Risk factors for venous thrombosis in the black population

Risk factors for venous thrombo-embolism (VTE) in the black population are poorly characterized. Of 142 black cases tested a genetic cause was identified in only 9.1%: 4.2% had protein C deficiency, 2.8% protein S deficiency, 0.7% antithrombin deficiency and 1.4% were heterozygous for FV Leiden. We hypothesised that elevated factor VIII levels constitute a candidate risk factor for venous thrombosis in the black population. Factor VIII (FVIII:C) levels were determined in 100 black patients with VTE and 100 black controls in a case-control study. Of the patients 34% had a FVIII:C above 228 IU/dL (the 90th centile value in normal blacks) compared to 10% controls. Relative to those with FVIII:C below this value, odds ratio (OR) for risk of VTE was 4.64 (95% CI 2.02-10.85). When FVIII:C below 150 IU/dL was used as a comparator, OR was 11.1 (95% CI 4.29-29.43). There was evidence for a dose-response relationship. We propose that raised FVIII:C is a major risk factor for VTE in black subjects with prevalence and odds ratio exceeding those reported for white subjects.     

Effects of pretreatment with clopidogrel on platelet and coagulation activation in patients undergoing elective coronary stenting

BACKGROUND: Current data suggest that pretreatment with clopidogrel (in addition to aspirin) prior to elective percutaneous coronary intervention (PCI) might be associated with a reduced incidence of subsequent adverse ischemic events. The aim of this placebo-controlled study was to find out whether an extended pretreatment period with clopidogrel before an elective PCI might confer a superior inhibition of the platelet activation and aggregation than clopidogrel given not until PCI. METHODS: Twenty patients with stable angina being already on aspirin were randomly assigned to receive the loading dose of 300 mg clopidogrel, either 24 h before or immediately after stent implantation. At several time points before and after PCI, the activation of both the platelet and the coagulation system was determined by measuring beta-thromboglobulin (beta-TG) and prothrombin fragment f1.2 (f1.2), respectively, in venous blood and in blood emerging from a microvascular injury (shed blood). RESULTS: Pretreatment with clopidogrel before PCI exhibited a slight reduction of beta-TG (from 178 to 139 ng/ml, p=0.085) and of f1.2 (from 0.81 to 0.75 nmol/l, p=0.045) in venous blood. Heparin administration (at the beginning of PCI) resulted in a 65% inhibition of ss-TG (from 10,590 to 2833 ng/ml) and 90% inhibition of f1.2 formation (from 38.7 to 4.2 nmol/l) in shed blood of patients with clopidogrel pretreatment. The extent of inhibition was, however, comparable to that observed in patients without clopidogrel pretreatment (beta-TG: from 8025 to 2812 ng/ml, 76% inhibition, p=0.47; f1.2: from 34.9 to 3.8 nmol/l, 86% inhibition, p=0.80). After PTT normalisation (6 h after PCI), levels of beta-TG and f1.2 both in venous blood and in shed blood did not differ between the two treatment regimens up to 48 h after PCI. CONCLUSION: Pretreatment with clopidogrel did not result in a pronounced inhibition of the platelet and coagulation system activation in patients on aspirin undergoing elective coronary stent implantation.     

The acute phase reaction explains only a part of initially elevated factor VIII:C levels: a prospective cohort study in patients with venous thrombosis

We determined in a prospective cohort of patients treated with vitamin K antagonists for venous thrombosis, the course of factor VIII (FVIII:C), C-reactive protein (CRP) and fibrinogen levels, to assess the influence of the acute phase reaction on FVIII:C levels. Second, we hypothesized that patients with preceding infectious symptoms might have higher levels of FVIII:C at baseline than patients without those.We included 75 patients. Blood was sampled at baseline, once during treatment (t = 1) and at the end of treatment (t = 2). Mean levels of FVIII:C were 207, 186 and 175 IU/dL (p for trend 0.003) at baseline, t = 1 and t = 2 respectively. Eight-eight percent of patients had an elevated FVIII:C at baseline, 75% at t = 1 and 72% at t = 2 (p for trend 0.045). Mean levels of FVIII:C were not different in patients with or without preceding infectious symptoms (206 versus 205 IU/dL respectively). A baseline CRP level below 62 mg/L could best distinguish between patients who will keep an elevated FVIII:C and those who will drop below 150 IU/dL.We conclude that FVIII:C levels are partially influenced by the acute phase reaction, especially in patients who keep a persistent elevated FVIII:C during treatment.Preceding infectious symptoms did not influence baseline FVIII:C levels.     

