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1.  Multimarker Approach in Discriminating Patients with Symptomatic and Asymptomatic Atherosclerotic Carotid Artery Stenosis 
Background and Purpose
Several circulating biomarkers have been implicated in carotid atherosclerotic plaque rupture and thrombosis; however, their clinical utility remains unknown. The aim of this study was to determine the role of a large biomarker panel in the discrimination of symptomatic (S) vs. asymptomatic (A/S) subjects in a contemporary population with carotid artery stenosis (CS).
Prospective sampling of circulating cytokines and blood lipids was performed in 300 unselected, consecutive patients with ≥50% CS, as assessed by duplex ultrasound (age 47-83 years; 110 with A/S and 190 with S) who were referred for potential CS revascularization.
CS severity and pharmacotherapy did not differ between the A/S and S patients. The median values of total cholesterol, low-density lipoprotein cholesterol, and lipoprotein(a) did not differ, but high-density lipoprotein (HDL) cholesterol was significantly higher (p<0.001) and triglycerides were lower (p=0.03) in the A/S-CS group than in the S-CS group. Interleukin-6 (IL-6) and high-sensitivity C-reactive protein were higher (p=0.04 and p=0.07, respectively) in the S-CS group. Circulating visfatin, soluble CD 40 receptor ligand, soluble vascular cell adhesion molecule, leptin, adiponectin, IL-1β, IL-8, IL-18, monocyte chemoattractant protein-1, myeloperoxidase, matrix metalloproteinases-8, -9, and -10, and fibrinogen were similar, but tissue inhibitor of matrix metalloproteinases-1 (TIMP) was reduced in S-CS compared to A/S-CS (p=0.02). Nevertheless, incorporation of TIMP and IL-6 did not improve the HDL-cholesterol receiver operating characteristics for S-CS status prediction. S-CS status was unrelated to angiographic stenosis severity or plaque burden, as assessed by intravascular ultrasound (p=0.16 and p=0.67, respectively). Multivariate logistic regression analysis revealed low HDL-cholesterol to be the only independent predictor of CS symptoms, with an odds ratio of 1.81 (95% confidence interval=1.15-2.84, p=0.01) for HDL <1.00 mmol/L (first quartile) vs. >1.37 (third quartile). In S-CS, osteoprotegerin and lipoprotein-associated phospholipase A2 (Lp-PLA2) were elevated in those with recent vs. remote symptoms (p=0.01 and p=0.02, respectively).
In an all-comer CS population on contemporary pharmacotherapy, low HDL-cholesterol (but not other previously implicated or several novel circulating biomarkers) is an independent predictor of S-CS status. In addition, an increase in circulating osteoprotegerin and Lp-PLA2 may transiently indicate S transformation of the carotid atherosclerotic plaque.
PMCID: PMC3722468  PMID: 23894240
carotid artery stenosis; biomarkers; circulating cytokines; risk factors; stroke risk; HDL-cholesterol
2.  Omega-3 Polyunsaturated Fatty Acids Increase Plasma Adiponectin to Leptin Ratio in Stable Coronary Artery Disease 
Cardiovascular Drugs and Therapy  2013;27(4):289-295.
Growing evidence suggests a cardioprotective role of omega-3 polyunsaturated fatty acids (PUFA). However, the exact mechanisms underlying the effects of omega-3 PUFA in humans have not yet been fully clarified.
We sought to evaluate omega-3 PUFA-mediated effects on adipokines in patients with stable coronary artery disease (CAD) undergoing elective percutaneous coronary intervention (PCI).
We conducted a prospective, double-blind, placebo-controlled, randomized study, in which adiponectin, leptin and resistin were determined at baseline, 3–5 days and 30 days during administration of omega-3 PUFA 1 g/day (n = 20) or placebo (n = 28).
As compared to controls administration of omega-3 PUFA resulted in increase of adiponectin by 13.4 % (P < 0.0001), reduction of leptin by 22 % (P < 0.0001) and increase of adiponectin to leptin (A/L) ratio by 45.5 % (P < 0.0001) at 30 days, but not at 3–5 days. Compared with placebo adiponectin was 12.7 % higher (P = 0.0042), leptin was 16.7 % lower (P < 0.0001) and A/L ratio was 33.3 % higher (P < 0.0001) in the omega-3 PUFA group at 30 days. Resistin decreased similarly in both groups after 1 month, without intergroup differences (P = 0.32). The multivariate model showed that the independent predictors of changes in adiponectin at 1 month (P < 0.001) were: omega-3 PUFA treatment, baseline platelet count, total cholesterol and those in leptin (P < 0.0001) were: omega-3 PUFA treatment and waist circumference. Independent predictors of A/L ratio changes (P < 0.0001) were: assigned treatment, current smoking and hyperlipidemia.
