Tissue Factor Pathway Inhibitor Activity Research Articles

Tissue Factor Pathway Inhibitor (TFPI) Beta and Gamma Isoforms Contribute to Bleeding in Hemophilia Mice

Key Points: Expression of any single TFPI isoform (α, β, or γ) is sufficient for mouse survival.Two-Kunitz domain TFPI isoforms, anchored β and circulating γ, each contribute to hemophilia bleeding.In vivo, TFPIβ is a more potent anticoagulant than TFPIα. Upon injury, tissue factor (TF) triggers coagulation by complexing with factor (F)VIIa to activate FIX and FX, leading to thrombin generation and a fibrin clot. Tissue factor pathway inhibitor limits TF-triggered coagulation. Humans and mice express transcripts encoding for 3-Kunitz domain TFPIα and membrane-anchored 2-Kunitz TFPIβ. Mice also express transcripts encoding untethered, 2-Kunitz TFPIγ. In humans, proteolysis of TFPIα and/or β produces “γ-like” 2-Kunitz TFPI. Hemophilia A and B, caused by deficiency in factors VIII and IX, respectively, are congenital X-linked recessive bleeding disorders that affect an estimated 1.1 million males worldwide. Hemophilia can cause excessive bleeding spontaneously or in response to trauma that can be life-threatening or significantly decrease the quality of life. Approved treatments for hemophilia include factor replacement, bypassing agents, the bispecific antibody Emicizumab, and recently approved gene therapies. Also in development are agents that reduce endogenous anticoagulant activity to counter the procoagulant deficiency of hemophiliacs, including monoclonal antibodies (Mab) to TFPI. These Mab target all TFPI isoforms to improve coagulation and correct bleeding in hemophilia patients, but with some demonstrated thrombosis risk. We investigated if TFPI-isoform-specific inhibition could provide equivalent efficacy which might reduce thrombotic risk. We generated TFPI-isoform-specific exon deletions in mice bred into hemophilia (FVIII-null) background. We find mice expressing any single TFPI isoform (α, β, or γ) appear healthy and reproduce. Our in vivo tail-vein re-bleeding assay is based on serial clot disruptions over 15 minutes, which we have shown is sensitive to anticoagulant and antiplatelet treatment, as well as factor replacement therapy. In this assay, we find C57Bl/6 WT mice clot 25.6 + 0.8 times (mean + SEM), whereas FVIII-null hemophilia mice clot 5.3 +0.4 times. In hemophilia mice, TFPIα-specific deletion is without impact, forming clots 5.1 + 0.8 times, whereas TFPIβ-specific or TFPIγ-specific deletion improves clot formation to 16.8 + 2.0 and 15.2 + 1.5 times, respectively (p<0001). Even hemophilia mice with heterozygotic deletion of the β exon form clots 11.8 + 0.8 times (p<0001). Thus, in vivo 2-Kunitz TFPI isoforms, anchored β and untethered γ each provides greater anticoagulant activity and impact on bleeding than 3-Kunitz TFPIα. Ex vivo plasma-based thrombin generation assays show that TFPIγ provides more anticoagulant activity than TFPIα. As shown in the figure, the deletion of TFPIγ increases thrombin generation to a greater extent than the deletion of TFPIα. The addition of an anti-TFPI antibody that effectively blocks TFPI activity shows a significant increase in endogenous thrombin potential in the plasma of TFPIαβ-deleted (γ-only) mice (370 nM.min), versus TFPIβγ-deleted (α-only) plasma (70 nM.min) and TFPIαγ-deleted (β-only) mice plasma (22 nM.min), (p<0.001). As expected, membrane-anchored TFPIβ is not detected in plasma. It is well established that 3-Kunitz TFPIα is a potent anticoagulant in plasma-based clotting time assays, while truncated 2-Kunitz TFPI, like mouse TFPIγ, appears inactive. Based on such assays, it was unexpected that TFPIγ-only expressing mice thrive, and in vivo, TFPIα is not the dominant endogenous anticoagulant. If mouse models are to be useful in providing insights into human hemostasis and TF-associated diseases, then it is critical to understand the similarities and differences between mice and humans. As humans do not express TFPIγ, the observed impact of depleting circulating 2-Kunitz TFPI on bleeding and thrombin generation in hemophilic mice is unlikely to translate to man. By contrast, our results demonstrating that, in vivo, TFPIβ is a more potent anticoagulant than TFPIα in mice has potential therapeutic implications in humans.

Longitudinal observations of TFPI levels in paediatric Haemophilia A patients.

Inhibition of tissue factor pathway inhibitor (TFPI) is a potential new mode of action to achieve haemostasis in haemophilia A and B patients. Knowledge about potential developmental changes of TFPI levels during childhood are a prerequisite to translate adult doses of TFPI inhibitors to doses in paediatric patients. In this study we present longitudinal data for total TFPI concentrations (TFPI-T) and TFPI activity (TFPI-A) from 48 paediatric Haemophilia A patients in the age range from 3 to 18 years (2-12 observations per patient). TFPI-T and TFPI-A tend to decrease over age during childhood. Lowest values were observed between 12 and<18 years. On average, TFPI-T and TFPI-A were lower in adolescent haemophilia patients than in adult haemophilia patients. In summary, the presented information on TFPI levels in children adds to the current knowledge of developmental haemostasis and it can be helpful in evaluating how children respond to haemophilia treatment including the new class of anti-TFPI compounds.

Hypercoagulability State Combined with Post-Treatment Hypofibrinolysis in Invasive Breast Cancer: A Seven-Year Follow-Up Evaluating Disease-Free and Overall Survival.

