Kristien Winckers

Early Atherosclerosis Exhibits an Enhanced Procoagulant State

BACKGROUND Thrombin generation in vivo may be important in regulating atherosclerotic progression. In the present study, we examined for the first time the activity and presence of relevant coagulation proteins in relation to the progression of atherosclerosis. METHODS AND RESULTS Both early and stable advanced atherosclerotic lesions were collected pairwise from each individual (n=27) during autopsy. Tissue homogenates were prepared from both total plaques and isolated plaque layers, in which the activity of factors (F) II, X, and XII and tissue factor was determined. Microarray analysis was implemented to elucidate local messenger RNA synthesis of coagulation proteins. Part of each specimen was paraffin embedded, and histological sections were immunohistochemically stained for multiple coagulation markers with the use of commercial antibodies. Data are expressed as median (interquartile range [IQR]). Tissue factor, FII, FX, and FXII activities were significantly higher in early atherosclerotic lesions than in stable advanced atherosclerotic lesions. Endogenous thrombin potential and thrombin-antithrombin complex values consolidated a procoagulant profile of early atherosclerotic lesions (endogenous thrombin potential, 1240 nmol/L x min [IQR, 1173 to 1311]; thrombin-antithrombin complex, 1045 ng/mg [IQR, 842.6 to 1376]) versus stable advanced atherosclerotic lesions (endogenous thrombin potential, 782 nmol/L x min [IQR, 0 to 1151]; thrombin-antithrombin complex, 718.4 ng/mg [IQR, 508.6 to 1151]). Tissue factor, FVII, and FX colocalized with macrophages and smooth muscle cells. In addition, multiple procoagulant and anticoagulant proteases were immunohistochemically mapped to various locations throughout the atherosclerotic vessel wall in both early and advanced atherosclerotic stages. CONCLUSIONS This study shows an enhanced procoagulant state of early-stage atherosclerotic plaques compared with advanced-stage plaques, which may provide novel insights into the role of coagulation during atherosclerotic plaque progression.

Vascular calcification: the price to pay for anticoagulation therapy with vitamin K-antagonists.

Vitamin K-antagonists (VKA) are the most widely used anti-thrombotic drugs with substantial efficacy in reducing risk of arterial and venous thrombosis. Several lines of evidence indicate, however, that VKA inhibit not only post-translational activation of vitamin K-dependent coagulation factors but also synthesis of functional extra-hepatic vitamin K-dependent proteins thereby eliciting undesired side-effects. Vascular calcification is one of the recently revealed side-effects of VKA. Vascular calcification is an actively regulated process involving vascular cells and a number of vitamin K-dependent proteins. Mechanistic understanding of vascular calcification is essential to improve VKA-based treatments of both thrombotic disorders and atherosclerosis. This review addresses vitamin K-cycle and vitamin K-dependent processes of vascular calcification that are affected by VKA. We conclude that there is a growing need for better understanding of the effects of anticoagulants on vascular calcification and atherosclerosis.

The role of tissue factor pathway inhibitor in atherosclerosis and arterial thrombosis

Tissue factor pathway inhibitor (TFPI) is the main inhibitor of tissue factor (TF)-mediated coagulation. In atherosclerotic plaques TFPI co-localizes with TF, where it is believed to play an important role in attenuating TF activity. Findings in animal models such as TFPI knockout models and gene transfer models are consistent on the role of TFPI in arterial thrombosis as they reveal an active role for TFPI in attenuating arterial thrombus formation. In addition, ample experimental evidence exists indicating that TFPI has inhibitory effects on both smooth muscle cell migration and proliferation, both which are recognized as important pathological features in atherosclerosis development. Nonetheless, the clinical relevance of these antithrombotic and atheroprotective effects remains unclear. Paradoxically, the majority of clinical studies find increased instead of decreased TFPI antigen and activity levels in atherothrombotic disease, particularly in atherosclerosis and coronary artery disease (CAD). Increased TFPI levels in cardiovascular disease might result from complex interactions with established cardiovascular risk factors, such as hypercholesterolemia, diabetes and smoking. Moreover, it is postulated that increased TFPI levels reflect either the amount of endothelial perturbation and platelet activation, or a compensatory mechanism for the increased procoagulant state observed in cardiovascular disease. In all, the prognostic value of plasma TFPI in cardiovascular disease remains to be established. The current review focuses on TFPI in clinical studies of asymptomatic and symptomatic atherosclerosis, coronary artery disease and ischemic stroke, and discusses potential atheroprotective actions of TFPI.

