Abstract Prothrombin complex concentrates (PCC) are used off-label to treat direct factor Xa inhibitor (FXaI)–associated bleeding or to optimize hemostasis before urgent surgery. The Global Assessment of Hemostatic TGA Indices Following the Use of Prothrombin Complex Concentrates for Major Bleeding or Urgent Surgery in Patients Treated with Factor Xa Inhibitors (GAUGE) study is a prospective observational cohort study of the effects of PCC on thrombin generation and hemostasis in FXaI-treated patients. FXaI-treated patients who presented with major bleeding or needed urgent surgery received PCC (50 IU/kg). Platelet-poor plasma was collected before and after PCC administration. Thrombin generation assay parameters, including lag time (LT), time to peak (TTP), peak thrombin generation (peak), endogenous thrombin potential (ETP), and mean velocity rate index (mVRI) were measured using calibrated automated thrombography. Hemostatic efficacy, thromboembolism, and mortality were adjudicated in duplicate. Multivariable log-linear regression was used to evaluate PCC effects on FXaI levels. Unsupervised k-means clustering was used to identify patients with the largest ETP increment. GAUGE included 101 episodes of FXaI-associated PCC use (FXaI-associated bleeding = 71; urgent surgery = 30). The median PCC dose was 48.0 IU/kg (interquartile range [IQR], 42.0-50.0). PCC did not affect FXaI levels (P> .05). PCC increased ETP (Δ868.9 ± 766.6 nM/min), peak (Δ85.7nM ± 108.2nM), and mVRI (Δ16.5 nM/min [IQR, 4.4-45.0]). PCC did not affect the LT or TTP (P> .05). Patients in cluster 1 had significantly lower pre-PCC FXaI levels (79 ng/mL [IQR, 55-93] vs 165 ng/mL [IQR, 77-219]; P = .009) and a greater ΔETP increment (P< .0001) than patients in cluster 2. Effective hemostasis was achieved in 50.0% of major bleeding events (95% confidence interval [CI], 38.4-61.6), and procedural hemostasis was normal in 76.7% of urgent surgeries (95% CI, 59.1-88.2). The 30-day risks of thromboembolism and mortality were 6.9% (95% CI, 3.4-13.6) and 12.9% (95% CI, 7.7-20.8), respectively. PCC increased quantitative thrombin generation without shortening LT or TTP, supporting a prohemostatic effect rather than true FXaI reversal.

BACKGROUND Lipoprotein(a) [Lp(a)] is a causal risk factor for coronary artery disease (CAD), with proposed prothrombotic properties besides proatherogenic effects. However, the association between Lp(a) and coagulation remains controversial so far. OBJECTIVE This study examined this relationship in subjects with or without angiographically documented CAD. METHODS Plasma levels of Lp(a) and coagulation biomarkers were assessed in clinically stable subjects undergoing elective coronary angiography. Subjects taking any anticoagulant therapy were excluded. The coagulation panel included coagulant activities of factors II, V, VII, VIII, IX, X, XI, and XII, von Willebrand factor antigen (vWF:Ag), thrombin generation assay (TGA), total tissue factor pathway inhibitor (TFPI), and activated factor VII-antithrombin (FVIIa-AT) complex. Lp(a) threshold values were defined according to the European Atherosclerosis Society consensus statement: normal <30 mg/dL, intermediate 30 to 50 mg/dL, and high >50 mg/dL. RESULTS Complete laboratory data were available for 383 subjects (males 75.3%; mean age 68.2 ± 9.7 years): 65 subjects had normal coronary arteries, 51 subjects had coronary stenosis <50%, and 267 subjects had coronary stenosis ≥50%. A modest yet significant increase in FV coagulant activity (FV:C) from low to high Lp(a) plasma levels was found and confirmed after adjustment for potential confounding factors. No differences were observed for all the other coagulant activities, vWF:Ag, total TFPI, FVIIa-AT levels, or TGA parameters. CONCLUSION In this pilot study, no major contribution of Lp(a) plasma levels was observed in modulating coagulation phenotype, as evaluated by multiple biomarkers. High Lp(a) plasma levels were associated with only a mild increase in FV:C.

A direct oral anticoagulant (DOAC) concentration threshold above which an impact on surgical hemostasis starts to occur is unknown. Thrombin generation assays (TGAs) provide a measure of the coagulation phenotype. This study aimed to determine whether preoperative TGA parameters are associated with postoperative bleeding, and whether this is partly due to residual DOAC levels.We conducted a nested case-control study using samples from apixaban/rivaroxaban-treated patients with atrial fibrillation from the PAUSE (Perioperative Anticoagulation Use for Surgery Evaluation) perioperative study. Cases were participants with postoperative major or clinically relevant nonmajor bleeding; controls were participants without bleeding. DOAC levels were measured using a chromogenic anti-Xa assay (BIOPHEN DiXaI; rivaroxaban/apixaban calibrators). TGA parameters were measured using calibrated automated thrombography.Generalized linear mixed models and causal mediation analyses were used to evaluate the relationship between DOAC levels, TGA parameters, and bleeding.Forty eight cases were matched to 474 controls. Residual DOAC levels were higher in cases than controls (p = 0.03) and each TGA parameter was correlated with residual DOAC levels (p<0.05). A longer lag time (LT; odds ratio [OR] = 1.319 per minute [95% confidence interval [CI]: 1.077-1.617]) and time-to-peak (TTP; OR = 1.154 per minute [95% CI: 1.028-1.296]) were associated with an increased odds of bleeding; higher peak (OR = 0.994 per nM [95% CI: 0.989-0.998]) and mean velocity rate index (mVRI; OR = 0.986 per nM/min [95% CI: 0.976-0.996]) were associated with a lower odds of bleeding. The effect of apixaban/rivaroxaban levels on bleeding was mediated by altered TGA parameters (LT, TTP, peak, mVRI).These findings support a measurable effect from low residual DOAC levels on thrombin generation and suggest a causal contribution of both toward bleeding.

