Thrombin-activatable Fibrinolysis Inhibitor Research Articles

Hypofibrinolysis is associated with the severity of Covid-19 infection: The role of obesity

Introduction: Obesity, by causing hypofibrinolysis and thrombotic complications, ought to be a risk factor in terms of disease severity and mortality in COVID-19 infection.We aim to investigate the effects of obesity on fibrinolytic system in coronavirus patients while studying the changes of major fibrinolytic inhibitors plasminogen activator inhibitor (PAI-1) and thrombin activatable fibrinolysis inhibitor (TAFI). Material and methods: Sixty-six patients and 21 healthy donors were investigated and observed until either their recovery or death. Body mass index (BMI) was calculated for each person and peripheral blood used for PAI-1 and TAFI measurements. PAI-1 antigen and TAFI activated/inactivated (TAFIa/i) were measured using ELISA kits. Results: PAI-1 antigen and TAFIa/i plasma levels are higher in patients than in control group (P<00001 for both).Patients needing ICU had higher TAFIa/i values than non-ICU patients. The 15 patients who died had higher TAFIa/i levels than those staying alive.Obese patients (BMI . 30) had higher PAI-1 levels than non-obese patients. TAFIa/i is associated with D-dimer, C-reactive protein (CRP), ferritin, creatinine and neutrophile count, whereas PAI-1 is associated only with the serum creatinine level. Conclusion: Our study shows that the levels of the major fibrinolytic inhibitors PAI 1 and TAFI increase in patients with COVID-19 infection, and proves for the first time that PAI 1 levels increase more in obese patients than in non-obese ones. It is also revealed that the plasma TAFI level is associated with the severity of COVID-19 infection, which suggests that the inhibitory treatments against TAFI should be effective in preventing thrombotic complications in the course of COVID-19 infection.

Relationship Between Polymorphism of Thrombin-Activatable Fibrinolysis Inhibitor Gene +1040C/T and a Cohort of Chinese Women With Recurrent Spontaneous Abortion.

The balance between coagulation and fibrinolysis is essential for a successful pregnancy. This study aimed to explore the genetic variant of +1040C/T in the coding region of thrombin-activatable fibrinolysis inhibitor (TAFI) gene in women with recurrent spontaneous abortion (RSA) and in unrelated healthy controls and to investigate the possible association between TAFI +1040C/T polymorphism and RSA. Peripheral blood samples were collected from 137 Chinese patients with RSA and 103 unrelated healthy Chinese controls. The TAFI +1040C/T polymorphism was analyzed using SNaPshot SNP typing after DNA extraction. The frequency of the C allele was lower in RSA patients compared with the controls (0.78 vs 0.84). A subanalysis of the TAFI +1040C/T polymorphism in the 2 populations of RSA women (groups 2RSA and >2RSA) showed that the +1040CT genotype was significantly higher and the +1040CC genotype was significantly lower than from that found in controls. The allele +1040C was associated with a reduced risk of RSA in both group 2RSA (OR = 0.418, 95%CI, 0.255-0.685) and group >2RSA (OR = 0.473, 95%CI, 0.274-0.819) compared with controls. Our data indicate a protective role for TAFI +1040C allele against RSA, and may be associated with the genetic susceptibility of RSA.

Spontaneous fibrinolysis and possibilities of its acceleration in pulmonary embolism

This review contains the data concerning the mechanisms of spontaneous fibrinolysis in pulmonary vessels and possibilities of its acceleration in pulmonary embolism. The spontaneous fibrinolysis system is known to be sequential and multifactorial, with the interaction of accelerators (t-PA and u-PA) and inhibitors (alpha-2-antiplasmin, PAI-1, TAFI). The fibrinolytic processes take place in case of prevailing reactions of accelerating factors over inhibiting ones. The endothelium of pulmonary vessels possesses pronounced antithrombogenic and profibrinolytic properties, therefore, the processes of fibrinolysis in the pulmonary vascular bed normally occur more intensively than in the vessels of the systemic circulation. The membrane proteins of the endothelium annexins A2 activate plasminogen, whereas thrombomodulin inhibits the activity of PAI-1. The main approaches to increase the fibrinolysis intensity in conditions of pulmonary embolism may be aimed at elevating the activity of fibrinolytic enzymes (enhancing the synthesis of annexins A2, the use of NMDA-receptor antagonists) and suppressing its inhibitors (the use of monoclonal antibodies to alpha-2-antiplasmin, as well as plasminogen activator inhibitor-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI). Promising directions for future research can be the synthesis of a new generation of tissue-type plasminogen activators, and investigations of the possibility of clinical application of antithrombin and thrombomodulin, angiotensin converting enzyme inhibitors and cortisol antagonists. To meet these challenges, it is necessary to develop new models of venous thrombosis and acute pulmonary embolism in different animal species, with the assessment of the changes in the venous haemodynamics and pulmonary microcirculation on the background of administration of a new class of fibrinolytic agents.