(1) Background: Cancer treatment, including chemotherapy, endocrine therapy, targeted therapy and radiotherapy, has been identified as an important independent risk factor for venous thromboembolism in cancer patients. The aim of the study was to evaluate the effect of adjuvant therapy on the coagulation and fibrinolysis components in invasive breast cancer. (2) Methods: Tissue factor pathway inhibitor (TFPI), tissue factor (TF), tissue plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1) antigen (concentration) and TFPI and TF activities were examined in the blood samples of 60 breast cancer patients treated by adjuvant chemotherapy, endocrine therapy, radiotherapy and immunotherapy. Blood samples were taken 24 h before primary surgery and 8 months after tumour removal surgery. (3) Results: Adjuvant therapy administrated to breast cancer patients significantly increased the concentration of plasma TF, the PAI-1 antigen and also the activity of TFPI and TF, but significantly decreased the level of the t-PA antigen. Combined chemotherapy and endocrine therapy, but not monotherapy, has an important effect on haemostatic biomarker levels. (4) Conclusions: Breast cancer patients receiving adjuvant therapy have an elevated risk of developing a hypercoagulability and hypofibrinolysis state leading to venous thromboembolism.

Endothelial-dependent S-Sulfhydration of tissue factor pathway inhibitor regulates blood coagulation

Tissue factor pathway inhibitor (TFPI) is an important regulator of coagulation and a link between inflammation and thrombosis. Here we investigated whether endothelial cell-driven oxidative post-translational modifications could have an impact on TFPI activity. We focused on S-sulfhydration, which is a hydrogen sulfide-dependent post-translational modification that, in endothelial cells, is regulated by the enzyme cystathionine γ-lyase (CSE). The study made use of human primary endothelial cells and blood from healthy individuals or subjects with atherosclerosis as well as from mice lacking endothelial CSE. TFPI was S-sulfhydrated in endothelial cells from healthy individuals and mice, while the loss of endothelial CSE expression/activity reduced its modification. Non-S-sulfhydrated TFPI was no longer able to interact with factor Xa, which facilitated the activation of tissue factor. Similarly, non-S-sulfhydratable TFPI mutants bound less protein S, while supplementation with hydrogen sulfide donors, preserved TFPI activity. Phenotypically, loss of TFPI S-sulfhydration increased clot retraction, suggesting that this post-translational modification is a new endothelial cell-dependent mechanism that contributes to the regulation of blood coagulation.

Protein S: a Central Regulator of Blood Coagulation.

Protein S is a central regulator of coagulation as it critically participates in down-regulation of both extrinsic and intrinsic pathways of the coagulation cascade. In this review, we aim to provide an update on protein S and its anticoagulant functions as a central hemostatic regulator. Electronic databases including, Google, Google Scholar, PMC, PubMed, Science Direct, and Scopus were rigorously searched using the terms protein S, hemostasis, natural anticoagulants, regulators of coagulation, and coagulation inhibitors for the completion of this descriptive review. Literature review shows that protein S is a potent cofactor for activated protein C (APC) in the regulation of the intrinsic pathway and a cofactor for tissue factor pathway inhibitor (TFPI) in the regulation of the extrinsic pathway. The strong association between protein S deficiency either hereditary or acquired and increased risk for venous thrombosis indicates the important and central role of protein S in controlling the initiation and propagation phase of coagulation cascade and that protein S is an important determinant for optimal activity of both APC and TFPI in coagulation regulation. Available evidence suggests that the role of protein S in the down-regulation of blood coagulation is mainly mediated through its high affinity binding to negatively charged phospholipid surfaces. This high affinity binding to negatively charged phospholipids helps bring the anticoagulant proteins to the membranes, resulting in efficient and targeted regulation of coagulation. In the shade of current COVID-19 pandemic, protein S deficiency has been found to be a leading cause of thrombotic complications associated with COVID-19.

COVID-19 Is Associated with Endothelial Dysfunction and Enhanced Plasma Thrombin Generation Despite Pharmacological Thromboprophylaxis

