis of hyperfibrinolysis. The Thrombodynamics-4D assay (TD4D) is a videomicroscopy system allowing the dynamic analysis of fibrin clot. Fibrinolysis is highlighted by a change in clot intensity. The aim of this observational study was to evaluate the TD4D as a tool to diagnose fibrinolysis during OLT. Thirty consecutive patients were included. We studied a subset of 41 samples from 13 patients who demonstrated hyperfibrinolysis during OLT by global fibrinolytic capacity studied by the Lysis Timer (GFC/LT) and/or euglobulin clot lysis time (ECLT) and/or EXTEM maximum lysis (EXTEM ML) on ROTEM. Three samples exhibited fibrinolysis. They exhibited significantly shorter ECLT, higher lysis on EXTEM graphs, shorter GFC/LT clot lysis time and higher t-PA activity values. After adding urokinase, 13 samples exhibited fibrinolysis. In conclusion, TD4D allows the dynamic analysis of fibrin clot formation and lysis. It only recognises the most severe forms of hyperfibrinolysis during OLT.

Lysis Timer: a new sensitive tool to diagnose hyperfibrinolysis in liver transplantation.

AIMS: Diagnosis of hyperfibrinolysis in orthotopic liver transplantation (OLT) remains challenging. Euglobulin clot lysis time (ECLT) is not adapted to clinical situations. ROTEM is specific but seldom sensitive to hyperfibrinolysis. The Lysis Timer assesses 'Global Fibrinolytic Capacity' in citrated plasma (GFC/LT). GFC/LT associates reagents for in vitro triggering of the clot (thrombin and calcium) and its lysis (tissue-plasminogenactivator (t-PA)), turbidity signal acquisition by the Lysis Timer, and dedicated software converting the digital signal into an optical curve. A visual check of the curves was systematic to ascertain the lysis time values calculated by the software. The primary aim of this prospective observational study was to evaluate the ability of GFC/LT to recognise hyperfibrinolysis during OLT. The secondary aim was to compare its results with ROTEM maximum lysis (EXTEM ML) and with standard laboratory tests. METHODS: Thirty consecutive adult patients undergoing OLT were included (NCT03012633). Standard laboratory tests, ROTEM, GFC/LT, ECLT and fibrinolysis parameters were assayed at five sample times. RESULTS: GFC/LT was correlated with ECLT, plasmin activator inhibitor 1 antigen and activity and t-PA activity (r=0.490, 0.681, 0.643 and -0.359, respectively). Hyperfibrinolysis was defined as ECLT ≤60 min. Receiver operating characteristic curve analysis showed that GFC/LT with a threshold of 31 min detected hyperfibrinolysis with a sensitivity of 0.88 (95% CI 0.73 to 0.96), a specificity of 0.68 (95% CI 0.56 to 0.78) and an area under the curve (AUC) of 0.85 (95% CI 0.74 to 0.94). EXTEM ML >12% did not detect hyperfibrinolysis (sensitivity 0.38 (95% CI 0.24 to 0.55), specificity 0.95 (95% CI 0.86 to 0.99) and AUC 0.60 (95% CI 0.46 to 0.75)). CONCLUSIONS: GFC/LT recognised hyperfibrinolysis during OLT with a significant agreement with the other tests of fibrinolysis. TRIAL REGISTRATION NUMBER: NCT03012633.

Evaluation of dabigatran, rivaroxaban and apixaban target-specific assays in a multicenter French study.

Dabigatran etexilate, rivaroxaban and apixaban (DOACs) are widely used and measurement of their concentration is desirable in certain clinical situations. Target-specific assays are available but limited information exists on their performance especially in their ability to accurately measure low and high concentrations. AIMS: To define, in a multicenter study, the precision and accuracy of DOAC measurements in daily practice. METHODS: 15 plasma samples (kindly provided by Hyphen-Biomed) spiked with 5 blinded concentrations of dabigatran, rivaroxaban or apixaban (targeted 0-40-100-250-500ng/mL, actual concentrations measured by HPLC-MS/MS), were sent to 30 haemostasis laboratories. DOAC concentration, PT and aPTT were measured once in each sample using local reagents. Interlaboratory precision was determined by its coefficient of variation (CV) and accuracy by its bias. RESULTS: 464 DOAC measurements were performed in the 30 laboratories using 4 dabigatran and 5 rivaroxaban/apixaban calibrated assays on 3 analysers. Inter-laboratory CVs were below 18% for concentrations ≥100ng/mL, and higher for concentrations ~40ng/mL; biases were below 8% for all drugs and concentrations. In DOAC-free samples, concentrations were all below the lower limit of quantification except for one value (dabigatran: 35ng/mL). Depending on the concentrations, significant differences were found between reagents in rivaroxaban and apixaban concentration values. PT and aPTT ratios displayed a low sensitivity to apixaban. CONCLUSION: Our results suggest that calibrated DOAC assays allow the reliable measurement of a wide range of drug concentrations, even though improvement of their performances is necessary, especially for measuring low concentrations.