D-dimer and factor VIII are independent risk factors for recurrence after anticoagulation withdrawal for a first idiopathic deep vein thrombosis

Background and objectives: To assess the predictive value of D-dimer (D-d) and Factor VIII (FVIII) in combination for recurrent venous thromboembolism (VTE) after vitamin K antagonist (VKA) therapy suspension. Design and methods: Consecutive outpatients with a first episode of idiopathic proximal deep vein thrombosis of the lower limbs were enrolled on the day of VKA suspension. After 30+/-10 days, D-d (cut-off value: 500 ng/mL), chromogenic FVIII activity and inherited thrombophilia were determined. Follow-up was 2 years. Results: Overall recurrence rate was 16.4% (55/336; 95% CI:13-21%). The multivariate hazard ratio (HR) for recurrence was 2.45 (95% CI: 1.24-4.99) for abnormal D-d and 2.76 (95% CI:1.57-4.85) for FVIII above the 75th percentile (2.42 U/mL) after adjustment for age, sex, thrombophilia, VKA duration and residual venous obstruction. When compared with normal D-d and FVIII, the multivariate HR was 4.5 (95% CI: 1.7-12.2) for normal D-d with FVIII above 2.42 U/mL and 2.7 (95% CI: 1.2-6.6) and 7.1 (95% CI:2.8-17.6) for abnormal D-d with FVIII, respectively, below and above 2.42 U/mL. Interpretation and conclusions: D-d and FVIII at 30+/-10 days after VKA withdrawal are independent risk factors for recurrent VTE.     

Venous thrombosis following the use of intermediate purity FVIII concentrate to treat patients with von Willebrand's disease

We describe four patients with von Willebrand's disease (VWD) who experienced venous thrombosis after treatment with an intermediate purity factor VIII (FVIII) concentrate (Haemate P3) was used to cover invasive or surgical procedures. Most patients had additional risk factors for venous thromboembolism (VTE) and it is difficult to be certain of the contribution of the concentrate to the VTE. In view of the recognised association between high factor VIII activity (FVIII:C) levels and VTE there is a physiological basis for this complication and it is important to consider this when administering FVIII containing concentrates to VWD patients.     

Influence of anticoagulant therapy with vitamin K antagonists on plasma levels of coagulation factor VIII

Vitamin K-antagonists (VKA) decreases vitamin K coagulation factors. To counterbalance this effect, it has been postulated that non-vitamin K proteins increase during VKA treatment. To investigate if VKA affect FVIII, a cohort of 1772 patients referred from Jan 1997 to Oct 2008 to our Thrombosis Center for a thrombophilia screening after at least 3 months from diagnosis of first venous thrombosis was studied. At the time of blood sampling, 1303 patients had discontinued VKA for at least one month, whereas the remaining 469 were still taking VKA. FVIII was significantly higher in patients on VKA than in those who had discontinued VKA (mean±SD: 144 ± 41 IU/dL and 134 ± 40 IU/dL, respectively, p < 0.0001), also after adjustment for sex, age, body mass index, thrombophilia and time elapsed from thrombosis in a multiple linear regression analysis. In order to avoid overestimation of FVIII levels, patients should be preferentially tested after VKA discontinuation.     

