Endogenous thrombin potential (ETP) in plasma from patients with AMI during antithrombotic treatment

Background

The beneficial impact of warfarin in preventing new events after AMI is well established. Decrease in thrombin generation seems to be the key element in anticoagulant treatment.

Objectives

The aims were to investigate the effect of warfarin and platelet inhibition on thrombin generation, assessed by the endogenous thrombin potential (ETP), and study the relation between coagulation parameters and ETP in patients with AMI.

Patients/Methods

In the present sub-study of the WARIS II trial, patients with AMI were randomly assigned to treatment with aspirin 160 mg/d (n = 57), aspirin 75 mg/d and warfarin (INR 2.0-2.5) (n = 68) or warfarin (INR 2.8-4.2) (n = 61). Fasting blood samples were collected from patients at discharge from hospital and after 6 weeks treatment.

Results

Correlation analyses showed that both ETP and peak thrombin levels were significantly correlated with Factor VII Ag (r = 0.38 and 0.36 respectively, p < 0.01 for both) and with F1+2 (r = 0.26 and 0.23 respectively, p = 0.01 for both) at baseline. Antithrombotic treatment for 6 weeks caused a highly significant inhibition of ETP in patients treated with warfarin (− 28% ± 5%, p < 0.001), and patients treated with aspirin/warfarin (− 24% ± 8%, p = 0.04). Similarly, peak thrombin levels were reduced in patients treated with warfarin (− 18% ± 7%, p = 0.049) and aspirin/warfarin (− 19% ± 5%, p = 0.029), whereas an increase (12% ± 4%, p = 0.029) occurred during aspirin treatment alone. F1+2 levels decreased by 64% and 58% in the warfarin and aspirin/warfarin groups, respectively (p = 0.001 for both).

Conclusions

In patients with AMI, warfarin significantly reduced the endogenous thrombin generation and the potential to generate thrombin in plasma ex vivo, whereas aspirin alone had no effect on thrombin generation in vivo or ex vivo, assessed by ETP.     

Activated protein C plasma levels in the fasting and postprandial states among patients with previous unprovoked venous thromboembolism

Introduction

The protein C anticoagulant system is of major importance in the regulation of thrombotic risk, but it is not known whether low plasma levels of activated protein C (APC) in vivo reflect a compromised anticoagulant situation with increased thrombotic risk. Previous studies have reported low, normal or increased plasma APC levels in unselected patients with venous thromboembolism (VTE).

Materials and methods

We performed a population-based, case-control study in patients with a previous history of unprovoked VTE and subjected the participants to a standard fat tolerance test (1 g fat/kg body weight) in order to promote physiological coagulation activation.

Results

VTE patients had higher BMI (28.3 ± 4.4 kg/m² versus 26.3 ± 3.9 kg/m², p = 0.045) and greater waist circumference (98.2 ± 12.5 cm versus 93.4 ± 13.4 cm, p = 0.041) than age and sex matched controls. APC levels were equal in fasting plasma (3.00 ± 0.74 ng/ml and 2.99 ± 0.60 ng/ml, p = 0.66) but higher in postprandial plasma (3.18 ± 0.57 ng/ml and 2.81 ± 0.38 ng/ml, p = 0.008) collected from VTE patients and controls, respectively. Endogenous thrombin generation in plasma following a standardized meal, assessed by thrombin-antithrombin complex (TAT), increased similarly in both groups, whereas APC increased only among the VTE patients during the postprandial state. Plasma levels of APC increased linearly with TAT in the postprandial state (p for linear trend = 0.012).

Conclusions

Our findings fail to support the hypothesis that low APC levels are linked to increased thrombotic risk in unprovoked VTE, and they suggest that plasma APC is a biomarker of thrombin generation.     

Platelet activation and endogenous thrombin potential in pre-eclampsia

Introduction

Platelets and the coagulation system may be involved in the pathogenesis of pre-eclampsia. We investigated whether platelet and coagulation activation markers, are elevated in pre-eclampsia.

Materials/methods

Case-control study in which activated platelets, platelet-monocyte/ neutrophil aggregates, platelet microparticles (measured by flow cytometry) and four markers of thrombin generation capacity (endogenous thrombin potential (ETP), peak height, lag time and time to peak) using the Calibrated Automated Thrombogram system were assessed in pregnant women of similar gestational age with (n = 46) and without (n = 46) pre-eclampsia, and in healthy non-pregnant women (n = 42).

