Tissue factor-induced Thrombin Generation in the Fasting and Postprandial State among Elderly Survivors of Myocardial Infarction

Introduction

Tissue factor (TF)-induced thrombin generation (TG) ex vivo has been suggested to be an important method to assess thrombotic risk. No studies have investigated the impact of postprandial lipemia on TF-induced TG. Since myocardial infarction (MI) is associated with elevated postprandial levels of triglycerides, we hypothesized a differential impact of postprandial lipemia on coagulation activation in MI-patients and healthy controls.

Material and Methods

Elderly survivors of acute MI (n = 44) and healthy age-and sex matched controls (n = 43) underwent a fat tolerance test (1 gram per kg body weight) to assess coagulation activation during postprandial lipemia.

Results

The incremental area under the curve (AUCi) for serum triglycerides was higher in MI-patients than in healthy age-and sex matched controls (5.64 ± 0.52 mmol/L?h and 3.94 ± 0.39 mmol/L?h, p = 0.012) during the postprandial phase. Subsequent endogenous activation of coagulation, assessed by FVIIa and thrombin generation (F1 + 2), was similar among groups and not related to levels of triglycerides during the postprandial phase. Healthy individuals had a gradual decline in TF-induced thrombin generation ex vivo, assessed by endogenous thrombin potential (ETP) (AUCi = - 542.4 ± 71.4 nM?min?h, p < 0.001), whereas MI-patients retained their ETP (AUCi = 127.4 ± 89.0 nM?min?h, p = 0.47) in plasma during the postprandial phase (p for group difference = 0.005).

Conclusions

MI-patients had elevated postprandial lipemia and retained their ability for TF-induced TG in plasma ex vivo in the postprandial phase, whereas the capacity gradually decreased in healthy individuals. Further studies are warranted to reveal underlying mechanism(s) and clinical implications.     

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.     

Plasma factor and inhibitor composition contributes to thrombin generation dynamics in patients with acute or previous cerebrovascular events

Introduction

More than 80% of cerebrovascular events are ischemic and largely thromboembolic by nature. We evaluated whether plasma factor composition and thrombin generation dynamics might be a contributor to the thrombotic phenotype of ischemic cerebrovascular events.

Materials and Methods

We studied (1) 100 patients with acute ischemic stroke (n = 50) or transient ischemic attack (TIA) (n = 50) within the first 24 hours from symptom onset, and (2) 100 individuals 1 to 4 years following ischemic stroke (n = 50) or TIA (n = 50). The tissue factor pathway to thrombin generation was simulated with a mathematical model using plasma levels of clotting factors (F)II, V, VII, VIII, IX, X, antithrombin and free tissue factor pathway inhibitor (TFPI).

Results

The plasma levels of free TFPI, FII, FVIII, and FX were higher, while antithrombin was lower, in the acute patients compared to the previous event group (all p ≤ 0.02). Thrombin generation during acute events was enhanced, with an 11% faster maximum rate, a 15% higher maximum level and a 26% larger total production (all p < 0.01). The increased thrombin generation in acute patients was determined by higher FII and lower antithrombin, while increased free TFPI mediated this effect. When the groups are classified by etiology, all stroke sub-types except cardioembolic have increased TFPI and decreased AT and total thrombin produced.

Conclusion

Augmented thrombin generation in acute stroke/TIA is to some extent determined by altered plasma levels of coagulation factors.     

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.     

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.     

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.     

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.     

Differences in thrombus structure and kinetics in patients with type 2 diabetes mellitus after non ST elevation acute coronary syndrome

Introduction

Despite optimal secondary prevention therapy following non-ST elevation acute coronary syndrome (NSTE-ACS), recurrent thrombotic events are more frequent in patients with type 2 diabetes mellitus (T2DM).

Materials and Methods

This exploratory study was aimed to evaluate quantitative and qualitative aspects of thrombus. In 28 patients with and without T2DM treated with aspirin and clopidogrel we assessed thrombus quantity using an ex-vivo chamber, platelet reactivity, thrombus ultrastructure and thrombus kinetics one week after NSTE-ACS.