BackgroundHospitalised patients with severe COVID-19 (requiring critical care level support) appear to be at increased risk of thrombosis despite standard pharmacological thromboprophylaxis. The magnitude of thrombotic risk in patients with COVID-19 of moderate severity (not requiring critical care) is less clear. The optimal approach to thromboprophylaxis (and the role of intensified thromboprophylaxis) remains to be determined.Evidence of endothelial dysfunction has been widely reported in COVID-19 (particularly in severe COVID) and this may contribute to hypercoagulability.AimTo assess differences in patterns of hypercoagulability and endothelial dysfunction between a group of patients with moderate COVID-19 and a group of age-matched hospitalized patients (SARS-CoV-2 PCR negative) receiving low molecular weight heparin (LMWH) thromboprophylaxis.MethodsBlood was collected from individuals admitted to hospital with COVID-19 of moderate severity (not requiring critical care level support) and a group of age-matched patients admitted with infective/inflammatory illness (SARS-CoV-2 PCR negative). All subjects received standard-dose LMWH thromboprophylaxis, with blood drawn at 12 hours post-dose (and with measurement of anti-FXa activity levels). Circulating levels of endothelial & fibrinolytic markers including ICAM, PAI-1, VCAM, soluble thrombomodulin (sTM), and tissue plasminogen activator (tPA) were determined by ELISA. Thrombin generation (TG) in platelet-poor plasma was assessed by calibrated automated thrombography in the presence of tissue factor (Final concentration, 1pM & 5pM), thrombomodulin (TM) (Final concentration, 6.25nM), and an inhibitory anti-tissue factor pathway inhibitor antibody (anti-TFPI; Final concentration 100μg/mL).Results14 COVID-19 positive subjects and 11 hospitalized controls were recruited. There were no differences in mean age (69.7±4.5 vs 61.6±4.7 years; p= 0.2) or mean Body mass index (25.7±1.1 vs 22.7±1.2 Kg/m2; p=0.1) between groups. No COVID-19 patient or control required critical care support. In the COVID group, radiological evidence of pneumonitis [diffuse (n=3) or peripheral infiltrates (n=7)] was present in the majority of cases. None of the COVID-19 cases were requiring supplemental oxygen at the time of recruitment.All controls were admitted with either respiratory or urinary infection [radiological evidence of pneumonia in 4/11; supplemental oxygen requirement in 2/11, (28-36% FiO2 via nasal cannula)].Plasma levels of sTM, ICAM, PAI-1 & VCAM were similar in both groups. Levels of t-PA were significantly higher in the COVID group (8.31±4.35 vs 4.91±2.37 ng/mL; p= 0.005). Despite similar plasma anti-Xa activity in both groups (0.06 vs 0.04 IU/mL; p=0.2), mean endogenous thrombin potential (ETP) was significantly higher in the COVID group (1929±119.7 vs 1528±138.9 nM*min; p=0.02), although peak thrombin was similar (173.6±26 vs 161.5±31nM). ETP-TM ratio was similar between groups (0.3±0.1 vs 0.2±0.1; p=0.3).Despite increased ETP, the lag time to thrombin generation was significantly prolonged in the COVID group (8.3±0.6 vs 5.8±0.5 mins, p= 0.006). This pattern has previously been observed in vascular diseases associated with altered plasma tissue factor pathway inhibitor (TFPI) activity. In the presence of an anti-TFPI antibody, the difference in lagtime between groups was attenuated (4.7±0.2 vs 3.5±0.1 mins; p= 0.002) and the difference in overall thrombin generation (delta TG) between both groups became significantly increased (Fig.1).ConclusionPlasma thrombin generation is enhanced in patients with non-severe COVID-19 despite pharmacological thromboprophylaxis. Endothelial dysfunction is also observed in this group and appears to modulate parameters of plasma thrombin generation. The clinical implications of these observations are not known although clinical studies of intensified thromboprophylaxis in attenuating thrombotic risk and other complications are ongoing.Fig 1. Inhibition of TFPI activity enhances thrombin generation in COVID-19.In the presence of an inhibitory anti-TFPI antibody, peak plasma thrombin generation was enhanced in COVID-19 in contrast to that observed among SARS-CoV-2 PCR negative hospitalised patients (339.6+25.2 vs 247.4+10.1, p=0.01). [Display omitted] DisclosuresMaguire: Actelion: Research Funding; Bayer Pharma: Research Funding. Ni Ainle: Daiichi-Sankyo: Research Funding; Actelion: Research Funding; Leo Pharma: Research Funding; Bayer Pharma: Research Funding. Kevane: Leo Pharma: Research Funding.

Severe thrombophilia in a factor V‐deficient patient homozygous for the Ala2086Asp mutation (FV Besançon)

BackgroundCoagulation factor V (FV), present in plasma and platelets, has both pro‐ and anticoagulant functions. ObjectiveWe investigated an FV‐deficient patient (FV:C 3%, FV:Ag 4%) paradoxically presenting with recurrent venous thrombosis (11 events) instead of bleeding. Methods/ResultsThrombophilia screening revealed only heterozygosity for the F2 20210G>A mutation. Although thrombin generation in the patient's platelet‐poor plasma was suggestive of a hypocoagulable state, thrombin generation in the patient's platelet‐rich plasma (PRP) was higher than in control PRP and extremely resistant to activated protein C (APC). This was partially attributable to the complete abolition of the APC‐cofactor activity of FV and a marked reduction of plasma tissue factor pathway inhibitor antigen and activity. The patient was homozygous for a novel missense mutation (Ala2086Asp, FVBesançon) that favors a “closed conformation” of the C2 domain, predicting impaired binding of FV(a) to phospholipids. Recombinant FVBesançon was hardly secreted, indicating that this mutation is responsible for the patient's FV deficiency. Model system experiments performed using highly diluted plasma as a source of FV showed that, compared with normal FVa, FVaBesançon has slightly (≤1.5‐fold) unfavorable kinetic parameters (Km, Vmax) of prothrombin activation, but also a lower rate of APC‐catalyzed inactivation in the presence of protein S. ConclusionsFVBesançon induces a hypercoagulable state via quantitative (markedly decreased FV level) and qualitative (phospholipid‐binding defect) effects that affect anticoagulant pathways (anticoagulant activities of FV, FVa inactivation, tissue factor pathway inhibitor α level) more strongly than the prothrombinase activity of FVa. A possible specific role of platelet FV cannot be excluded.

The Impact of Anticoagulant Activity of Tissue Factor Pathway Inhibitor Measured by a Novel Functional Assay for Predicting Deep Venous Thrombosis in Trauma Patients: A Prospective Nested Case-Control Study.

ObjectiveDeep venous thrombosis (DVT) is a common complication in patients with traumatic injury. Tissue factor pathway inhibitor (TFPI) is a natural anticoagulant protein in the extrinsic coagulation pathway. However, the relationship between DVT after trauma and the anticoagulant activity of TFPI remains unclear. In this prospective study, we investigated the role of TFPI in trauma patients with DVT to evaluate whether the anticoagulant activity of TFPI measured by a new functional assay can be used to help predict the risk of DVT. Patients and methods: This prospective nested case-control study enrolled trauma patients and healthy volunteers. Forty-eight trauma patients diagnosed with DVT and forty-eight matched trauma patients without DVT were included in the study. 120 healthy volunteers were also included as controls. Blood samples and case information were collected at admission. Patients accepted angiography before surgery to diagnose DVT. The parameters examined included TFPI anticoagulant activity, free-TFPI antigen, blood cell counts, and routine clinical coagulation tests. Results: For the parameters of TFPI anticoagulant activity, three were markedly increased in the DVT group compared to the non-DVT group (TFPI initial anticoagulant time ratio, P = .022; TFPI whole anticoagulant time ratio, P = .048; and TFPI anticoagulant rate, P = .034). The free-TFPI antigen concentration also showed a significant increasing trend in trauma patients with DVT compared with trauma patients without DVT (P = .035). Multivariate logistic regression analysis identified four independent factors for the development of DVT (TFPI initial anticoagulant time ratio, free-TFPI antigen, prothrombin time, and red blood cell count). We calculated the TFPI correlation coefficient and found that the area under the receiver operating characteristic curve was .821. Conclusions: A novel functional assay was developed to measure the anticoagulant activity of TFPI. The anticoagulant activity of TFPI can be used as a potential biomarker for diagnosing DVT in trauma patients.