In high risk stable coronary patients after PCI omega-3 PUFA supplementation improves adipokine profile in circulating blood. This might be a novel, favourable mechanism of omega-3 PUFA action.
Electronic supplementary material
The online version of this article (doi:10.1007/s10557-013-6457-x) contains supplementary material, which is available to authorized users.
PMCID: PMC3709088  PMID: 23584593
Omega-3 polyunsaturated fatty acids; Adiponectin; Leptin; Resistin; Stable coronary artery disease; Percutaneous coronary intervention
3.  Active tissue factor and activated factor XI in patients with acute ischemic cerebrovascular events 
Elevated factor (F)XI and tissue factor (TF) have been reported to occur in patients with acute ischemic stroke (AIS). We sought to investigate whether circulating activated FXI (FXIa) and TF on admission can predict clinical outcomes in patients with acute cerebrovascular events.
Materials and methods
In the observational study we evaluated 205 consecutive patients aged 70 years or less within the first 72 hours of acute event, including 140 with AIS and 65 with transient ischemic attack (TIA). Plasma TF and FXIa activity were determined on admission in clotting assays by measuring the response to inhibitory monoclonal antibodies.
Active TF and FXIa activity were detected in 58 (28.9%) and 132 (64.4%) patients on admission, respectively. Active TF was detected in 45 of the 136 AIS patients with available TF levels (33.1%) and 13 of the 65 acute TIA patients (20%; p=0.05). Corresponding values for FXIa were 99 of the 140 (70.7%) and 33 of the 65 (50.8%; p=0.006), respectively. Patients with detectable TF were more frequently female and hypertensive, while subjects with detectable FXIa had more often diabetes and higher levels of fibrinogen, C-reactive protein, and interleukin-6 (all p<0.05). Patients with detectable FXIa but not TF had higher NIHSS score, higher modified Rankin scale score and lower Barthel Index at discharge (all p<0.05).
Circulating active TF and FXIa occur frequently in acute cerebrovascular ischemic events. Active FXIa in plasma might be useful as a novel risk marker of worse functional outcomes in patients with acute cerebrovascular events.
PMCID: PMC3189427  PMID: 21707613
acute cerebrovascular events; functional outcomes; coagulation; factor XI; stroke; tissue factor
4.  From Principle to Practice: Bridging the Gap in Patient Profiling 
PLoS ONE  2013;8(1):e54728.
The standard clinical coagulation assays, activated partial thromboplastin time (aPTT) and prothrombin time (PT) cannot predict thrombotic or bleeding risk. Since thrombin generation is central to haemorrhage control and when unregulated, is the likely cause of thrombosis, thrombin generation assays (TGA) have gained acceptance as “global assays” of haemostasis. These assays generate an enormous amount of data including four key thrombin parameters (lag time, maximum rate, peak and total thrombin) that may change to varying degrees over time in longitudinal studies. Currently, each thrombin parameter is averaged and presented individually in a table, bar graph or box plot; no method exists to visualize comprehensive thrombin generation data over time. To address this need, we have created a method that visualizes all four thrombin parameters simultaneously and can be animated to evaluate how thrombin generation changes over time. This method uses all thrombin parameters to intrinsically rank individuals based on their haemostatic status. The thrombin generation parameters can be derived empirically using TGA or simulated using computational models (CM). To establish the utility and diverse applicability of our method we demonstrate how warfarin therapy (CM), factor VIII prophylaxis for haemophilia A (CM), and pregnancy (TGA) affects thrombin generation over time. The method is especially suited to evaluate an individual's thrombotic and bleeding risk during “normal” processes (e.g pregnancy or aging) or during therapeutic challenges to the haemostatic system. Ultimately, our method is designed to visualize individualized patient profiles which are becoming evermore important as personalized medicine strategies become routine clinical practice.
PMCID: PMC3556038  PMID: 23372761
5.  Circulating activated factor XI and active tissue factor as predictors of worse prognosis in patients following ischemic cerebrovascular events 
Thrombosis research  2011;128(5):e62-e66.
Elevated factor (F)XI is associated with an increased risk for ischemic stroke. Activated FXI (FXIa) and tissue factor (TF) have not been studied following stroke. The aim of the current study was to evaluate circulating FXIa and TF in patients with prior cerebrovascular events.
We studied 241 patients, including 162 after ischemic stroke and 79 after transient ischemic attack (TIA), recruited 6 months to 4 years (median, 36 months) after the events. Plasma TF and FXIa activity at discharge at the time of index event were determined in clotting assays by measuring the response to inhibitory monoclonal antibodies.