Formalised assessment of systemic thrombogenicity in patients with Polycythemia vera suffered an ischemic stroke

Introduction. Thrombosis diagnosis and prevention in patients with Polycythemia vera (PV) suffered an ischemic stroke (IS) are still open. The aim was to find the reasons for systemic thrombogenicity and to compare the applicability of the main scales assessing thrombosis risk in patients with PV suffered IS.Materials and methods. We followed up 127 people (42-75 y.o.), of which 68 were patients with PV suffered IS (group I) and 59 non-PV-patients with ischemic stroke (group II). Clinical study included common blood analysis, rheological properties of erythrocytes, coagulation and endothelial parameters, cytokines, inflammation markers, angiogenesis markers, and testing for the V617F mutation in the JAK2 gene. The follow up included common and neurological examinations as well, and the assesment of thrombosis risk factors with both Caprini scale and CHA2DS2-VASc scale. All patients were examined twice as in the acute period of IS as well as in 16-18 months.Results. Between the groups no significant differences were found for the NIHSS average score and for Bartel index as well.The Caprini score belonged to the “very high risk” (score &gt; 6) in both groups in the acute period of IS. At the same time, the score “8-10 points” prevailed in group II (68%) whereas the score “11-12 points” prevailed in group I.In the acute time of IS the CHA2DS2-VASc score revealed 12% of patients from both groups who had a score of “3-4 points” (moderate risk of thrombosis).In group I thrombotic complications rate correlated significantly with the JAK2V617F gene allelicloading (r = 0.236; p &lt; 0.05), and the development of recurrence cerebrovascular disorders correlated significantly with Caprini score (r = 0.241; p &lt; 0.05), but not with CHA2DS2-VASc score.Aiming to predict thrombotic complications in PV-patients the threshold (cut off) points were established for those markers as factor VIII, factor VII, red blood cell deformability, thrombin activated fibrinolysis inhibitor (TAFI), red blood cell count, white blood cell count, t-PA, VEGF-A, p-thrombomodulin, and ADAMTS-13.This pattern of parameters showed the odds ratio of thrombotic complications 10.3 (95% CI 7.6-13.8) in PV-patients in thelong-term period.At the end of the follow up the Caprini score showed a trend towards a decreasing in total while the CHA2DS2-VASc score remained virtually unchanged.Conclusion. We assume the accurate assessment of thrombotic risk in patients with Polycythemia vera suffered an ischemic stroke requires a proposed pattern of parameters including the test for JAK2V617F allelicloading and the calculation of Caprini score but not CHA2DS2-VASc score. Final results may provoke to change standard antithrombotic therapy in those patients towards its intensification due to pathogenetic featues of cancer-associated thrombosis.

Activated platelet-based inhibition of fibrinolysis via thrombin-activatable fibrinolysis inhibitor activation system

AbstractOur previous real-time imaging studies directly demonstrated the spatiotemporal regulation of clot formation and lysis by activated platelets. In addition to their procoagulant functions, platelets enhanced profibrinolytic potential by augmenting the accumulation of tissue-type plasminogen activator (tPA) and plasminogen, in vivo in a murine microthrombus model, and in vitro in a platelet-containing plasma clot model. To clarify the role of thrombin-activatable fibrinolysis inhibitor (TAFI), which regulates coagulation-dependent anti-fibrinolytic potential, we analyzed tPA-induced clot lysis times in platelet-containing plasma. Platelets prolonged clot lysis times in a concentration-dependent manner, which were successfully abolished by a thrombomodulin-neutralizing antibody or an activated TAFI inhibitor (TAFIaI). The results obtained using TAFI- or factor XIII–deficient plasma suggested that TAFI in plasma, but not in platelets, was essential for this prolongation, though its cross-linkage with fibrin was not necessary. Confocal laser scanning microscopy revealed that fluorescence-labeled plasminogen accumulated on activated platelet surfaces and propagated to the periphery, similar to the propagation of fibrinolysis. Plasminogen accumulation and propagation were both enhanced by TAFIaI, but only accumulation was enhanced by thrombomodulin-neutralizing antibody. Labeled TAFI also accumulated on both fibrin fibers and activated platelet surfaces, which were Lys-binding-site-dependent and Lys-binding-site-independent, respectively. Finally, TAFIaI significantly prolonged the occlusion times of tPA-containing whole blood in a microchip-based flow chamber system, suggesting that TAFI attenuated the tPA-dependent prolongation of clot formation under flow. Thus, activated platelet surfaces are targeted by plasma TAFI, to attenuate plasminogen accumulation and fibrinolysis, which may contribute to thrombogenicity under flow.