BACKGROUND:Activated factor VII-antithrombin complex (FVIIa-AT) is an indirect plasma biomarker of the interaction between tissue factor (TF) and FVIIa. High FVIIa-AT levels have been associated with an increased risk of mortality. METHODS:We investigated the genetic determinants of FVIIa-AT plasma levels by a candidate gene approach in a cohort of 610 subjects with (n = 478) or without (n = 132) angiographically-demonstrated coronary artery disease (CAD). RESULTS:Plasma concentration of FVIIa-AT did not differ between CAD and CAD-free subjects, but predicted the mortality risk in CAD during a median follow-up of 64-months. Among 7 polymorphisms in 4 candidate genes, codifying for FVII, TF, Endothelial Protein C Receptor (EPCR), and Low-density lipoprotein receptor-Related Protein 1 (LRP1), none was associated with CAD. Three of them were independently associated with FVIIa-AT plasma concentration. F3 -603 A > G polymorphism predicted mortality in CAD consistent with the influence on FVIIa-AT levels, with the G allele-carriers having both higher FVIIa-AT concentration (86.4 with 95 %CI 82.4-90.5 versus 76.7 with 95 %CI 71.0-82.8 pM) and higher mortality risk (HR 1.92 with 95 %CI 1.08-3.41) as compared with AA homozygotes. Conversely, the F7 -323 A1/A2, the strongest genetic predictor of FVIIa-AT variability, and EPCR Ser219Gly polymorphisms were associated with FVIIa-AT levels but were not predictive of mortality. CONCLUSIONS:Our results indicate that FVIIa-AT plasma levels are influenced by distinct genetic determinants linked to either TF or FVII expression. The heterogeneous association of FVIIa-AT-related polymorphisms with mortality risk in CAD suggests a predominant role of TF expression rather than FVII levels in the setting of secondary cardiovascular prevention.

A DOAC concentration threshold above which an impact on surgical hemostasis starts to occur is unknown. Thrombin generation assays (TGAs) provide a measure of the coagulation phenotype. This study aimed to determine whether preoperative TGA parameters are associated with postoperative bleeding, and whether this is partly due to residual DOAC levels. We conducted a nested case-control study using samples from apixaban/rivaroxaban-treated patients with atrial fibrillation from the PAUSE perioperative study. Cases were participants with postoperative major or clinically relevant non-major bleeding; controls were participants without bleeding. TGA parameters were measured using Calibrated Automated Thrombography (5 pM tissue factor). Generalized linear mixed models and causal mediation analyses were used to evaluate the relationship between DOAC levels, TGA parameters, and bleeding. Forty-eight cases were matched to 474 controls. Residual DOAC levels were higher in cases than controls (p = 0.03) and TGA parameters were correlated with residual DOAC levels (p < 0.05). A longer lag time (LT; OR = 1.319 per minute [95%CI 1.077-1.617]) and time-to-peak (TTP; OR = 1.154 per minute [95%CI 1.028-1.296]) were associated with an increased odds of bleeding; higher peak (OR = 0.994 per nM [95%CI 0.989-0.998]) and higher mean velocity rate index (mVRI; OR = 0.986 per nM/min [95%CI 0.976-0.996]) were associated with a lower odds of bleeding. The effect of apixaban/rivaroxaban levels on bleeding was mediated by altered TGA parameters (LT, TTP, peak, mVRI). TGA parameters are associated with postoperative bleeding and the impact of DOAC levels on bleeding is mediated by effects on thrombin generation.

Background: lipoprotein(a) [Lp(a)] is an established risk factor for cardiovascular disease. Among the molecular mechanisms underlying this association, beyond proatherogenic and proinflammatory properties, a prothrombotic diathesis has also been proposed, usually related to the homology between apolipoprotein(a) and plasminogen which supports a potential antifibrinolytic role. Moreover, other Lp(a)-related mechanisms of haemostatic imbalance have been advocated, from platelet activation to tissue factor (TF) expression. However, the link between Lp(a) and coagulation remains controversial so far. Aims: the aim of this study was to investigate the relationship of Lp(a) plasma levels with coagulation phenotype, evaluated by the assessment of both individual coagulation factors and global coagulation test such as thrombin generation assay (TGA), in a cardiovascular cohort of subjects with angiographic documentation of coronary artery vessels. Material and methods: plasma levels of Lp(a) and a large panel of coagulation biomarkers were assessed in clinically stable subjects undergoing elective coronary angiography. Subjects taking any anticoagulant therapy were excluded from this analysis. The coagulation panel included coagulant activities of factors II, V, VII, VIII, IX, X, XI and XII, as well as TGA, which explores the individual's overall plasma propensity to form a blood clot when triggered by TF exposure ex-vivo, and activated factor VII-antithrombin (FVIIa-AT) complex, which is considered an indirect marker of TF expression. Lp(a) threshold values were defined according to the European Atherosclerosis Society consensus statement: normal <30 mg/dL, intermediate 30-50 mg/dL, and high >50 mg/dL. Results: laboratory data were available for 384 subjects (males 75.3%; mean age 68.2±9.7 years): 65 subjects had normal coronary arteries (CAD-free), 52 subjects had coronary lesions with stenosis <50% (CAD-borderline), and 267 subjects had coronary lesions with stenosis ≥50% (CAD). Most of patients (87.0%) were taking lipid-lowering therapies. Subjects with intermediate-high Lp(a) plasma levels were more represented in CAD than in CAD-free (12.0%-24.0% versus 7.7%-18.5%, respectively). As regards the correlation with coagulation biomarkers, in the whole study population a progressive and mild increase in coagulant activity of FV (FV:C) from low to high Lp(a) plasma levels was found (84.3±18.7%, 88.8±18.3%, and 90.1±18.7%, P=0.010), while there was no difference for the other individual coagulant activities. No difference was detected for either FVIIa-AT plasma levels or TGA parameters. The association of Lp(a) levels with FV:C was confirmed by multiple linear regression models after adjustment for sex, age, CAD diagnosis, and renal function (standardized beta coefficient=0.101, P=0.047), plasma lipids, including LDL cholesterol and apolipoprotein B (standardized beta coefficient=0.107, P=0.046), and the other coagulant activities (standardized beta coefficient=0.133, P=0.004). Conclusions: in this pilot analysis within a cohort of cardiovascular patients we did not find a major contribution of Lp(a) plasma levels in modulating coagulation phenotype, which was assessed by means of different laboratory biomarkers. High Lp(a) plasma levels were associated only with a mild increase of FV:C. This association appears independent of some potential confounding factors but deserves further validation and elucidation by both larger epidemiological/clinical studies and more in-depth biological/biochemical investigations.