Results

The percentage of, CD62P+ platelets (p = 0.013), CD62P+ platelet microparticles (p = 0.029) and platelet-monocyte aggregates (p = 0.019) were significantly higher in women with pre-eclampsia than the pregnant controls. Both groups of pregnant women had significantly higher ETP and peak height (p  < 0.001) than the healthy non pregnant group and the women with pre-eclampsia had significantly higher ETP and peak height (p < 0.001) than the normotensive pregnant controls.

Conclusion

In the most comprehensive laboratory analysis to date, we found evidence of both platelet and coagulation activation in women with pre-eclampsia.     

Increased thrombin generation in splanchnic vein thrombosis is related to the presence of liver cirrhosis and not to the thrombotic event

Introduction

In recent years there have been increasing evidence associating liver disease with hypercoagulability, rather than bleeding. The aim of the study was to evaluate the haemostatic potential in patients with liver disease.

Patients and methods

We measured thrombin generation in the presence and absence of thrombomodulin in patients with portal vein thrombosis (PVT, n = 47), Budd-Chiari syndrome (BCS, n = 15) and cirrhosis (n = 24) and compared the results to those obtained from healthy controls (n = 21). Fifteen patients with PVT and 10 patients with BCS were treated with warfarin and were compared to an equal number of patients with atrial fibrillation matched for prothrombin time-international normalized ratio. We assessed resistance to thrombomodulin by using ratios [marker measured in the presence/absence of thrombomodulin].

Results

There were no differences in thrombin generation between patients on warfarin treatment and their controls. Cirrhotic patients generated more thrombin in the presence of thrombomodulin and exhibited thrombomodulin resistance compared to controls [p = 0.006 for endogenous thrombin potential (ETP) and p < 0.001 for peak thrombin and both ratios ETP and peak] and patients with non-cirrhotic PVT (p = 0.001, p = 0.006, p < 0.001, p < 0.001 for ETP, peak, ratio ETP, ratio peak, respectively). The patients with cirrhotic PVT exhibited higher ETP (p = 0.044) and peak (p = 0.02) in the presence of thrombomodulin than controls, as well as thrombomodulin resistance (ETP and peak ratios: p = 0.001).

Conclusions

Hypercoagulability and thrombomodulin resistance in patients with cirrhosis were independent of the presence of splanchnic vein thrombosis. The hypercoagulability in patients with cirrhotic PVT could have implications for considering longer or more intensive treatment with anticoagulants in this group.     

Depressive symptoms, perceived social support, and prothrombotic measures in patients with venous thromboembolism

Introduction

Psychosocial factors have been associated with both a prothrombotic state and an increased risk of venous thromboembolism (VTE). We examined the relation of depressive symptoms and social support with D-dimer, an integrative measure of enhanced coagulation activity, and several additional prothrombotic measures in patients with VTE.

Methods

We studied 173 patients with a previous deep venous thrombosis and/or pulmonary embolism (mean age ± SD 45 ± 14 years, 55% men). Clinical and lab assessments took place ≥ 3 months after VTE and ≥ 1 month after discontinuation of oral anticoagulants. The patients rated depressive symptoms and social support by validated questionnaires.

Results

After adjusting for sociodemographic and medical covariates, interactions emerged between depressive symptoms and social support for D-dimer (p = 0.012) and aPTT (p = 0.002). As opposed to patients with high levels of social support, those with low levels of social support showed a direct association of depressive symptoms with D-dimer (r = 0.19, p = 0.014) and an inverse relationship with aPTT (r = -0.14, p < 0.09). Depressive symptoms were associated with levels of thrombin-antithrombin complex (r = 0.19, p = 0.016). Greater social support was associated with prolonged aPTT (r = 0.16, p = 0.046). There were no significant associations of depressive symptoms and social support with D-dimer, fibrinogen, FII:C, FV:C, FVII:C, FVIII:C, FX:C, INR, and thrombin time.

