METHODS FOR IDENTIFYING WHETHER PATIENTS WITH ACUTE DECOMPENSATED HEART FAILURE (ADHF) EXHIBIT A HYPERCOAGULABLE STATE

Abstract

Subjects with heart failure (HF) are at higher risk of developing thrombosis. The inventors thus determined the thrombin generating capacity in patients with acute decompensated heart failure (ADHF). Their prospective study included 34 ADHF patients and 30 control inpatients without HF. Compared to controls, endogenous thrombin potential (ETP) was higher in ADHF patients at admission using platelet-rich plasma and remained increased during the hospitalization period. Soluble markers of endothelial dysfunction including von Willebrand factor, factor VIII and endothelial protein C receptor were higher in ADHF patients at admission. The plasma levels of DNA-histone complexes were elevated significantly in ADHF patients at admission compared to controls. Higher concentrations of annexin-V/tissue factor (TF)-positive eEVs were also found also in ADHF patients at admission compared to controls. The findings demonstrate that methods of determining the thrombin generating capacity in patients with acute decompensated heart failure (ADHF), which combine measurements of endogenous thrombin potential and endothelial dysfunction, are particularly valuable for stratifying the patients and thus for orienting treatments and following-up of the patients.

Claims

1. A method for identifying whether a patient with acute decompensated heart failure (ADHF) exhibits a hypercoagulable state comprising i) determining thrombin generation capacity from a blood sample obtained from the patient, ii) determining the level of at least one soluble biomarker of endothelial dysfunction from the blood sample obtained from the patient, iii) comparing the thrombin generation capacity determined at step i) and the level determined at step ii) with their respective predetermined reference values and iv) concluding that the patient exhibits a hypercoagulable state when both the thrombin generation capacity and the level of soluble marker of endothelial dysfunction are higher than their respective predetermined reference values.

2. The method of claim 1 wherein the blood sample is a platelet-rich plasma (PRP) sample or a platelet-poor plasma sample.

3. The method of claim 1 wherein the thrombin generation capacity is determined by a calibrated automated thrombogram (CAT) assay.

4. The method of claim 3 which comprises determining the Endogenous Thrombin Potential (ETP) in the blood sample obtained from the patient and comparing said value with a predetermined reference value.

5. The method of claim 3 wherein activated protein C (APC) is added to the blood sample and the APC concentration inhibiting thrombin generation by 50% is determined and compared to a predetermined reference value.

6. The method of clam 1 wherein the at least one soluble biomarker of endothelial dysfunction selected from the group consisting of von Willebrand factor, factor VIII, endothelial protein C receptor, tissue factor pathway inhibitor, endothelium-derived extracellular vesicles and circulating DNA-histone complexes in the blood sample obtained from the patient.

7. The method of claim 6 further comprising determining the level of endothelial TF+and/or AnnV+extracellular vesicles.

8. (canceled)

9. A method of treating a hypercoagulable state in a patient with acute decompensated heart failure (ADHF), comprising i) determining thrombin generation capacity and the level of at least one soluble biomarker of endothelial dysfunction from a blood sample from the patient, ii) comparing the thrombin generation capacity and the level with their respective predetermined reference values, and, when both the thrombin generation capacity and the level are higher than their respective predetermined reference values, iii) administering an anti-coagulant to the patient.

10. The method of claim 9 wherein the anti-coagulant is selected from the group consisting of factor Xa inhibitors, DNAses, and P-selectin antagonists.

11. The method of claim 9, wherein the patient is at risk of rehospitalization.

Description

FIGURES

[0028] FIG. 1. Acute decompensated heart failure increases plasma thrombin generation. Representative thrombin generation curve in platelet-rich plasma. Thrombin generation was triggered by 0.5 pM tissue factor and monitored with the calibrated automated thrombogram assay.

[0029] FIG. 2. Acute decompensated heart failure impairs activated protein C sensitivity. Inhibition of endogenous thrombin potential (ETP) by activated protein C (APC) in platelet-rich plasma.

