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Background and Aims: Tissue factor (TF) and activated factor XI (FXIa) have been associated with acute coronary syndrome, ischemic stroke and venous thromboembolism. Their predictive value in stable coronary artery disease (CAD) is unclear. We investigated whether active TF and FXIa were associated with clinical outcomes in CAD patients in long-term observation., Methods: In 124 stable patients with multivessel CAD, we assessed the presence of circulating, active TF and FXIa by measuring a response of thrombin generation to respective inhibitory antibodies. We recorded the composite endpoint of myocardial infarction (MI), stroke, systemic thromboembolism and cardiovascular death during follow-up (median 106 months, interquartile range 95-119)., Results: Circulating FXIa and active TF were detected in 40% and 20.8% of the 120 patients (aged 65.0 [57.0-70.3] years, men, 78.3%), who completed follow-up. The composite endpoint occurred more frequently in patients with detectable active TF and FXIa present at baseline (hazard ratio [HR] 4.02, 95% confidence interval [CI] 2.26-7.17, p < 0.001 and HR 6.21, 95% CI 3.40-11.40, p < 0.001, respectively). On multivariate analysis FXIa, but not active TF, was an independent predictor of the composite endpoint, as well as MI, stroke/systemic thromboembolism, and cardiovascular death, when analyzed separately., Conclusions: To our knowledge, this study is the first to show that circulating FXIa predicts arterial thromboembolic events in advanced CAD, supporting a growing interest in FXIa inhibitors as novel antithrombotic agents., (Copyright © 2022. Published by Elsevier B.V.)

Introduction: Plasma thrombin generation kinetics as measured by the calibrated automated thrombogram (CAT) assay is a predictor of symptomatic venous thromboembolism after trauma. We hypothesized that data from a new prototype assay for measurement of thrombin generation kinetics in fresh whole blood (near patient testing of thrombin generation), will correlate with the standard CAT assay in the same patients, making it a potential tool in the future care of trauma patients., Methods: Patients were enrolled from June 2018 to February 2020. Within 12 hours of injury, blood samples were collected simultaneously for both assays. Variables compared and correlated between assays were lag time, peak height, time to peak, and endogenous thrombin potential. Data are presented as median with interquartile range (IQR). Spearman and Pearson correlations were estimated and tested between both assays; a P value of <0.05 was considered to be significant., Results: A total of 64 trauma patients had samples analyzed: injury severity score = 17 (IQR), 10-26], hospital length of stay = 7.5 (IQR), 2-18) days, age = 52 (IQR, 35-63) years, 71.9% male, and 42.2% of patients received a transfusion within 24 hours of injury. Thrombin generation parameters between plasma and whole blood were compared and found that all parameters of the two assays correlate in trauma patients., Conclusion: In this pilot study, we have found that a novel point-of-care whole blood thrombin generation assay yields results with modest but statistically significant correlations to those of a standard plasma thrombin generation assay. This finding supports studying this device in a larger, adequately powered study., (© 2021 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis (ISTH).)

Hemostatic tests have been utilized to clarify the blood coagulation potential. The novel thrombin generation (TG) assay of this study provides explicit information and is the most physiologically-relevant hemostatic test ex vivo. We describe how this assay allows for TG under a number of relevant circumstances. First, whole blood (WB) from healthy individuals was analyzed ± 5 pM tissue factor (TF) and ± contact pathway inhibition. Without an exogenous initiator TG was decreased and delayed, but addition of 5 pM TF shortened the lag phase and increased peak thrombin. Additional experiments included fresh WB from a trauma patient analyzed for endogenous activity and TG from healthy donors subjected to TG antagonists which prolonged the lag phase whereas TG agonists consistently shortened the lag phase in a dose dependent manner. Lastly, platelet-poor plasma was reconstituted with packed red blood cells and TG was monitored in the presence and absence of both TF as an activator and PCPS as a phospholipid surface. Our data illustrate the potential that this continuous TG assay has in the evaluation of disorders relevant to blood coagulation and in the monitoring of treatments administered in response to these disorders., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Introduction: An elevated procoagulant activity observed in trauma patients is, in part, related to tissue factor (TF) located on blood cells and microparticles. However, analysis of trauma patient plasma indicates that there are other contributor(s) to the procoagulant activity. We hypothesize that factor (F)XIa and FIXa are responsible for an additional procoagulant activity in burn patients., Methods: Multiple time-point plasma samples from 56 burn patients (total number of samples was 471; up to 20 time-points/patient collected in 3 weeks following admission) were evaluated in a thrombin generation assay using inhibitory antibodies to TF, FIXa and FXIa., Results: Due to the limited volume of some samples, not all were analyzed for all three proteins. At admission, 10 of 53 patients (19%) had active TF, 53 of 55 (96%) had FXIa and 48 of 55 (87%) had FIXa in their plasma. 34 patients of 56 enrolled (61%) showed TF activity at one or more time-points. All patients had FXIa and 96% had FIXa at one or more time-points. Overall, TF was observed in 99 of 455 samples analyzed (22%), FXIa in 424 of 471 (90%) and FIXa in 244 of 471 (52%). The concentration of TF was relatively low and varied between 0 and 2.1pM, whereas that of FXIa was higher, exceeding 100pM in some samples. The majority of samples with FIXa had it at sub-nanomolar concentrations. No TF, FXIa and FIXa activity was detected in plasma from healthy individuals., Conclusions: For the first time reported, the majority of plasma samples from burn patients have active FXIa and FIXa, with a significant fraction of them having active TF. The concentration of all three proteins varies in a wide range., (Copyright © 2017 Elsevier Ltd and ISBI. All rights reserved.)