Conclusions

Depressive symptoms are associated with enhanced coagulation activity in patients with VTE, particularly so in those who perceive low levels of social support. Conversely, high levels of social support may contribute directly and through buffering the effect of depressive symptoms to attenuated clotting activity in VTE patients.     

Continuous thrombin infusion leads to a bleeding phenotype in sheep

Background

In addition to a recognized role in the coagulation cascade and haemostasis, thrombin is known to have multiple functions. We aimed to establish an ovine model to study thrombin effects in vivo.

Methods

Thrombin (0.0004-0.42 IU/kg/min) was continuously infused in Austrian Mountain Sheep over five hours in the dose escalation study (n = 5 animals; 15 experiments). In the dose verification study animals received 0.42 IU/kg/min of thrombin vs. saline solution in a cross-over design (n = 3 animals; 7 experiments).

Results

Thrombin at an infusion rate of 0.42 IU/kg/min decreased fibrinogen levels by 75% (p < 0.001) and increased degradation products of the fibrinogen beta-chain as shown in a proteomic analysis. Thrombin decreased platelet counts by 36% (p = 0.006), prolonged thrombin time by 70% (p = 0.012) and activated partial thromboplastin time by 32%. Interestingly, thrombin infusion significantly increased the activity of coagulation factors V and X (p < 0.05) and decreased the activity of the coagulation factors VIII and XIII (p < 0.05). Accordingly, thrombin displayed predominantly anti-coagulant and anti-platelet effects: 1) thrombin prolonged clotting time/clot formation time 7-fold (p = 0.019) and induced a 65% decrease in maximal clot firmness (p < 0.001); 2) thrombin reduced collagen- induced platelet aggregation by 85% and prolonged collagen/adenosine diphosphate closure time 3-fold; and 3) thrombin caused lung haemorrhage but not thromboembolism.

Conclusion

Protracted intravenous infusion of thrombin over a period of five hours offers a new experimental model to study thrombin effects in a large animal species.     

Markers of endothelial and platelet activation are associated with high on-aspirin platelet reactivity in patients with stable coronary artery disease

Introduction

Aspirin inhibits the cyclooxygenase-1 (COX-1) mediated thromboxane A2 synthesis. Despite COX-1 inhibition, in patients with coronary artery disease (CAD), platelets can be activated through other mechanisms, like activation by thrombin.

Materials and Methods

At baseline in this cross-sectional substudy of the ASCET trial, 1001 stable CAD patients, all on single aspirin treatment, were classified by the PFA100® method, as having high on-aspirin residual platelet reactivity (RPR) or not. Markers of hypercoagulability, endothelial and platelet activation as related to RPR, were evaluated to explore the potential mechanisms behind high on-aspirin RPR.

Results

Altogether, 25.9% (n = 259) of the patients were found to have high on-aspirin RPR. S-thromboxane B₂ levels were very low and did not differ between patients having high on-aspirin RPR or not. Patients with high on-aspirin RPR had significantly higher levels of von Willebrand Factor (vWF) (124 vs 100%, p < 0.001, platelet count (236 vs 224 × 10⁹/l, p = 0.008), total TFPI (68.4 vs 65.5 ng/ml, p = 0.005) and ß-thromboglobulin (ß-TG) (33.3 vs 31.3 IU/ml, p = 0.041) compared to patients with low on-aspirin RPR. No significant differences between the groups were observed in levels of endogenous thrombin generation (ETP), pro-thrombin fragment 1+2 (F1+2), D-dimer, soluble TF (sTF) or P-selectin (all p > 0.05).

Conclusions

The high on-aspirin RPR as defined by PFA100® seems not to be due to increased thrombin activity as evaluated with ETP, sTF, F1+2 or D-dimer. The elevated levels of platelet count, ß-TG, TFPI and especially vWF might be explained by increased endothelial and platelet activation in these patients.     

Different effects of enoxaparin and unfractionated heparin on some thrombogenesis markers during hemodialysis: a cross-over study

Background

Unfractionated heparin (UFH) and low molecular weight heparin constitute fundamental anticoagulants during hemodialysis (HD). We aimed to investigate the effect of UFH and enoxaparin on plasma levels of prothrombin fragment 1 + 2 (PF 1 + 2) and thrombin/antithrombin complex (TAT) as markers of intravascular thrombogenesis during HD.