[0030] FIG. 3. Levels of circulating markers of endothelial activation such as von Willebrand factor (VWF) are elevated patients with acute decompensated heart failure.

[0031] FIG. 4. The concentration of procoagulant endothelium-derived extracellular vesicles is elevated in patients with acute decompensated heart failure. Extracellular vesicle-related procoagulant activities rely on phospholipid-dependent potential detected by annexin V binding.

[0032] FIG. 5. Plasma levels of DNA-histone complexes are elevated in patients with acute decompensated heart failure.

EXAMPLES

Example 1

[0033] Material & Methods

[0034] Collection of Blood Samples

[0035] Venous blood samples were collected into Monovette (Sarstedt, Nmbrecht, Germany) syringes containing 1/10 volume of 0.10.sup.6 M sodium citrate. All analyses were performed within two hours after blood collection. Platelet-Rich Plasma (PRP) was prepared by blood centrifugation at 190 g for 10 min at 20 C. The PRP was recovered and adjusted to 150 G/L by the addition of platelet-poor plasma obtained by centrifugation of the remaining blood at 1750 g for 10 min at 20 C. Platelet-free Plasma (PFP) was obtained from platelet-poor plasma centrifuged at 13000 g for 30 min at 4 C. PFP was immediately frozen at 80 C. and thawed 15 min at 37 C. when needed. For extracellular vesicle enumeration and characterization, blood samples were subjected to two successive centrifugations at 2500g for 15 min and the platelet-depleted plasma (PDP) was prepared and stored at -80 C. until use.

[0036] Thrombin Generation Assays

[0037] Calibrated automated thrombogram (CAT) was performed at 37 C. in a microtiter plate fluorometer (Fluoroskan Ascent, ThermoLabsystems, Helsinki, Finland) using a dedicated software program (Thrombinoscope BV, Maastricht, The Netherlands)..sup.10 Coagulation was triggered with 0.5 pM recombinant human tissue factor (TF) (Innovin, Dade Behring). For measurements in PFP, a mixture of phosphatidyl-choline (PC)/serine (PS)/ethanolamine (PE) (PC/PS/PE, 60/20/20, mole %) at a final concentration of 4 M was added. Thrombin generation curves were recorded in triplicate using PRP in the absence or in the presence of in-house activated protein C (APC) at several final concentrations (6.7, 13.9, 25 and 65 nM) and PFP in the absence or in the presence of in-house APC at several final concentrations (0.85, 1.7, 6.7 and 13.9 nM). The total amount of thrombin activity (i.e. the endogenous thrombin potential, ETP) was assessed as the area under the curve. The APC concentration that produced a 50% inhibition of ETP was defined as IC50-APC..sup.11

[0038] Hemostatic Variables and Endothelial Markers

[0039] All measurements were performed in platelet-poor plasma. Soluble, soluble EPCR, free tissue factor pathway inhibitor (TFPI), von Willebrand factor (VWF) and factor VIII (FVIII) were measured by ELISA kits (Asserachrom, Diagnostica Stago, Asnieres, France). Cell-derived extracellular vesicles

[0040] The fluorescent antibodies, CD41-phycoerythrin (CD41-PE), CD144-PE, CD14-PE and allophycocyanin-EPCR were purchased from BD Pharmingen. The other antibody against TF, clone TF9-10H10, allophycocyanin-TF was from Biotechne, and annexin-V (AnnV)-FITC from Fisher Scientific. MPs were enumerated by high-sensitivity flow cytometry using a standardized procedure:.sup.12 plasma (20 L) was incubated with the optimal concentration of specific antibody plus 10 L of AnnV-FITC. Stained samples were analyzed on a GALLIOS instrument (Beckman-Coulter, Miami, Fla., USA).

[0041] Results

[0042] Compared to controls, endogenous thrombin potential (ETP) was higher in ADHF patients at hospital admission using platelet-rich and platelet-poor plasmas and remained increased during the hospitalization period (FIG. 1).