Results

T2DM was associated with increased thrombus [14861 (8003 to 30161) vs 8908 (6812 to 11996), μ²/mm, median (IQR), p = 0.045] and platelet reactivity. In addition, diabetic thrombus showed lower visco-elastic tensile strength [(− 0.2(− 1.7 to 0.7) vs 1.0(− 0.9 to 3.3), p = 0.044)] and was more resistant to autolysis [(27.8(11.7 to 70.7) vs 78.8(68.5 to109.6) mm/min, p = 0.002)]. On SEM, fibrin fibres in diabetes were thinner, with higher lateral interlinkage and mesh-like organisation. Thrombus quantity correlated inversely with thrombus retraction (r = − 0.450 p = 0.016) but not with platelet reactivity (r = 0.153, p = 0.544).

Conclusions

Despite optimal antiplatelet therapy, T2DM patients after NSTE-ACS developed increased thrombus of lower tensile strength and slower retraction. SEM revealed loosely arranged fibrin fibres. Our data showed significant differences in the magnitude as well as structural and mechanistic characteristics of thrombus in patients with T2DM.     

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.     

Personality traits and childhood trauma as correlates of metabolic risk factors: the Netherlands Study of Depression and Anxiety (NESDA)

Objective

Personality and childhood trauma may affect cardiovascular disease (CVD) risk. However, evidence for an association with metabolic risk factors for CVD is limited and ambiguous. Moreover, despite their interrelatedness, personality and childhood trauma were not yet studied simultaneously. Therefore, we aimed to explore whether personality and childhood trauma are correlates of metabolic risk factors.

Methods

Among 2755 participants of the Netherlands Study of Depression and Anxiety (NESDA), we investigated through linear regression models whether Big Five personality traits (i.e., extraversion, openness, agreeableness, neuroticism and conscientiousness) and childhood trauma type (i.e., emotional neglect, and psychological, physical and sexual abuse) were correlates of metabolic risk factors (i.e., lipids, waist circumference (WC), glucose and blood pressure). Basic covariates (i.e., age, sex and income level), lifestyle, severity of depressive symptoms and years of education were taken into account.

Results

Openness was the most robust favorable correlate, and sexual abuse was the most robust unfavorable correlate of lipids and WC, and of overall metabolic risk (β = −.070; p < .001 and β = .035; p = .04, respectively).

Conclusions

People with a low openness trait and those who experienced childhood sexual abuse are at higher risk of dyslipidemia and abdominal obesity.     

Stent implantation in the superficial femoral artery: Short thrombelastometry-derived coagulation times identify patients with late in-stent restenosis

Introduction

The mechanisms of restenosis, the recurrence of luminal narrowing, are complex and incompletely understood to date. Thrombin, the pivotal enzyme in haemostasis, presumably contributes to the formation of in-stent restenosis (ISR). It was therefore the aim of our study to investigate whether blood coagulation/thrombin generation plays a critical role in the formation of ISR in peripheral artery disease patients with stent angioplasty in the superficial femoral artery.

Materials and Methods

We aimed to examine in this retrospective study whether patients with high-degree restenosis (50-75% lumen diameter reduction, n = 20) are in a hypercoaguable state implying enhanced readiness to generate thrombin compared to patients with low-degree restenosis (< 50% lumen diameter reduction, n = 14).

Results

The coagulation tests calibrated automated thrombography, activated partial thromboplastin time, platelet aggregation, platelet adhesion, fibrinogen, and microparticles’ procoagulant activity did not indicate a different coagulation status in the two patient groups. However, the thrombelastometry-derived value Coagulation Time (CT) was significantly shorter in the high-degree restenosis group (p = 0.012), indicating a hypercoagulable state of patients with high-degree restenosis. Under our experimental conditions, CTs shorter than 444.5 s identify patients at high risk (sensitivity = 95%) for luminal narrowing.

Conclusions

Our study supports the assumption that blood coagulation/thrombin generation plays a critical role in the development of ISR in peripheral arteries after stent insertion and that the thrombelastometry-derived CT might be a suitable value to identify peripheral artery disease patients at risk for development of high-degree in-stent restenosis in the superficial femoral artery.     

Circulating tissue factor positive microparticles in patients with acute recurrent deep venous thrombosis

Introduction

Circulating tissue factor positive microparticles (MPTF) were reported in a wide range of diseases with thrombotic tendency. Though D-dimer assay had a high negative predictive value for deep venous thrombosis (DVT) recurrence, there are currently no reliable positive predictors for recurrent DVT. We therefore quantified MPTF in patients with acute recurrent DVT to determine whether MPTF levels could be used to predict recurrent DVT.