Genetic background of type I protein C deficiency in Finland.

In contrast to other populations the usually rare type II form of protein C deficiency is as common in Finland as type I deficiency. We recently reported that a single mutation explained virtually all cases of type II protein C deficiency in Finland, indicating strong founder effect. We now investigated in the same population the genetic background of type I protein C deficiency. Thirty-eight apparently unrelated families were studied. They represent the vast majority of all families with type I deficiency in Finland. A genetic defect was identified in 23 (61%) families who carried 13 different mutations. Only three of the 13 mutations have been reported in other populations. Unlike in type II deficiency, considerable heterogeneity in mutations was found in type I deficiency. Our results indicate interesting differences in mutational histories of these two different forms of protein C deficiency in Finland.

Pharmacodynamic markers in the early clinical assessment of otamixaban, a direct factor Xa inhibitor.

This manuscript reports the assessment of pharmacodynamic (PD) markers of anti-coagulation in the first-in-man study with the novel direct Factor Xa (FXa) inhibitor, otamixaban, with a brief description of safety and pharmacokinetic (PK) findings. The study comprised ten consecutive parallel groups of healthy male subjects (6 active, 2 placebo per group). Eight groups received escalating intravenous doses of otamixaban as 6-hour infusions (1.7 to 183 microg/kg/h) and two groups received a bolus dose (30 or 120 microg/kg) with a 6-hour infusion (60 or 140 microg/ kg/h, respectively). PD markers included anti-FXa activity and clotting time measurements, i.e. activated Thromboplastin Time (aPTT), Prothrombin Time (PT), Heptest Clotting Time (HCT), and Russell's Viper Venom-induced clotting Time (RVVT). In addition, Endogenous Thrombin Potential (ETP) was assessed in the bolus-plus-infusion dose groups. Otamixaban was well tolerated. Otamixaban plasma concentrations increased with escalating dose, were maximal at the end-of-infusion (C(eoi)), and decreased rapidly as the infusion was stopped. Anti-FXa activity coincided with otamixaban plasma concentrations and clotting time measurements followed the same pattern. Maximal changes from baseline at C(eoi) were 1.9 +/- 0.2 for aPTT, 2.0 +/- 0.2 for PT, 5.1 +/- 0.6 for HCT, and 4.5 +/- 1.2 for RVVT. Otamixaban inhibited thrombin generation (24% decrease in ETP) and a delay in thrombin generation was noticed in vitro at high concentrations.

Diagnosis accuracy of a new challenger for thrombosis exclusion, the Stratus CS DDMR.

BACKGROUND: The diagnostic accuracy of a new Point of Care, rapid and quantitative D-dimer assay (Stratus CS DDMR from Dade Behring) was evaluated. METHODS: Vidas test from bioMerieux was used as reference method in 279 patients recruited from a management study in progress in our institution. RESULTS: Both assays show comparable reproducibility (2.9% with the Stratus CS DDMR and 4.4% with the D-dimer Vidas in the cut-off range) and good correlation (R(2)=0.9057). The overall test performance as assessed by the area under the curve of the ROC curves is 0.801 for the Stratus CS DDMR assay and 0.798 for the D-dimer Vidas assay. By using assay regression curves, likelihood ratios or test agreement approaches, a 440-450 ng/ml value is evidenced as the threshold value for the Stratus CS DDMR, which nears at best the performances of the D-dimer Vidas 500 or 550 ng/ml threshold value. This proposed exclusion value ensures a 95% sensitivity and a 45% specificity. The few false negative results using the two assays only evidenced sub-popliteal thromboses, that would not have been considered as having thrombosis if an above-the-knee test had been performed. In these conditions, sensitivity would have been 100%. CONCLUSIONS: A new quantitative D-dimer assay, the Stratus CS DDMR, demonstrated performances comparable with those of the DD Vidas test.