[0043] The activated protein C (APC) concentration inhibiting thrombin generation in platelet- rich plasma by 50% was also higher at hospital admission (FIG. 2).

[0044] Soluble markers of endothelial dysfunction including von Willebrand factor (FIG. 3), factor VIII, free tissue factor pathway inhibitor and the soluble forms of endothelial protein C receptor and thrombomodulin were higher in ADHF patients at hospital admission.

[0045] Higher concentrations of annexin-V/tissue factor (TF)-positive endothelium-derived extracellular vesicles were found in ADHF patients at hospital admission compared to controls (FIG. 4).

[0046] Plasma levels of DNA-histone complexes as an indirect measure of the highly complex neutrophil death signaling pathway (NETosis) are elevated in ADHF patients at hospital admission (FIG. 5).

Example 2

[0047] Methods

[0048] Patients and Controls

[0049] The study was a single center prospective study of 34 consecutive patients admitted to a cardiology department for ADHF (all in New York Heart Association NYHA functional class III or IV) defined in accordance with the European Society of Cardiology guidelines as the rapid onset/progression of HF symptoms and signs secondary to abnormal cardiac function requiring hospital admission. Blood was collected at hospital admission, at the day of discharge and after hospital discharge following at least six weeks of clinical stability. Hemodynamic measurements were a routine part of clinical patient assessment and the HF therapy during hospital stay and at discharge was at the discretion of the treating physician. A group of 30 patients with stable chronic HF (CHF) recruited from outpatient clinics was also included to identify differences with the acute phase of decompensated HF. The study included 30 control in patients (CT) with preserved ventricular function referred to the department of cardiology for a scheduled medical assessment and who did not experience HF but had similar patterns of comorbidities, risk factors (eg, hypertension, diabetes and smoking) and background medication to clarify the potential confounding contribution of hospital stay. Exclusion criteria for all study groups were factors that could affect coagulation: infectious and inflammatory disorders, neoplasia, serum creatinine >200 mol/L, use of steroid drugs, acute coronary syndrome, atrial fibrillation and all comorbidities requiring therapeutic anticoagulation. Exclusion criteria during hospital stay were worsening HF requiring assist device or heart transplant. All ADHF patients received a venous thromboembolism (VTE) prophylaxis (enoxaparin 4000 IU once daily). Blood collection was performed before VTE prophylaxis at admission and 36 hours after the last enoxaparin injection at the day of discharge. No VTE prophylaxis was administered in CT and CHF patients. The study protocol was approved by the local ethics committee (Comite de Protection des Personnes agreement 15/10/2014). All study patients provided written informed consent.

[0050] Collection of Blood Samples

[0051] Venous blood samples were collected into Monovette (Sarstedt, Nmbrecht, Germany) syringes containing 1/10 volume of 0.106 M sodium citrate. All analyses were performed within two hours after blood collection. Platelet-Rich Plasma (PRP) was prepared by blood centrifugation at 190 g for 10 min at 20 C. The PRP was recovered and adjusted to 150 G/L for CAT by the addition of platelet-poor plasma obtained by centrifugation of the remaining blood at 1750 g for 10 min at 20 C. Platelet-free Plasma (PFP) was obtained from platelet-poor plasma centrifuged at 13000 g for 30 min at 4 C. PFP was immediately frozen at 80 C. and thawed 15 min at 37 C. when needed. For EV enumeration and characterization, blood was subjected to two successive centrifugations at 2500 g for 15 min and the platelet- depleted plasma (PDP) was prepared and stored at 80 C. until use.