Active TF was detected in 25 (10.4%) of the patients, while FXIa activity (median, 37.5 [IQR 397] pM) was found in 64 (26.7%) of the patients (p<0.01). The prevalence of active TF and FXIa was higher in subjects with previous stroke compared with those with a history of TIA (13 vs 5.1%, p=0.05, and 34% vs 11.4%, p<0.0001, respectively). Patients with circulating FXIa were younger and had higher fibrinogen and interleukin-6 compared to the remainder. Patients with detectable TF or FXIa activity had higher NIHSS score, higher modified Rankin scale and lower Barthel Index than the remaining subjects (all p<0.05).
Circulating active TF and FXIa can occur in patients with cerebrovascular ischemic events ≥6 months after the events. The presence of these factors is associated with worse functional outcomes, which highlights the role of persistent hypercoagulable state in cerebrovascular disease.
PMCID: PMC3205247  PMID: 21820158
6.  Type 2 diabetes as a modifier of fibrin clot properties in patients with coronary artery disease 
Altered fibrin clot structure has been reported both in patients with coronary artery disease (CAD) and those with type 2 diabetes mellitus (DM2). The aim of the present study was to evaluate plasma fibrin clot permeability and susceptibility to lysis in patients with DM2 and CAD. We studied 132 consecutive CAD patients, including 67 subjects with DM2, scheduled for elective coronary artery bypass grafting surgery. Ex vivo plasma fibrin clot permeability (Ks) and lysis time (t50%) induced by 1 μg/mL recombinant tissue plasminogen activator (tPA), along with plasma levels of plasminogen activator inhibitor-1 (PAI-1), thrombin activatable fibrinolysis inhibitor (TAFI), tPA, von Willebrand factor (vWF), P-selectin, soluble CD40 ligand (sCD40L), were measured. Diabetic and non-diabetic patients did not differ in regard to demographics and remaining cardiovascular risk factors. Concomitant DM2 was associated with higher glucose (+24.3 %, p < 0.001), fibrinogen (+9.0 %, p = 0.037), PAI-1 (+58.7 %, p < 0.001), tPA (+24.0 %, p < 0.001) and P-selectin (+12.2 %, p < 0.001). Compared with the non-diabetic group, the CAD patients with DM2 had lower Ks (-6.1 %, p = 0.02) and prolonged t50% (+5.1 %, p = 0.04). Multiple regression analysis of the whole study group showed that vWF, PAI-1, fibrinogen and DM2 were the independent predictors of t50% (R2 = 0.58, p < 0.001), while only vWF was an independent predictor of Ks (R2 = 0.22, p < 0.001). This study indicates that DM2 is potent enough to unfavorably affect plasma fibrin clot characteristics despite abnormal clot phenotype typically observed in CAD. Of note, platelet and endothelial markers appear to contribute to fibrin clot properties in CAD concomitant with DM2.
PMCID: PMC3549239  PMID: 23086579
Coronary artery disease; Diabetes mellitus; Fibrin clot; Fibrinolysis; Platelet activation
7.  Architecture of fibrin network inside thrombotic material obtained from the right atrium and pulmonary arteries: flow and location matter 
Pulmonary embolectomy is a treatment option in selected patients with high-risk pulmonary embolism (PE). Efficiency of thrombus degradation in PE largely depends on the architecture of its fibrin network, however little is known about its determinants. We present the case of a 56-year-old woman with high-risk PE and proximal deep-vein thrombosis, whose thrombotic material removed during embolectomy from the right atrium and pulmonary (lobar and segmental) arteries has been studied using scanning electron microscopy (SEM). SEM images showed that distally located thrombi are richer in densely-packed fibrin fibers and contain more white cells and less erythrocytes than the proximal ones and the atrial thrombus. Fibrin fibers alignment along the flow vector was observed in the thrombi removed from high-velocity flow pulmonary arteries, and not in the atrial thrombus. The content of denser fibrin network and platelet aggregates was increased in segmental thromboemboli. Our findings describe the relation between thrombus architecture and location, and might help to elucidate thrombus resistance to anticoagulant therapy in some PE patients.
PMCID: PMC3529871  PMID: 23080213
Pulmonary embolism; Right atrium; Thrombus architecture; Fibrin
8.  Thrombin generation in chronic obstructive pulmonary disease: dependence on plasma factor composition 
Thrombosis research  2011;128(4):e24-e28.
Chronic obstructive pulmonary disease (COPD) is associated with an increased risk for thromboembolic events. We investigated thrombin generation profiles in COPD patients and their dependence on plasma factor/inhibitor composition.