Haemostaseome-associated SNPs: has the thrombotic phenotype a greater influence than ethnicity? GMT study from Aquitaine including Basque individuals.

The Genetic Markers for Thrombosis (GMT) study compared the relative influence of ethnicity and thrombotic phenotype regarding the distribution of SNPs implicated in haemostasis pathophysiology ("haemostaseome"). We assessed 384 SNPs in three groups, each of 480 subjects: 1) general population of Aquitaine region (Southwestern France) used as control; 2) patients with venous thromboembolism from the same area; and 3) autochthonous Basques, a genetic isolate, who demonstrate unusual characteristics regarding the coagulation system. This study sought to evaluate i) the value of looking for a large number of genes in order to identify new genetic markers of thrombosis, ii) the value of investigating low risk factors and potential preferential associations, iii) the impact of ethnicity on the characterisation of markers for thrombosis. We did not detect any previously unrecognised SNP significantly associated with thrombosis risk or any preferential associations of low-risk factors in patients with thrombosis. The sum of Ï°² values for our 110 significant SNPs demonstrated a smaller genetic distance between patients and controls (321 cumulated Ï°² value) than between Basques and controls (1,570 cumulated Ï°² value). Hence, our study confirms the genetic particularity of Basques especially regarding a significantly lower expression of the non-O blood group (p< 0.0004). This is mitigated by a higher prevalence of factor II Leiden (p< 0.02) while factor V Leiden prevalence does not differ. Numerous other differences covering a wide range of proteins of the haemostaseome may result in an overall different genetic risk for venous thromboembolism.

Rivaroxaban and apixaban in orthopaedics: is there a difference in their plasma concentrations and anticoagulant effects?

The aim of this study was to improve knowledge of what happens in the coagulation of orthopaedic patients under rivaroxaban and apixaban, in order to finalize and cross-validate effective measurement methods and to provide arguments for helping to reference one or the other drug in our central pharmacy. One hundred and two patients undergoing total hip or knee replacement were included. Half of them received rivaroxaban and the other half received apixaban. Blood samples (n = 244 with each drug) were taken at Cmax preoperatively and twice a week, apart from the day of the patient's discharge, when Ctrough concentration was targeted. Routine coagulation parameters, and functional and liquid chromatography tandem mass spectrometry assays for measurement of circulating concentrations were studied. The LC-MS/MS assay and the functional assays carried out in patients under routine conditions were highly correlated, apart from low concentrations (<30 ng/ml), which were affected by the variable individual potential to inhibit the exogenous bovine Xa used in the functional assays. After 1 week of treatment, the drugs differed: Cmax and Ctrough were closer when apixaban was taken twice daily (83 ±â€Š39 and 58 ±â€Š17 ng/ml) than with rivaroxaban taken once a day (113 ±â€Š67 and 13 ±â€Š20 ng/ml). Rivaroxaban had a greater influence on routine coagulation tests and reduced the maximum thrombin concentration more efficiently, as assessed by the thrombin generation test. Although rivaroxaban and apixaban present apparently similar constant rates, they exhibit significant differences in their concentrations and anticoagulant effects when studied ex vivo in orthopedic patients.

Protein C and protein S assessment in hospital laboratories: which strategy and what role for DNA sequencing?

This paper presents a critical assessment of protein C (PC) and protein S (PS) functional and immunological approaches with regard to DNA sequencing in a large hospital recruitment for thrombosis exploration in more than 1700 consecutive patients. After examination of clinical status and PC and PS phenotype, a genotypic study was implemented for 17 PC-deficient and 28 PS-deficient patients (activity < 70%). Sixty-five percent of the genotyped PC-deficient patients were found to have heterozygous mutations. Among the < 70% values, decreases in PC activity without gene mutation were always slight (mean value 64 +/- 7%) while patients presenting a PC gene mutation had a mean 50 +/- 17% activity (P < 0.05). Among the eight PC mutations found, only one has previously been described. A novel mutation in the promoter region (-1522), located in the HNF-1 site and associated with the Y226H heterozygous mutation, was found in a 9-month-old girl with 4% PC activity. Determination of PS functional activity was considerably improved by contemporaneous measurement of calibration and samples in a single step. Only 50% of the genotyped PS-deficient patients demonstrated heterozygous alterations of the gene. The benefit of sequencing to identify putative causal mutations was only 39% in PS-deficient women, while it was 90% in men. Among the nine PS mutations found, six have not yet been published. In the present paper, we explain our methodological choices and diagnostic strategy.