[0052] Thrombin Generation Assays

[0053] CAT was performed at 37 C. using a dedicated software program (Thrombinoscope BV, Maastricht, The Netherlands) as reported previously. Coagulation was triggered with 0.5 pM recombinant human tissue factor (TF) (Innovin, Dade Behring). For measurements in PFP, a home-made mixture of phosphatidyl-choline/serine/ethanolamine (60/20/20, mole %) at a final concentration of 4 M was added. Thrombin generation curves were recorded in triplicate in the absence or in the presence of in-house APC at several final concentration (6.7, 13.9, 25 and 65 nM for PRP and 0.85, 1.7, 6.7 and 13.9 nM for PFP). The total amount of thrombin activity (i.e. the endogenous thrombin potential, ETP) was assessed as the area under the curve. The APC concentration that produced a 50% inhibition of ETP was defined as IC50-APC.

[0054] Quantification of Hemostatic Variables and Endothelial Markers

[0055] All measurements were performed in PDP. Soluble thrombomodulin (TM), soluble endothelial protein C receptor (EPCR), free tissue factor pathway inhibitor (TFPI), von Willebrand factor (VWF) and factor VIII (FVIII) were measured by ELISA kits (Asserachrom, Diagnostica Stago, Asnieres, France).

[0056] Measurement of Endothelium-Derived Extracellular Vesicles

[0057] The fluorescent antibodies, CD144-phycoerythrin (CD144-PE) and allophycocyanin EPCR were purchased from BD Pharmingen. The other antibody against TF, clone TF9-10H10, allophycocyanin-TF was from Biotechne, and annexin-V (AnnV)-FITC from Fisher Scientific. EVs were enumerated by high-sensitivity flow cytometry using a standardized procedure: plasma (20 L) was incubated with the optimal concentration of specific antibody plus 10 L of AnnV-FITC. Stained samples were analyzed on a GALLIOS instrument (Beckman-Coulter, Miami, Fla., USA).

[0058] Determination of Nucleosome Plasma Levels

[0059] Circulating levels of nucleosomes were measured in PDP by a specific ELISA using a pair of antibodies directed against the histone and DNA components of nucleosomes (Cell

[0060] Death Detection-ELISA plus, Roche Diagnostics, Sigma-Aldrich). Because the manufacturer did not provide a standard curve, a reference material containing high amounts of endogenous nucleosomes was used and nucleosome values were reported in arbitrary units.

[0061] Statistical Analysis

[0062] Categorical data are presented as numbers (%); continuous data are presented as meansstandard error (SD). Differences in categorical values were performed using Pearson's 2 test or non-parametric Fisher's exact test as appropriate. Continuous variables were compared using the using Wilcoxon tests. The variation of the studied variables in ADHF patients throughout the follow-up period was assessed by using the Friedman test. Correlations were assessed by calculating the Spearman correlation coefficient. Alpha risk was fixed to 5% for all analysis. Analyses were done using SAS 9.4 (SAS Institute, Cary, N.C., USA).

[0063] Results

[0064] Baseline Clinical Characteristics

[0065] The ADHF group at baseline was similar to the non-HF cardiology patients (CT) group with regard to the sex-ratio, comorbidities and risks factors (Table 1). ADHF had decreased left ventricular ejection fraction and increased left ventricular end diastolic volume and pulmonary arterial pressures. Serum B-type natriuretic peptide (BNP) and C-reactive protein (CRP) levels were higher in ADHF patients than in controls. No significant difference was found in the use of active cardiovascular medication except for diuretics, which were more often used in ADHF patients. Five ADHF patients died within three months after hospital discharge without available biological follow-up. Control CHF patients differed substantially from ADHF patients with respect to hypertension and diabetes, which were more frequent in ADHF patients. As expected, levels of BNP and CRP were higher in ADHF patients than in CHF patients. Patients with CHF and ADHF presented similar reduced kidney function, as measured by GFR (<60 mL/min), compared with controls. Leukocyte and platelet counts as well as fibrinogen levels were not different in all groups.