Background: Thromboinflammation is recognized as a central pathological process in sepsis. Sepsis-induced inflammation, triggered by bacterial infection, is tightly linked to a hypercoagulable state and endothelial cell activation. The failure of natural anticoagulant pathways—due to overwhelming thromboinflammation and/or consumption of natural coagulation inhibitors—contributes to the development of disseminated intravascular coagulation (DIC) and clinical deterioration. Excessive expression of tissue factor (TF) by activated endothelial cells and monocytes plays a key role in this process. Release of procoagulant microparticles (MP) is related with amplification of blood borne hypercoagulability in patients with sepsis. The identification of septic patients at risk of deterioration aiming to prompt administration of antithrombotic treatment remains an unmet need. To this aim, the identification of new biomarkers of hypercoagulability is a promising strategy. Among emerging candidates, TF-bearing microparticles (TF⁺-MPs) represent a particularly compelling biomarker with both mechanistic and clinical relevance. Aim: The ROADMAP-Sepsis study evaluated the clinical relevance of TF⁺ -MP activity quantification in relation to systemic inflammation, hypercoagulability, endothelial cell dysfunction and 20-day mortality in patients with sepsis, stratified by DIC status. Methods: In this prospective, observational study, 114 ICU patients with sepsis (according to the Sepsis-3 criteria: documented infection and SOFA score ≥2) and without prior antithrombotic treatment were enrolled. Patients were stratified according to the ISTH-DIC score into a DIC (score ≥5; n=65) and non-DIC groups (n=49). Blood samples were collected within 2 hours of ICU admission into citrated tubes (Vacutainer®). Platelet-poor plasma (PPP) was prepared using standardized protocols for MP analysis. TF⁺-MP activity was measured using a TF-dependent FXa generation assay (CY-Quant MV-TF Activity, Biocytex, France), and total MP concentration was assessed with the ZYMUPHEN MP kit (Hyphen Biomed, France). D-dimer, fibrin degradation products (FDPs), soluble thrombomodulin (sTM), tissue factor pathway inhibitor (TFPI), CRP, IL-1β , Procalcitonin (PCT), and TNF-α , were measured with ELISA kits (Diagnostica Stago). Thrombin generation (TG) in PPP was analyzed by calibrated automated thrombogram (CAT®, Stago) with 5 pM TF PPP-Reagent®. The primary clinical endpoint was 20-day mortality. Results: The DIC group had a significantly higher ISTH score compared to the non-DIC group (p < 0.001). Total MP concentrations did not differ significantly between groups and showed no correlation with DIC or SOFA scores, nor with D-dimer, FDPs, sTM, or TFPI levels. In contrast, TF ⁺-MP activity was significantly elevated in the DIC group versus the non-DIC group (p < 0.001). TF⁺ -MP activity strongly correlated with the ISTH DIC score (r² = 0.9; p = 0.003), SOFA score (r² = 0.8; p = 0.001), D-dimer (p = 0.001), FDP (p = 0.01), and sTM (p = 0.02), but not with TFPI levels. Thrombin generation parameters differed markedly: the DIC group had a significantly longer lag-time and reduced mean rate index (MRI) and Peak of thrombin generation (p = 0.001). Lag time was positively correlated with DIC and SOFA scores, while endogenous thrombin potential (ETP) and peak values were negatively correlated. Inflammatory markers (IL-1β , CRP, TNF-α and PCT) were significantly elevated in the DIC group (p < 0.01). TF⁺ -MP activity correlated significantly with IL-1 β, TNF-α , CRP, and PCT levels. TF⁺ -MP activity was significantly higher in non-survivors compared to survivors at 20 days (p < 0.005), while total MP concentrations did not differ. Conclusion: The ROADMAP-Sepsis study highlights TF⁺ -MP activity as a central mediator of thromboinflammation and a key contributor to the development of DIC, endothelial dysfunction, and increased sepsis severity. Unlike total microparticle levels, TF ⁺-MP activity showed strong correlations with inflammatory markers, markers of hypercoagulability, and 20-day mortality. These findings support the clinical relevance of TF⁺-MP activity as a prognostic biomarker for identifying septic patients at high risk of deterioration. Ongoing studies from our group are focused on validating its incorporation into predictive scoring systems to guide risk stratification and therapeutic decision-making.

Background: Tissue factor (TF), the main initiator of the coagulation cascade, plays a role in cancer progression and prognosis. Activated factor VII-antithrombin complex (FVIIa-AT) is considered an indirect marker of TF exposure by reflecting TF-FVIIa interaction. Objectives: To assess the link between FVIIa-AT plasma levels, TF messenger RNA (mRNA) expression, and survival in cancer. Methods: TF pathway-related coagulation biomarkers were assessed in 136 patients with cancer (52 with hepatocellular carcinoma, 41 with cholangiocarcinoma, and 43 with colon cancer) undergoing surgical intervention with curative intent. TF mRNA expression analysis in neoplastic vs nonneoplastic liver tissues was evaluated in a subgroup of 91 patients with primary liver cancer. Results: FVIIa-AT levels were higher in patients with cancer than in 136 sex- and agematched cancer -free controls. In patients with cancer, high levels of FVIIa-AT and total TF pathway inhibitor were associated with an increased mortality risk after adjustment for confounders, but only FVIIa-AT remained a predictor of mortality by including both FVIIa-AT and total TF pathway inhibitor in Cox regression (hazard ratio, 2.80; 95% CI, 1.23-6.39; the highest vs the lowest quartile). This association remained significant even after adjustment for extracellular vesicle-associated TF-dependent procoagulant activity. In the subgroup of patients with primary liver cancer, patients with high TF mRNA levels had an increased mortality risk compared with that for those with low TF mRNA levels (hazard ratio, 1.92; 95% CI, 1.03-3.57), and there was a consistent correlation among high FVIIa-AT levels, high TF mRNA levels, and increased risk of mortality. Conclusion: High FVIIa-AT levels may allow the identification of patients with cancer involving high TF expression and predict a higher mortality risk in liver cancer.