The procoagulant activity (PA) of stored units of red blood cells (RBC) increases over time, which is related to the expression/exposure of tissue factor (TF). However, there is a discrepancy between the TF measured and changes in PA observed, suggesting that other blood components contribute to this activity. Our goal was to evaluate changes in PA of stored RBCs and to determine possible contributors to it. RBC units from 4 healthy donors were prepared and stored at 4 °C. On selected days, RBC aliquots were reconstituted with autologous plasma and tested in the thromboelastography assay. Corresponding supernatants were tested in a clotting assay. For all donors, the clotting time (CT) of reconstituted RBC units decreased from ∼3000-4000s on day 1 to ∼1000-1600s on day 30, with the most dramatic changes occurring between days 1 and 5. Anti-TF antibody slightly prolonged the CT. The concentration of TF did not change significantly over time and was within the range of 0.3-2.3 pM. Bovine lactadherin (LTD) prolonged the CT of the RBC (by 2.4-3.4-fold in days 3-5 and by 1.3-1.8-fold at day 30). Anti-TF antibody together with LTD had a cumulative effect on the CT prolongation. CT of supernatants responded to both anti-TF and anti-FXIa antibodies. Three contributors to the PA of stored RBC were identified, i.e. FXIa in solution and phosphatidylserine and TF exposed on blood cells and microparticles. Failure of LTD and antibodies to completely eliminate PA suggests that other components of blood could contribute to it., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We have adapted the corn-trypsin inhibitor whole-blood model to include EA.hy926 as an endothelium surrogate to evaluate the vascular modulation of blood coagulation initiated by relipidated recombinant tissue factor (rTf) and a cellular Tf surrogate, lipopolysaccharide (LPS)-stimulated THP1 cells (LPS-THP-1). Compared with bare tubes, EA.hy926 with rTf decreased the rate of thrombin formation, ITS accumulation, and the production of fibrinopeptide A. These phenomena occurred with increased rates of factor Va (fVa) inactivation by cleavages at R(506) and R(306). Thus, EA.hy926 provides thrombin-dependent protein C activation and APC fVa inactivation. Comparisons of rTf with LPS-THP-1 showed that the latter gave reduced rates for TAT formation but equivalent fibrinopeptide A, and fV activation/inactivation. In the presence of EA.hy926, the reverse was obtained; with the surrogate endothelium and LPS-THP-1 the rates of TAT generation, fibrinopeptide release, and fV activation were almost doubled, whereas cleavage at R(306) was equivalent. These observations suggest cooperativity between the 2 cell surrogates. These data suggest that the use of these 2 cell lines provides a reproducible quasi-endothelial quasi-inflammatory cytokine-stimulated monocyte system that provides a method to evaluate the variations in blood phenotype against the background of stable inflammatory cell activator and a stable vascular endothelial surrogate.

The presence of antibodies (Abs) in hemophilia A patients can potentially influence the therapeutic qualities of factor VIII (fVIII) administration. Much work has been focused on the presence of inhibitory antibodies, whereas the quantitation of noninhibitory anti-fVIII antibodies has been largely undetermined. Our objective was to develop a sensitive and specific fluorescence-based immunoassay (FLI) for the quantitation of anti-fVIIIAbs in human plasma. Affinity-purified human anti-fVIIIAb, isolated from a hemophilia A subject, was used as a calibrator with a detectability limit of 40 (+/-1.5) pM. The calibrator and the human plasma anti-fVIIIAb were captured on recombinant fVIII (rfVIII)- coupled microspheres and probed with mouse anti-human Ig-R-phycoerythrin. Plasma samples from 150 healthy donors and 39 inhibitor-negative hemophilia A subjects were compared with 4 inhibitor-positive hemophilia A plasma samples with inhibitor titers of 1 BU/mL (94.6 +/- 0.8 nM), 11 BU/mL (214.3 +/- 7.1 nM), 106 BU/mL (2209.4 +/- 84.9 nM), 140 BU/mL (2417.7 +/- 3.8 nM) as measured by the Nijmegen method. We also describe the validation of a mouse anti-human fVIIIAb as a surrogate calibrator. Four healthy individuals (3%) showed detectable anti-fVIIIAb in the range of 0.6 to 6.2 nM, whereas 13 (33%) of the 39 inhibitor-free hemophilia A subjects were positive for anti-fVIIIAb in the range of 0.5 to 20 nM. The method may be useful for therapeutic management of hemophilia A patients.