[0066] In vitro Thrombin Generation is Accelerated and Increased in ADHF Patients at Hospital Admission

[0067] At hospital admission, ETP in the presence of platelets (PRP adjusted at the same platelet count) was higher whereas lag-time and time to peak were markedly but not significantly shorter for ADHF patients compared to controls (Table 2 and data not shown). In absence of platelets (PFP), thrombin peak, ETP and velocity were increased and lag-time and time to peak were shortened for ADHF patients compared to controls (Table 2 and data not shown). Compared to controls, ETP was increased in CHF patients only in the presence of platelets. There was no significant difference in ETP between CHF and ADHF patients at admission in the presence of platelets whereas ETP was higher in the absence of platelets of ADHF patients. Increase in ETP and/or thrombin peak values was maintained on the day of discharge but was lost at the post-discharge time-point (Table 2 and data not shown).

[0068] APC Sensitivity is Impaired in ADHF Patients at Hospital Admission

[0069] ETP in the presence of APC was consistently higher at hospital admission compared to controls (data not shown). The APC concentration (IC50-APC) that gave half maximum inhibition of ETP without APC was significantly increased in the presence of platelets at hospital admission compared to controls (Table 2). The IC50-APC in the absence of platelets tended to increase in ADHF patients as compared to controls but did not reach statistical significance. No differences were found between CHF patients and controls or ADHF patients at admission in the presence of platelets whereas IC50-APC in the absence of platelets was increased in ADHF compared to CHF patients. When ETP (without APC) and IC50-APC are combined using their arithmetic product, a procoagulant phenotype was confirmed by a near threefold value at hospital admission compared to the controls (Table 2). This parameter tended to increase in PRP of CHF patients as compared to controls but did not reach statistical significance. Compared to CHF patients, ETPIC50-APC values were increased in the absence of platelets of ADHF patients at admission. ETP values in the presence of APC were shifted downwards on the post-discharge time-point compared to admission (data not shown). At hospital discharge, and even more so at the post-discharge time-point, the arithmetic product ETPIC50-APC was decreased.

[0070] Soluble Markers of Endothelial Activation are Elevated at the Acute Decompensated Phases of HF

[0071] ADHF patients had higher plasma levels of sEPCR, sTM, VWF, free TFPI and FVIII at hospital admission versus controls (Table 2 and data not shown). The increase in FVIII paralleled the increase in VWF as revealed by similar values of the FVIII/VWF ratio. Levels of these factors were significantly higher at admission of ADHF patients compared with plasma levels in the CHF group. Only FVIII and thus FVIII/VWF ratio was elevated in CHF patients compared to controls. Elevation in these factors was maintained on the day of discharge but was lost on the postdischarge time-point except for free TFPI. Compared to controls, the concentrations of AnnV+eEVs, TF+eEVs and AnnV+/TF+eEVs was significantly higher in ADHF patients at admission (Table 2 and data not shown). The concentration of TF+eEVs in CHF patients significantly increased compared to controls. No difference in the concentrations of EPCR+eEVs and AnnV+/EPCR+eEVs was denoted between ADHF patients and controls or CHF patients (Table 2). The concentration of AnnV+eEVs and TF+eEVs in ADHF patients significantly decreased over the hospital course and was reduced on the post-discharge time- point compared to admission.

[0072] Circulating Nucleosomes are Elevated in ADHF Patients at Hospital Admission

[0073] We measured the plasma levels of nucleosomes in a subset of ADHF patients including all those followed up after discharge. Circulating nucleosomes were increased in ADHF patients at admission compared with controls and CHF patients (data not shown). At the day of discharge, levels of nucleosomes remained elevated whereas a significant decline was evident on the post-discharge time-point. Nucleosome levels at hospital admission were significantly correlated with IC50-APC and ETPIC50-APC in the presence of platelets (r=0.59; p=0.013 and r=0.63; p=0.006 respectively) and AnnV+eEVs (r=0.76; p=0.0004).