Background: The role of human plasma fatty acid (FA) composition in coagulation cascade has received limited attention so far, despite evidence that the coagulation is deeply influenced by lipid binding. Aims: To assess the relationships between plasma FAs and thrombin generation parameters in a single-center, angiographically-controlled, cohort of subjects with or without coronary artery disease (CAD). Methods: Plasma FA concentrations were measured by a gas-chromatographic method in clinically stable subjects undergoing to elective coronary angiography who were also characterized for thrombin generation assay. Thrombin generation was studied using the Calibrated Automated Thrombogram assay (CAT®, Diagnostica Stago, France) The following parameters of thrombogram were analyzed: the lag-time of thrombin generation, the time to reach the peak of thrombin (ttPeak), the thrombin peak (Peak), the endogenous thrombin potential (ETP) that reflects the total amount of thrombin activity. Subjects with any acute illness, including acute coronary syndrome, in the month before blood collection and coronary angiography were excluded from this study. Results: The plasma FA profile was available for 245 subjects (males 73.9%; mean age 68.1±10.3 years): 54 with normal coronary arteries (CAD-free group) and 191 with coronary lesions with stenosis ≥50% (CAD group), of whom 51 with history of previous myocardial infarction (MI). Thirty-nine subjects taking oral anticoagulant therapies were excluded from further analysis on thrombin generation. In the 206 subjects not taking oral anticoagulants the saturated FAs 20:0, 22:0, and 24:0 were inversely correlated with peak of thrombin (R=-0.200 with P=0.004, R=-0.151 with P=0.031, and R=-0.182 with P=0.009, respectively) and endogenous thrombin potential (R=-0.217 with P=0.002, R=-0.170 with P=0.015, and R=-0.202 with P=0.004, respectively), while the saturated FA 26:0 correlated inversely with peak of thrombin (R=-0.208, P=0.003) and directly with time-to-peak (R=0.190, P=0.006). Defining very long-chain saturated FAs (VLSFAs) as those with 20 carbons or more, the sum of their levels remained inversely associated with both peak of thrombin and endogenous thrombin potential after adjustment for gender, age, CAD diagnosis, renal function and other traditional cardiovascular risk factors by linear regression analysis (standardized beta coefficient=-0.465 with P<0.001 and standardized beta coefficient=-0.400 with P=0.001, respectively). In the whole study population subjects with CAD with or without MI had lower levels of VLSFAs than CAD-free subjects (1.43±0.34, 1.58±0.40, and 1.68±0.43 g/100g, respectively, P=0.005 by ANOVA). Stratifying the study population on the basis of FA levels in CAD-free subjects, the prevalence of subjects within the highest VLSFA quartile (≥2.06 g/100g) decreased progressively from CAD-free to CAD without MI and CAD with MI (24.1%, 11.4%, and 3.9%, respectively, P=0.003). Conclusions: This preliminary analysis showed an inverse correlation between VLSFA and thrombin generation, thereby indicating a potential antithrombotic effect of high VLSFA levels. Additionally, high VLSFA levels were linked to a lower prevalence of CAD and MI, consistently with the recent studies indicating that elevated VLSFA concentrations are associated with favourable cardiovascular outcomes.

Introduction: Sickle cell disease (SCD) is linked to hypercoagulability and risk of venous thromboembolism. It is characterised by high concentrations of erythrocyte-derived microparticles (EC-d-MP). In addition, in SCD endothelial cells and monocytes can be activated, which in turn are sources of tissue factor bearing MPs (TF-MP). Their impact on the thrombin generation process remains unclear. Aim: We hypothesised that in SCD TF-MP and procoagulant phospholipids from endothelial cells and monocytes are implicated in blood hypercoagulability and potentially enhance thrombin generation . Materials and methods: Consecutive patients with steady state SCD (n=68) and 31 age and sex-matched patients in painful crisis (n=31) and 30 healthy individuals (controls) were included. Flow cytometric analysis was performed with a precise number of standard beads (Megamix®) to determine MVs count, and specific conjugated antibodies were used to determine the cellular origin of the MVs. Microparticles derived from erythrocytes (RBd-MP), platelets (Pltd-MP), monocyte (Mod-MP), endothelial cell (ECd-MP) were identified using an anti-CD235a, CD61/CD41a, CD14, CB106 and CD41 monoclonal antibodies and FITC labelled annexin V. Total TF-MP was evaluated by CY-Quant MV-TF activity (BioCytex ,Marseille, France). Cellular origin of TF on MPs, samples were triple labelled with Cy5-labeled annexin V, a cell type-specific PE-labeled MoAb against either CD14 or CD144 and a FITC- MoAb against TF. Thrombin generation (TG) in platelet poor plasma was measured by CAT assay using PPP-reagent 5pM in presence and absence of thrombomodulin (TM) . Procoagulant phospholipid dependent activity was assessed by the Procoag-PPL assay, TM and TFPI were measured by Asserachrom TM and Asserachrom TFPI (Stago, Asnieres, France). Results: The mean age of SCD patients in painful crisis and steady state were 25.1 ± 9.6 (range: 18-39 years) and 26.3 ± 8.1 years (range: 18-40 years), respectively. The mean age in the control group was 27.4 ± 7.2 years (range: 16-36 years). Total MPs were significantly elevated for both groups of patients with SCD in steady as compared to the controls (p<0.001). A significant difference was observed between the steady state patients and crisis patients (p<0.001). In both groups of SCD patients, RBd-MP, Pltd-MP expressing or not PS, ECd-MP and Mod-MP were significantly increased as compared to the control group (p<0.001). The RBd-MP, RBd-MP-PS+, Ed-MP and Mod-MP were significantly elevated in patients in crisis compared to those at steady state SCD (p<0.001, p<0.001, p<0.05, p<0.001 respectively). The TF-MPs were derived from endothelial cells and monocytes but not from red blood cells or platelets. Patients either in crisis or at steady state SCD showed significantly higher total TF-MPs T as compared to the control group (p<0.001). Patients in crisis had significantly higher TF-MP as compared to those at steady state SCD (p<0.001). Mod-MP-TF+ and Ed-MP-TF+ were significantly elevated in both patient groups as compared to the control group (p<0.001). Patients in crisis had significantly higher MP levels as compared to those at steady state SCD (p<0.001). TG was significantly increased in SCD patients as compared to the control. Among thrombogram parameters, the mean rate index (MRI) and Peak of thrombin were significantly higher in the SCD-patient group as compared to the control group (p<0.01). The MRI was significantly increased in patients with crisis as compared to those at steady state SCD (p<0.01). The lag-time and the ETP were not significantly different between SCD patients and the control group. The ETP +/- TM ratio increased significantly in both patients' groups as compared to the controls (p<0.001), with a significant difference between steady state and crisis (p<0.01). The TM, and TFPI and the PPL-ct were significantly decreased in patients as compared to the controls (p<0.01). The PPL-ct was inversely correlated with the levels of Rb-MP/PS+ and Pd-MP/PS+.( p<0.02).The Rb-MP/PS+ were positively correlated with MRI (p<0.02). The concentration of the total MPs-TF+, Mod-MP-TF+ was positively correlated with MRI and Peak (p<0.01). Conclusion: In SCD patients the TF bearing MPs derived from endothelial cells or monocytes could, contribute to thrombotic events and may be involved in painful crisis. Circulating MVs TF + may be considered as a potential biomarker for disease severity in SCD patients.

