BACKGROUND: Glanzmann thrombasthenia (GT) is caused by an inherited defect of platelet αIIbβ3 integrin. Concizumab,a monoclonal antibody specific for Tissue Factor Pathway Inhibitor (TFPI), abolishes its anticoagulant effect.
OBJECTIVE: To evaluate the in vitro ability of concizumab to improve haemostasis in GT.
METHODS: The effects of concizumab were evaluated in whole blood or platelet-rich plasma (PRP) from GT patients (n=5-9) using a thrombin generation assay (TGA), rotational thromboelastometry (ROTEM), a global fibrinolytic capacity assay and a flow-chamber assay (T-TAS). Washed platelets (WP) and 20 nM recombinant activated factor VIIa (rFVIIa) were included for comparison.
RESULTS: The lag time in the TGA was significantly longer (+85%, p<0.0001) in GT patients than in controls. WP, rFVIIa and concizumab each significantly improved thrombin generation profiles. The ROTEM clotting time was significantly longer in GT patients than in controls (677 s vs 523 s; p=0.03). However, CT improved after adding WP, rFVIIa or concizumab. Under flow, occlusive thrombi were present in all healthy controls after 10 min, whereas platelet-fibrin depositions were not seen in GT patients. Sub-occlusive or occlusive thrombi formed when GT blood was mixed with WP, rFVIIa or concizumab. Clots in GT PRP were more susceptible to fibrinolysis and were improved by WP, rFVIIa or concizumab.
CONCLUSIONS: Concizumab enhanced thrombin generation, decreased the ROTEM CT, improved thrombus formation under flow and reduced clot lysis. Our results demonstrate the potential of concizumab for subcutaneous prophylaxis in GT patients.

UNASSIGNED: Several studies have examined parameters of increased thrombogenicity in COVID-19, but studies examining their association with long-term outcome and potential effects of antiviral agents in hospitalized patients with COVID-19 are scarce.
UNASSIGNED: To evaluate plasma levels of hemostatic proteins during hospitalization in relation to disease severity, treatment modalities, and persistent pulmonary pathology after 3 months.
UNASSIGNED: In 165 patients with COVID-19 recruited into the NOR-Solidarity trial (NCT04321616) and randomized to treatment with hydroxychloroquine, remdesivir, or standard of care, we analyzed plasma levels of hemostatic proteins during the first 10 days of hospitalization (n = 160) and at 3 months of follow-up (n = 100) by enzyme immunoassay.
UNASSIGNED: Our main findings were as follows: (i) tissue plasminogen activator (tPA) and tissue factor pathway inhibitor (TFPI) were increased in patients with severe disease (ie, the combined endpoint of respiratory failure [Po2-to-FiO2 ratio, <26.6 kPa] or need for treatment at an intensive care unit) during hospitalization. Compared to patients without severe disease, tPA levels were a median of 42% (P < .001), 29% (P = .002), and 36% (P = .015) higher at baseline, 3 to 5 days, and 7 to 10 days, respectively. For TFPI, median levels were 37% (P = .003), 25% (P < .001), and 10% (P = .13) higher in patients with severe disease at these time points, respectively. No changes in thrombin-antithrombin complex; alpha 2-antiplasmin; a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; or antithrombin were observed in relation to severe disease. (ii) Patients treated with remdesivir had lower levels of TFPI than those in patients treated with standard of care alone. (iii) TFPI levels during hospitalization, but not at 3 months of follow-up, were higher in those with persistent pathology on chest computed tomography imaging 3 months after hospital admission than in those without such pathology. No consistent changes in thrombin-antithrombin complex, alpha 2-antiplasmin, ADAMTS-13, tPA, or antithrombin were observed in relation to pulmonary pathology at 3 months of follow-up.
UNASSIGNED: TFPI and tPA are associated with severe disease in hospitalized patients with COVID-19. For TFPI, high levels measured during the first 10 days of hospitalization were also associated with persistent pulmonary pathology even 3 months after hospital admittance.