Novel risk factors for premature peripheral arterial occlusive disease in non-diabetic patients: a case-control study.

BACKGROUND: This study aimed to determine the prevalence of genetic and environmental vascular risk factors in non diabetic patients with premature peripheral arterial disease, either peripheral arterial occlusive disease or thromboangiitis obliterans, the two main entities of peripheral arterial disease, and to established whether some of them are specifically associated with one or another of the premature peripheral arterial disease subgroups. METHODS AND RESULTS: This study included 113 non diabetic patients with premature peripheral arterial disease (diagnosis <45-year old) presenting either a peripheral arterial occlusive disease (N = 64) or a thromboangiitis obliterans (N = 49), and 241 controls matched for age and gender. Both patient groups demonstrated common traits including cigarette smoking, low physical activity, decreased levels of HDL-cholesterol, apolipoprotein A-I, pyridoxal 5'-phosphate (active form of B6 vitamin) and zinc. Premature peripheral arterial occlusive disease was characterized by the presence of a family history of peripheral arterial and carotid artery diseases (OR 2.3 and 5.8 respectively, 95% CI), high lipoprotein (a) levels above 300 mg/L (OR 2.3, 95% CI), the presence of the factor V Leiden (OR 5.1, 95% CI) and the glycoprotein Ia(807T,837T,873A) allele (OR 2.3, 95% CI). In thromboangiitis obliterans group, more patients were regular consumers of cannabis (OR 3.5, 95% CI) and higher levels in plasma copper has been shown (OR 6.5, 95% CI). CONCLUSIONS: According to our results from a non exhaustive list of study parameters, we might hypothesize for 1) a genetic basis for premature peripheral arterial occlusive disease development and 2) the prevalence of environmental factors in the development of thromboangiitis obliterans (tobacco and cannabis). Moreover, for the first time, we demonstrated that the 807T/837T/873A allele of platelet glycoprotein Ia may confer an additional risk for development of peripheral atherosclerosis in premature peripheral arterial occlusive disease.

Coagulation parameters in patients receiving dabigatran etexilate or rivaroxaban: two observational studies in patients undergoing total hip or total knee replacement.

INTRODUCTION: Dabigatran and rivaroxaban have recently been added to the armamentarium for thromboprophylaxis in orthopedic surgery. Although this is their first licensed indication, others will soon follow. Owing to their claimed predictable anticoagulant response that dispenses with the need for monitoring coagulation, their effects are poorly described in routine cases. However, interpreting blood coagulation results and evaluating whether a treatment is properly targeted in the case of untoward incidents will become a common concern for clinicians. METHODS: Eighty patients undergoing total hip or knee replacement were included in two studies. Forty of them received dabigatran (study 1) and 40 rivaroxaban (study 2). Blood samples (n = 176 and 166) were taken preoperatively and twice a week from the first postoperative day. RESULTS: Dabigatran increased aPTTr about two-fold and PT about 1.2-fold, and it was mostly an initiation-phase modulator of thrombin generation. Mean circulating concentrations as measured by a diluted thrombin time were 105 ± 85 ng/mL at T(max) in samples from patients receiving the full dosing. They depended significantly on renal function, body weight and gender. Rivaroxaban increased aPTTr and PTr around 1.5 fold and modified the initiation and amplification phases of thrombin generation, with a lowered and prolonged thrombin burst. Mean circulating concentrations as measured by an antiXa test were 117 ± 78 ng/mL at T(max). With both drugs, routine coagulation tests, thrombin generation curves and functionally determined concentrations exhibited high interindividual variability. CONCLUSION: Routine coagulation tests are altered in patients receiving dabigatran or rivaroxaban, but their alterations poorly reflect the circulating concentrations as determined by functional approaches.