Materials and Methods

Microparticles (MPs) were isolated from plasma of initial DVT patients (n = 25), recurrent DVT patients (n = 25) and sex- and age-matched healthy individuals (n = 25), stained with annexin V, cell-specific monoclonal antibodies (MoAbs) and a MoAb directed against tissue factor (TF), and analyzed by flow cytometry. We also determined the plasma procoagulant activity with a Human TF Chromogenic Activity Assay Kit.

Results

We found total MPTF to be elevated in recurrent DVT patients versus normal individuals (P = 0.001). The number of monocyte-derived MPTF in both initial and recurrent DVT was higher than in normal individuals (P < 0.01, respectively). The platelet and endothelial cell derived MPTF in recurrent DVT were significantly increased relative to other MPTF (P < 0.05), although there was no difference between initial DVT patients and normal individuals. We demonstrated elevated procoagulant activity of platelet-free plasma in DVT patients relative to normal individuals, and a positive correlation with MPTF.

Conclusions

The elevated MPTF could be a potentially predictor for DVT recurrence. Further studies are needed to validate its sensitivity and specificity.     

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.     

The relationship between aortic aneurysm sac thrombus volume on coagulation, fibrinolysis and platelet activity

Aim

Abdominal aortic aneurysm (AAA) is associated with chronic mural inflammation and a pro-thrombotic diathesis. It has been suggested that both may be related to biologically active intra-sac thrombus. The aim of this study was to examine the relationship between thrombin generation, fibrinolysis, platelet activity and AAA sac thrombus volume.

Methods

30 patients (29 men) of median (IQR) age 75 (71-82) years with an infra-renal AAA > 5.5 cm in antero-posterior diameter were prospectively studied. AAA, lumen and thrombus volumes were calculated using a CT workstation (Vitrea). Plasma thrombin-antithrombin (TAT), plasminogen activator inhibitor (PAI)-1, and soluble (s) P-selectin were measured as biomarkers of coagulation, fibrinolysis and platelet activity, respectively

Results

Median (IQR) AAA total, lumen and thrombus volumes were 188 (147-247) cm³, 80 (54.3-107) cm³ and 97.6 (63-127) cm³ respectively.TAT levels were significantly higher (median, QR, 7.15 [4.7-31.3] μg/L, p = < 0.001) and sP-selectin levels significantly lower (median, IQR, 80.5 [68-128] ng/ml, p = < 0.0001) than the normal range. PAI-1 levels (median, IQR, 20.9 [8.4-50.7] ng/ml) were normal. There was no correlation between AAA thrombus volume and PAI-1 (r = − 0.25, p = 0.47), sP-Selectin (r = 0.26, p = 0.43) or TAT plasma levels (r = − 0.21, p = 0.54).

Conclusion

The present study confirms that patients with AAA demonstrate haemostatic derangement, but the extent of the haemostatic derangement does not correlate with AAA sac thrombus volume.     

Increased heparanase level and procoagulant activity in orthopedic surgery patients receiving prophylactic dose of enoxaparin

Background

Orthopedic hip and knee surgeries are followed by a hypercoagulable state. Heparanase is implicated in inflammation, coagulation activation and angiogenesis. Recently, heparanase was shown to directly interact with tissue factor (TF) and to enhance the generation of factor Xa (Nadir et al., Haematologica, 2010). In addition, an assay assessing heparanase procoagulant activity has been lately developed (Nadir et al., Thromb Res, 2011). In the present study heparanase level and procoagulant activity in patients undergoing orthopedic surgery were assessed.

Methods

The study group included 50 orthopedic patients. 31 patients underwent hip surgery and 19 had knee operation. 15 individuals suffered from traumatic hip fractures and 35 had osteoarthrosis of hip or knee joints. All patients received prophylactic dose of enoxaparin starting 6-8 hours post operation and lasting for 5 weeks. Plasma samples were drawn preoperatively and at 1 hour, 1 week and 1 month post operation. Samples were tested for heparanase levels by ELISA and TF/heparanase complex activity, TF activity, heparanase procoagulant activity, factor Xa and thrombin levels using chromogenic substrates.