BACKGROUND: Befovacimab (formerly BAY 1093884) is a fully human monoclonal antibody able to bind to tissue factor pathway inhibitor (TFPI) and developed as a non-replacement therapy for individuals with haemophilia A/B, with or without inhibitors.
OBJECTIVE: To assess the safety of multiple escalating doses of befovacimab in individuals with severe haemophilia A/B with or without inhibitors.
METHODS: In this non-randomised, open-label Phase 2 study (NCT03597022), adult males with <1% factor VIII or <2% factor IX and ≥4 bleeds in the previous six months were enrolled in three dose cohorts (100/225/400 mg). Participants received befovacimab subcutaneously once weekly. The primary endpoint was safety; secondary endpoints included annualised bleeding rate (ABR) and pharmacokinetics/pharmacodynamics (PK/PD) of befovacimab.
RESULTS: A total of 24 participants (n = 8 in each dose cohort) were treated for 2-47 weeks. Patients treated with 100 mg and 225 mg doses of befovacimab demonstrated improved bleeding control compared with pre-study bleeding rates, with a dose-dependent effect. Dosing was suspended and the study prematurely terminated following three drug-related thrombotic serious adverse events (SAEs): two at the 225 mg dose and one at the 400 mg dose. These occurred in the absence of bleeding episodes or concomitant use of replacement/bypass therapies. No laboratory abnormalities were observed, and PK/PD data did not show correlation between SAE occurrence and levels of circulating befovacimab or free TFPI.
CONCLUSIONS: Despite favourable initial results from preclinical and clinical studies, a positive safety profile of befovacimab was not confirmed. The lack of SAE-related laboratory abnormalities or differentiating PK/PD characteristics in participants experiencing SAEs raises concerns about the predictability of thrombosis following befovacimab treatment and emphasises the need for further investigation into the therapeutic window of anti-TFPI treatment.

OBJECTIVE: Deep 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.

BACKGROUND: Sepsis typically results in enhanced coagulation system activation and microthrombus formation. Microparticle (MP) production promotes coagulation and enhances pro-coagulation. This study investigated how circulating MP levels and tissue factor-bearing MP (TF+-MP) activity caused coagulation in patients with septic disseminated intravascular coagulation (DIC).
METHODS: Thirty patients with septic DIC and 30 healthy controls were studied from December 2017 to March 2019. Patient blood samples were collected at enrolment (day 1) and on days 3 and 5; DIC scores and Sequential Organ Failure Assessment (SOFA) scores were recorded. TF+-MP activity was measured using TF-dependent factor Xa generation experiments. Circulating MP concentrations were determined by MP capture assay. Clotting factor activity, antithrombin level, soluble thrombomodulin, and serum tissue factor pathway inhibitor (TFPI) concentrations were measured.
RESULTS: Patients with septic DIC had lower circulating MP levels than healthy control patients. Circulating MP levels in patients with septic DIC were positively correlated with DIC scores and negatively correlated with coagulation factors, but TF+-MP activity did not correlate with clotting factor levels and TFPI.
CONCLUSIONS: In patients with septic DIC, circulating MP levels are important in promoting coagulation activation and increasing clotting factor consumption. TF+-MP activity may not be the main form of active TF.

BACKGROUND: Tissue factor pathway inhibitor (TFPI) is an endogenous protein that inhibits the extrinsic (tissue factor) pathway and negatively regulates thrombin production during coagulation. Inhibiting TFPI may become a useful target for haemophilia drug development to allow greater thrombin generation without use of the intrinsic (contact) pathway.
OBJECTIVE: The in vitro effects of befovacimab, a humanized TFPI neutralizing antibody, were studied in whole blood and plasma samples from patients with severe FVIII deficiency.
METHODS: Blood and plasma obtained from participants was supplemented in vitro with befovacimab (0.5, 1, 5, 10 and 100 nM) or recombinant factor VIII (rFVIII) 5-, 10- and 40% and analysed using rotational thromboelastometry (ROTEM), thrombin generation assay (TGA) and the dilute prothrombin time (dPT) assay. The in vitro coagulation effects of befovacimab were compared to samples supplemented with rFVIII.
RESULTS: Befovacimab induced consistent pro-coagulant responses in ROTEM parameters including reduction in clotting times and increases in α-angle; induced reductions in dPT clotting time; and improvements in TGA parameters (reduced lag time and increased thrombin generation parameters). There was a modest concentration-dependent response generally from 0.5- to 10 nM, after which, the pharmacodynamic effect plateaued through the 100 nM concentration. Befovacimab concentrations of 5 to 10 nM showed pro-coagulant activity comparable to blood samples supplemented with rFVIII 10-40%.
CONCLUSIONS: Befovacimab has modest dose-response effects from 0.5 to 10 nM with minimal improvement with higher concentrations. In vitro befovacimab blood concentrations of 5 to 10 nM had pro-coagulant effects similar to blood supplemented with rFVIII 10- to 40%.