Methods

We enrolled 22 chronic HD patients, who were randomly assigned to either iv enoxaparin (n = 11) or UFH (n = 11) anticoagulation, and followed prospectively for 12 weeks before crossing over to the alternate therapy for further 12 weeks. Plasma levels of PF 1 + 2 and TAT were measured by immunoassay at the start, at 10 and 180 min of HD session after each period of evaluation.

Results

The baseline PF 1 + 2 and TAT levels were comparable under enoxaparin and UFH treatment. PF 1 + 2 significantly decreased during both UFH (χ² ANOVA = 9.82, P = 0.007) and enoxaparin (χ² ANOVA = 29.40, P < 10^− 6) anticoagulated HD, while over-HD TAT levels changes differed depending on the type of heparin. The switch from enoxaparin to UFH treatment was connected with a significantly higher PF 1 + 2 after 10 and 180 min as well as higher TAT concentration after 180 min of HD. Only during enoxaparin anticoagulated HD 34% PF 1 + 2 decrease and TAT levels after 180 min of HD was closely associated with heparin dosage.

Conclusion

Single bolus of enoxaparin ensures efficient and convenient anti-thrombotic protection during HD procedure. Enoxaparin mean dose of 0.67 mg/kg, which is generally lower than manufacturer's instructions, can be recommended for over-dialytic regular use.     

Increased thrombin generation in inflammatory bowel diseases

Background

Inflammatory bowel diseases (IBD) are characterized by an increased thrombotic risk of uncertain etiology. Endogenous thrombin potential (ETP), a parameter of the thrombin generation curve, represents a new tool in the evaluation of thrombotic and bleeding disorders.

Aims

To study ETP in IBD patients and to correlate the results with clinical and biochemical features.

Methods

Seventy-four IBD patients (37 ulcerative colitis and 37 Crohn's disease) and 74 sex- and age-matched healthy individuals. ETP was measured upon activation of coagulation with small amounts of tissue factor and phospholipids in the presence or absence of thrombomodulin; results were expressed as nM thrombin·minutes.

Results

Mean±SD ETP values were significantly higher in patients (1,499 ± 454) than controls (1,261 ± 385) (p < 0.001) only when the test was performed in the presence of thrombomodulin. ETP evaluated as ratio (with/without thrombomodulin), taken as an index of hypercoagulability, was significantly higher in patients (0.69 ± 0.14) than controls (0.62 ± 0.18) (p < 0.006). Patients with increased C-reactive protein (CRP) had significantly higher mean ETP (1,721 ± 458) than those with normal CRP (1,357 ± 394) or controls (1,261 ± 385) (p < 0.001). Patients who at the time of blood sampling were classified as having a clinically active disease had ETP higher than those who were quiescent (1,655 ± 451 versus 1,388 ± 427, p < 0.001) or controls (1,261 ± 385, p < 0.001).

Conclusions

ETP measured in the presence of thrombomodulin or as ratio (with/without thrombomodulin) is increased in IBD patients, mainly in those with increased CRP or active disease. It may be considered as a candidate test for prospective studies aimed at assessing the risk of thrombosis in IBD patients.     

The plasma levels of activated thrombin activatable fibrinolysis inhibitor and thrombomodulin in Behçet Disease and their association with thrombosis

Objective

To investigate the plasma levels of activated thrombin activatable fibrinolysis inhibitor (aTAFI) and thrombomodulin (TM) in Behçet disease (BD) and their relationship with thrombosis.

Methods

Plasma aTAFI and TM levels were measured by ELISA in 89 patients with BD (18 having venous thrombosis) and in 86 healthy controls.

Results

Compared with healthy controls, the BD group had significantly lower levels of aTAFI (13.49 ± 8.88 µg/ml vs. 26.76 ± 11.57 µg/ml, p < 0.0001) and significantly higher levels of TM (3.26 ± 1.85 ng/ml vs. 2.6 ± 0.69 ng/ml, p = 0.0003). Neither aTAFI, nor TM levels differed significantly between BD patients with and without thrombosis (p > 0.05). Despite a tendency to positive correlation (r = 0.37, p = 0.0004) between plasma levels of aTAFI and TM in healthy controls, there was a tendency for negative correlation (r = -0.51, p < 0.0001) between these two parameters in BD patients.