TABLE-US-00001 TABLE 1 Baseline patient's characteristics p-value p-value p-value CT CHF CT vs ADHF CT vs CHF vs (N = 30) (N = 30) CHF (N = 34) ADHF ADHF Demography Male gender 22 (73%) 23 (77%) 0.77 22 (65%) 0.46 0.30 Age (years) 65 15 69 12 0.33 73 17 0.03 0.17 Risk factors Hypertension 18 (60%) 16 (53%) 0.60 27 (79%) 0.09 0.03 Diabetes 8 (27%) 5 (17%) 0.34 15 (44%) 0.15 0.02 Smoking 8 (27%) 12 (40%) 0.27 7 (21%) 0.57 0.09 Previous history Valvular cardiopathy 4 (13%) 4 (13%) 1.00 8 (24%) 0.35 0.35 Ischemic cardiopathy 14 (47%) 18 (60%) 0.30 13 (38%) 0.50 0.08 Implantable cardiac defibrillator 0 (0%) 14 (47%) <0.0001 3 (9%) 0.10 0.0006 Cardiac resynchronisation therapy 0 (0%) 3 (10%) 0.24 4 (12%) 0.12 1.0 Chronic kidney disease 3 (10%) 3 (10%) 1.00 9 (27%) 0.11 0.12 Concomitant medications Beta-blockers 17 (57%) 25 (83%) 0.009 27 (79%) 0.07 0.29 ACE Inhibitors 17 (57%) 26 (87%) 0.003 29 (85%) 0.02 0.35 Diuretics 3 (10%) 17 (57%) <0.0001 32 (94%) <0.0001 0.001 Statins 17 (57%) 23 (77%) 0.052 22 (65%) 0.62 0.13 Aspirin 22 (73%) 24 (83%) 0.35 24 (71%) 0.64 0.16 Clopidogrel 7 (23%) 5 (17%) 0.56 13 (38%) 0.23 0.08 Echocardiography LVEDV (mL) 91 9 166 63 0.0001 132 51 0.02 0.07 LVESV (mL) 57 10 102 36 0.002 82 39 0.07 0.10 LVEF (%) 59 3 31 8 <0.0001 35 15 <0.0001 0.22 sPAP (mmHg) 33 4 44 7 <0.0001 45 10 <0.0001 0.76 Biology at admission BNP (ng/L) 101 11 549 1015 <0.0001 1432 1468 <0.0001 <0.0001 Creatinine (mg/L) 11.4 0.5 10.9 0.5 0.21 13.2 1.0 0.50 0.19 GFR (mL/min/1.73 m.sup.2) 69 14 57 12 0.0002 55 19 0.002 0.50 Glycemia (g/L) 1.0 0.2 1.1 0.3 0.34 1.2 0.4 0.03 0.29 CRP (mg/L) 5.6 2.9 7.2 6.5 0.28 14.9 10.6 <0.0001 <0.0001 ASAT (UI/L) 19 5 22 20 0.0004 36 34 0.24 0.002 ALAT (UI/L) 17 5 19 13 0.43 38 63 0.09 0.03 Fibrinogen (g/L) 2.7 0.7 2.5 0.8 0.42 2.9 1.1 0.38 0.15 Leukocyte count (10.sup.3/mm.sup.3) 6.4 1.9 6.4 1.6 0.52 6.8 2.2 0.94 0.44 Platelet count (10.sup.3/mm.sup.3) 215 107 197 107 0.57 201 81 0.52 0.86 Results are mean SD or frequency (percent). ACE indicates angiotensin-converting enzyme; LVEDV, left ventricular end diastolic volume; LVESV, left ventricular end systolic volume; LVEF, left ventricular ejection fraction; sPAP, pulmonary arterial pressures; BNP, B-type natriuretic peptide; GFR, glomerular filtration rate; CRP, C-reactive protein; ASAT, aspartate aminotransferase; ALAT, alanine aminotransferase.