Patients with Antineutrophil Cytoplasmic Antibodies Associated Vasculitis in Remission Are Hypercoagulable

Objectives. The risk of venous thromboembolism (VTE) is increased in patients with antineutrophil cytoplasmic antibodies (ANCA) associated vasculitides (AAV) as compared to healthy subjects. The mechanisms underlying this increased occurrence of VTE are not completely understood. We hypothesize that AAV patients in remission are more procoagulant than healthy controls. Methods. Patients with AAV in remission and no VTE for the last 6 months were included. Patients with severe renal impairment (serum creatinine > 250 μmol/l) were excluded. Age and sex matched healthy controls were included. The endogenous thrombin potential (ETP) was determined together with hemostatic variables: fibrinogen, D-dimers, factor VIII (FVIII), tissue factor pathway inhibitor (TFPI), protein C, and free protein S. Results. Thirty-one patients were included. In 27 patients not taking anticoagulants, ETP was measured and found to be elevated: 137.1% as compared to a median of 90.0% for healthy controls (p < 0.01). Fibrinogen and D-dimer levels were not elevated in patients (median 3.5 g/l and 279 μg/l, respectively). FVIII and TFPI levels were also significantly increased in patients as compared to healthy controls (159% vs 137%; 122.5% vs 101%, respectively), whereas protein C and free protein S levels were not elevated (126.5% vs 118.6% and 124.6% vs 118.3%, respectively). Conclusion. Patients with AAV in remission are more procoagulant than healthy controls, as indicated by an increased ETP. The increased FVIII level measured in these patients suggests persistence of endothelial activation and/or dysfunction. This endothelial dysfunction may cause a continuous low-grade procoagulant state.

Increased tissue factor pathway inhibitor activity is associated with myocardial infarction in young women: results from the RATIO study

Summary.  Background: The tissue factor pathway inhibitor (TFPI)/protein S anticoagulant system is a potent inhibitor of blood coagulation. TFPI and protein S are major determinants of thrombin generation (TG) tests determined at low tissue factor (TF) and at high TF concentrations in the presence of activated protein C (APC). Both TFPI and protein S protect against venous thrombosis, but the importance of the TFPI/protein S system in arterial thrombosis remains unclear. Objectives: To investigate the influence of the TFPI/protein S anticoagulant system on the risk of myocardial infarction (MI) in young women. Methods: The RATIO study is a case–control study in women under 50 years of age, including 205 patients and 638 controls. TFPI and protein S were quantified using ELISA. The TFPI/protein S activity (nTFPIr) and the APC sensitivity ratio (nAPCsr) were determined using TG tests. Odds ratios (ORs) adjusted for putative confounders and corresponding 95% confidence intervals (95% CI) were determined. Results: Women with MI had higher TFPI levels than controls (135.9 ± 40% vs. 124.2 ± 41%), resulting in increased TFPI/protein S activities and increased APC sensitivity. Furthermore, an increased TFPI activity was associated with MI [nTFPIr: adjusted OR Q1 vs. Q4 = 2.1 (95%CI 1.1–4.1)]. Additionally, an increased APC sensitivity was associated with MI [nAPCsr: adjusted OR Q1 vs. Q4 = 1.7 (95% CI 0.9–3.2)] Conclusion: Women with MI had increased TFPI levels compared with controls. Consequently, the TFPI/protein S activity and APC sensitivity are increased in women with MI. Whether this increase in TFPI activity acts as a compensating mechanism for an increased procoagulant state or is a marker of endothelial damage remains to be investigated.

Associations Between Thrombin Generation and the Risk of Cardiovascular Disease in Elderly Patients: Results From the PROSPER Study

BACKGROUND Hypercoagulability may be an important contributor to the pathophysiology of atherosclerosis and atherothrombosis. As thrombin fulfills a central role in coagulation and links to several cellular mechanisms involved in arterial disease, we hypothesized that thrombin generation is associated with cardiovascular events in elderly patients. METHODS We studied the relationship between plasma thrombin generation and incident coronary heart disease (CHD) and stroke in the PROspective Study of Pravastatin in the Elderly at Risk (PROSPER). From this multicenter prospective cohort, 4,932 samples of subjects (70-82 years) with pre-existing vascular disease or risk factors were available for thrombin generation measurements. RESULTS Within the 3.2 years of follow-up incident stroke and CHD was observed in 227 and 545 subjects, respectively. Baseline thrombin generation was significantly decreased in subjects with incident stroke compared with subjects without: normalized peak height 71.1±40.8% versus 82.3±44.9%, p = .0002, and normalized endogenous thrombin potential 79.1±23.3% versus 87.0±24.8%, p < .0001 (mean and SDs). Thrombin generation was independently and inversely associated with stroke risk: hazard ratio 0.71 (95%CI: 0.60-0.85), 0.68 (95%CI: 0.58-0.79), for normalized peak height and normalized endogenous thrombin potential, respectively (all p < .001). In subjects with incident CHD, thrombin generation was comparable to subjects without a coronary event. Only an increased normalized peak height was significantly associated with incident CHD (hazard ratio 1.17 [95% CI: 1.06-1.28], p = .002). CONCLUSIONS We demonstrate that a delayed and decreased thrombin generation is a strong and independent predictor for stroke in elderly people at increased risk of vascular disease. However, no convincing consistent association could be demonstrated between thrombin generation and incident CHD.