Conclusion

The plasma aTAFI and TM levels do not seem to be related with the presence of thrombosis observed in BD. Increased plasma TM levels in BD may simply reflect endothelial cell activation and dysfunction.     

Plasma activity of individual coagulation factors, hemodilution and blood loss after cardiac surgery: A prospective observational study

Background

Hemodilution and consumption of coagulation factors during cardiopulmonary bypass has been suggested to contribute to bleeding complications after cardiac surgery. The aim was to describe the activity of individual coagulation factors after CABG in relation to hemodilution and postoperative bleeding.

Materials and Methods

Plasma concentrations of fibrinogen and plasma activity of FII, FV, FVII, FVIII, FIX, FX, FXI and FXIII adjusted for hemodilution were analysed in 57 CABG patients before, and 2 h and 24 h after surgery. Postoperative bleeding was registered and correlations to coagulation factor activity were calculated.

Results

Adjusted plasma concentration of fibrinogen (-14 ± 6%), and plasma activity of FII (-9 ± 6%), FV (-13 ± 8%), FX (-13 ± 7%) and FXIII (-9 ± 14%) were reduced two hours after surgery compared to baseline (all p < 0.001). FVII (+ 3 ± 12%, p = 0.34) and FXI (+ 1 ± 19%, p = 0.50) were unchanged, while FVIII (+ 23 ± 44%, p = 0.006) and FIX (+ 23 ± 17%, p < 0.001) increased. Twenty-four hours after surgery fibrinogen (+ 45 ± 27%), FVIII (+ 93 ± 66%) and FIX (+ 33 ± 26%) were all increased (all p < 0.001), while FVII (-37 ± 14%, p < 0.001), FXI (-4 ± 18%, p = 0.02) and FXIII (-6 ± 15%, p = 0.004) were decreased.Median postoperative blood loss was 380 ml/12 h. There were significant inverse correlations between postoperative blood loss and fibrinogen concentration 2 h after surgery (r = -0.33, p = 0.019) and between postoperative blood loss and pre- and postoperative FXIII activity (r = -0.34, p = 0.009 and r = -0.41, p = 0.003, respectively), but not between blood loss and any of the other factors.

Conclusions

There is a marked dissociation in plasma activity of individual coagulation factors after CABG. Plasma concentration of fibrinogen and factor XIII activity correlates inversely to postoperative blood loss after CABG.     

Coagulation activation and ultrasound characteristics in patients with carotid artery disease

Introduction

Elevated levels of markers for thrombin activation are associated with plaque echogenicity and degree of stenosis in patients with carotid artery stenosis. The Activated Protein C-Protein C Inhibitor (APC-PCI) complex reflects activation of the Protein C system and is a measure of thrombin generation. The aim of the present study was to examine APC-PCI complex in patients undergoing thrombendartherectomy for carotid artery stenosis, and to relate the findings to clinical characteristics and plaque morphology as determined by ultrasound.

Materials and Methods

Blood was obtained from 125 patients (39 female, median age 71 years) with carotid artery stenosis admitted from September 2005 to May 2007. The APC-PCI complex was measured using a sandwich immunofluorometric method and compared to an age- and sex-matched healthy control-group. Clinical and demographic characteristics, routine laboratory markers and ultrasound characteristics were analysed using univariate and multivariate analysis.

Results

APC-PCI complex concentration was significantly increased in patients with carotid artery stenosis (median 0.21 µg/L; 10th to 90th percentile 0.15-0.36) compared to a healthy control-group (0.19 µg/L; 0.11-0.31; P = .009). There was no significant difference in APC-PCI-values between asymptomatic (n = 48) and symptomatic (n = 77) patients with carotid artery stenosis (0.22 vs. 0.20 µg/L; p = 0.626). Patients with minor stroke (n = 31) had a higher median APC-PCI-concentration (0.27 µg/L; 0.15-0.63) than patients with amaurosis fugax (0.19 µg/L; 0.15-0.36) or transient ischemic attack (0.21 µg/L; 0.12-0.36) (p = 0.016). No association was found between APC-PCI-values and the degrees of carotid artery stenosis or the time from the latest neurological symptoms to blood sampling. Patients with echolucent plaques had significantly lower APC-PCI concentrations (0.20 µg/L; 0.14-0.35 vs. 0.24 µg/L; 0.15-0.60; p = 0.043), according to the Gray-Weale classification.