Factors (f) (fII, fV, fVII, fVIII, fIX, fX), antithrombin, protein C (PC) and free tissue factor pathway inhibitor (fTFPI) from 60 COPD patients (aged 64.2±10.1 years; a mean forced expiratory volume in 1 second [FEV1], 55.6 ± 15.8% of predicted values) were compared with those for 43 controls matched for age, sex, weight and smoking. Patients receiving anticoagulation were excluded. Using each individual’s plasma coagulation protein composition, tissue factor-initiated thrombin generation was assessed computationally.
COPD patients had higher fII (115±16 vs 102±10%, p<0.0001), fV (114±19 vs 102±12%, p=0.0002), fVII (111±15 vs 102±17%, p=0.002), fVIII (170±34 vs 115±27%, p<0.0001), and fIX (119±21 vs 107±17%, p=0.003), and lower fTFPI (17.7±3.2 vs 18.9±3.2 ng/ml, p=0.047) compared with controls, while fX, antithrombin, and PC were similar in both groups. Computational thrombin generation profiles showed that compared with controls, COPD patients had higher maximum thrombin levels (+28.3%, p<0.0001), rates of thrombin generation (+46.1%, p<0.0001) and total thrombin formation (+14.4%, p<0.001), together with shorter initiation phase of thrombin generation (p<0.0001) and the time to maximum thrombin levels (p<0.0001). Thrombin generation profiles in COPD patients can be normalized via correction of fII, fVIII, fIX and TFPI. The severity of COPD and inflammatory markers were not associated with thrombin generation profiles.
Prothrombotic phenotype in COPD patients is largely driven by increased prothrombin, fVIII, fIX, and lower fTFPI.
PMCID: PMC3183323  PMID: 21624643
coagulation factors; COPD; thrombin
10.  Activated factor XI and tissue factor in aortic stenosis: Links with thrombin generation 
In our previous studies we showed that a significant proportion of patients with various cardiovascular diseases have active tissue factor (TF) and factor (F)XIa in their plasma.
To evaluate these two proteins in plasma from patients with aortic stenosis (AS) and established their relationship with the severity of the disease.
Fifty-four consecutive patients with AS, including 38 (70.4%) severe AS patients, were studied. Plasma FXIa and TF activity were determined in clotting assays by measuring the response to inhibitory monoclonal antibodies.
TF activity was detectible in plasma from 14 of 54 patients (25.9%), including 13 of 38 with severe AS (34.2%) and 1 of 16 (6.25%) with moderate AS (p=0.052). FXIa activity was found in 12 (22.2%) patients, mostly in individuals with severe AS (11 of 38, 28.9%, p=0.067). All 12 patients with circulating FXIa had active TF in their plasma as well. Severe AS patients with detectable TF had higher maximal (111±20 vs 97±16 mm Hg, p=0.02) and mean (61±12 vs 53±8 mm Hg, p=0.02) transvalvular gradient, compared with those without such activity in plasma. In severe AS patients with detectable active TF, prothrombin fragment 1.2, a thrombin generation marker, was higher than in patients without TF (375±122 vs. 207±64 pM, p<0.001).
Detectable FXIa and TF activity was observed for the first time in AS patients, primarily in severe ones. This activity correlates with thrombin generation in those patients.
PMCID: PMC3146974  PMID: 21519234
Aortic stenosis; factor XIa; tissue factor; prothrombin fragment 1.2
Journal of Thrombosis and Haemostasis  2009;7(Suppl 1):181-186.
We have developed an integrated approach that combines empirical and computational methodologies to define an individual’s thrombin phenotype. We have evaluated the process of thrombin generation in healthy individuals and individuals with defined pathologies (hemophilia A and acute coronary syndrome) in order to develop general criteria relevant to assess an individual’s propensity for hemorrhage or thrombosis. Three complementary hypotheses have emerged from our work: 1) compensation by the ensemble of other coagulation proteins in individuals with specific factor deficiencies can “normalize” an individual’s thrombin generation process and represents a rationale for their unexpected phenotype; 2) individuals with clinically unremarkable factor levels may present thrombin generation profiles typical of individuals with hemostatic complications; and 3) in some hemostatic disorders a specific pattern of expression of a small ensemble of coagulation factors may be sufficient to explain the overall phenotype.
PMCID: PMC3395063  PMID: 19630796
12.  Activated factor XI and tissue factor in chronic obstructive pulmonary disease: links with inflammation and thrombin generation 
Thrombosis research  2011;127(3):242-246.
