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Introduction: Patients with coronary artery disease (CAD) display a prothrombotic fibrin clot phenotype, involving low permeability and resistance to lysis. The determinants of this phenotype remain elusive. Circulating tissue factor (TF) and activated factor XI (FXIa) are linked to arterial thromboembolism. We investigated whether detectable active TF and FXIa influence fibrin clot properties in CAD. Methods: In 118 CAD patients (median age 65 years, 78% men), we assessed Ks, an indicator of clot permeability, and clot lysis time (CLT) in plasma‐based assays, along with the presence of active TF and FXIa. We also analysed proteins involved in clotting and thrombolysis, including fibrinogen, plasminogen activator inhibitor‐1 (PAI‐1) and thrombin activatable thrombolysis inhibitor (TAFI). During a median 106 month (interquartile range 95–119) follow‐up, myocardial infarction (MI), stroke, systemic thromboembolism (SE) and cardiovascular (CV) death were recorded. Results: Circulating TF and FXIa, detected in 20.3% and 39.8% of patients, respectively, were associated with low Ks and prolonged CLT. Solely FXIa remained an independent predictor of low Ks and high CLT on multivariable analysis. Additionally, fibrinogen and PAI‐1 were associated with low Ks, while PAI‐1 and TAFI—with prolonged CLT. During follow‐up low Ks and prolonged CLT increased the risk of MI and the latter also a composite endpoint of MI, stroke/SE or CV death. Conclusions: To our knowledge, this study is the first to show that circulating FXIa is associated with prothrombotic fibrin clot properties in CAD, suggesting additional mechanisms through which FXIa inhibitors could act as novel antithrombotic agents in CAD. [ABSTRACT FROM AUTHOR]

Tissue factor (TF), an initiator of blood coagulation in vivo, is expressed in a variety of cells. Sufficient natural TF has been isolated to clone and express recombinant proteins ranging from full-length TF to its extracellular domain. Because of the limited availability of natural TF, recombinant proteins have been used as surrogates. Despite the differences in their post-translational modifications, it has been accepted that membrane-anchored recombinant TFs are quite similar to the natural TF. Recent studies, however, have shown that post-translational modifications play an important role in TF-triggered thrombin generation. [ABSTRACT FROM AUTHOR]

Abstract: Tissue factor (TF) is a transmembrane protein which, in complex with factor (F)VIIa, initiates blood coagulation. Numerous studies have determined TF epitopes and individual amino acids which play an important role in the TF/FVIIa complex formation and its activity towards natural substrates. However the subject of cell-surface TF activity remains controversial. It has been almost commonly accepted that TF on the cell surface has low (if any) activity, i.e. is encrypted and requires specific conditions/reagents to become active, i.e. decrypted. One of the leading theories suggests that cell membrane lipid composition plays a crucial role in TF decryption, whereas another assigns the key role to the formation of the Cys186-Cys209 disulfide bond. Despite a number of studies published from several laboratories, the role of this bond in the activity of the TF/FVIIa complex remains elusive and controversial. One of the causes of this controversy could be related to the lack of specificity of the reagents used for the cell treatment leading to possible alterations in other cell surface proteins and cell membrane environment. In conclusion, the influence of the Cys186-Cys209 this bond on cell surface TF function remains unclear. [Copyright &y& Elsevier]

Abstract: Tissue factor is a membrane protein, which in a complex with factor VIIa initiates in vivo blood coagulation. Due to the scarcity of natural tissue factor protein, most studies have relied upon recombinant tissue factor forms. However, there have been only cursory experimental comparisons of natural and recombinant tissue factor proteins. Our preliminary data suggested that placental tissue factor in a complex with factor VIIa was more efficient activator of factor X than the recombinant protein. After deglycosylation, both forms of tissue factor showed almost an identical activity in the extrinsic factor Xase. Analyses using tryptic digestion and mass-spectrometry revealed that the levels of glycosylation and the composition of carbohydrates present in natural placental tissue factor were different than those in its recombinant counterpart. These data indicate that natural and recombinant tissue factor proteins differ in their posttranslational modifications and that these differences translate into different cofactor activity. Thus the use of recombinant tissue factor proteins for the quantitation of natural tissue factor is misleading. [Copyright &y& Elsevier]