Conclusions

Patients with carotid artery disease exhibit increased concentrations of APC-PCI compared to a healthy control-group, particularly those patients with echogenic plaques, who have significantly higher APC-PCI levels than patients with echolucent plaques.     

Enhanced thrombin formation and fibrinolysis during acute Puumala hantavirus infection

Introduction

Nephropathia epidemica (NE) is a viral hemorrhagic fever with renal syndrome associated with thrombocytopenia and mild bleeding. We assessed activation of coagulation and fibrinolysis during the acute phase of NE.

Materials and methods

19 hospital-treated patients were involved. Plasma levels of D-dimer, prothrombin fragments 1 + 2 (F1 + 2), activated partial thromboplastin time (APTT), prothrombin time (PT%), thrombin time (TT), fibrinogen, antithrombin (AT), protein S free antigen (PS), protein C (PC) and complete blood count (CBC) were measured three times during the acute phase and once at 32-54 days after the onset of fever (recovery phase). Laboratory abnormalities were evaluated by the disseminated intravascular coagulation (DIC) scoring advocated by the International Society of Thrombosis and Haemostasis (ISTH).

Results

APTT was prolonged and D-dimer and F1 + 2 increased during the acute phase of NE. AT, PC and PS decreased, and TT was shortened, all implying increased thrombin generation. Acutely F1 + 2 was 3.4-fold and D-dimer even 24-fold higher compared with the recovery phase (median 726 vs 213 pmol/l, and median 4.8 vs 0.2 mg/l, respectively, p < 0.001 for both). Platelet count correlated with AT, PC, and PS (r = 0.73, r = 0.81, and r = 0.71, respectively, p < 0.001 for all) as well as with fibrinogen (r = 0.72, p < 0.001). Only five patients fulfilled the ISTH diagnosis of DIC.

Conclusions

During acute NE thrombocytopenia was associated with decreased natural anticoagulants, shortened thrombin time and enhanced fibrinolysis. Augmented thrombin formation and fibrinolysis characterize this hantavirus infection.     

Activation of coagulation in amniotic fluid during normal human pregnancy

Introduction

Amniotic fluid (AF) is an important medium for fetal development which exhibits high procoagulant activities; however, the role of these procoagulants during pregnancy has not been elucidated and might be associated with pregnancy complications. The current study aimed to evaluate factor X (FX) activation and its association with tissue factor (TF), tissue factor pathway inhibitor (TFPI) and coagulation activation markers in AF during normal human pregnancy.

Methods

Activation of FX and concentration of TF, free TFPI, D-dimer and prothrombin fragments (F1 + 2) were evaluated in AF samples obtained for chromosome analysis from 91 women with normal pregnancy: 65 samples were taken from patients at 16-20 weeks of gestation, 9 samples were drawn at 21-30 weeks and 17 samples−after 30 weeks of gestation.

Results

Activation of FX in AF significantly increased during normal pregnancy (from 65 ± 41 to 205 ± 80 equivalent RVV ng/mg total protein, P < 0.0001). TF and TFPI levels in AF also rose with gestational age. In contrast, the AF concentration of D-dimer and F1 + 2, markers of coagulation activation significantly decreased when expressed per mg total protein. Levels of free TFPI correlated with TF (r = 0.5, P < 0.001), and were 8-fold higher than those of TF during pregnancy.

Conclusion

High levels of TFPI might be associated with the inhibition of procoagulant activity in amniotic fluid during normal pregnancy, which may account for the rarity of clinical amniotic fluid embolism.     

Anticoagulation therapy with combined low dose aspirin and warfarin following mechanical heart valve replacement

Introduction

This study was designed to evaluate safety and efficacy of combined low dose aspirin and warfarin therapy following mechanical heart valve replacement.

Methods

A total of 1 496 patients (686 males, mean age 35 ± 8.5 years) undergoing mechanical heart valvular replacement were randomly divided into study (warfarin plus 75-100 mg aspirin) or control (warfarin only) group. International normalized ratio (INR) and prothrombin time was maintained at 1.8-2.5 and 1.5-2.0 times of the normal value, respectively. Thromboembolic events and major bleedings were registered during follow up.