Persistent high factor VIII activity leading to increased thrombin generation - a prospective cohort study

Introduction

A persistently elevated level of factor VIII (FVIII) is an independent risk factor for venous thromboembolism (VTE). Although the pathophysiology of VTE is unclear, the involvement of thrombin generation (TG) has been postulated. Consequently this study was designed to (i) investigate the relationships between FVIII, Thrombin generation test (TGT) parameters and D-dimer in VTE patients, (ii) determine whether elevated levels of FVIII and increased TG in these patients are transient or sustained.

Patients and Methods

After an initial period of anticoagulation had been completed 91 VTE patients and 52 healthy controls were recruited. FVIII levels were determined by one-stage clotting (FVIII:C) and chromogenic (FVIII:Ch) assays. The potential to generate thrombin was measured using the Calibrated Automated Thrombogram (CAT) and D-Dimer was by immuno-turbidometric assay.

Results

Patients' FVIII:C levels and FVIII:Ch, exhibited good agreement (rs = 0.94; p < 0.0001), although FVIII:C exhibited a mean bias of -6%. FVIII:Ch show a significant correlation with TGT Peak Thrombin (rs = 0.30; p = 0.004) and Peak Thrombin was found to be significantly higher (p = 0.04) in patients with FVIII > 200 iu/dL. Furthermore elevated levels of FVIII and increased thrombin generation parameters appeared to be consistent over time.

Conclusion

Our data suggests that high FVIII leading to increased TG confers a significant risk of recurrent VTE and therefore we speculate that these patients may benefit from prolonged anticoagulation therapy.     

Effect of edoxaban on markers of coagulation in venous and shed blood compared with fondaparinux

Edoxaban, an oral direct factor Xa (FXa) inhibitor, is in phase III clinical development for stroke prevention in atrial fibrillation and treatment of venous thromboembolism. The shed blood model allows for study of activated coagulation at a site of standardised tissue injury due to local release of tissue factor. The objective of this study was to evaluate the effect of three doses of edoxaban on markers of coagulation in shed and venous blood versus placebo and a standard prophylactic dose of fondaparinux. A total of 100 healthy male subjects were randomised to receive single doses of one of five treatments: subcutaneously administered fondaparinux 2.5 mg; orally administered edoxaban 30, 60, or 120 mg; or placebo. The primary objective was measurement of blood coagulation markers prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin (TAT) complex, and platelet activation marker β-thromboglobulin (β-TG), in venous and shed blood. Secondary objectives included pharmacokinetics, shed blood volume, and safety of edoxaban. Single doses of edoxaban caused rapid and significant decreases of F1+2, TAT, and β-TG in the shed blood model, indicating inhibition of thrombin generation and platelet activation. Inhibition was significantly less for fondaparinux versus edoxaban. Baseline-corrected F1+2, TAT, and β-TG values demonstrated sustained inhibition up to 24 hours for shed blood in the edoxaban groups but no significant inhibition in venous blood. Overall, edoxaban treatments were well tolerated. In conclusion, single oral doses of edoxaban 30, 60, or 120 mg caused rapid and sustained inhibition of coagulation up to 24 hours in the shed blood model.     

The effect of dalteparin on coagulation activation during pregnancy in women with thrombophilia. A randomized trial