Increased cardiovascular mortality and risk of venous thromboembolism are serious extra-pulmonary complications of chronic obstructive pulmonary disease (COPD). Previously, circulating active tissue factor (TF) and factor XIa (FXIa) have been reported to be associated with acute coronary syndromes.
To measure plasma FXIa and active TF, prothrombin fragment 1.2 (F1.2), and markers of systemic inflammation (C-reactive protein [CRP], interleukin-6 [IL-6], tumor necrosis factor α [TNFα] and matrix metalloproteinase 9 [MMP-9]) in 60 patients with documented stable COPD free of previous thromboembolic events.
In-house clotting assays using inhibitory monoclonal antibodies against FXIa and TF.
FXIa was detected in 9 (15%) and TF activity in 7 (11.7%) COPD patients. Subjects positive for FXIa and/or TF (n=10; 16.7%) had higher F1.2 (median [interquartile range], 398 [216] vs 192 [42] pM, p<0.000001), fibrinogen (5.58 [2.01] vs 3.97 [2.47] g/L, p=0.0007), CRP (14.75 [1.20] vs 1.88 [2.95] mg/L, p<0.000001), IL-6 (8.14 [4.74] vs 2.45 [2.24] pg/mL, p=0.00002), and right ventricular systolic pressure (47 [15] vs 38 [12] mmHg, p=0.023), and lower vital capacity (66 [15] vs 80 [17] % predicted, p=0.04) than COPD patients without detectable FXIa and TF. COPD severity was not associated with the presence of circulating FXIa and active TF.
This is the first study to show that active FXIa and TF are present in stable COPD patients, who exhibit enhanced systemic inflammation and thrombin generation. Our findings suggest a new prothrombotic mechanism which might contribute to elevated risk of thromboembolic complications in COPD.
PMCID: PMC3042502  PMID: 21236471
Chronic obstructive pulmonary disease; tissue factor; factor XIa; inflammation
13.  Anticoagulants and the Propagation Phase of Thrombin Generation 
PLoS ONE  2011;6(11):e27852.
The view that clot time-based assays do not provide a sufficient assessment of an individual's hemostatic competence, especially in the context of anticoagulant therapy, has provoked a search for new metrics, with significant focus directed at techniques that define the propagation phase of thrombin generation. Here we use our deterministic mathematical model of tissue-factor initiated thrombin generation in combination with reconstructions using purified protein components to characterize how the interplay between anticoagulant mechanisms and variable composition of the coagulation proteome result in differential regulation of the propagation phase of thrombin generation. Thrombin parameters were extracted from computationally derived thrombin generation profiles generated using coagulation proteome factor data from warfarin-treated individuals (N = 54) and matching groups of control individuals (N = 37). A computational clot time prolongation value (cINR) was devised that correlated with their actual International Normalized Ratio (INR) values, with differences between individual INR and cINR values shown to derive from the insensitivity of the INR to tissue factor pathway inhibitor (TFPI). The analysis suggests that normal range variation in TFPI levels could be an important contributor to the failure of the INR to adequately reflect the anticoagulated state in some individuals. Warfarin-induced changes in thrombin propagation phase parameters were then compared to those induced by unfractionated heparin, fondaparinux, rivaroxaban, and a reversible thrombin inhibitor. Anticoagulants were assessed at concentrations yielding equivalent cINR values, with each anticoagulant evaluated using 32 unique coagulation proteome compositions. The analyses showed that no anticoagulant recapitulated all features of warfarin propagation phase dynamics; differences in propagation phase effects suggest that anticoagulants that selectively target fXa or thrombin may provoke fewer bleeding episodes. More generally, the study shows that computational modeling of the response of core elements of the coagulation proteome to a physiologically relevant tissue factor stimulus may improve the monitoring of a broad range of anticoagulants.
PMCID: PMC3220702  PMID: 22125631
14.  Plasma factor and inhibitor composition contributes to thrombin generation dynamics in patients with acute or previous cerebrovascular events 
Thrombosis research  2010;126(4):262-269.
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).
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.
Augmented thrombin generation in acute stroke/TIA is to some extent determined by altered plasma levels of coagulation factors.
PMCID: PMC2946539  PMID: 20709367
stroke; thrombin generation; factor composition; computational modeling
15.  Does Diabetes Accelerate the Progression of Aortic Stenosis through Enhanced Inflammatory Response within Aortic valves? 
Inflammation  2011;35(3):834-840.