Results

Heparanase levels were significantly higher 1 hour and 1 week post operatively compared to preoperative levels (p < 0.05, p < 0.005, respectively). The most dramatic changes were observed in heparanase procoagulant activity reaching a 2 fold increase 1 week postoperatively and 1.7 fold increase 1 month after surgery (p < 0.0001, p < 0.0001, respectively). Levels of factor Xa and thrombin did not significantly change.

Conclusions

Heparanase is involved in coagulation activation of orthopedic surgery patients. Heparanase procoagulant activity is highest 1 week postoperatively and remains high 1 month after operation. Considering extending prophylactic anticoagulant therapy or evaluating heparanase procoagulant activity may potentially prevent late thrombotic events.     

Tissue plasminogen activator (tPA) activity is a novel and early marker of asymptomatic LEAD in type 2 diabetes

Introduction

Lower extremity arterial disease (LEAD) is often one of the first signs of a generalized atherosclerotic disease in type 1 and type 2 diabetic subjects.

Materials and methods

We studied 143 diabetic subjects at 30-70 years of age, M/F 69/74, 74 with type 1 and 69 with type 2 diabetes, without previously known or suspected lower extremity arterial disease. The relationship between early asymptomatic lower extremity arterial disease and blood levels of HbA1c, lipids and fibrinolysis markers (tPA-activity, tPA mass, PAI-1 activity, tPA-PAI-1 complex) was assessed. In parallel, a group with non-diabetic subjects (n = 80) was studied.

Results

35 (24%) diabetic subjects were classified as having sign(s) of LEAD, defined as having at least one reduced peripheral blood pressure measurement, 28% in type 1 vs 20% in type 2 diabetic subjects (p = NS). In univariate logistic regression analyses age, glycemic level (HbA1c), male gender (only in type 1 diabetic subjects), hypertension and tPA activity (only in type 2 diabetic subjects) were positively associated with LEAD. When markers of fibrinolysis were entered into a multivariate model adjusting for age, hypertension, and HbA1c, only tPA activity remained independently associated with LEAD (p = 0.01) and this was also found in type 2 diabetic subjects (p = 0.05). In type 1 diabetic subjects the increase in odds ratio was non-significant.

Conclusions

Tissue plasminogen activator (tPA) activity may be an independent and early marker for asymptomatic lower extremity arterial disease in diabetic subjects, particularly in type 2 diabetes. Thus an altered fibrinolytic activity could be an early marker of atherosclerosis development in the lower extremities but the cause-effect relationship remains unclear.     

Liporetro-D-peptides - a novel class of highly selective thrombin inhibitors

Introduction

Plasma serine protease thrombin plays a key role in coagulation, haemostasis and thromboembolic diseases. Direct thrombin inhibitors could be beneficial for future anticoagulant therapy. We have synthesized and studied liporetro-D-peptides - efficient thrombin inhibitors resistant to enzymatic degradation.

Materials and Methods

Compounds X-D-Arg-D-Phe-OMe, where X = residue of lauric or myristic acid or 9-fluorenylmethoxycarbonyl, have been synthesized by conventional peptide synthesis in solution and their comparative inhibitory analysis in relation to thrombin, factor X, plasmin and trypsin has been conducted.

Results

Modification of the synthetic liporetro-D-peptides with the myristic acid residue was the most successful one. This modification has dramatically increased the inhibition efficacy (Ki = 0,17 μM) and selectivity toward the chosen target enzyme, thrombin, in comparison to factor X, plasmin and trypsin (more than 600, 900, and 5000-fold, respectively).

Conclusions

Our findings establish an important role of the fatty moiety in the structure of peptide inhibitors with regards to their potency and selectivity toward thrombin.     

Role of serotonergic-related systems in suicidal behavior: Data from a case-control association study

Objective

To investigate whether functional polymorphisms directly (HTR2A and SLC6A4 genes) or indirectly (IL-1 gene complex, APOE and ACE genes) related with serotonergic neurotransmission were associated with suicidal behavior.

Subjects and methods

227 suicide attempters, 686 non-suicidal psychiatric patients, and 420 healthy controls from a homogeneous Spanish Caucasian population were genotyped using standard methods.