Low-molecular-weight heparin (LMWH) is increasingly being used for prophylaxis of venous thromboembolism (VTE) and prevention of pregnancy associated morbidity in pregnant women with thrombophilia. We sought to determine if the administration of prophylactic doses of LMWH downregulates coagulation activation in high risk pregnant women with thrombophilia. This sub-study was planned as part of a randomized open label controlled trial (Thrombophilia in Pregnancy Prophylaxis Study [TIPPS]) in which patients at high risk of pregnancy complications with confirmed thrombophilia are randomized to receive either dalteparin (5,000 units/day until 20 weeks then 5,000 units q12h until 37 weeks or onset of labor) or no treatment. Blood samples were collected at baseline, day 7-9 (after starting study drug), week 20 (before increasing study drug), week 36 (prior to stopping study drug) and at the time of admission to the labor and delivery unit. Samples were not drawn at fixed times in relation to drug injection. These samples were analyzed for levels of thrombin-antithrombin complexes (TAT), prothrombin fragments 1 + 2 (F1.2), D-dimer and anti-Xa activity. Generalized linear mixed models were used for statistical analysis and model results were controlled for age, smoking status, type of thrombophilia and predisposing risk factors. The effect of dalteparin on TAT levels was defined as the primary outcome. Of 198 patients eligible, 114 were enrolled in TIPPS. Ninety-one were eligible for the TIPPS coagulation activation sub-study and randomized. Thirty-nine patients were analyzed in the treatment group (dalteparin) and 46 patients in the control group (no intervention). Levels of coagulation activation factors F1.2, TAT and D-dimer increased significantly throughout pregnancy in both groups (p < 0.0001). Dalteparin prophylaxis resulted in a significant increase in anti-Xa activity through pregnancy (p < 0.0001) compared to controls. Dalteparin had no significant effects on the levels of TAT, F1.2 and D-dimer throughout pregnancy in thrombophilic women. A post-hoc Monte Carlo power analysis revealed that our study had 100% and 88% power to detect reductions in TAT values on treatment of 50% and 25%, respectively. Prophylaxis with dalteparin at doses used in this study did not reduce coagulation activation in high risk thrombophilic women during pregnancy.     

Elevated factor VIII in hereditary haemorrhagic telangiectasia (HHT): association with venous thromboembolism

Hereditary haemorrhagic telangiectasia (HHT) causes chronic nasal and gastrointestinal haemorrhage. Prothrombotic agents are commonly used for severe haemorrhage. Thrombotic risks have not been defined. In order to identify prothrombotic variables in HHT patients, and assess their potential functional significance, a pilot ELISA-based study comparing plasma proteins in healthy individuals with HHT to age/sex-matched non-HHT controls was validated in a full study of 309 consecutive HHT-affected individuals. In the pilot study, factor VIII (FVIII) and von Willebrand factor antigen concentrations were elevated in the HHT group compared to non-HHT controls (p<0.0013, Mann-Whitney). Service laboratory measurements confirmed high FVIII:Ag in 125 HHT-affected individuals with no recent ill-health, intervention or venous thromboemboli. FVIII:Ag levels increased with age. Logistic regression also suggested an age-independent association with HHT-associated pulmonary arteriovenous malformations (AVMs). No association was demonstrated between FVIII:Ag and acute phase response, disseminated intravascular coagulation, ABO group, pulmonary artery pressure, or markers of HHT haemorrhage. Elevated FVIII:Ag were associated with shortened activated partial thromboplastin times (APTTs), and VTE:VTE affected 20/309 (6.5%) HHT-affected individuals, at median age 61(36-71) years. Four VTE occurred in factorV Leiden heterozygotes in the months following PAVM-associated brain abscess. The strongest association with VTE was with log-transformed FVIII:Ag measured 10-132 months from VTE (odds ratio 2.41, 95% confidence intervals 1.254, 4.612, p=0.008). Age made no additional contribution to VTE risk once adjusted for FVIII:Ag. In conclusion, HHT-related elevation of FVIII:Ag levels may influence thrombotic risk in HHT. Individualised risk-benefit considerations may be helpful in HHT management.     

Absolute annual incidences of first events of venous thromboembolism and arterial vascular events in individuals with elevated FVIII:c. A prospective family cohort study