Characterisation of a novel thrombomodulin c.1487delC,p.(Pro496Argfs*10) variant and evaluation of therapeutic strategies to manage the rare bleeding phenotype

IntroductionA novel variant in the thrombomodulin (TM) gene, c.1487delC,p.(Pro496Argfs*10), referred to as Pro496Argfs*10, was identified in a family with an unexplained bleeding disorder. The Pro496Argfs*10 variant results in loss of the transmembrane and intracellular segments of TM and is associated with an increase in soluble TM (sTM) in the plasma. The aim of this study was to characterise the effect of elevated sTM on thrombin generation (TG) and fibrinolysis, and to evaluate therapeutic strategies to manage the patients. MethodsPlasma samples were obtained from two patients carrying the variant. TG was triggered using 5 pM tissue factor and measured using the Calibrated Automated Thrombogram. A turbidity clot lysis assay was used to monitor fibrinolysis. TM antigen was quantified by ELISA. ResultsPatients with the Pro496Argfs*10 variant had significantly elevated plasma sTM compared to controls (372.6 vs. 6.0 ng/ml). TG potential was significantly lower in patients but was restored by inhibition of activated protein C (APC) or addition of activated Factor VII (FVIIa) or platelet concentrates. In vitro experiments suggested that activated prothrombin complex concentrates (APCC) posed a risk of thrombosis. The time to 50% lysis was significantly prolonged in patients compared to controls, 69.7 vs. 42.3 min. Clot lysis time was shortened by inhibition of activated thrombin activatable fibrinolysis inhibitor (TAFIa). ConclusionsOur data demonstrate that increased sTM enhances APC generation and reduces TG. Simultaneously, the rate of fibrinolysis is delayed due to increased TAFI activation by sTM. Treatment with platelet or FVIIa concentrates may be beneficial to manage this rare bleeding disorder.

Developing Transient Gene Therapies to Decrease the Stability of Thrombi for Coagulopathy and Thrombosis

Thrombotic disorders are prevalent and dangerous. Thrombosis involves the formation of unwanted blood clots inside blood vessels, which can impair blood flow and lead to severe cardiovascular events, such as heart attack, stroke, and pulmonary embolism. Current therapies for thrombosis are unsatisfactory in that they require frequent re-administration, and are associated with a significant bleeding risk, a consequence of inhibiting the coagulation cascade upstream of fibrin generation. Coagulation proteins factor XIII (FXIII) and thrombin activatable fibrinolysis inhibitor (TAFI) are enzymes that act to stabilize clots, downstream of fibrin generation and polymerization. Though this makes them ideal targets to reduce the burden of thrombosis while maintaining hemostasis, no inhibitors for either protein are currently available in the clinic. As we have recently published (Strilchuk, et al. Blood. 2020), lipid nanoparticles can be used to deliver siRNA to knock-down FXIII-B and achieve depletion of FXIII-A from circulation in mice and rabbits. FXIII-A depletion causes less antiplasmin to be crosslinked to clots, resulting in clots that are more susceptible to fibrinolysis. In the current abstract, we have expanded on preliminary data showing that while clots are weaker, bleeding is not enhanced in mice or rabbits after minor or major injury. We have also leveraged the same biological tools to knock-down TAFI for days after a single administration. In the short term, these novel RNA agents will be valuable tools in investigating the biology of thrombotic disorders. The ultimate goal is to develop these agents into precise and long-acting prophylactic therapies for thrombotic disorders, that are safer and more effective than current standards of care. Disclosures No relevant conflicts of interest to declare.

S62798, a TAFIa inhibitor, accelerates endogenous thrombolysis in a murine model of pulmonary thromboembolism