Abstract: Tissue factor (TF) is the major physiological initiator of blood coagulation. It exists as an integral membrane protein that upon injury to the blood vessel becomes exposed to the blood stream and initiates a series of enzymatic reactions that cause blood to clot. TF is also found within circulating blood cells, requiring specific signaling events to promote its expression. In this review we will discuss current controversies concerning the structure–activity relationships of TF and contributions of TF to the hemostatic process, the potential roles of intravascular TF, including non-cell-bound TF and cell-expressed TF and the overall relationship between TF function and hemorrhage control. [Copyright &y& Elsevier]

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. 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). 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. 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. [Display omitted] • The prognostic role of active tissue factor (TF) and activated FXI (FXIa) in coronary artery disease (CAD) is unclear. • We investigated the impact of active TF and FXIa on long-term outcomes in CAD patients. • FXIa and, to a lesser extent, active TF were associated with cardiovascular events in CAD. • Targeting FXIa might represent a promising preventive strategy in high-risk CAD patients. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Background: Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms. Methods: Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results. Results: Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography. Conclusion and significance: Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF. [Copyright &y& Elsevier]

Abstract: Background: Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties. Methods: The status of disulfides in recombinant TF1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function. Results: In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function. Conclusion: The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis. General significance: Results of this study advance our knowledge on TF structure/function relationships. [Copyright &y& Elsevier]

Abstract: Background: Tissue factor (TF) is a single polypeptide integral membrane glycoprotein composed of 263 residues and is essential to life in its role as the initiator of blood coagulation. Previously we have shown that the activity of the natural placental TF (pTF) and the recombinant TF (rTF) from Sf9 insect cells is different (Krudysz-Amblo, J. et al (2010) J. Biol. Chem. 285, 3371–3382). Methods: In this study, using mass spectrometry, we show by quantitative analysis that the extent of glycosylation varies on each protein. Results and conclusions: Fractional abundance of each glycan composition at each of the three glycosylation sites reveals the most pronounced difference to be at asparagine (Asn) 11. This residue is located in the region of extensive TF-factor VIIa (FVIIa) interaction. Carbohydrate fractional abundance at Asn11 revealed that glycosylation in the natural placental TF is much more prevalent (~76%) than in the recombinant protein (~20%). The extent of glycosylation on Asn124 and Asn137 is similar in the two proteins, despite the pronounced differences in the carbohydrate composition. Additionally, 77% of rTF exists as TF des-1, 2 (missing the first two amino acids from the N-terminus). In contrast, only 31% of pTF is found in the des-1, 2 form. Conclusion: These observations may attribute to the difference in the ability of TF–FVIIa complex to activate factor X (FX). General significance: Structural and functional comparison of the recombinant and natural protein advances our understanding and knowledge on the biological activity of TF. [Copyright &y& Elsevier]

Abstract: Introduction: Increased cardiovascular mortality and risk of venous thromboembolism are serious extra-pulmonary complications of chronic obstructive pulmonary disease (COPD). Previously, circulating active tissue factor (TF) and factor XIa (FXIa) have been reported to be associated with acute coronary syndromes. Objective: To measure plasma FXIa and active TF, prothrombin fragment 1.2 (F1.2), and markers of systemic inflammation (C-reactive protein [CRP], interleukin-6 [IL-6], tumor necrosis factor α [TNFα] and matrix metalloproteinase 9 [MMP-9]) in 60 patients with documented stable COPD free of previous thromboembolic events. Methods: In-house clotting assays using inhibitory monoclonal antibodies against FXIa and TF. Results: FXIa was detected in 9 (15%) and TF activity in 7 (11.7%) COPD patients. Subjects positive for FXIa and/or TF (n=10; 16.7%) had higher F1.2 (median [interquartile range], 398 [216] vs 192 [42] pM, p<0.000001), fibrinogen (5.58 [2.01] vs 3.97 [2.47] g/L, p=0.0007), CRP (14.75 [1.20] vs 1.88 [2.95] mg/L, p<0.000001), IL-6 (8.14 [4.74] vs 2.45 [2.24] pg/mL, p=0.00002), and right ventricular systolic pressure (47 [15] vs 38 [12] mmHg, p=0.023), and lower vital capacity (66 [15] vs 80 [17] % predicted, p=0.04) than COPD patients without detectable FXIa and TF. COPD severity was not associated with the presence of circulating FXIa and active TF. Conclusions: This is the first study to show that active FXIa and TF are present in stable COPD patients, who exhibit enhanced systemic inflammation and thrombin generation. Our findings suggest a new prothrombotic mechanism which might contribute to elevated risk of thromboembolic complications in COPD. [Copyright &y& Elsevier]