Elevated levels of factor VIII:c (elevated FVIII:c) are associated with an increased risk for venous thromboembolism (VTE) and arterial vascular events, and are at least in part determined genetically. We prospectively followed 192 asymptomatic individuals with elevated FVIII:c (>150%) and 340 with normal levels for an average duration of 31 months (range 7 to 56 months) to investigate the risk of VTE and arterial vascular events. Participants were first degree relatives of consecutive patients with elevated FVIII:c and VTE or arterial vascular events before the age of 50 years. The incidences of VTE were 1.25% (0.46-2.73) per year in the subjects with elevated FVIII:c, versus 0.23% (0.03-0.82) in those with normal levels (OR 5.5 [1.1-27.3]). The annual incidences of arterial vascular events were 1.04% (0.34-2.42) and 0.23% (0.03-0.82) in relatives with and without elevated levels of FVIII:c, respectively (OR: 4.5 [0.9-23.5]). After adjustment for age, smoking, known diabetes mellitus, hyperlipidemia, and hypertension, the odds ratio for any event was 3.7 (1.1-13.1). In conclusion, asymptomatic individuals with elevated FVIII:c levels and a positive family history of VTE or arterial vascular events before the age of 50 appear to have a high annual incidence of first VTE and arterial vascular events. Elevated FVIII:c may be a common risk factor for both clinical entities.     

Markers of activated coagulation in patients with factor V Leiden and/or G20210A prothrombin gene mutation

The activated protein C (APC) resistance phenotype associated with an abnormal factor V Leiden (FVL), and the G20210A prothrombin gene mutation are the most common findings in patients with venous thromboembolism (VTE). In a group of 210 patients, we compared the levels of markers of coagulation activation in carriers of FVL (71 heterozygous, 30 homozygous), G20210A prothrombin mutation (88 heterozygous) or both mutations combined (21 heterozygous), in order to assess whether these markers allow identification of a group of patients with a higher risk of thrombosis; they were also compared to normal values. A total of 143 patients had a personal history of VTE and 67 were asymptomatic. None of them had other hereditary causes of thrombophilia or an antiphospholipid syndrome. None were currently treated with either anticoagulant or hormonal treatment. Pregnant women were excluded.No significant difference between the four groups of patients could be found in the levels of F1+2, TAT and DDI. Levels were all significantly higher than the control values (p<0.05).The levels of F1+2 and TAT were similar in patients with or without a history of VTE, regardless of the type of mutation. DDI levels were significantly higher in patients with a history of VTE than in asymptomatic subjects (443+/-248 vs. 333+/-222 ng/ml, p=0.02) but with only 57% sensitivity and specificity. In conclusion, our study confirms the hypercoagulable state found in mutation carriers and points out the inability of F1+2 and TAT assays to identify a group of subjects at higher risk of thrombosis, within carriers of genetic risk factors. Although the sensitivity and specificity of DDI assay are low, high DDI concentrations tend to be associated with the risk of VTE.     

The pharmacokinetic diversity of two von Willebrand factor (VWF)/ factor VIII (FVIII) concentrates in subjects with congenital von Willebrand disease. Results from a prospective, randomised crossover study

The pharmacokinetic (PK) profiles of von Willebrand factor (VWF) /factor VIII (FVIII) concentrates are important for treatment efficacy and safety of von Willebrand disease (VWD) patients. This prospective, head-to-head, randomised crossover study compared the PK profile of a new, high purity, human plasma-derived (pd)VWF/FVIII concentrate, Wilate, with the PK profile of an intermediate purity (pd)VWF/FVIII concentrate, Humate-P, in VWD patients. Subjects with inherited VWD were randomised to a single intravenous dose (40 IU/kg VWF ristocetin cofactor activity [VWF:RCo]) of Wilate or Humate-P in Period 1, and switched to the other study drug in Period 2. Each period was preceded by a washout time of ≥ 7 days. Coagulation factor parameters were analysed at multiple time-points. Of 22 randomised subjects, 20 had evaluable PK profiles, which indicated comparability for VWF antigen and VWF:RCo between Wilate and Humate-P. The reported VWF:RCo average and terminal t1/2 of 10.4 and 15.8 hours (h), respectively, for Wilate and 9.3 h and 12.8 h for Humate-P, were not statistically different. Also, the mean VWF:RCo in vivo recoveries (Wilate 1.89, Humate-P 1.99 IU/dl per IU/kg) were similar between the two replacement therapies. Wilate showed parallel decay curves for VWF:RCo and FVIII clotting activity (FVIII:C) over time, while FVIII:C of Humate-P displayed a plateau between 0 and 12-24 h. This study demonstrated bioequivalent PK properties for VWF between Wilate and Humate-P. The PK profile of Wilate, combined with the 1:1 VWF/FVIII ratio, theoretically should facilitate dosing and laboratory monitoring of VWF replacement to prevent bleeding in individuals with VWD.     