TABLE-US-00002 TABLE 2 Thrombin generation parameters and circulating markers of endothelial activation p-value p-value p-value p-value ADHF ADHF CT vs ADHF CT vs CHF vs ADHF post- Ad-Dis- CT CHF CHF admission ADHF Ad ADHF Ad discharge discharge PostDis* Platelet-rich plasma (PRP) N 30 30 32 27 13 ETP (nM .Math. min) 983 162 1147 260 0.007 1293 463 0.09 0.25 1375 451 1100 398 0.053 Lag-time (min) 17.1 7.3 12.7 4.1 0.02 13.6 6.4 0.54 0.50 13.7 3.6 15.4 4.2 0.33 Thrombin peak (nM) 81 44 100 65 0.44 100 58 0.16 0.72 112 53 68 38 0.007 Time to peak (min) 27.7 12.9 21.7 8.2 0.07 21.4 8.4 0.08 0.97 21.3 6.0 26.7 8.0 0.03 Velocity (nM/min) 16.2 18.2 17.7 18.1 0.89 16.9 14.4 0.42 0.61 18.0 12.0 7.9 6.6 0.02 IC.sub.50-APC (nM) 6.9 3.2 8.9 5.0 0.14 14.3 137 0.008 0.23 9.8 5.5 8.0 4.0 0.11 ETP IC.sub.50-APC 6956 3862 10174 6232 0.058 18460 19555 0.0002 0.06 13891 9917 8051 4848 0.03 Platelet-free plasma (PFP) N 29 30 28 29 13 ETP (nM .Math. min) 806 331 932 385 0.28 1150 412 0.004 0.08 1179 427 900 510 0.03 Lag-time (min) 10.9 6.4 8.4 3.6 0.16 3.9 1.3 0.0001 <0.0001 4.7 1.2 5.0 2.0 0.006 Thrombin peak (nM) 149 103 192 105 0.12 237 98 0.003 0.12 229 105 169 85 0.43 Time to peak (min) 13.3 6.1 11.4 4.0 0.25 6.5 1.5 <0.0001 <0.0001 7.6 2.0 7.9 2.1 0.006 Velocity (nM/min) 66.1 69.2 85.0 64.9 0.11 107.8 64.5 0.005 0.11 99.1 64.0 61.8 33.0 0.43 IC.sub.50-APC (nM) 0.8 0.6 0.7 0.6 0.53 2.3 3.5 0.12 0.03 1.4 2.7 0.6 0.5 0.058 ETP IC.sub.50-APC 781 808 604 591 0.39 3133 5503 0.03 0.004 1901 3892 434 448 0.02 Circulating markers of endothelial activation N 30 28 27 27 11 sEPCR (ng/mL) 162 79 168 63 0.39 245 83 0.0003 0.003 239 89 169 46 0.048 sTM (pg/mL) 157 35 163 58 0.72 284 100 <0.0001 <0.0001 274 114 146 27 0.0002 Free TFPI (ngmL) 10.9 2.5 11.9 2.8 0.16 13.9 4.1 0.002 0.055 12.8 3.3 12.3 2.4 0.62 FVIII/VWF ratio 0.46 0.26 0.65 0.21 0.001 0.46 0.25 0.91 0.002 0.32 0.14 0.42 0.16 0.48 Endothelium-derived extracellular vesicles N 30 22 32 33 13 AnnV.sup.+/TF.sup.+ (EVs/L) 57 44 85 76 0.36 258 321 0.003 0.045 116 116 61 49 0.12 EPCR.sup.+ (EVs/L) 87 54 104 92 0.99 128 153 0.72 0.66 122 122 98 76 0.48 EPCR.sup.+/TF.sup.+ (EVs/L) 69 54 55 44 0.34 116 151 0.24 0.07 107 114 64 33 0.27 Values are means SD. ETP indicates the endogenous thrombin potential in the absence of activated protein C (APC); IC.sub.50-APC, the APC concentration needed to reduce ETP by 50%; EPCR, endothelial protein C receptor; TM, thrombomodulin; VWF, von Willebrand factor; FVIII, coagulation factor VIII; TFPI, tissue factor pathway inhibitor; EVs extracellular vesicles, AnnV, annexin V; TF, tissue factor. *Friedman test for quantitative variables throughout the follow-up period.

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