Background The link between blood hypercoagulability, endothelial cell activation and infertility or in vitro fertilization (IVF) failure is a puzzling issue. Activation of platelets, endothelial cells and blood coagulation, proinflammatory and angiogenetic potential play important role in implantation and early embryonic development. Biomarkers of cellular hypercoagulability are associated with high risk of IVF failure. Soluble Leukemia Inhibitory Factor (LIF), an interleukin 6 class cytokine expressed in the trophectoderm of the developing embryo is an important regulator in the establishment of pregnancy. Endoglin (Eng), a co-receptor for TGF-β1 and TGF-β3 is implicated in vascular complications of pregnancy and particularly in preeclampsia. Aim: The prospective longitudinal monocentric observational cohort study ROADMAP-IVF, evaluated the synergistic prognostic value of LIF and Eng with the biomarkers of cellular hypercoagulability in predicting the endometrial receptivity of fresh IVF cycles. Materials and Methods. The ROADMAP-IVF enrolled 40 women eligible for IVF with normal blood count, PT, aPTT, Fg, renal and liver function. The control group (CG) consisted of 30 healthy women with history of uncomplicated pregnancies. Exclusion criteria: Use of anticoagulant or antiplatelet agents during the last 30 days before inclusion. Known cardiovascular disease, active cancer. Active corticosteroid treatment. Primary end-point: Echographically documented pregnancy at 7 weeks from implantation. Blood was collected on day 2 (D2) from the natural cycle and 6 days after hormone treatment initiation corresponding to D8 of the cycle. Procoagulant phospholipid-dependent clotting time (PPL-ct), tissue factor (TF), thrombomodulin (TM), von Willebrand factor (vWF) and D-Dimers (DDi) were measured with assays from Diagnostica Stago (Asnieres, France). P- and E-Selectin, LIF and Eng were measured with ELISA. Thrombin generation (TG) with the TF 5 pM PPP-Reagent ® was assessed on Calibrated Automated Thrombogram (Stago, France). The cut-off values for studied biomarkers have been selected on the basis of ROC analysis. Univariate and multivariate logistic regression analysis examined the associations between the biomarkers and the study outcome. Results: 40 women were enrolled in the study. In 11 women pregnancy was confirmed with ultrasound and in 23 women no pregnancy was echographically documented. Age was not significantly different between the IVF (41 ys; range 22 - 48 ys) and the control group (40 ys; range 20 - 46 ys). At D2 and D8 the DDi and LIF were significantly higher in IVFG as compared to the CG. At D2 most of the patients had PPL-ct, DDi and MRI of TG higher than the upper normal limit (UNL). At D8 only TF was significantly increased as compared to the CG. Analytical data are presented in Table 1. Age did not correlate with the levels of the studied biomarkers. Significant correlations (p<0.05) were found between DDi and ETP (r=0.35); TM, TF and P-Selectin (r=0.4) at D2; TF, LIF and Eng (r=0.5) at D2 and D8; P-Selectin and PPL-ct (r=-0.4) at D2; P-Selectin and TM (r=0.4), E-Selectin (r=0.5), LIF (0.4) at D2; E-Selectin and P-Selectin (r=0.5), TF (r=0.3), and LIF (r=0.4) at D2. The ROC analysis showed that IVF failure was associate with DDi increase at D2 (AUC = 0.64); P-Selectin increase at D2(AUC = 0.57) and D8 (AUC = 0.61); TM increase at D8 (AUC = 0.61); TF increase at D8 (AUC = 0.57); LIF increase at D8 (AUC = 0.59); ETP increase at D2 (AUC = 0.67) and D8 (AUC = 0.67); Peak increase at D2 (AUC = 0.64) and D8 (AUC = 0.61). Prolongation of PPL-ct on D8 was predictive for positive pregnancy outcome(AUC 0.64). Conclusion The ROADMAP-IVF study showed that at least one out of four women eligible for IVF present biological evidence of activation of platelets, endothelial cells, blood coagulation or LIF upregulation. Hormone treatment administration does not significantly alter the proinflammatory or the hypercoagulable state. Among the studied biomarkers the levels of LIF, DDi, P-Selectine, TM and TF as well as the status of thrombin generation and PPL-ct showed a significant predictive value for the IVF outcome, particularly when measured at 6 days after hormone treatment administration. These findings will lead to the elaboration of an risk assessment model for IVF failure combining the selected biomarkers of hypercoagulability with LIF.