Results

Patients were followed up for 24 ± 9 months. The average dose of warfarin in the study and control group was 2.92 ± 0.87 mg and 2.89 ± 0.79 mg, respectively (p > 0.05). The overall thromboembolic events in study group were lower than in control group (2.1% vs. 3.6%, p = 0.044). No statistically significant differences were found in hemorrhage events (3.5% vs. 3.7%, p > 0.05) or mortality (0.3% vs 0.4%, p > 0.05) between the two groups.

Conclusions

Following mechanical valve replacement, combined low dose aspirin and warfarin therapy was associated with a greater reduction in thromboembolism events than warfarin therapy alone. This combined treatment was not associated with an increase in the rate of major bleeding or mortality.     

Intra- and inter-rater reliability of motor unit number estimation and quantitative motor unit analysis in the upper trapezius

Objective

To collect normative data and assess the intra- and inter-rater reliability of decomposition-enhanced spike-triggered averaging (DE-STA) motor unit number estimation (MUNE) and quantitative MU analysis obtained using decomposition-based quantitative electromyography (DQEMG) in the upper trapezius (UT).

Methods

In 10 control subjects, the experimental protocol was performed twice by the same examiner, and once by a second examiner.

Results

Mean MUNE values were 339 ± 121 (rater 1a), 320 ± 131 (rater 1b), and 262 ± 115 (rater 2) MUs. Intra- and inter-rater reliability was good for maximum CMAP (ICC = 0.77 and 0.79, respectively) and moderate for MUNE (ICC = 0.69 and 0.73, respectively), with poor inter-rater reliability for mean S-MUP (ICC = 0.42). Significant differences between rater 1a and 2 were found for mean S-MUP (p = 0.014) and MUNE (p = 0.002), and moderate to good levels of reliability found for quantitative needle-detected MUP parameters.

Conclusions

Various components of the protocol may have contributed to mean S-MUP variability, and may require particular attention in a large, proximal muscle like the UT.

Significance

This study has established preliminary data using DQEMG in a novel muscle which may be relevant to study in patients with ALS.     

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.     

The Influence of Direct Thrombin Inhibitors on the Formation of Platelet-leukocyte Aggregates and Tissue Factor Expression

Introduction

High concentrations of platelet-monocyte aggregates (PMAs) have been found in patients with myocardial infarction (MI). Oral direct thrombin inhibitors (DTIs) are under evaluation as long-term antithrombotic treatment. The aim was to evaluate whether DTIs affect the formation of platelet-leukocyte aggregates, TF expression and procoagulant microparticles (MPs).

Material and Methods

DTIs were added to an experimental whole blood model before platelet activation with thrombin or ADP. The concentrations of PMAs, platelet-granulocyte aggregates (PGAs), the amount of platelets bound per leukocyte and MPs were investigated by flow cytometry. TF mRNA and activity were recorded in all settings. TF activity was evaluated in a MI population treated with or without an oral DTI.

Results

In vitro, thrombin and ADP increased the formation of PMAs and PGAs as well as TF mRNA expression. DTIs reduced the amount platelets bound to monocytes (p = 0.02) and to granulocytes (p = 0.001) upon thrombin stimulation together with a reduction of TF mRNA. In contrast, the ADP-induced formation of PMAs, PGAs and TF mRNA was not affected by the DTIs. Both thrombin and ADP stimulation increased the amount of TF-expressing MPs, which was effectively inhibited by the DTIs (p = 0.02-0.002). In the MI population, the DTI reduced the TF activity (p < 0.001).

Conclusion

DTIs modulate the formation of PMAs, PGAs and the TF production therein. Together with a reduction of procoagulant MPs, these results may contribute to the clinical benefit found of oral DTIs. Targeting different mechanisms in platelet and coagulation activation may be of importance due to the lack of effect of DTIs on ADP-induced platelet-leukocyte aggregates and TF production.     