Abstract Pulmonary embolism (PE) is the third leading cause of cardiovascular death in western countries. The enhancement of fibrinolysis constitutes a promising approach to treat thrombotic diseases. In patients, venous thrombosis and thromboembolism risks are associated with increased plasma levels of TAFI (Thrombin Activatable Fibrinolysis Inhibitor) antigen as well as the active form TAFIa. S62798 is a competitive, selective and potent human TAFIa inhibitor (IC50±SD=11.2±0.4nM). It is however less potent on mouse TAFIa (IC50±SD=270±39nM). Here, we tested the ability of S62798 to enhance endogenous fibrinolysis in a mouse model of pulmonary thromboembolism. Human Tissue Factor (TF) was injected in C57Bl6 male mice. Ten minutes later, mice (n=4 to 14 per group) were treated (IV) with S62798 (from 0.01 to 100mg/kg) or vehicle (0.9% NaCl). Ten or twenty minutes (min) later, mice were anesthetized and lungs were collected, homogenized and pulmonary fibrin was quantified by ELISA. Results are expressed as ratio of geometric mean of pulmonary fibrin (μg/mL): tested treatment/ vehicle [95% confidence interval (CI)]. Ten minutes after S62798 treatment, pulmonary fibrin deposition was dose-dependently decreased with a Minimal Effective Dose of 0.04mg/kg [90% prediction interval 0.037 - 0.051] and an ED50 of 0.03mg/kg [95% CI: 0.01; 0.06]. Mice were then treated with 0.1mg/kg S62798 or vehicle (10 min after TF induction) and fibrin deposition in lungs was quantified 10 and 20 minutes post S62798 treatment. The level of pulmonary fibrin deposition was significantly decreased (p&amp;lt;0.0001) compared to vehicle group (ratio 0.31 [0.21; 0.45] at 10 min; 0.35 [0.24; 0.51] at 20 min). Finally, the effect of S62798 (1mg/kg) in combination with heparin was evaluated (n=10/group). When administered 10 min before TF injection, heparin (2000IU/kg) significantly (p&amp;lt;0.0001) decreased pulmonary fibrin level (20 min post TF: ratio 0.03 [0.01; 0.05]). When treatment was done in a curative setting (10 min post TF), heparin alone had no effect (p=0.85) on fibrin deposition (ratio 0.96 [0.65; 1.43]) whereas a similar significant (p&amp;lt;0.0001) decreased pulmonary fibrin deposition was observed in response to S62798 alone or associated with heparin (ratio 0.27 [0.18; 0.40] (S62798 alone) and 0.29 [0.20; 0.43] (S62798+heparin)). In this model, curative S62798 treatment, alone or associated to heparin, accelerated clot degradation by potentiating endogenous fibrinolysis and thus decreased pulmonary fibrin deposition. Due to its capacity to enhance endogenous fibrinolysis, S62798, which has completed phase I studies, is expected to be a therapeutic option for intermediate high risk PE patients on top of anticoagulants. With early recanalization, S62798 should rapidly reduce pulmonary artery pressure and resistance, with concomitant improvement in right ventricular function, preserving cardiac function, and reducing acute PE-related morbidity and mortality in these patients. Funding Acknowledgement Type of funding source: None

Favourable safety profile of S62798, a potent TAFIa (activated thrombin-activatable fibrinolysis inhibitor) inhibitor, in first-in-man study in healthy subjects

Abstract Background Thromboembolic diseases, such as venous thromboembolic diseases represent a significant public health burden. S62798, a selective and potent inhibitor of activated thrombin-activatable fibrinolysis inhibitor (TAFIa), is being developed as a fibrinolysis enhancer for the treatment of thromboembolic diseases, including pulmonary embolism. TAFIa inhibits the fibrinolysis by limiting plasmin generation. Objectives This double-blind, randomised, placebo-controlled first-in-man study was conducted to investigate the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of single ascending doses of S62798 in healthy subjects. Methods Young male (18–40 years) subjects were randomised 3:1 to single ascending doses of S62798 ranging from 3.2 mg to 320 mg or placebo, administered as an intravenous bolus followed by a 7 hour 2-step infusion (part 1), or to 30 mg or 40 mg or placebo administered as a 1-min bolus (part 2). Follow-up ranged from 6 to 13 days post S62798 administration depending on cohorts. Exclusion criteria included bleeding disorders, recent bleeding, and abnormal blood coagulation parameters. Anticoagulant/antiplatelet treatments were not allowed for 30 days prior to and during the study. Safety was assessed by recording adverse events (AE), physical examination, vital signs, biochemical, haematological and haemostasis parameters (prothrombin time, activated partial thromboplastin time, thrombin clotting time, D-Dimers and fibrinogen). Plasma and urine samples for PK assessment were collected pre-dose and up to day 13 to assess the full PK profile. In part 1, ex vivo TAFIa inhibition (spiking of purified human TAFIa) and in vitro clot lysis time were assessed at pre-dose, during and after infusion. The study was performed in France in accordance with the declaration of Helsinki revised in Fortaleza. EudraCT number: 2016–002108–25. Results A total of 56 subjects were randomized to part 1 and 16 to part 2. Subjects were (mean ± SD) 29.5 ±- 5.7 years old with a BMI of 23.9±2.6 kg/m2 in part 1 and 31.4±5.2 years old with a BMI of 23.9±2.5 kg/m2 in part 2. All doses of S62798 administered as a bolus and infusion or a single bolus were well tolerated, and no serious adverse events or discontinuations resulting from AEs occurred during the study. No effects of S62798 on haemostasis parameters were observed. Cardiac safety assessed by 12-lead 24h Holter ECG showed no relevant abnormalities. Plasma exposure of S62798 increased proportionally with the dose. S62798 inhibits TAFIa activity and decreases clot lysis time (reflecting TAFIa activity inhibition) rapidly and dose-dependently in all treated groups. Conclusions S62798, a potent TAFIa inhibitor, has a favourable safety profile with a linear PK. PD results on TAFIa inhibition and clot lysis assay suggest that S62798 has relevant properties to pursue clinical development as an enhancer of endogenous fibrinolysis for the treatment of thromboembolic diseases Funding Acknowledgement Type of funding source: Private company. Main funding source(s): IRIS