High prevalence of elevated clotting factor VIII in chronic thromboembolic pulmonary hypertension

Chronic thromboembolic pulmonary hypertension (CTEPH) is an enigmatic disorder lacking signs, symptoms and classical risk factors for venous thromboembolism. The objective of the prospective case controlled study, carried out at the Pulmonary Hypertension Unit, University Hospital Vienna, Austria, was to investigate whether plasma FVIII is elevated in CTEPH patients. The study examined 122 consecutive patients diagnosed with CTEPH. Plasma FVIII was measured and compared with plasma FVIII of healthy controls (n = 82) and of patients with nonthromboembolic pulmonary arterial hypertension (PAH, n = 88). Results show that CTEPH patients had higher FVIII levels than controls (233 +/- 83IU/dl versus 123 +/- 40IU/dl, p < 0.0001) and PAH patients (158 +/- 61IU/dl, p < 0.0001). Plasma FVIII one year after surgery (212 +/- 94IU/dl) was statistically unchanged compared with preoperative values (FVIII: 226 +/- 88IU/dl, n = 25). FVIII > 230IU/dl was more prevalent in CTEPH patients (41%) than in controls (5%, p < 0.0001) and PAH patients (22%, p = 0.022). We can conclude that elevated plasma FVIII is the first prothrombotic factor identified in a large proportion of CTEPH patients.     

Thromboprophylaxis following caesarean section--a comparison of the antithrombotic properties of three low molecular weight heparins--dalteparin, enoxaparin and tinzaparin.

Pharmacological thromboprophylaxis is increasingly being used after caesarean section to prevent venous thromboembolism. Although a variety of low molecular weight heparins (LMWH) have been used no comparative study exists on their effects on the haemostatic system in this situation. Furthermore, their antithrombotic effect may be mediated through effects other than their inhibitory effect on activated factor X. We compared the plasma anti-factor Xa activity, plasma concentration of tissue factor pathway inhibitor (TFPI) and the reduction in plasma thrombin-antithrombin (TAT) complex concentration in 30 women randomised to receive either dalteparin 5,000 IU anti-Xa once daily (n = 10), enoxaparin 4,000 IU anti-Xa once daily (n = 10) or tinzaparin 50 IU/kg anti-Xa (average dose 3,650 anti-Xa units) once daily (n = 10) following caesarean section. Sampling occurred at 0, 1, 3, 6, 12 and 24 h relative to time of dosing. All preparations produced an increase in mean anti-Xa assay (p < 0.0001), a reduction in mean TAT (p < 0.05) and an increase in mean TFPI concentration (p <0.05). Analysis of variance (ANOVA) revealed a significant difference between the LMWHs in terms of mean anti-factor Xa activity (p < 0.005) and reduction in plasma TAT concentration (p < 0.005). Post hoc analysis indicated that the anti-Xa values of the groups receiving enoxaparin and dalteparin were significantly higher than those of the group receiving tinzaparin (p < 0.05), but not significantly different from each other. Post hoc analysis of the reduction in plasma TAT concentration showed the reduction to be significantly less in the group receiving enoxaparin compared to the dalteparin and tinzaparin groups (p < 0.05), which did not differ significantly from each other. There was no significant difference between treatment groups with regard to plasma concentration of TFPI. These findings demonstrate that LMWHs differ in their effects on haemostatic parameters including thrombin generation as assessed by TAT. The increase in TFPI may be an additional mediator of LMWH's antithrombotic effects. Although these findings demonstrate that LMWHs differ in their haemostatic effects, this does not necessarily infer a clinical difference between these agents.     