Backround: Thromboembolism occurs in about 10% of patients with multiple myeloma (MM). The incidence of VTE is higher in newly diagnosed MM patients as compared to those with relapsed or refractory disease and it is higher during the first 3 to 6 months after the initial diagnosis and treatment initiation. Some of the treatments administered to patients with MM are independent risk factors for VTE. Immunomodulatory agents (IMiDs) among anti-myeloma treatments stand out as having a considerable prothrombotic effect. Recognizing the significant risk associated with the use of immunomodulatory agents (the International Myeloma Working Group (IMWG) 2014 statement, and the European Myeloma Network Guidelines in 2015 both included guidance on the prevention of VTE in MM patients who receive IMiDs. The prospective, longitudinal observational study ROADMAP-MM CAT was designed to explore alternative strategies for the development of risk stratification tools in patients with multiple myeloma. To this target we evaluated the baseline profile of hypercoagulability in multiple myeloma patients with various disease status. Blood borne hypercoagulabity partly consisted of enhanced thrombin generation, a common phenomenon in patients with malignancies . It remains to be seen whether this phenomenon appears for multiple myeloma patients during the physical course of the disease. Aim: We conducted a study to explore the relationship between stages of MM and alterations of various thrombosis-related biomarkers in patients with MM and their relationship with MM therapy. Materials and Methods Patients with MM (n=162) were recruited and stratified to the following groups: 59 newly diagnosed treatment-naïve patients (ND), 49 patients receiving IMiDs (IM), 52 in complete remission (CR) and 12 patients in partial remission on IMiDs (PR/IM). Patients on anticoagulant treatment were excluded from the study. The control group (CG) consisted of 30 healthy age and sex-matched individuals. Samples of platelet-poor plasma (PPP) were assessed for thrombin generation (TG) with the TF 5pM PPP-Reagent® on Calibrated Automated Thrombogram (Diagnostica Stago, France). Plasminogen activator inhibitor-1 (PAI-1), soluble endothelial protein C receptor (sEPCR), and soluble vascular cell adhesion molecule-1 (sVCAM-1), Procoag-PPL were measured using antibody-based ELISA kits (Invitrogen International Inc., CA, USA, and Diagnostica Stago, France ). TF expression MPC-derived microparticles (MPC-dMPs) were detected using a Zymuphen MP-TF activity kit (Hyphen BioMed, Neuville sur Oise, France). The upper and lower normal limits (LNL and UNL) were calculated by the mean±2 SD. Results A total of 162 patients were enrolled (age 66.0±12.0 yrs; 53% male). Distribution of disease stage was as follows: 32% ISS I, 23% ISS II, 45% ISS III. Bone disease was present in 71% of patients and 19% of patients had high risk cytogenetic lesions. Patients with ongoing MM (ND, IM, PR/IM) had significantly lower Peak, ETP and MRI as compared to the CG. In contrast, patients in CR had Peak, ETP, MRI values similar to the CG. Patients with PR had lower ETP and MRI values as compared to the CR group. In ND 9% had TG >UNL and 22% had TGUNL and 67% had TGUNL and 35% had TGUNL and 12% had TG

Background: Hypercoagulability is a common blood alteration in newly diagnosed chemotherapy naïve patients with multiple myeloma. Multiple myeloma (MM) figures among malignancies that significantly increase the risk of venous thromboembolism (VTE). The rate of VTE is higher at the time of diagnosis and during the first months following initiation of first line therapy; approximately 10% of newly diagnosed MM (NDMM) will develop a VTE. Despite adequate thromboprophylaxis as per guidelines, the risk of residual VTE is not eliminated and remains as high as 12%. The optimization of VTE prevention in patients with MM is an unmet need. The exact mechanism of the increased risk of VTE is not yet fully understood. The identification of the procoagulant potential of cancer cells, which is related principally to tissue factor (TF) expression, attracts particular interest. Aim: We conducted a longitudinal prospective observational study, to explore the relationship of MM with cellular and plasma hypercoagulability as well as tissue factor positive microparticles, aiming to identify the most relevant biomarkers, which could be used in a Risk Assessment Model (RAM) for VTE in combination with clinical risk factors. Methods: Newly diagnosed patients (182) with multiple myeloma (NDMM) were prospectively enrolled. Patients were followed up for 12 months and the primary end-point was symptomatic objectively confirmed VTE. Patients were risk stratified and received thromboprophylaxis (none, aspirin on low molecular weight heparin (LMWH) based on previously published recommendations by the IMWG and EMN. Prior to treatment initiation and thromboprophylaxis initiation baseline biomarkers were obtained: Thrombin generation (TGT) in citrated PPP was assessed with the Thrombogram-Thrombinoscope® assay using PPP-reagent® 5 pm TF by Diagnostica Stago. The levels of P-Selectin and heparanase in plasma were measured with ELISA Kit (Cusabio Biotech and R&D Systems respectively). The procoagulant phospholipids clotting time was measured with STA-Procoag-PPL®, Levels of Factor VIIa were measured by Staclot VIIa-rTF®, D-Dimers (DDi) by Liatest D-Di (Diagnostica Stago, France), and Tissue Factor activity (TFa) by specific clotting based home test. TF expression MPC-derived microparticles (MPC-dMPs) were detected using a Zymuphen MP-TF activity kit (Hyphen BioMed, Neuville sur Oise, France). These were compared against values in a population of healthy individuals (n=30). Results: The distribution of patients enrolled in the study is as follows: Median age was 67 years (36-86) and 52% of the population was male. Median time to follow up was 7 months (1-12 months). The control group (30 healthy individuals). The overall rate of symptomatic VTE during follow-up was 11.5% (n = 21 patients). Eleven out of 21 events (52%) occurred within 3 months from treatment initiation. Six of these patients did not receive any thromboprophylaxis; six patients were on aspirin at the time of the event and three were on LMWH.At inclusion, patients showed significantly increased levels of TFa, FVIIa, D-Dimers and FM, and significantly shorter Procoag-PPL® as compared to the group of healthy individuals. Levels of P-selectin and TM were significantly lower in patients as compared to healthy individuals. The levels of heparanase were not significantly different in the group of patients as compared to the healthy individuals. Overall thrombin generation was attenuated in patients compared to healthy individuals. Lag-time and ttPeak were significantly increased and Peak, MRI, and ETP were significantly lower as compared to the control group, MP-TF activity measured in NDMM significantly increased 1.46± 0.38 pg/mL compared to control subjects 0.19 ± 0.05 pg/mL (Table 1). Multivariate logistic regression analysis demonstrated that ETP, Procoag-PPL® and MP-TF were independently associated with VTE occurrence. (Table 2) Conclusion: The prospective ROADMAP-CAT-MM study demonstrates the presence of pronounced cellular hypercoagulability in newly diagnosed chemotherapy naïve patients with MM characterized by decreased Procoag-PPL® clotting time, enhanced endothelial cell activation, and exhausted thrombin generation. The Procoag-PPL clotting time and the ETP and MP-TF were found to be independently associated with the risk of VTE and can be prospectively incorporated into a RAM for VTE in MM.