Overexpression of Platelet Tfpiα Rescues TFPI Deficient Embryos from Mid-Gestational, but Not Late Gestational Lethality

Tissue Factor Pathway Inhibitor (TFPI) is an alternatively spliced Kunitz-type protease inhibitor required for murine embryonic development. On a mixed 129-C57BL/6 background, deletion of the first Kunitz (K1) domain of TFPI (Tfpitm1Gjb; Tfpi-/-) results in complete intrauterine lethality that occurs in about 60% of Tfpi-/- embryos during mid-gestation with the remaining 40% dying during late gestation. We recently discovered that this late gestation lethality is associated with severe cerebrovascular defects that are completely absent in Tfpi-/- embryos harboring genetic deletion of Factor V (FV), indicating that regulation of FV-dependent procoagulant activity by TFPI is necessary for proper cerebral vascular development. In mice, TFPIα is the only isoform present in platelets, TFPIβ is the major isoform on endothelium, and TFPIγ is the major isoform in plasma. All isoforms inhibit the tissue factor-Factor VIIa (TF-FVIIa) catalytic complex and Factor Xa (FXa) via the K1 and K2 domains, respectively. TFPIα is distinct from other isoforms as it contains a basic C-terminus that enables inhibition of early forms of prothrombinase (FXa-FVa). To better understand the biological activities of platelet TFPIα (pTFPIα), a transgenic mouse model expressing TFPIα under control of the platelet specific murine Gp1ba promoter was generated on the C57BL/6NCrl background. The transgene (Tg) was bred onto the Tfpitm1Gjb congenic C57BL/6J (B6J) background to examine how overexpression of pTFPIα alters embryonic survival of Tfpi-/- mice. Out of 407 offspring derived by crossing Tfpi+/- females to Tfpi+/- Tg+ males, the expected 50% Tg transmission ratio was observed in Tfpi+/+ and Tfpi+/- mice at wean, yet no Tfpi-/- Tg+ or Tfpi-/- Tg0 mice were produced. When examining Tg function in assays measuring TF-FVIIa mediated generation of FXa, it was found that Tfpi+/+ and Tfpi+/- Tg+ mice had 4- to 5-fold increased platelet TFPI inhibitory activity over Tg0 littermates, while plasma TFPI activity and antigen were unaltered indicating functional expression and storage of transgenic TFPIα within platelets. To study the impact of pTFPIα overexpression on Tfpi-/- survival, 120 late gestation embryos derived from Tfpi+/- x Tfpi+/- Tg+ backcrosses were harvested between E14.5-18. In comparison to the 5% frequency of Tfpi-/- 129-C57BL/6 embryos observed at this time interval, the Tfpi alleles were normally distributed with 28% Tfpi+/+, 49% Tfpi+/- and 23% Tfpi-/-. Of the Tfpi-/- embryos, 86% were Tg+ and 14% were Tg0. However, only 21% of Tfpi+/+ embryos were Tg+ (50% expected) suggesting that the Tg caused lethality in Tfpi+/+ embryos, a finding not observed in the weaning age cohort. Given this, we hypothesized that underlying genetic factors were complicating survival outcomes. PCR-based DNA walking uncovered that non-independent assortment played a major role in the disproportionate embryo frequencies, as the Tg integrated into an intergenic region on chromosome 2, ~36.4 megabases proximal to, and in linkage with, the endogenous Tfpi locus. Additionally, by intercrossing Tfpi+/- mice on the congenic B6J background, we found that 14% of 359 embryos harvested between E14.5-16.5 were Tfpi-/- , which was higher than the 5% observed in 129-C57BL/6 embryos, indicating that strain specific genetic interactions modulate Tfpi-/- mid-gestational lethality. After accounting for recombination rates, male breeder haplotypes, and expected frequencies at late gestation, pTFPIα overexpression completely suppressed mid-gestational lethality of Tfpi-/- embryos. Histological analyses revealed that the Tg had no effect on development of cerebrovascular defects in Tfpi-/- embryos during late gestation consistent with their lack of survival to wean. In conclusion, underlying genetic factors complicated our analyses illustrating the importance of meticulously characterizing transgenic mouse models to avoid spurious interpretation of results. After accounting for these unexpected genetic effects, our analyses indicated that pTFPIα overexpression totally rescued Tfpi-/- embryos from mid-gestational lethality. However, late gestational lethality persisted likely because overexpression of pTFPIα did not prevent the severe cerebrovascular developmental defects occurring at this stage of development in Tfpi-/- embryos. Disclosures Mast: Novo Nordisk: Honoraria, Research Funding.

Relation between Tissue Factor Pathway Inhibitor Activity and Cardiovascular Risk Factors and Diseases in a Large Population Sample.