Diabetes predisposes to aortic stenosis (AS). We aimed to investigate if diabetes affects the expression of selected coagulation proteins and inflammatory markers in AS valves. Twenty patients with severe AS and concomitant type 2 diabetes mellitus (DM) and 40 well-matched patients without DM scheduled for valve replacement were recruited. Valvular tissue factor (TF), TF pathway inhibitor (TFPI), prothrombin, C-reactive protein (CRP) expression were evaluated by immunostaining and TF, prothrombin, and CRP transcripts were analyzed by real-time PCR. DM patients had elevated plasma CRP (9.2 [0.74–51.9] mg/l vs. 4.7 [0.59–23.14] mg/l, p = 0.009) and TF (293.06 [192.32–386.12] pg/ml vs. 140 [104.17–177.76] pg/ml, p = 0.003) compared to non-DM patients. In DM group, TF−, TFPI−, and prothrombin expression within valves was not related to demographics, body mass index, and concomitant diseases, whereas increased expression related to DM was found for CRP on both protein (2.87 [0.5–9]% vs. 0.94 [0–4]%, p = 0.01) and transcript levels (1.3 ± 0.61 vs. 0.22 ± 0.43, p = 0.009). CRP-positive areas were positively correlated with mRNA TF (r = 0.84, p = 0.036). Diabetes mellitus is associated with enhanced inflammation within AS valves, measured by CRP expression, which may contribute to faster AS progression.
PMCID: PMC3332381  PMID: 21935671
aortic stenosis; diabetes mellitus; C-reactive protein; inflammation; Medicine & Public Health; Pathology; Rheumatology; Pharmacology/Toxicology; Internal Medicine
16.  Active tissue factor and activated factor XI in circulating blood of patients with systolic heart failure due to ischemic cardiomyopathy 
Elevated clotting factors and thrombin generation have been reported to occur in patients with heart failure (HF). Circulating activated factor XI (FXIa) and active tissue factor (TF) can be detected in acute coronary syndromes and stable angina.
We investigated circulating FXIa and active TF and their associations in patients with systolic HF due to ischemic cardiomyopathy.
In an observational study, we assessed 53 consecutive patients, aged below 75 years, with stable HF associated with documented coronary artery disease (CAD). Atrial fibrillation (LA), recent thromboembolic events, and current anticoagulant therapy were the exclusion criteria. Plasma TF and FXIa activity was determined in clotting assays by measuring the response to inhibitory monoclonal antibodies.
Coagulant TF activity was detected in 20 patients (37.7%), and FXIa in 22 patients (41.5%). Patients with detectable TF activity and/or FXIa were younger, had a history of myocardial infarction more frequently, significantly higher F1+2 prothrombin fragments, larger LA and right ventricular diastolic diameter, and higher right ventricular systolic pressure than the remaining subjects (P ≤0.01 for all). Circulating FXIa was positively correlated with F1+2 levels (r = 0.69; P <0.001).
Circulating active TF and FXIa occurred in about 40% of patients with systolic HF due to ischemic cardiomyopathy. The presence of these factors was associated with enhanced thrombin formation. Associations between both factors and LA diameter and right ventricular parameters might suggest that TF and FXIa predispose to thromboembolic complications of HF.
PMCID: PMC3168988  PMID: 20864906
activated factor XI; coronary artery disease; systolic heart failure; thrombin generation; tissue factor
17.  Blood Coagulation Dynamics in Hemostasis 
Hamostaseologie  2009;29(1):7-16.
Our studies involve computational simulations, a reconstructed plasma/platelet proteome, whole blood in vitro and blood exuding from microvascular wounds. All studies indicate that in normal hemostasis, the binding of tissue factor (Tf) with plasma factor (f) VIIa (extrinsic fXase complex) results in the INITIATION PHASE of the procoagulant response. This phase is negatively regulated by tissue factor pathway inhibitor (TFPI) in combination with antithrombin (AT) and the protein C (PC) pathway. The synergy between these inhibitors provides a threshold-limited reaction in which a stimulus of sufficient magnitude must be provided for continuation of the reaction. With sufficient stimulus, the fXa produced activates some prothrombin. This initial thrombin activates the procofactors and platelets required for presentation of the intrinsic fXase (fVIIIa-fIXa) and prothrombinase (fVa-fXa) complexes which drive the subsequent PROPAGATION PHASE; continuous downregulation of which is provided by AT and the thrombin-thrombomodulin-PC complex. FXa generation during the PROPAGATION PHASE is largely (>90%) provided by the intrinsic fXase complex. Tf is required for the INITIATION PHASE of the reaction but becomes non-essential once the PROPAGATION PHASE has been achieved. The PROPAGATION PHASE catalysts (fVIIIa-fIXa and fVa-fXa) continue to drive the reaction as blood is resupplied to the wound site by flow. Ultimately, the control of the reaction is governed by the pro- and anticoagulant dynamics and the supply of blood reactants to the site of a perforating injury. Our systems have been utilized to examine the qualities of hypothetical and novel antihemorrhagic and anticoagulation agents in epidemiologic studies of venous and arterial thrombosis and the hemorrhagic pathology.