Background The ROADMAP-EOP study aimed to identify clinically relevant biomarkers of hypercoagulability for the identification of pregnant women at risk of early onset preeclampsia worsening. Methods The ROADMAP-EOP observational single center retrospective case–control study was conducted in Greece (Centre for Human Reproduction, Genesis Athens Clinic, Athens, Greece) from July 2020 to July and enrolled pregnant women diagnosed with EOP stratified in mild EOP group (n = 34) and severe EOP group (n = 15) as well as women with uncomplicated pregnancy (control group; n = 35). All women were assessed with thromboelastometry (ROTEM®), Calibrated Automated Thrombogram®, tissue factor activity (TFa), procoagulant phospholipid dependentclotting time (Procoag-PPL®), Proteins S (PS), TFPI, D-dimer, antithrombin (AT), thrombomodulin (TM), fibrinogen, prothrombin time (PT) and activated partial thromboplastin time (aPTT). The primary study end-point was severe earlyonset preeclampsia. Principal component analysis (PCA) was performed. Results The PCA analysis showed that a score composed of the lag-time, ttPeak and Procoag-PPL accurately predicted severe EOP (sensitivity 71.4%, specificity 61.8%, and AUC of the ROC analysis 0.953). Conclusion The pilot ROADMAP-EOP shows that activation of endothelial cells and blood hypercoagulability are driven events in the worsening of EOP. Among a large panel of biomarkers and coagulation assays, thrombingeneration test and procoagulant phospholipid dependent clotting time emerged as clinically relevant for the evaluation of the risk of severe EOP. This methodology for the development of a new clinic-biological risk assessment model for prompt identification of pregnant women at risk of severe EOP must be validated in a large multi-centerprospective study.

Background: Patients with COVID 19 disease have haemostatic dysfunction and are at higher risk of thrombotic complications. Although age and sex are a major risk factor for outcome. Even though the incidence of COVID 19 seems to be similar in both genders, men are at a higher risk of a worse outcome, with the odds up to 2.8 times higher than those for women. It is not known whether sex hormones contribute to this preposition. The real impact of sex hormone levels on the severity of COVID 19 remains to be demonstrated. Objectives: We aimed to investigate the impact of variations in sex hormone levels on the hypercoagulability and severity of COVID 19 in women and men according to age. Methods: 186 patients with symptomatic newly diagnosed Covid 19 were enrolled in the study. Specially we included postmenaupausal women (>50 years) and age matched men. Participants were divided into two groups: moderate Covid-19 (ARDS) and severe Covid-19. The control group consisted of 60 healthy individuals, with the same age as patients. Biomarkers of hypercoagulability and endothelial cell activation: Procoagulant phospholipid (PPL) TFPI, D-Dimer, Soluble thrombomodulin , Tissue factor activity, Thrombin generation (TG) with the Calibrated Automated Thrombogram (CAT) were all measured with assays were from Diagnostica Stago, Asnières, France. Total testosterone, estradiol, and progesterone were measured by ELISA method (Abcam, France). IL-6, Complement C3a and C5a by Human IL-6 ELISA kit, ThermoFisher (Scientific Asnieres-sur-Seine, France). The Upper Normal Limit (UNL) and the Lower Normal Limit (LNL) for each studied biomarker were defined in the control group as follows: UNL= mean + 2 standard deviation (SD), and LNL: = mean - 2 SD. Results: The levels of biomarkers of hypercoagulability, endothelial cell activation and inflammation in patients and controls are reported in Table 1. Thrombogram was marked by significantly prolonged lag time, prolonged time to peak and decreased ETP compared to the control group. The comparison between females and males showed that the lag-time was over the UNL in 18% of men over 50 years versus 8% in men under 50 years, and in 10% and 0% of women respectively. ETP values were below the LNL in 82% of men over 50 years versus 63% for men under 50 years and in 54% and 92% for women respectively. In the same way, the peak of thrombin was over the UNL in 39% and 15% for men over 50 and men under 50 versus 12% and 0% for women respectively. The TFPI were over the UNL in 99% of men over 50 years and in 84% of men under 50 years versus 91% and 87% for women respectively. D-Di increased in 88% of patients under or over 50 years independently of the group. The PPL were shorter than the LNL in 90 % and 80% of men over and under 50 years respectively, and for women in 86% and 77% respectively. The levels of TM were significantly lower in women for the 2 groups in comparison with men over or under 50 years. The levels of C3a, were above the UNL in 86%, and 63% for men over and under 50 years respectively and in 55% and 87% of women over and under 50 years respectively. The testosterone levels in males both younger and older than 50 years were significatively below the lower limit of the normal range of each age group. The estradiol levels were in normal range for women but significantly increased men (Table2). Stratification by the severity of COVID-19 showed that the testosterone levels were significantly lower in men who developed ARDS or severe COVID-19 than in those who did not (p>0.05). In men, the estradiol levels were significantly increased but the difference was not significant between those with severe or mild COVID-19. Males who developed severe COVID-19 had a lower progesterone level. No statistically significant difference in testosterone or progesterone levels were found in the women compared to normal range, the estradiol level was lower in females with ARDS (Table 3.)Relationship analysis of sex hormones with markers of immune activation, testosterone levels and estradiol/testosterone were correlated with, IL-6, fibrinogen, and D-Di. Conclusion: Our study highlights that disturbance in circulating sex hormone levels is a hallmark of critical COVID-19 in males and reinforces the concept that determination of testosterone and E2/T ratio in association with biomarkers of hypercoagulability upon diagnosis might lead to an a accurate risk assessment tool for prompt identification of patients at high risk of COVID-19 worsening. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Background: In patients with severe COVID-19, activation of blood coagulation and endothelial cells orchestrated with complement activation and cytokine storm leads to disease worsening and death. Hypercoagulable state and endothelial cell activation and fi brinolytic unbalance are common alterations in patients with COVID-19 hospitalized either at the conventional medical ward or at the intensive care unit (ICU). Two systematic reviews multivariate The ROADMAP-postCOVID-19 study offers an analysis of a large panel of biomarkers of endothelial cell activation and hypercoagulability in survivors of COVID-19. The study documents that two months after symptoms' onset, activation of endothelial cells, in vivo thrombin generation, and fi brin lysis are frequent phenomena up to two months from COVID-19 symptom onset. The data presented herein allow to propose that evaluation of blood hypercoagulability (using D-dimer for instance) and endothelial cell activation could offer the possibility of prompt identi fi cation of COVID-19 survivors at risk of post-COVID-19 vascular complications. The fi ndings of the present study underline the need for a thorough evaluation of a potential correlation between the sustained hypercoagulability and endothelial cell activation with the symptomatology of long-COVID-19 to apply anticipated diagnostic and therapeutic strategies. The clinical relevance of the identi fi ed biomarkers of endothelial activation and hypercoagulability in the assessment of the risk of long COVID-19 must be investigated in a prospective study.