Long-term therapy with low-molecular-weight heparin in cancer patients with venous thromboembolism

Long-term therapy with low-molecular-weight heparin (LMWH) is the treatment of choice for cancer patients with venous thromboembolism (VTE). However, the ideal doses of LMWH have not been thoroughly studied. We used the RIETE Registry data to assess the influence of the daily LMWH dosage on outcome during the first three months after VTE. We used propensity score-matching to compare patients who received <150 vs. those receiving ≥150 UI/kg/day LMWH. Up to July 2010, 3,222 cancer patients with VTE received long-term therapy with fixed doses of LMWH. Of these, 1,472 (46%) received <150 IU/kg/day (mean, 112 ± 28), and 1,750 received ≥150 IU/kg/day (mean, 184 ± 32). Results of the propensity score matching involved 1269 matched pairs. During follow-up, the incidence of pulmonary embolism (PE) recurrences was similar (1.2% vs. 1.9%), but patients receiving <150 IU/kg/day LMWH had a lower incidence of fatal PE than those treated with ≥150 IU/kg/day (0.2% vs. 1.0%; p=0.004). Multivariate analysis confirmed that patients receiving <150 IU/kg/day LMWH had a lower risk for fatal PE (odds ratio [OR]: 0.2; 95% confidence interval [CI]: 0.06-0.8) and for major bleeding (OR: 0.6; 95% CI: 0.3-1.0) than those treated with ≥150 IU/kg/day. In real life, one in every two cancer patients with VTE received lower doses of LMWH than those used in randomised trials, with large variations from patient to patient. Unexpectedly, patients treated with <150 IU/kg/day LMWH had fewer fatal PE cases and fewer major bleeding events than those receiving ≥150 IU/kg/day LMWH. This finding, however, should be validated in prospective clinical trials.     

Inhibition of thrombin generation by the oral direct thrombin inhibitor ximelagatran in shed blood from healthy male subjects

Ximelagatran, an oral direct thrombin inhibitor, whose active form is melagatran, was studied using a model of thrombin generation in humans. Healthy male volunteers (18 per group) received ximelagatran (60 mg p.o.), dalteparin (120 IU/kg s.c.) or a control (water p.o.). Shed blood, collected after incision of the forearm with standardised bleeding time devices at pre-dose, and at 2, 4 and 10 h post-dosing, was analysed for markers of thrombin generation. Statistically significant reductions (p < 0.05) in levels of prothrombin fragment 1+2 (F1+2) and thrombin-antithrombin complex (TAT) in shed blood were detected at 2 and 4 h post-dosing in both the ximelagatran and dalteparin groups. Shed blood F1+2 and TAT levels had returned to pre-dose levels at 10 h post-dosing. Using a shed blood model, we demonstrate that the reversible thrombin inhibitor melagatran and, therefore, oral administration of ximelagatran, inhibits thrombin generation in humans after acute activation of coagulation.     

Clinical efficacy of a novel VWF-containing FVIII concentrate, Wilate(®), in the prophylaxis and treatment of bleeding episodes in previously treated haemophilia A patients

The new-generation coagulation factor VIII (FVIII)/von Willebrand factor (VWF) concentrate, Wilate^®, is effective in the therapy of von Willebrand disease for which it was originally developed. The ratio of haemostatic components (FVIII to VWF) of this high-purity plasma-derived concentrate is 1:1, with VWF naturally stabilising FVIII. Bleeding episode control in patients with haemophilia A (HA) is generally managed by replacement therapy with recombinant or plasma-derived FVIII. When complexed with VWF as in the physiological situation, the product may also show a lower incidence of inhibitor development in patients with HA. The clinical efficacy, safety and tolerability of Wilate^® were therefore evaluated in 81 previously treated individual patients with severe HA. Data from 5 good clinical practice (GCP) prospective clinical studies were pooled and assessed with regards to the prevention and treatment of bleeding and during surgical procedures. Haemostatic efficacy was excellent or good in 96.7% of 1495 rated treatments of bleeding episodes. The average dose per treatment was approximately 30 IU FVIII/kg. In surgical settings, the global haemostatic efficacy of Wilate^® was generally rated excellent or good for use during and after operations with a mean total dose of FVIII of 848.6 IU/kg ± 578.5 IU/kg administered peri-operatively. Overall, tolerability of Wilate^® was rated very good or good by patients and physicians. Rarely occurring adverse events were mild in intensity and no anti-FVIII inhibitors were detected. Wilate^® also displayed a good viral safety profile with no seroconversions occurring in response to treatment. These data show that Wilate^® is an efficacious treatment option in the management of patients with severe HA.     