Determinants of plasma fibrin clot lysis measured using three different assays in healthy subjects

IntroductionSeveral methods for measuring fibrinolytic capacity in plasma have been developed yielding frequently inconsistent results. We investigated which factors determine fibrinolytic capacity in three plasma-based assays. Material and methodsIn 80 apparently healthy controls (aged 43 ± 10 years, 50 women [62.5%]) we evaluated fibrinolysis using three assays: (1) by Pieters et al. (CLT2018), (2) by Lisman et al. (CLT), and (3) by Carter et al. (Lys50). Coagulation factors and fibrinolytic proteins, including histidine-rich glycoprotein (HRG) and γ′-fibrinogen, were determined. Regression models were performed to identify determinants of lysis times. ResultsPositive correlations were observed between CLT2018 and both CLT (r = 0.73) and Lys50 (r = 0.61), as well as between CLT and Lys50 (r = 0.46, all p < 0.001). The main determinants of both CLT2018 and CLT were plasminogen activator inhibitor-1 (PAI-1), followed by thrombin-activatable fibrinolysis inhibitor (TAFI) and α2-antiplasmin. Histidine-rich glycoprotein was a predictor of the longest-normal CLT2018 alone (OR 1.04, 95% CI 1.02–1.06). α2-Antiplasmin and fibrinogen levels, followed by PAI-1 and TAFI determined Lys50. After adjustment for age, sex, and body mass index, C-reactive protein (CRP) was an independent predictor of the top quartiles of the three lysis times. ConclusionsWe showed that apart from PAI-1, TAFI, and α2-antiplasmin, several other factors, in particular CRP, can affect the results of global fibrinolysis tests used in research. Our findings may help understand why the choice of a specific fibrinolysis assay can affect the presence and/or magnitude of intergroup differences in fibrinolytic capacity in a given disease state.

Anabolic-Androgenic Steroid Abuse Impairs Fibrin Clot Lysis.

Abuse of anabolic-androgenic steroids (AASs) is suspected to increase the risk of cardiovascular disease (CVD) and cardiovascular mortality in otherwise healthy individuals. AAS abuse may increase the incidence of CVD by altering the hemostatic balance toward a procoagulant state. Studies on the effect of AAS abuse on the fibrinolytic system, however, have either demonstrated a profibrinolytic effect or no effect of AAS abuse, but the overall effect of AAS on fibrinolysis has not been addressed so far. This cross-sectional study investigated the effect of AAS on fibrin clot lysis, fibrin structure, and the hemostatic proteins, potentially affecting these measures in current and former AAS abusers and healthy age-matched controls. The study population consisted of 37 current and 33 former AAS abusers, along with 30 healthy age-matched controls. Fibrin clot lysis, fibrin structure properties, fibrinogen, coagulation factor XIII (FXIII) plasminogen, plasmin inhibitor, plasminogen activator inhibitor-1 (PAI-1), and thrombin activatable fibrinolysis inhibitor (TAFI) were determined. Fibrin clot lysis was significantly reduced in participants abusing AAS compared with former abusers and controls (p < 0.001). Plasma fibrinogen, plasminogen, and plasmin inhibitor were significantly increased in current abusers (p < 0.05). No significant differences were observed with respect to measures of fibrin structure properties, PAI-1, and TAFI (p > 0.05). In conclusion, AAS abuse depresses fibrin clot lysis. This effect is not associated with alterations in fibrin structure but is rather caused by increased plasma concentrations of fibrinogen, FXIII, and plasmin inhibitor. These findings suggest that AAS abuse may be associated with increased thrombotic disease.

Prediction-Determining Outcomes and Their Predictors in Atrial Fibrillation Patients Receiving Multicomponent Antithrombotic Therapy in Real Clinical Practice.