Results

There were no differences in genotype frequencies between the three groups. The −1438A/G [χ² (df) = 9.80 (2), uncorrected p = 0.007] and IL-1α −889C/T [χ² (df) = 8.76 (2), uncorrected p = 0.013] genotype frequencies between impulsive and planned suicide attempts trended toward being different (not significant after Bonferroni correction). Suicide attempts were more often impulsive in the presence of −1438G/G or IL-1α −889C/T or C/C genotypes. There was interaction between the polymorphism 5-HTTLPR and age [LRT (df) = 6.84 (2), p = 0.033] and between the polymorphisms APOE and IL-1RA (86 bp)_n [LRT (df) = 12.21 (4), p = 0.016] in relation to suicide attempt lethality.

Conclusion

These findings further evidence the complexity of the association between genetics and suicidal behavior, the need to study homogenous forms of the behavior and the relevance of impulsive and aggressive traits as endophenotypes for suicidal behavior.     

Evaluation of the procoagulant activity of endogenous phospholipids in the platelet-free plasma of children with sickle cell disease using functional assays

Background

The mechanisms of hypercoagulability in sickle cell disease (SCD) are poorly understood.

Objective

We aimed to explore the procoagulant activity of endogenous phospholipids (ePL) in the platelet-free plasma of SCD children.

Methods

A factor Xa clotting time (XACT), thrombin generation (TG) and a capture-based assay for the detection of procoagulant microparticles (PMP) were used. Forty three SCD children (35 SS, 6 SC and 2 Sβ +) were evaluated at steady-state and compared to 20 controls. Fourteen patients were also evaluated during vaso-occlusive crisis. TG was performed using 10pM tissue factor without addition of exogenous phospholipids. A control condition was also performed using 10pM tissue factor and 4 μM phospholipids. Percentages of the test/control conditions were calculated for the peak height (% peak), endogenous thrombin potential (% ETP) and velocity index (% VI).

Results

XACT times were shorter, PMP levels, peak height and velocity index of thrombin generation were higher in SCD patients than controls. Lag time and ETP were not different between the two groups. % peak, % ETP and % VI were higher in patients than controls. Significant correlations were found between PMP levels and XACT, also between PMP levels and peak height, velocity index, ETP and their respective percentages to the control condition, but not with lag time. Double heterozygous patients showed intermediate values for XACT and TG parameters. No significant difference was observed when comparing patients at steady-state versus vaso-occlusive crisis.

Conclusion

High procoagulant activity of ePL was observed in the platelet-free plasma of SCD children, probably borne by procoagulant microparticles. This may contribute to a high hemostatic potential and predisposition to thrombotic complications in these patients.     

Microparticle-associated tissue factor activity correlates with plasma levels of bacterial lipopolysaccharides in meningococcal septic shock

Introduction

The plasma level of bacterial lipopolysaccharides (LPS) is associated with activation of the coagulation system, inhibition of fibrinolysis and the nature of the clinical presentation and outcome in patients with meningococcal disease. Tissue factor (TF)-bearing microparticles (MPs) appear to contribute to the pathogenesis of disseminated intravascular coagulation (DIC). The aim of this study was to investigate the relationship between MP-associated TF activity and the level of bacterial LPS in plasma from patients with meningococcal septic shock and meningitis.

Materials and methods

MPs isolated from citrated plasmas were assessed for TF-dependent activity with both a plasma-based thrombin generation assay (CAT) and whole blood-based thromboelastometry (ROTEM). The LPS level was measured using a chromogenic Limulus amebocyte lysate assay.

Results

MPs obtained from patients with meningococcal septic shock initiated significantly more efficient and TF-dependent thrombin generation in the CAT assay compared to MPs from patients with meningococcal meningitis. Differences in MP-associated TF activity between the septic shock patients and the meningitis patients were also evident when MPs were added to whole blood using ROTEM. The level of plasma LPS in patients with septic shock (range 2–2,100 EU/mL) was correlated with thrombogram parameters in the CAT assay; lagtime (r_s = − 0.84), time to peak (r_s = − 0.83), peak (r_s = 0.85) and ETP (r_s = 0.83).

Conclusions

MPs obtained from patients with meningococcal septic shock displayed more efficient TF-dependent thrombin generation and clot formation compared to MPs from meningitis patients. MP-associated TF activity was closely associated with plasma LPS levels in the septic shock group.