Tissue factor pathway inhibitor (TFPI) is a potent anticoagulant protein in the extrinsic coagulation pathway. In the present study, we aim to identify the cardiovascular determinants for total TFPI activity and its association with cardiovascular disease (CVD) and total mortality. Total TFPI activity was assessed in a selection of the population-based Gutenberg Health Study (n = 5,000). Statistical analysis was performed to identify the determinants for total TFPI activity as well as the associations with CVD and mortality. Multivariable linear regression analysis identified smoking (β 0.095 [0.054-0.136]) as a positive determinant for total TFPI activity, while diabetes (β -0.072 [-0.134 to -0.009]), obesity (β -0.063 [-0.101 to -0.024]), and history of coronary artery disease (CAD) were negatively associated with total TFPI activity, independent of age, sex, and the remaining cardiovascular risk factors. After adjustment for lipoprotein levels, the association between total TFPI activity levels and obesity and CAD was lost. The analysis additionally revealed a strong positive association between total TFPI activity levels and low-density lipoprotein (β 0.221 [0.204-0.237]). The Cox regression models revealed that a higher total TFPI activity, above 97.5th percentile of the reference group, was associated with an increased mortality risk (hazard ratio = 2.58 [95% confidence interval: 1.49-4.47]), independent of age, sex, and cardiovascular risk profile. In the Gutenberg Health Study population-based cohort, the highest percentage of total TFPI correlated with an increased mortality risk. While elevated TFPI may reflect endothelial cell activation, the associations between total TFPI activity and obesity and CAD, points to additional mechanistic interactions.

Increased tissue factor activity promotes thrombin generation at type 1 diabetes onset in children

ObjectiveIn type 1 diabetes (T1D), a prothrombotic status due to elevated coagulation factors coincides with metabolic derailment. In a previous study, we discovered altered thrombin generation profiles in children with T1D. These alterations are potentially most pronounced at T1D onset and ameliorated after insulin treatment. We tested this hypothesis in a longitudinal study, measuring thrombin generation together with coagulation parameters in children at T1D onset and during follow‐up.Materials and methodsTwenty‐three children (12 female, age: 9.4 [2.7‐17.3] years; median [range]) were tested at T1D onset and after long‐term insulin treatment. Thrombin generation was measured using calibrated automated thrombography. Tissue factor (TF) activity and tissue factor pathway inhibitor (TFPI) activity were measured using enzyme‐linked immunosorbent assay (ELISA).ResultsA procoagulant shift was observed in thrombin generation traces at T1D onset compared to follow‐up (time to peak: 5.67 [4.11‐7.67] min vs 6.39 [4.89‐10.44] min, P < .001). These alterations at T1D onset coincided with increased TF activity (5.18 [0.01‐12.97] pmol/L vs 2.67 [0.04‐10.41] pmol/L, P < .05) and increased TFPI activity (0.051 [0.038‐0.074] U/mL vs 0.035 [0.026‐0.056] U/mL, P < .05).ConclusionThe procoagulant shift in thrombin generation at T1D onset is a result of increased TF activity, but this effect is partially counterbalanced by increased TFPI levels. Elevated TF and TFPI levels hint to a fragile hemostatic balance at the endothelial lining of blood vessels. Additional prothrombotic stimuli may tip over this balance explaining the increased thrombotic risk of children with T1D.

Inhibitors of the blood coagulation process in patients with essential thrombocythemia.

: The aim of the study was to assess the activity of protein C, protein S and tissue factor pathway inhibitor in relation to the risk factors for thrombotic complications in patients with essential thrombocythemia.The study group consisted of 45 newly diagnosed patients with essential thrombocythemia. Protein S activity was determined by chromogenic method. Activities of protein C and tissue factor pathway inhibitor (TFPI) were determined using ELISAs.Significantly lower protein C and protein S activity but higher TFPI activity were found in patients with ET in comparison with the control group. TFPI activity was higher in women as compared to men, and in patients over 60 years of age compared with patients below 60 years of age. TFPI activity was higher in patients with leukocytes count at least 11 g/l than in patients with leukocytes count below 11 g/l and the difference almost reached statistical significance. Significantly lower protein C activity was found in patients with the JAK2V617F mutation, in comparison with essential thrombocythemia patients JAK2V617F (-).The reduced protein C and protein S activity may be one of the pathogenic factors of increased prothrombotic state in essential thrombocythemia patients. The decreased protein C activity in patients with the JAK2 V617F mutation seems to confirm the significant role of this mutation in the pathogenesis of thrombotic complications in essential thrombocythemia patients. Significantly increased TFPI activity in essential thrombocythemia patients above 60 years of age and with leukocyte count above 11 g/l expresses the activation of the compensatory mechanism for increased prothrombotic activity.

Is Neovascularization a Leading Mechanism of Intra-plaque Hemorrhage and Carotid Plaque Destabilization?