Aim Searching for clinical, angiographic, and biochemical predictors of cardiovascular complications (CVC) and hemorrhagic complications in patients with atrial fibrillation (AF) receiving a multicomponent antithrombotic therapy (MAT) for an elective percutaneous coronary intervention (PCI). Patients with ischemic heart disease (IHD) and AF who require MAT for PCI are at a high risk of thrombotic complications (stroke, systemic embolism, coronary events) and hemorrhage. This warrants searching for new risk factors determining prediction of the outcome.Materials and methods This study included 207 patients (146 males aged 70.1±8.3 years) with IHD and AF who received direct oral anticoagulants (DOAC) as a part of their MAT therapy. Median duration of the follow-up was 12 [8.0; 12.0] months. The efficacy endpoint was a sum of CVCs combining cardiovascular death, ischemic stroke, venous thromboembolic complications, acute coronary syndrome (ACS), and requirement for an unscheduled PCI. "Coronary events", including ACS and requirement for an unscheduled PCI were analyzed separately. The safety endpoint was BARC type 2-5 bleeding. Upon admission, biomarkers (growth-differentiation factor 15 (GDF-15), D-dimer, thrombin-activated fibrinolysis inhibitor (TAFI), and plasminogen activator inhibitor-1 (PAI-1)) were measured for all patients. Searching for prognostically significant indexes was performed with the Cox proportional hazards regression.Results Incidence of all CVCs was 16.4 %. Independent predictors of CVC included the DOAC treatment at a reduced dose (odds ratio (OR) 2.5 at 95 % confidence interval (CI) 1.02-6.15; p=0.0454), GDF-15 &gt;1191 pg /ml (OR 3.76 at 95 % CI, 1.26-11.18; p=0.0172), PAI-1 &gt;13.2 U/ml (OR 2.67 at 95 % CI, 1.13-6,26; p=0.0245). Incidence of coronary complications was 9.2 %. Independent predictors of coronary complications included a SYNTAX index &gt;26.5 (OR 4.5 at 95 % CI, 1.45-13.60; p=0.0090), PCI for chronic coronary occlusion (OR 3.21 at 95 % CI, 1.10-9.33; p=0.0326), a GDF-15 &gt;1191 pg/ml (ОR 4.70 at 95 % CI, 1.32-16.81; p=0.0172). Incidence of BARC type 2-5 bleeding was 26.1 %. The only independent predictor for hemorrhage complications was the total PRECISE-DAPT score &gt;30 (ОR 3.22; 95 % CI, 1.89-5.51; р&lt;0.0001).Conclusion Three independent predictors of CVC were identified for patients with IHD and AF treated with MAT following an elective PCI: treatment with a reduced dose of DOAC, GDF-15 &gt;1191 pg /ml, and PAI-1&gt;13.2 U/ml. Independent predictors of coronary complications included a SYNTAX index &gt;26.5, PCI for chronic coronary occlusion, and GDF-15 &gt;1191 pg/ml. The factor associated with a risk of bleeding was the total PRECISE-DAPT score &gt;30.

Increased levels of histidine-rich glycoprotein are associated with the development of post-thrombotic syndrome

Denser fibrin networks which are relatively resistant to lysis can predispose to post-thrombotic syndrome (PTS). Histidine-rich glycoprotein (HRG), a blood protein displaying antifibrinolytic properties, is present in fibrin clots. We investigated whether HRG may affect the risk of PTS in relation to alterations to fibrin characteristics. In venous thromboembolism (VTE) patients, we evaluated plasma HRG levels, plasma clot permeability, maximum absorbance, clot lysis time and maximum rate of increase in D-dimer levels released from clots after 3 months of the index event. We excluded patients with cancer and severe comorbidities. After 2 years of follow-up, 48 patients who developed PTS had 18.6% higher HRG at baseline. Baseline HRG positively correlated with clot lysis time, maximum absorbance, and thrombin-activatable fibrinolysis inhibitor (TAFI) activity but was inversely correlated with plasma clot permeability and maximum rate of increase in D-dimer levels released from clots. On multivariate regression model adjusted for age, fibrinogen and glucose, independent predictors of PTS were recurrent VTE, baseline HRG level, and TAFI activity. VTE recurred in 45 patients, including 30 patients with PTS, and this event showed no association with elevated HRG. Our findings suggest that increased HRG levels might contribute to the development of PTS, in part through prothrombotic fibrin clot properties.

A new pedigree with thrombomodulin‐associated coagulopathy in which delayed fibrinolysis is partially attenuated by co‐inherited TAFI deficiency

BackgroundThrombomodulin‐associated coagulopathy (TM‐AC) is a rare bleeding disorder in which a single reported p.Cys537* variant in the thrombomodulin gene THBD causes high plasma thrombomodulin (TM) levels. High TM levels attenuate thrombin generation and delay fibrinolysis. ObjectivesTo report the characteristics of pedigree with a novel THBD variant causing TM‐AC, and co‐inherited deficiency of thrombin‐activatable fibrinolysis inhibitor (TAFI). Patients/methodsIdentification of pathogenic variants in hemostasis genes by next‐generation sequencing and case recall for deep phenotyping. ResultsPedigree members with a previously reported THBD variant predicting p.Pro496Argfs*10 and chain truncation in TM transmembrane domain had abnormal bleeding and greatly increased plasma TM levels. Affected cases had attenuated thrombin generation and delayed fibrinolysis similar to previous reported TM_AC cases with THBD p.Cys537*. Coincidentally, some pedigree members also harbored a stop‐gain variant in CPB2 encoding TAFI. This reduced plasma TAFI levels but was asymptomatic. Pedigree members with TM‐AC caused by the p.Pro496Argfs*10 THBD variant and also TAFI deficiency had a partially attenuated delay in fibrinolysis, but no change in the defective thrombin generation. ConclusionsThese data extend the reported genetic repertoire of TM‐AC and establish a common molecular pathogenesis arising from high plasma levels of TM extra‐cellular domain. The data further confirm that the delay in fibrinolysis associated with TM‐AC is directly linked to increased TAFI activation. The combination of the rare variants in the pedigree members provides a unique genetic model to develop understanding of the thrombin‐TM system and its regulation of TAFI.