PMCID: PMC3152749  PMID: 19151839
Thrombin; blood coagulation; hemostasis; numerical models; phenotype variability
18.  The “normal” factor VIII concentration in plasma 
Thrombosis research  2010;126(2):119-123.
The quantitation of factor (F)VIII by activity-based assays is influenced by the method, procedure, the quality and properties of reagents used and concentrations of other plasma proteins, including von Willebrand factor (VWF).
To compare FVIII concentrations measured by activity-based assays with those obtained by an immunoassay and to establish the influence of plasma dilution on the FVIII clotting activity (FVIIIc).
The APTT, a chromogenic assay (Coatest) and two in-house immunoassays were used. Albumin-free recombinant FVIII was used as the calibrator in all assays.
For a group of 44 healthy individuals (HI), the mean value observed for FVIII antigen (FVIIIag; 1.22±0.56 nM; S.D.) is substantially higher than that for FVIIIc (0.65±0.29 nM) and the chromogenic assay (FVIIIch; 0.50±0.23 nM). A positive correlation between FVIIIag and VWFag with R2=0.20 was observed. Since plasma VWF has an inhibitory effect on FVIIIc, we evaluated the influence of plasma dilutions on FVIIIc in HI (n=105). At a 4-fold dilution, estimates of FVIIIc by clotting assay were much lower than FVIIIag (0.77±0.31 vs. 1.14±0.48 nM). At 10- and 25-fold dilutions, the estimated FVIIIc increased to 0.87±0.36 and 0.94±0.44 nM, respectively.
1) In plasma, FVIIIag is higher than FVIIIc and FVIIIch; and 2) Real FVIII concentrations in plasma can be estimated by measuring FVIIIag.
PMCID: PMC2910827  PMID: 20451961
Factor VIII antigen; Factor VIII activity; von Willebrand factor; APTT assay; Coatest assay; Immunoassays
20.  Fibrinogen Beta-Chain -C148T Polymorphism is Associated with Increased Fibrinogen, C-Reactive Protein, and Interleukin-6 in Patients Undergoing Coronary Artery Bypass Grafting 
Inflammation  2011;35(2):429-435.
The fibrinogen beta-chain (FGB) -C148T polymorphism is linked with plasma fibrinogen concentration in the general population. We examined whether the -C148T polymorphism is associated with pre- and early postoperative levels of fibrinogen, C-reactive protein (CRP), and interleukin-6 (IL-6) in 243 consecutive patients undergoing coronary artery bypass grafting (CABG) surgery. Plasma inflammatory markers were measured prior to and 5–7 days after surgery. The -C148T polymorphism was analyzed with the restriction fragment-length polymorphism method. The genotype distribution was as follows: CC—142 (58%), CT—85 (35%), and TT—16 (7%). Carriers of the -148T allele had higher preoperative plasma fibrinogen (4.42 ± 0.14 vs. 4.07 ± 0.11 mg/L, p = 0.04) and CRP levels (7.49 ± 1.2 vs. 4.26 ± 1.0 mg/L, p = 0.04) compared with non-carriers; 5 to 7 days after CABG, patients carrying -148T allele had increased CRP (70.4 ± 5.0 vs. 51.6 ± 4.25 mg/L, p = 0.005) and IL-6 levels (22.34 ± 2.64 vs. 15.53 ± 2.28 pg/L, p = 0.05), but not fibrinogen, compared with the remaining subjects. In-hospital nonfatal stroke occurred more frequently in -148T allele carriers (4% vs. 0%, p = 0.02). No genotype-associated differences were found in the occurrence of postoperative myocardial infarction and death. Presence of the -148T allele has also been associated with longer intensive care stay and intubation time (p = 0.01). Multivariate analysis identified the CT+TT genotype as an independent predictor of pre- and postoperative CRP levels. The results indicate that the presence of the -148T FGB allele determines higher pre- and postoperative levels of inflammatory markers, which might be associated with in-hospital clinical outcomes.
PMCID: PMC3314811  PMID: 21499712
CABG; -C148T FGB polymorphism; inflammation; CRP; fibrinogen; IL-6
21.  Hyperglycemia Is Associated With Enhanced Thrombin Formation, Platelet Activation, and Fibrin Clot Resistance to Lysis in Patients With Acute Coronary Syndrome  
Diabetes Care  2008;31(8):1590-1595.