Introduction: The accelerating carotid plaque growth can cause fibrous cap rupture followed by embolization to brain vessels. Important role in this process is ascribed to thrombotic factors and their inhibitors although another mechanism based on rupture of deficient vessels formed inside the plaque within the neovascularization process is also proposed. Interestingly, the second sequence of events provoke intra-plaque hemorrhage without a fibrous cap rupture. To determine whether the neovascularization is a main process responsible for carotid plaque destabilization the current study was conducted in asymptomatic and symptomatic patients with carotid stenosis. Methods: There were 121 individuals included to the study. Among them, study group consisted of 86 consecutive patients undergoing carotid endarterectomy (23 symptomatic, 63 asymptomatic, mean age 68±7,4 years) and 35 individuals without any symptoms and signs of atherosclerosis served as a control group. The concentration of vascular endothelial growth factor (VEGF) and its receptors, sVEGFR1, sVEGFR2, and tissue factor (TF) and tissue factor pathway inhibitor (TFPI) (ELISA immunoassay) in plaque homogenates and blood plasma was measured. In addition, activity of TF and TFPI (actichrome assay) were also measured. Concentration and activity results of all tested parameters were recounted for the milligram of protein contained in the plasma and plaques homogenates. Results: Analyzing patients with symptomatic and asymptomatic carotid stenosis the only statistically significant difference was found in the concentration of VEGF in plaque homogenates which was significantly higher in symptomatic plaques (302,9 vs 182,1 pg/ml; p=0,005 and 88,0 vs 67,9 pg/mg protein; p=0,05). The plaques homogenate’s TF level recounted for the milligram of protein was nearly 300-fold higher and TF activity was 5-fold higher in comparison with their plasma concertation and activity in the study group. The concentration and activity of TFPI was also higher in the plaque homogenates then in the plasma. The activity of TFPI in plaque homogenates recounted for the milligram of protein was 25-fold higher than in the plasma of the study patients. The VEGF concentration in plaque homogenates was significantly higher than in the plasma of study patients. There was no difference in VEGF concentration in the plasma of the study group and the control group. The lowest concentration of VEGF-R1 recounted for the milligram of protein was observed in the plasma of the control group. Higher concentration of VEGF-R1 recounted for the milligram protein was found in the plasma of the study group, but the distinctly highest concentration of VEGF-R1 (more than 10-fold higher) was observed in the study group plaque homogenates. Reverse characteristic showed the concentration of VEGF-R2, the lowest level was observed in the plaque homogenates and the highest in the plasma of the control group. Conclusion: The neovascularization process might be a leading mechanism of intra-plaque hemorrhage and carotid plaque destabilization. The plaque homogenates and blood plasma of patients with severe carotid stenosis have a strong prothrombotic profile. The plaque homogenates of patients with carotid stenosis in contrary to plasma possess signs of outspread angiogenetic activity.

Effect of hormone replacement therapy on atherogenic lipid profile in postmenopausal women

BackgroundWomen develop cardiovascular disease (CVD) approximately 7–10 years later than men, but progress with similar risk after menopause. Recent studies suggest that hormone replacement therapy (HRT) is cardioprotective when initiated early after menopause, but the mechanisms involved are still unclear. ObjectiveIn the current study, we aimed to examine the effects of HRT treatment on the plasma atherogenicity in postmenopausal women. We studied the total lipid profile in blood samples collected in a randomized, double-blinded, placebo controlled clinical trial of women with a history of venous thrombosis (VT), the EVTET study. MethodsOne-hundred and forty postmenopausal women <70 years were included in EVTET and randomized either to active treatment (one tablet of 2 mg estradiol and 1 mg norethisterone acetate daily) (n = 71) or placebo (n = 69). Blood samples were taken at baseline and after 3 months and subjected to routine assessment of hemostatic factors and lipids. ResultsOur study show that HRT compared to placebo significantly reduced plasma levels of Lp(a), ApoA1, ApoB, total cholesterol (TC), HDL-C, LDL-C, TC/HDL-C and LDL-C/HDL-C ratio at 3 months. No effect was observed on ApoB/ApoA1 ratio or triglycerides. The change in Lp(a) was significantly and inversely correlated with the change in estradiol (r = −0.32; P = 0.001) and positively correlated to the change in lipids, tissue factor pathway inhibitor activity and antigen, protein C and fibrinogen (r between 0.26 and 0.45, p < 0.01). ConclusionIn sum, this study confirms a strong effect of HRT on atherogenic lipids with a large reduction in the pro-thrombotic Lp(a), suggesting an overall favorable effect on thrombogenicity after HRT replacement therapy in post-menopausal women at risk of VT.

Activation of TF-Dependent Blood Coagulation Pathway and VEGF-A in Patients with Essential Thrombocythemia

Background and objectives: Recent studies suggest that a vascular endothelial growth factor (VEGF-A) may be involved in the thrombotic process by stimulating the expression of tissue factor in vascular endothelial cells. Tissue factor (TF) can also stimulate the transcription of the gene encoding VEGF-A. The relationship between coagulation and angiogenesis in myeloproliferative neoplasms is not fully understood. The aim of this study was to evaluate the concentration of TF in relation to VEGF-A in the blood of patients with essential thrombocythemia (ET). Patients and methods: The study group consisted of 130, newly diagnosed patients with ET (mean age 61 years). The control group consisted of 35 healthy volunteers (mean age 51 years). Concentrations of VEGF-A, TF, and tissue factor pathway inhibitor (TFPI) were analysed using immunoenzymatic methods. TF and TFPI activities were performed using chromogenic assays. Results: The median concentration of TF Ag was 3-fold higher and the TF activity was more than 15-fold higher in ET patients than in normal individuals. There were no statistically significant differences in the TFPI concentration and activity between groups. VEGF-A was significantly increased in patients with ET (p < 0.000001). Analysis of correlations revealed a positive correlation between VEGF-A and TF Ag as well as a positive correlation between VEGF-A and TFPI activity. Conclusions: The simultaneous increase of TF concentration and activity, VEGF-A in the blood of patients with ET, as well as a positive correlation between the concentration of TF and VEGF-A demonstrates the coexistence of TF-dependent coagulation and activation of angiogenesis.

Evaluation of plasma levels of tissue factor and tissue factor pathway inhibitor in patients with psoriasis.

IntroductionPsoriasis is a chronic, recurrent, inflammatory skin disorder with systemic involvement. It has recently been established that psoriasis is associated with an increased cardiovascular risk. Chronic skin-specific inflammation may promote atherosclerosis. Myocardial infarction or stroke can also be a result of underlying haemostasis disorders. Disorders in fibrinolysis and thrombosis in patients with psoriasis have been observed by many authors.AimThis study points to the key role played by the tissue factor (TF) and tissue factor pathway inhibitor (TFPI) in the extrinsic pathway of blood coagulation and the potential influence of microvascular disorders in inflamed psoriatic skin on TF and TFPI activity.Material and methodsThe study included 47 patients with active psoriasis vulgaris, hospitalized in the Dermatological Ward of the Regional Specialist Hospital, Research and Development Centre in Wroclaw, as well as 18 people from the control group.ResultsThere were significant differences in the blood concentrations of TF and TFPI in patients with psoriasis when compared to the control group. A low TFPI concentration in psoriatic patients may indicate an increased risk of atherosclerosis. Interpretation of a decreased level of TF in patients with psoriasis is difficult because it seems to be at odds with observations among patients with other atherosclerosis risk factors such as hypertension, hyperlipidaemia, diabetes or smoking.ConclusionsIt appears that further studies are necessary to explain this problem, perhaps to include an evaluation of TF levels in psoriatic skin.