Hypofibrinolytic state and high thrombin generation may play a major role in SARS‐COV2 associated thrombosis

BackgroundThirty percent of Covid‐19 patients admitted to intensive care units present with thrombotic complications despite thromboprophylaxis. Bed rest, obesity, hypoxia, coagulopathy, and acute excessive inflammation are potential mechanisms reported by previous studies. Better understanding of the underlying mechanisms leading to thrombosis is crucial for developing more appropriate prophylaxis and treatment strategies. ObjectiveWe aimed to assess fibrinolytic activity and thrombin generation in 78 Covid‐19 patients. Patients and MethodsForty‐eight patients admitted to the intensive care unit and 30 patients admitted to the internal medicine department were included in the study. All patients received thromboprophylaxis. We measured fibrinolytic parameters (tissue plasminogen activator, PAI‐1, thrombin activatable fibrinolysis inhibitor, alpha2 anti‐plasmin, and tissue plasminogen activator‐modified ROTEM device), thrombin generation, and other coagulation tests (D‐dimer, fibrinogen, factor VIII, antithrombin). Results and ConclusionsWe observed two key findings: a high thrombin generation capacity that remained within normal values despite heparin therapy and a hypofibrinolysis mainly associated with increased PAI‐1 levels. A modified ROTEM is able to detect both hypercoagulability and hypofibrinolysis simultaneously in Covid‐19 patients with thrombosis.

Reductions in plasmin inhibitor and fibrinogen predict the improved fibrin clot lysis 6 months after obesity surgery.

Prothrombotic and metabolic variables are decreased after obesity surgery, and fibrin clot lysis is increased. It is unknown how fibrinolytic variables are affected, and whether fibrinolytic and metabolic changes predict the enhanced clot lysis. Study aims were to determine fibrinolytic biomarkers before and 6 months after Roux-en-Y gastric bypass (RYGB) and to identify predictors of the RYGB-induced increase in clot lysis. Women (n = 42) and men (n = 18) with obesity underwent RYGB, and factor XIII (FXIII), thrombin activatable fibrinolysis inhibitor (TAFI), plasminogen and plasmin inhibitor (PI) were measured before and 6 months after surgery. Regression analyses identified determinants of the RYGB-induced increase in clot lysis among changes in fibrinogen and in fibrinolytic and metabolic variables. Results showed that after RYGB, FXIII, TAFI, plasminogen and PI were reduced (P < .0005). Reductions in PI (β = -0.59) and fibrinogen (β = -0.35), together with age (β = -0.22) and male sex (β = 0.22), predicted the enhanced clot lysis with the model explaining 56% (P < .0005). Predictors of the reduction in PI were reductions in cholesterol (β = 0.37) and glucose (β = 0.29), together with male sex (β = -0.28), whereas reductions in fibrinogen were predicted by lowering of interleukin-6 (IL-6) (β = 0.32). In conclusion, fibrinolytic variables were reduced 6 months after RYGB. Targeting PI and fibrinogen, by reducing metabolic variables such as glucose, cholesterol and IL-6, has a profibrinolytic effect in obesity.

Plasma fibrin clot proteomics in patients with acute pulmonary embolism: Association with clot properties