Levels of heparin-releasable TFPI are increased in first-ever lacunar stroke patients

Objectives: New insights in the pathophysiology of lacunar stroke (LS) suggest that it is caused by increased permeability of the blood-brain barrier due to endothelial activation. Because endothelial cells are the major production and storage site of tissue factor pathway inhibitor (TFPI), this protein can be used as marker of endothelial activation. In this observational study we measured the different pools of TFPI, as a marker of endothelial function, in first-ever lacunar stroke patients. Methods: We determined antigen levels of total and free full-length (FL) TFPI using ELISA in 149 patients and 42 controls. Heparin-releasable free FL TFPI was determined in a random subset of 17 patients and 15 controls. By brain MRI, we classified LS patients as having isolated lacunar infarct (ILA) or silent ischemic lesions (SILs). Results: Plasma levels of total TFPI were highest in patients with SILs compared with those with ILA, but this association disappeared after correction for age and levels of low-density lipoprotein cholesterol. However, levels of heparin-releasable free FL TFPI were higher in patients than in controls. Conclusions: Although ambient plasma levels of total TFPI were not different in subtypes of LS, the increased levels of heparin-releasable TFPI in patients suggest a role of endothelial activation in the pathogenesis of LS.

Impaired tissue factor pathway inhibitor function is associated with recurrent venous thromboembolism in patients with first unprovoked deep venous thrombosis.

Based Clinical Practice Guidelines. Chest 2012; 141(Suppl. 2):e152S–84S.2 van den Besselaar AMHP, Poller L, Tripodi A, WHO ExpertCommittee on Biological Standardization. Guidelines for thrombo-plastins and plasma used to control oral anticoagulant therapy .WHO Technical Report Series no. 889. Geneva: WHO, 1999: 64–93.3 Leichsenring I, Plesch W, Unkrig V, Kitchen S, Kitchen DP, MacleanR, Dikkeschei B, van den Besselaar AM. Multicentre ISI assignmentand calibration of the INR measuring range of a new point-of-caresystem designed for home monitoring of oral anticoagulation therapy.Thromb Haemost 2007; 97: 856–61.

Platelet full length TFPI-α in healthy volunteers is not affected by sex or hormonal use

Background Only 10% of plasma TFPIα (TFPI) exists in the full length form, the rest circulates as a C-terminally truncated form. However, blood platelets exclusively contain full length TFPI, which is released at the site of injury upon platelet activation, and which could play an important local regulatory role in thrombin generation and prevention of thrombosis. Methods The anticoagulant activities of full length and truncated TFPI were investigated using thrombin generation assays. Blood samples were obtained from 30 healthy volunteers (10 male subjects, 10 female subjects, and 10 females using oral contraceptives). Platelet TFPI was released in platelet rich plasma and in platelet isolates using convulxin or thrombin, and measured by free TFPI ELISA and thrombin generation assays. Results Full length TFPI and platelet TFPI were much more potent inhibitors of thrombin generation than truncated TFPI, which was virtually inactive. Although mean plasma TFPI antigen levels decreased from men (0.30 nM) to women (0.20 nM) to women using oral contraceptives (0.11 nM), no relevant differences were found in platelet TFPI among those subgroups. Conclusions Platelets release similar amounts of TFPI regardless of plasma TFPI concentrations and is unaffected by sex or oral contraceptive use. We speculate that platelet TFPI is important to prevent systemic coagulation and thrombosis and restrict thrombus formation to the site of the growing platelet plug. The stable contribution of platelet TFPI to the anticoagulant potential in plasma is likely to become particularly relevant under conditions of low plasma TFPI levels in combination of oral contraceptives use.

Suppressive Role of Tissue Factor Pathway Inhibitor-α in Platelet-Dependent Fibrin Formation under Flow Is Restricted to Low Procoagulant Strength

Tissue factor pathway inhibitor-alpha (TFPI-α) is a Kunitz-type serine protease inhibitor, which suppresses coagulation by inhibiting the tissue factor (TF)/factor VIIa complex as well as factor Xa. In static plasma-phospholipid systems, TFPI-α thus suppresses both factor Xa and thrombin generation. In this article, we used a microfluidics approach to investigate how TFPI-α regulates fibrin clot formation in platelet thrombi at low wall shear rate. We therefore hypothesized that the anticoagulant effect of TFPI-α in plasma is a function of the local procoagulant strength-defined as the magnitude of thrombin generation under flow, due to local activities of TF/factor VIIa and factor Xa. To test this hypothesis, we modulated local coagulation by microspot coating of flow channels with 0 to 100 pM TF/collagen, or by using blood from patients with haemophilia A or B. For blood or plasma from healthy subjects, blocking of TFPI-α enhanced fibrin formation, extending from a platelet thrombus, under flow only at <2 pM coated TF. This enhancement was paralleled by an increased thrombin generation. For mouse plasma, genetic deficiency in TFPI enhanced fibrin formation under flow also at 0 pM TF microspots. On the other hand, using blood from haemophilia A or B patients, TFPI-α antagonism markedly enhanced fibrin formation at microspots with up to 100 pM coated TF. We conclude that, under flow, TFPI-α is capable to antagonize fibrin formation in a manner dependent on and restricted by local TF/factor VIIa and factor Xa activities.