Background. Hypercoagulable state and endothelial cell activation are common alterations in patients with COVID-19. Nevertheless, the hypothesis of persistent hypercoagulability and endothelial cell activation following recovery from COVID-19 remains an unresolved issue. Objectives. To investigate the persistence of endothelial cell activation and hypercoagulability after recovery from COVID-19. Patients/Methods. COVID-19 survivors (n = 208) and 30 healthy individuals were enrolled in this study. The following biomarkers were measured: procoagulant phospholipid-dependent clotting time (PPL-ct), D-Dimer, fibrin monomers (FM), free Tissue factor pathway inhibitor (free-TFP)I, heparinase, and soluble thrombomodulin (sTM). Antibodies against SARS-CoV-2 (IgG and IgA) were also measured. Results. The median interval between symptom onset and screening for SARS-CoV-2 antibodies was 62 days (IQR = 22 days). Survivors showed significantly higher levels of D-Dimers, FM, TFPI, and heparanase as compared to that of the control group. Survivors had significantly shorter PPL-ct. Elevated D-dimer was associated with older age. Elevated FM was associated with female gender. Elevated heparanase was independently associated with male gender. Decreased Procoag-PPL clotting time was associated with female gender. One out of four of COVID-19 survivors showed increase at least one biomarker of endothelial cell activation or hypercoagulability. Conclusions. Two months after onset of COVID-19, a significant activation of endothelial cells and in vivo thrombin generation persists in at least one out of four survivors of COVID-19. The clinical relevance of these biomarkers in the diagnosis and follow-up of patients with long COVID-19 merits to be evaluated in a prospective clinical study.

Background: In patients with severe COVID-19, activation of blood coagulation and endothelial cells orchestrated with complement activation and cytokine storm leads to disease worsening and death. Hypercoagulable state and endothelial cell activation and fi brinolytic unbalance are common alterations in patients with COVID-19 hospitalized either at the conventional medical ward or at the intensive care unit (ICU). Two systematic reviews multivariate The ROADMAP-postCOVID-19 study offers an analysis of a large panel of biomarkers of endothelial cell activation and hypercoagulability in survivors of COVID-19. The study documents that two months after symptoms' onset, activation of endothelial cells, in vivo thrombin generation, and fi brin lysis are frequent phenomena up to two months from COVID-19 symptom onset. The data presented herein allow to propose that evaluation of blood hypercoagulability (using D-dimer for instance) and endothelial cell activation could offer the possibility of prompt identi fi cation of COVID-19 survivors at risk of post-COVID-19 vascular complications. The fi ndings of the present study underline the need for a thorough evaluation of a potential correlation between the sustained hypercoagulability and endothelial cell activation with the symptomatology of long-COVID-19 to apply anticipated diagnostic and therapeutic strategies. The clinical relevance of the identi fi ed biomarkers of endothelial activation and hypercoagulability in the assessment of the risk of long COVID-19 must be investigated in a prospective study.

Introduction: Multiple myeloma (MM) is an hematological disease linked to a high thrombotic risk, estimated at around 10%/year, irrespective to disease onset. An important proportion of MM patients presents at least one of the well-defined vascular risk factors, such as diabetes, cardiac disease, hypertension or hyperlipidemia, which contribute to thrombotic risk. Thrombin Generation Test (TGT) could be an useful biomarker reflecting the prothrombotic profile of MM patients. Aim: To evaluate the potential correlation between vascular risk factors and TGT in MM patients. Materials and methods: This is a monocentric, prospective, non-interventional study on a cohort of 82 MM patients referred to the Hemostasis Center at Tenon University Hospital. Patients receiving already anticoagulant treatment of any kind were excluded. Thrombin generation was performed at their first visit using the Calibrated Thrombogram Assay (CAT®,Stago, Genevilliers, France), according to manufacturer's instructions. We collected all TGT parameters: lag time (LT), Endogenous Thrombin Potential (ETP) and Mean Rate Index (MRI).We also measured factor VIII activity (FVIII). Results were statistically analyzed using a two-sample t-test. Results: Among 82 patients with a male/female ratio of 44/35 and a median age at 65.2 years, 39 patients (48%) presented at least one cardiovascular risk factor (group A) and 42 had no vascular risk factors (group B) The LT value was reduced but not reaching a statistical difference between groups A and B (3 vs 3.95 sec, p= 0.8). MRI representing velocity was statistically shorter in group A (100 vs 118 nmol/min , p< 0.001). Surprisingly, ETP was significantly reduced in group A (1392 vs 1717 nmol/l x min, p < 0.001). FVIII levels were increased in group A (216% vs 192 %, p<0.05). Conclusion: MM hypercoagulability can be easily identified with MRI, a chronometric TGT parameter. Increased FVIII levels observed in vascular patients are also facilitating an accelerated thrombin generation and are correlated with an endothelial activation. ETP decrease, reflecting the link between both content and container, must be seen as an adaptative response. Thus, endothelial activation could lead to TFPI release and this particular TGT profile in high vascular risk MM patients with a hypercoagulable state. Disclosures No relevant conflicts of interest to declare.