It has been reported that 476 proteins can be detected in plasma fibrin clots from patients with venous thromboembolism. Plasma fibrin clots proteomic composition in relation to their properties has not been studied in acute pulmonary embolism (PE). Clots generated from plasma of 20 PE patients and 20 healthy controls were assessed using mass spectrometry, clot permeability (Ks), and clot lysis time (CLT). The proteomic composition of plasma fibrin clots from acute PE patients differed from that of control subjects in regard to 198 clot-bound proteins. In the acute PE group, we observed increased clot-bound fibrinogen, apolipoprotein B-100, platelet glycoprotein Ib, lipopolysaccharide-binding protein, and histones H3 + 4 and reduced fibronectin, α2-antiplasmin, α2-macroglobulin, factor (F)XIII, histidine-rich glycoprotein, antithrombin, von Willebrand Factor, plasminogen, and prothrombin. Among PE patients, low Ks (≤3.83 × 10−9 cm2) was associated with increased clot-bound C-reactive protein, kininogen-1, protein S, β-2-microglobulin, and thromboxane-A synthase when compared with patients having Ks > 3.83 × 10−9 cm2. Ks correlated inversely with FIX and FV, thrombin-activatable fibrinolysis inhibitor, complement C1s, C7, C8, and apolipoprotein A-I. The specific protein composition in plasma fibrin clots from acute PE patients is associated with denser clot formation. Several proteins unrelated to the coagulation system can modulate fibrin phenotype in acute thrombotic states. SignificanceOur study significantly advances the field of thrombosis and hemostasis. The plasma fibrin clot proteomics findings fill the gap of knowledge about the presence and the role of other proteins to the plasma fibrin clot in the acute phase of pulmonary embolism, aside fibrinogen, which is the main component of fibrin. The reported methodology, which involves the sample preparation using Multienzyme Digestion-Filter Aided Sample Preparation (MED FASP), data acquisition with the Quadrupole-Orbitrap mass spectrometer, and data analysis using the advanced tools such as MaxQuant, Total Protein Approach and Perseus, allows to gain not only the qualitative, but also the quantitative insights into the microworld of proteins entangled among the fibrin network. By comparing the clots formed from plasma of patients with acute pulmonary embolism with the clots from healthy control, we provide the specific protein composition associated with unfavorable clot properties observed in this disease. Moreover, our findings emphasize that several proteins unrelated to the coagulation system, can modulate fibrin phenotype in acute thrombotic states.

Endothelial cells orchestrate COVID-19 coagulopathy

Endothelial cells orchestrate COVID-19 coagulopathy

Similar hemostatic responses to hypovolemia induced by hemorrhage and lower body negative pressure reveal a hyperfibrinolytic subset of non-human primates.

To study central hypovolemia in humans, lower body negative pressure (LBNP) is a recognized alternative to blood removal (HEM). While LBNP mimics the cardiovascular responses of HEM in baboons, similarities in hemostatic responses to LBNP and HEM remain unknown in this species. Thirteen anesthetized baboons were exposed to progressive hypovolemia by HEM and, four weeks later, by LBNP. Hemostatic activity was evaluated by plasma markers, thromboelastography (TEG), flow cytometry, and platelet aggregometry at baseline (BL), during and after hypovolemia. BL values were indistinguishable for most parameters although platelet count, maximal clot strength (MA), protein C, thrombin anti-thrombin complex (TAT), thrombin activatable fibrinolysis inhibitor (TAFI) activity significantly differed between HEM and LBNP. Central hypovolemia induced by either method activated coagulation; TEG R-time decreased and MA increased during and after hypovolemia compared to BL. Platelets displayed activation by flow cytometry; platelet count and functional aggregometry were unchanged. TAFI activity and protein, Factors V and VIII, vWF, Proteins C and S all demonstrated hemodilution during HEM and hemoconcentration during LBNP, whereas tissue plasminogen activator (tPA), plasmin/anti-plasmin complex, and plasminogen activator inhibitor-1 did not. Fibrinolysis (TEG LY30) was unchanged by either method; however, at BL, fibrinolysis varied greatly. Post-hoc analysis separated baboons into low-lysis (LY30 <2%) or high-lysis (LY30 >2%) whose fibrinolytic state matched at both HEM and LBNP BL. In high-lysis, BL tPA and LY30 correlated strongly (r = 0.95; P<0.001), but this was absent in low-lysis. In low-lysis, BL TAFI activity and tPA correlated (r = 0.88; P<0.050), but this was absent in high-lysis. Central hypovolemia induced by either LBNP or HEM resulted in activation of coagulation; thus, LBNP is an adjunct to study hemorrhage-induced pro-coagulation in baboons. Furthermore, this study revealed a subset of baboons with baseline hyperfibrinolysis, which was strongly coupled to tPA and uncoupled from TAFI activity.

A case of thrombomodulin mutation causing defective thrombin binding with absence of protein C and TAFI activation

AbstractThrombomodulin functions as an anticoagulant through thrombin binding and protein C activation. We herein report the first case of hereditary functional thrombomodulin deficiency presenting with recurrent subcutaneous hemorrhage and old cerebral infarction. The patient had a homozygous substitution of glycine by aspartate at amino acid residue 412 (Gly412Asp) in the thrombin-binding domain of the thrombomodulin gene (designated thrombomodulin-Nagasaki). In vitro assays using a recombinant thrombomodulin with the same mutation as the patient showed a total lack of thrombin binding and activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI). Marked clinical and laboratory improvement was obtained with recombinant human soluble thrombomodulin therapy.