Low plasma TFPI levels have been associated with an increased risk of DVT; however its association with TFPI gene polymorphisms is controversial and not yet studied in India. The aim of our study was to analyze prevalence of TFPI gene polymorphisms, evaluate their effects on its plasma levels and determine its association with DVT. Plasma level and genetic polymorphisms (33T>C, 399C>T and 536C>T) of TFPI were screened in subjects (100 DVT patients and 100 controls). Mean TFPI level in patients was significantly lower than controls (Patients: 33.55±11.72ng/ml, Controls: 48.05±13.68ng/ml, p<0.001). DVT patients had significantly higher prevalence of 399C>T (p=0.001, ORa: 5.69, CI: 1.14-28.46) and lower prevalence of 33T>C polymorphism (p<0.001, ORa: 0.239, CI: 0.065-0.871). The wild type (TT genotype) of 33T>C and variant form (CT and TT genotype) of 399C>T polymorphism was significantly associated with low TFPI levels. TFPI 536C>T polymorphism was absent in all subjects. In conclusion, dual nature of TFPI gene polymorphisms were established in our association study; 33T>C being protective and 399C>T as an important risk factor in Indian DVT patients, probably mediated by alteration in TFPI levels. These findings may prove a vital role in risk stratification and treatment of DVT.

Tissue factor pathway inhibitor (TFPI) is the main physiological inhibitor of TF-induced blood coagulation process, and may play essential roles in the pathogenesis of major adverse cardiac events. This study was designed to determine whether the variation of TFPI was related with coronary artery disease (CAD) in the Han Chinese populations.
A total of 1271 patients with coronary atherosclerosis and 1287 normal individuals from northern China were enrolled in the present study. Four tagging single-nucleotide polymorphisms (SNPs) (rs7586970, rs6434222, rs10153820 and rs8176528) from TFPI were selected and genotyped by direct sequencing. And the genotypes of the above SNPs were determined in all these participants.
In the populations from Beijing and Harbin, no significant case-control differences in the frequencies of TFPI polymorphism (rs10153820 and rs8176528) were observed between CAD patients and controls. Meanwhile, two SNPs of TFPI (rs7586970 and rs6434222) were found to be associated with CAD in both groups. In stratified analyses based on gender, smoking, hypertension, diabetes mellitus and hyperlipidemia, we further determined that the investigated genetic variations of the TFPI genes seemed to be related with diabetes mellitus in CAD patients.
Genetic variations of the TFPI genes seem to be related with CAD, which likely cooperate with metabolic risk factor (diabetes mellitus) and play critical roles in the pathogenesis of coronary artery disease.

BACKGROUND: Prophylaxis with either intravenous (i.v.) factor VIII (FVIII) or FIX is the gold standard of care for patients with severe hemophilia. A monoclonal antibody (concizumab) targeting tissue factor pathway inhibitor (TFPI) that can be administered subcutaneously (s.c.) has the potential to alter current concepts of prophylaxis in hemophilia.
OBJECTIVE: To evaluate the safety and describe the pharmacokinetics and pharmacodynamics of single-dose concizumab in healthy volunteers and patients with hemophilia A or B.
METHODS: In this first human dose, phase 1, multicenter, randomized, double-blind, placebo-controlled trial escalating single i.v. (0.5-9000 μg kg(-1) ) or s.c. (50-3000 μg kg(-1) ) doses of concizumab were administered to healthy volunteers (n = 28) and hemophilia patients (n = 24).
RESULTS: Concizumab had a favorable safety profile after single i.v. or s.c. administration. There were no serious adverse events and no anti-concizumab antibodies. No clinically relevant changes in platelets, prothrombin time, activated partial thromboplastin time, fibrinogen, or antithrombin were found. A dose-dependent procoagulant effect of concizumab was seen as increased levels of D-dimers and prothrombin fragment 1 + 2. Nonlinear pharmacokinetics of concizumab was observed due to target-mediated clearance. A maximum mean AUC0-∞ of 33 960 h μg mL(-1) and a maximum mean concentration of 247 μg mL(-1) was measured at the highest dose.
CONCLUSIONS: Concizumab showed a favorable safety profile after i.v. or s.c. administration and nonlinear pharmacokinetics was observed due to target-mediated clearance. A concentration-dependent procoagulant effect of concizumab was observed, supporting further study into the potential use of s.c. concizumab for hemophilia treatment.