Tissue factor (TF) is an integral membrane protein essential for hemostasis. During the past several years, a number of studies have suggested that physiologically active TF circulates in blood at concentrations greater than 30 pM either as a component of blood cells and microparticles or as a soluble plasma protein. In our studies using contact pathway-inhibited blood or plasma containing activated platelets, typically no clot is observed for 20 minutes in the absence of exogenous TF. An inhibitory anti-TF antibody also has no effect on the clotting time in the absence of exogenous TF. The addition of TF to whole blood at a concentration as low as 16 to 20 fM results in pronounced acceleration of clot formation. The presence of potential platelet TF activity was evaluated using ionophore-treated platelets and employing functional and immunoassays. No detectable TF activity or antigen was observed on quiescent or ionophore-stimulated platelets. Similarly, no TF antigen was detected on mononuclear cells in nonstimulated whole blood, whereas in lipopolysaccharide (LPS)-stimulated blood a significant fraction of monocytes express TF. Our data indicate that the concentration of physiologically active TF in non-cytokine-stimulated blood from healthy individuals cannot exceed and is probably lower than 20 fM.

Tissue factor-induced blood coagulation was studied in 20 individuals, for varying periods of time during 54 months, in contact pathway-inhibited whole blood at 37 degrees C and evaluated in terms of the activation of various substrates. After quenching over time with inhibitors, the soluble phases were analyzed for thrombin-antithrombin III (TAT) complex formation, prothrombin fragments, platelet activation (osteonectin release), factor Va generation, fibrinopeptide (FP) A and FPB release, and factor XIII activation. TAT complex formation, for 35 experiments, showed an initiation phase (up to 4.6 +/- 0.6 minutes) in which thrombin was generated at an average rate of 0.93 +/- 0.3 nM/min catalyzed by about 1.3 pM prothrombinase yielding approximately 26 nM thrombin. During a subsequent propagation phase, thrombin was generated at a rate of 83.9 +/- 3.8 nM/min by about 120 pM prothrombinase, reaching ultimate levels of 851 +/- 53 nM. Clot time, determined subjectively, occurred at 4.7 +/- 0.2 minutes and correlated with the inception of the propagation phase. The thrombin concentrations associated with the transitions to rapid product formation are 510 +/- 180 pM for platelet activation (1.9 +/- 0.2 minutes), 840 +/- 280 pM for factor XIII activation and factor Va generation (2.2 +/- 0.6 minutes), 1.3 +/- 0.4 nM for FPA release (2.5 +/- 0.7 minutes), 1.7 +/- 0.5 nM for FPB release and prethrombin 2 (2.8 +/- 0.8 minutes), 7.0 +/- 2.2 nM for thrombin B chain (3.6 +/- 0.2 minutes), and 26 +/- 6.2 nM for the propagation phase of TAT formation (4.6 +/- 0.6 minutes). These results illustrate that the initial activation of thrombin substrates occurs during the initiation phase at less than 2 nM thrombin (0.2%). Most thrombin (96%) is formed well after clotting occurs.

The ability of factor VIIa to initiate thrombin generation and clot formation in blood from healthy donors, blood from patients with hemophilia A, and in anti-factor IX antibody-induced ("acquired") hemophilia B blood was investigated. In normal blood, both factor VIIa-tissue factor (TF) complex and factor VIIa alone initiated thrombin generation. The efficiency of factor VIIa was about 0.0001 that of the factor VIIa-TF complex. In congenital hemophilia A blood and "acquired" hemophilia B blood in vitro, addition of 10 to 50 nM factor VIIa (pharmacologic concentrations) corrected the clotting time at all TF concentrations tested (0-100 pM) but had little effect on thrombin generation. Fibrinopeptide release and insoluble clot formation were only marginally influenced by addition of factor VIIa. TF alone had a more pronounced effect on thrombin generation; an increase in TF from 0 to 100 pM increased the maximum thrombin level in "acquired" hemophilia B blood from 120 to 480 nM. Platelet activation was considerably enhanced by addition of factor VIIa to both hemophilia A blood and "acquired" hemophilia B blood. Thus, pharmacologic concentrations of factor VIIa cannot restore normal thrombin generation in hemophilia A and hemophilia B blood in vitro. The efficacy of factor VIIa (10-50 nM) in hemophilia blood is dependent on TF.