Lipid-Mediated Relation between Tissue Factor Pathway Inhibitor Activity and Cardiovascular Risk Factors and Diseases in a Large Population Sample

▪Background: Tissue factor pathway inhibitor (TFPI), a Kunitz-type serine protease, is a potent anticoagulant protein in the extrinsic coagulation pathway and acts by inhibiting both the FXa and the Tissue Factor-FVIIa complex. In contrast to total and free TFPI antigen levels, the reference values and clinical determinants of total TFPI activity have not yet been studied in detail in the general population. In the present study, we aim to identify the cardiovascular determinants for total TFPI activity and investigate its association with cardiovascular disease (CVD) and total mortality, in a population at large.Methods: For this study, the first 4779 subjects of the population-based Gutenberg Health Study were examined in a highly standardized setting. Total TFPI activity was assessed in platelet poor plasma by the Actichrome TFPI activity assay (American Diagnostica, Stamford, CT, USA). Sex-specific nomograms were developed to demonstrate the relation of total TFPI activity with age. Multivariable regression analysis was performed to assess the determinants of total TFPI activity and to evaluate the association with CVD. Cox-regression models, adjusted for age, sex, cardiovascular risk factors (CVRFs) and CVD, were calculated to investigate the association between total TFPI activity and total mortality.Results: The multivariable linear regression analysis identified smoking (β, 0.0952 [0.0541 to 0.136],) as a positive determinant for total TFPI activity, while diabetes (β, -0.0716 [-0.134 to -0.00905],), obesity (β, -0.0627 [-0.101 to -0.024],) and history of coronary artery disease (CAD) were negatively associated with TFPI activity, independent of age, sex and the remaining CVRFs. After adjustment for high-density lipoprotein levels (HDL), low-density lipoproteins (LDL) and triglycerides, the association between TFPI activity levels and obesity and CAD was lost. The analysis additionally reveals a strong positive association between TFPI activity levels and LDL (β, 0.221 [0.204 to 0.237],). The Cox regression models revealed that a higher TFPI activity, above 97.5th percentile of the reference group, was associated with an increased mortality risk (HR = 2.58, [95% CI: 1.49/4.47], p=0.00074) independent of age, sex, CVRFs, CVD, oral contraceptives and hormonal replacement therapy. After additional adjustment for LDL levels, this relation became stronger.Conclusion: To the best of our knowledge, this is the first large, epidemiological study of the general population, revealing an increased mortality risk in individuals with higher TFPI activity, independent of CVRFs and CVD and emphasized by LDL-levels. Additionally, it demonstrates the negative association between total TFPI activity levels and CVD, particularly CAD. Furthermore, an association between TFPI activity and obesity, mediated through lipoprotein particles is explored. Lastly, it describes the age- and sex-related reference range of total TFPI activity levels in a large population-based data set. DisclosuresNo relevant conflicts of interest to declare.

Pharmacodynamics, Pharmacokinetics and Safety of Bay 1093884, an Antibody Directed Against Human TFPI, in Patients with Factor VIII or IX Deficiency (With and Without Inhibitors): A Phase 1 Study

Background: BAY 1093884 is a fully human monoclonal antibody directed against the K1 and K2 domains of human tissue factor pathway inhibitor (TFPI). TFPI regulates the initiation of coagulation and is important for regulation of normal hemostatic response. In factor (F)VIII and FIX deficiency (hemophilia A [HA] and B [HB], respectively), lack of adequate amplification (FXa generation) in tandem with intact regulation contributes to reduced clotting. Inhibition of TFPI has the potential to normalize coagulation in hemophilia even in the absence of FVIII or FIX and it is expected to result in normalization of thrombin generation and consequently improvement in clot formation. TFPI is an attractive therapeutic target as it is unaffected by FVIII or FIX inhibitors and can be targeted in both HA and HB. A potential advantage of targeting TFPI is that downstream mechanisms that protect from excessive coagulation (including antithrombin), remain intact.Methods: BAY 1093884 was evaluated in a multicenter, open-label study in patients with severe HA or HB, with or without inhibitors. Safety, pharmacokinetics (PK) and pharmacodynamics (PD) were assessed after single intravenous (IV) (0.3 and 1 mg/kg) and subcutaneous (SC) (1, 3, and 6 mg/kg) doses. A multiple-dose (MD) cohort received BAY 1093884 150 mg SC once weekly for 6 weeks. Total and free TFPI protein levels and TFPI activity were measured. Thrombin generation assay (TGA) parameters were measured using a calibrated automated thrombogram. Clot formation was assessed using rotational thromboelastometry (ROTEM). A mechanistic physiology-based PK/PD (PBPK/PD) model was developed based on single-dose PK data observed in the study, and used to determine dosing in the MD cohort. Data for HA, HB, Japanese and inhibitor patient subgroups were analyzed separately.Results: Thirty-two patients were enrolled; 30 with HA and 2 with HB, of which 4 (3 HA, 1 HB) were included in the MD cohort. At least 4 patients were treated in each administration method/dose group. Clot formation using ROTEM was assessed in 6 patients. Total and free TFPI-related parameters showed dose-dependent effects, with the concentrations being below the lower limit of quantification for longer duration at higher doses. Improvements in TGA parameters were observed following both IV and SC administration; duration of improvement was longer at higher doses. BAY 1093884 led to shortening of the clotting time in all 6 patients tested. Dose-dependent increases in plasma concentration exceeding dose proportionality were seen following IV and SC administration, consistent with expectations of target-mediated drug disposition for BAY 1093884. Moderate accumulation was observed after MD. Limited information on subgroups (patients with inhibitors [n=4], patients with HB [n=2] and Japanese origin [n=1]) suggests that the PK and PD data for these subgroups were consistent with those from non-inhibitor HA patients. The PBPK/PD model adequately described the concentration time course and total TFPI following single-dose IV and SC administration of BAY 1093884; the PK and PD data from the MD cohort were as predicted by the model. The model also supported that the PK/PD of BAY 1093884 are independent of inhibitor status. No serious adverse events, deaths, or adverse events leading to discontinuation were observed. Fibrinogen and platelet counts remained within normal range at most visits and no meaningful changes in levels of antithrombin, protein C, or FV were seen. The mechanisms for anticoagulation and fibrinolysis were intact.Conclusions: Overall, BAY 1093884 gave no safety concerns in the dose range evaluated. Results suggest that BAY 1093884 shifts hemostatic balance towards greater thrombin generation and clot formation as intended. PK/PD characteristics were similar for inhibitor and HB patients as compared with HA patients, potentially supporting use in all of these patient types. The PBPK/PD model developed can be used for dose selection in future studies. Treatment efficacy will be evaluated in a future study. DisclosuresChowdary:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Swedish Orphan Biovitrum AB (Sobi): Honoraria; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Freeline: Consultancy; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bayer: Honoraria. Lissitchkov:Novo Nordisk: Other: Investigator fees as a participant of the clinical trial. Willmann:Bayer: Employment. Schwers:Bayer: Employment. Michaels:Bayer: Employment. Shah:Bayer: Employment.