Prognostic values of the factor Xa-activated clotting time and endogenous thrombin potential in patients suspected of having disseminated intravascular coagulation

Background

Widespread coagulation activation and intravascular fibrin formation are clinical features of disseminated intravascular coagulation (DIC). The endogenous thrombin potential (ETP) has been shown to be a useful marker for hypo- or hypercoagulability. The factor Xa-activated clotting time (XACT) represents plasma levels of procoagulant phospholipids. We investigated whether the ETP and XACT would be good prognostic markers in patients suspected of having DIC and whether these markers would show a significant correlation with the thrombin-antithrombin complex (TAT), a marker of in vivo coagulation activation.

Methods

One hundred twenty-nine patients suspected of having DIC were enrolled for the study. The TAT was measured by ELISA. The ETP and XACT were measured by calibrated automated thrombinography. The 28-day mortality was used as a predictor of clinical outcomes.

Results

In overt DIC, higher XACT (9.67 vs. 7.33 min) and higher TAT (26.15 vs. 11.56 ng/ml) results were obtained from the nonsurvivors than from the survivors. ETP levels were lower in the overt DIC group than in the no overt DIC group. In receiver operating characteristic analysis, which was conducted to predict the 28-day mortality, the areas under the receiver operating characteristic analysis curve were as follows: 0.71 (95% CI: 0.62-0.78) for the XACT, 0.70 (0.61-0.77) for the TAT, and 0.64 (0.55-0.72) for the ETP. For the diagnosis of overt DIC, the area under the curve of XACT, TAT and ETP were 0.77 (0.69-0.84), 0.64 (0.55-0.72) and 0.73 (0.64-0.80), respectively. The odds ratio of the XACT for the relative risk of 28-day mortality was 9.60 (3.53-26.11), and that of the TAT was 5.18 (2.11-12.72) and that of the ETP 7.66 (1.67-35.17). For the diagnosis of overt DIC, the odds ratio of XACT, TAT and ETP were 37.35 (4.86-286.89), 4.89 (1.93-12.43) and 4.89 (1.98-12.09), respectively. There was a negative correlation between the TAT and ETP (r = − 0.223, P = 0.012) and a positive correlation between the TAT and XACT (r = 0.251, P = 0.004).

Conclusion

Our results suggest that the XACT and ETP may be useful diagnostic and prognostic markers for the DIC. Among various markers, the XACT serves as a good prediction of the 28-day mortality in patients suspected of having DIC.     

Fibrin clot properties in women heterozygous for factor V Leiden mutation: Effects of oral contraceptives

Introduction

Oral contraceptives (OC) in the presence of factor V Leiden mutation (FVL) markedly increase the risk of venous thromboembolism (VTE). Little is known about the OC and FVL-related alterations in fibrin clot properties.

Subjects and Methods

Plasma fibrin clot permeability (K_s) and efficiency of lysis, reflected by clot lysis time (CLT) and the rate of D-dimer release from clots (D-D_rate) induced by recombinant tissue plasminogen activator (tPA) were determined in 25 women with a family history of VTE who were heterozygous for FVL [FVL(+/−) - twice, on third-generation OC and after their discontinuation. Female non-carriers of FVL, matched for demographics, using OC and after their discontinuation served as controls (n = 25). All participants had no personal history of VTE.

Results

OC discontinuation in FVL(+/−) women resulted in shortened CLT (− 9%), and increased K_s (+ 4%) and D-D_rate (+ 1.4%; all p < 0.01). Alterations in fibrin clot properties were associated with decreased prothrombin fragments 1 + 2 (F1 + 2) (− 8%), plasminogen activator inhibitor-1 (PAI-1) antigen (− 11%), and thrombin activatable fibrinolysis inhibitor (TAFI) activity (− 20%; all p < 0.01). During OC use FVL(+/−) carriers compared with non-carriers had higher platelet count, activity of PAI-1, TAFI, and tPA, as well as prolonged CLT and higher D-D_max, along with lower D-D_rate and K_s. Multiple regression analysis adjusted for fibrinogen and age, showed that PAI-1 antigen and TAFI activity independently predicted CLT in FVL(+/−) women on OC.

Conclusion

FVL(+/−) is associated with hypofibrinolysis in apparently healthy women and third-generation OC administration unfavorably alters plasma clot characteristics in female FVL(+/−) carriers with a family history of thrombotic events.