OBJECTIVE—Acute hyperglycemia on admission for acute coronary syndrome worsens the prognosis in patients with and without known diabetes. Postulated mechanisms of this observation include prothrombotic effects. The aim of this study was to evaluate the effect of elevated glucose levels on blood clotting in acute coronary syndrome patients.
RESEARCH DESIGN AND METHODS—We studied 60 acute coronary syndrome patients within the first 12 h after pain onset, including 20 subjects with type 2 diabetes, 20 subjects with no diagnosed diabetes but with glucose levels >7.0 mmol/l, and 20 subjects with glucose levels <7.0 mmol/l. We determined generation of thrombin-antithrombin complexes (TATs) and soluble CD40 ligand (sCD40L), a platelet activation marker, at the site of microvascular injury, together with ex vivo plasma fibrin clot permeability and lysis time.
RESULTS—The acute coronary syndrome patients with no prior diabetes but elevated glucose levels had increased maximum rates of formation and total production of TATs (by 42.9%, P < 0.0001, and by 25%, P < 0.0001, respectively) as well as sCD40L release (by 16.2%, P = 0.0011, and by 16.3%, P < 0.0001, respectively) compared with those with normoglycemia, whereas diabetic patients had the highest values of TATs and sCD40L variables (P < 0.0001 for all comparisons). Patients with hyperglycemia, with no previously diagnosed diabetes, had longer clot lysis time (by ∼18%, P < 0.0001) similar to that in diabetic subjects, but not lower clot permeability compared with that in normoglycemic subjects.
CONCLUSIONS—Hyperglycemia in acute coronary syndrome is associated with enhanced local thrombin generation and platelet activation, as well as unfavorably altered clot features in patients with and without a previous history of diabetes.
PMCID: PMC2494657  PMID: 18487475
23.  Factor XIII Val34Leu polymorphism and γ-chain cross-linking at the site of microvascular injury in healthy and coumadin-treated subjects 
Fibrin cross-linking by activated factor (F)XIII is essential for clot stability. In vitro, a common Leu34 polymorphism of the FXIII A-subunit increases the rate of thrombin-mediated FXIII activation, but not cross-linking activity upon complete FXIII activation. The effect of FXIII Val34Leu polymorphism on fibrin(ogen) cross-linking in vivo when vascular injury triggers the blood coagulation has not been studied yet. Using quantitative immunoblotting with antibodies raised against FXIII A-subunits, fibrinogen and γ-γ-dimers, the rates of FXIIIA cleavage and fibrin(ogen) cross-link formation in the fluid phase of 30-second blood samples collected at the site of microvascular injury were compared in the Leu34-positive and −negative healthy individuals and patients on long-term oral anticoagulation. In addition to accelerated FXIII activation, in healthy subjects the presence of FXIII Leu34 allele was associated with increased soluble γ-γ-dimer formation by 40% (1355±17 μg/L for Leu34 carriers vs 804.3±17 μg/L for Leu34 non-carriers; p=0.028) at the site of microvascular injury. This solution phase effect was abolished in coumadin-treated patients (369.4±75.9 μg/L for Leu34 carriers vs 290.5±35.9 μg/L for Leu34 non-carriers; p>0.05). The present study indicates that the Leu34 allele affects soluble γ-γ-dimer formation in untreated individuals, but not in those receiving acenocoumarol. Our data may help elucidate the impact of the FXIII Val34Leu polymorphism on fibrin crosslinking in vivo and its modulation by oral anticoagulants.
PMCID: PMC1307169  PMID: 16102108
factor XIII; fibrin; crosslinking; oral anticoagulation
24.  Reduced coronary flow reserve in Anderson-Fabry disease measured by transthoracic Doppler echocardiography 
Coronary flow reserve was assessed in a patient with Anderson-Fabry disease complicated by symmetric left ventricular hypertrophy. Coronary flow reserve was measurable in all three major coronary arteries providing an opportunity to compare regional coronary flow reserve from different vascular beds. In this patient all the three vascular beds supplied diffusely hypertrophied myocardium. Coronary flow disturbances in small intramyocardial perforating arteries were visible. The coronary flow reserve was reduced to a similar level (around to 2.0) in all three major arteries. In our patient with Anderson-Fabry disease, the coronary vasodilatation was blunted in a diffuse pattern corresponding to the myocardial hypertrophy distribution. In small intramyocardial arteries coronary flow was also disturbed. Accordingly, retrograde systolic flow and accelerated anterograde diastolic flow were documented.
PMCID: PMC1097744  PMID: 15857518

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