Children with Inflammatory Bowel Disease Exhibit Insensitivity to Tissue Factor Pathway Inhibitor

Background: Patients with inflammatory bowel diseases (IBD) exhibit chronic inflammation of the digestive tract associated with a prothrombotic shift in the plasmatic coagulation system as well as heightened platelet reactivity and preactivation. Recently, microbial and platelet-derived inorganic polyphosphate has been shown to inhibit tissue factor pathway inhibitor (TFPI), thus, influencing the hemostatic balance during inflammation. We hypothesized that polyphosphate plays a role in the pathophysiology of inflammatory bowel disease resulting in refractoriness to TFPI activity.Aims: We aimed to determine the hemostatic sensitivity to exogenous TFPI in IBD patients and healthy controls.Methods: Plasma from pediatric patients with active Crohn's disease (CD) (N=10) or ulcerative colitis (UC) (N=10) and age-matched healthy controls (N=20) was spiked with recombinant TFPI (150 ng/ml). Thrombin generation with/without exogenous TFPI was performed using Calibrated Automated Thrombography. Differences in lag time of thrombin generation with/without TFPI were calculated (∆lag time).Results: Without addition of exogenous TFPI, IBD patients exhibited a shorter lag time than controls (IBD: 2.65±0.41 min; Controls: 3.72±0.48 min, P<0.001) Addition of exogenous TFPI prolonged the lag time significantly in healthy controls (∆lag time= 4.69±0.61 min; P<0.001), while the lag time was just slightly prolonged in IBD patients (CD: ∆lag time=0.92±0.37 min; P<0.001; UC: ∆lag time=0.34±27 min; P<0.05).Conclusion: Plasma samples from pediatric IBD patients exhibit refractoriness towards the anticoagulant activity of exogenous TFPI. This hyposensitivity potentially extends to the action of endogenous TFPI which adds to the prothrombotic phenotype associated with IBD. Further studies are needed to determine potential associations with disease activity and the susceptibility to develop thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Role of ADTRP (Androgen-Dependent Tissue Factor Pathway Inhibitor Regulating Protein) in Vascular Development and Function.

BackgroundThe physiological function of ADTRP (androgen‐dependent tissue factor pathway inhibitor regulating protein) is unknown. We previously identified ADTRP as coregulating with and supporting the anticoagulant activity of tissue factor pathway inhibitor in endothelial cells in vitro. Here, we studied the role of ADTRP in vivo, specifically related to vascular development, stability, and function.Methods and ResultsGenetic inhibition of Adtrp produced vascular malformations in the low‐pressure vasculature of zebrafish embryos and newborn mice: dilation/tortuosity, perivascular inflammation, extravascular proteolysis, increased permeability, and microhemorrhages, which produced partially penetrant lethality. Vascular leakiness correlated with decreased endothelial cell junction components VE‐cadherin and claudin‐5. Changes in hemostasis in young adults comprised modest decrease of tissue factor pathway inhibitor antigen and activity and increased tail bleeding time and volume. Cell‐based reporter assays revealed that ADTRP negatively regulates canonical Wnt signaling, affecting membrane events downstream of low‐density lipoprotein receptor‐related protein 6 (LRP6) and upstream of glycogen synthase kinase 3 beta. ADTRP deficiency increased aberrant/ectopic Wnt/β‐catenin signaling in vivo in newborn mice and zebrafish embryos, and upregulated matrix metallopeptidase (MMP)‐9 in endothelial cells and mast cells (MCs). Vascular lesions in newborn Adtrp −/− pups displayed accumulation of MCs, decreased extracellular matrix content, and deficient perivascular cell coverage. Wnt‐pathway inhibition reversed the increased mmp9 in zebrafish embryos, demonstrating that mmp9 expression induced by Adtrp deficiency was downstream of canonical Wnt signaling.ConclusionsOur studies demonstrate that ADTRP plays a major role in vascular development and function, most likely through expression in endothelial cells and/or perivascular cells of Wnt‐regulated genes that control vascular stability and integrity.