Hemostatic changes following the modified Fontan operation (total cavopulmonary connection)

Thromboembolism is a serious complication after Fontan operation, which may be caused by alterations of the coagulation system. We therefore investigated pro- and anticoagulant factors in 20 patients aged 4 to 21 years, 4 to 63 months following total cavopulmonary connection. Furthermore we compared markers of thrombin activation and fibrinolysis and in vitro clotting and clot-lysis to age-matched healthy subjects. Compared to results of age-matched controls, the Fontan operated individuals had significant decreases in levels of protein C (0.88 U/ml in controls, 0.67 U/ml in patients; p <0.001) and protein S (1.05 in controls, 0.93 U/ml in patients; p <0.05). Moreover, half of the patients had high values of FVIII (>1.5 IU/ml), which are associated with an increased thrombotic risk. These changes may result in enhanced generation of thrombin and plasmin, indicated by our finding of increased thrombin-antithrombin III (TAT) and plasmin-antiplasmin (PAP) levels and a similar trend in prothrombin fragments F1+2. Clot lysis tests, global coagulation tests, red blood cell count, liver enzymes AST, ALT, but not GGT, were generally within the normal ranges.     

Factor VIII activation by factor VIIa analog (V158D/E296V/M298Q) in tissue factor-independent mechanisms

Factor (F)VIIa with tissue factor (TF) is a primary trigger of blood coagulation. The recombinant (r)FVIIa analog, NN1731 (V158D/E296V/ M298Q) containing a thrombin/FIXa-mimicking catalytic domain, is ~30-fold more effective on activated platelets without TF, but ~1.2-fold with TF, than rFVIIa for FX activation. We have recently demonstrated the FVIIa/TF-dependent FVIII activation in the early coagulation phase. We assessed the action of NN1731 on FVIII activation. NN1731/TF increased FVIII activity ~2.9-fold within 30 seconds, followed by rapid inactivation, and was slightly more active than rFVIIa/TF. NN1731-catalysed activation, however, was enhanced ~6-fold at 5 minutes (min), and its peak level persisted for ~30 min. NN1731/TF proteolysed FVIII at Arg???, Arg3?2, and Arg33?, similar to rFVIIa/TF, but cleavage by NN1731 alone was much slower at Arg33? than at Arg??? and Arg3?2. The Km and Vmax for NN1731/TF-catalysed activation were ~1.8-fold lower and ~2.3-fold greater than rFVIIa/TF. The Km for NN1731 alone was ~1.3-fold lower than rFVIIa, whilst the Vmax was ~7.9-fold greater, indicating that the efficiency of FVIII activation by NN1731 and NN1731/TF was ~11- and ~4-fold greater, respectively, than equivalent reactions with rFVIIa. In SPR-based assays, NN1731 bound to FVIII and the heavy chain (Kd; 0.62 and 1.9 nM) with ~1.4- and ~3.1-fold higher affinity than rFVIIa, and the A2 domain contributed to this increase. Von Willebrand factor moderated NN1731-catalysed activation more significantly than NN1731/TF. In conclusion, NN1731 was a greater potential than rFVIIa in up-regulating FVIII activity, and the TF-independent FVIII activation might represent a potential extra mode of its enhanced haemostatic effect.