Tissue Factor-induced Thrombin Generation Research Articles

Increased contact activated endogenous thrombin potential in pregnant women with preeclampsia.

Preeclampsia is a worldwide contributor to maternal and fetal morbidity and mortality. Women with preeclampsia are in a hyper-coagulable state with increased risk of thromboembolic disease later in life compared with normal pregnant women. The contact system (CAS) in plasma can mediate thrombin generation and is an important contributor to thrombus growth, but the activation of CAS during pregnancy complicated by preeclampsia is not yet elucidated, and CAS may play a role in the pathophysiology of preeclampsia. Therefore, the aim of the study is to address thrombin generation, and in particular, the capacity of the CAS-mediated pathway in patients with preeclampsia compared with pregnant controls. One hundred and seventeen women with preeclampsia and matched controls were included. The project was registered at www.clinicaltrials.gov as NCT04825145. CAS and tissue factor induced thrombin generation, proteins C and S, antithrombin, and histidine-rich glycoprotein (HRG) were assessed. Women with preeclampsia had significantly increased CAS and tissue factor-induced endogenous thrombin potential (ETP), and HRG compared with controls, P = 0.022, P = 0.024, and P = 0.02, respectively. The concentrations of protein C and antithrombin were significantly reduced in the preeclampsia group, P = 0.024 and P < 0.0001, respectively. No significant difference in the concentration of protein S was detected, P = 0.06. This study demonstrates a significant increased CAS-induced ETP and an overall decrease of important regulators of coagulation in women with preeclampsia compared with controls. These aspects can contribute to the hyper-coagulable state characterizing preeclampsia.

Diversity of Plasmatic Thrombin Pools Regulate Thrombin Generation and Blood Clotting: Interference By a Novel Nanobody

Background: Thrombin has a multitude of roles in the initiation and propagation of (anti)coagulation, in platelet activation and fibrin clot formation. The assessment of thrombin generation (TG) and thrombin-dynamics, combined with multi-omics data, has provided in-depth understanding of the plasma factors contributing to the (in)activation of thrombin in triggered plasma and whole-blood. By default, the generated thrombin is considered to act as a single pool. Here, we present evidence for at least two major proteolytically active thrombin pools with non-overlapping functions in blood clotting. Methods: A novel Syn-Nb-AF106 anti-fibrin nanobody (Nb106) was characterized, targeting a species-conserved thrombin-binding site exposed by fibrinopeptide A cleavage from fibrinogen (Sun et al, this meeting). Thrombin generation was assessed by calibrated automated thrombography using various triggers with control (multi-donor) and (reconstituted) coagulation factor-deficient plasmas, platelet-rich plasma or whole blood. Fibrin clot formation was evaluated by scanning electron microscopy (SEM). Clot retraction was macroscopically assessed. Results: In tissue factor-triggered thrombin generation,we discovered that Nb106 dose-dependently reduced the thrombin peak level and endogenous thrombin potential (ETP) to 54-62%, without affecting kinetic thrombin generation parameters. We observed a similar reduction in platelet-free plasma, platelet-rich plasma and whole blood. Biochemically, Nb106 displaced thrombin from a binding site on cleaved fibrinogen (Sun et al., abstract). Nb106 inhibited tissue factor-induced thrombin generation, using plasma deficient in factor IX, XI or XII, but not using plasmas deficient in fibrinogen or antithrombin. Reconstitution experiments showed that the inhibition depended on the fibrinogen concentration. A-specific control nanobodies were without effect. Together, the fibrin(ogen)-dependent and thrombin-inhibiting effect indicated that Nb106 operates by releasing proteolytically-active thrombin from fibrin to allow inactivation by antithrombin. Time-dependent, late spiking experiments revealed that Nb106 reduced the generation of thrombin only when added at 0-15 minutes after trigger, but no longer after 30 minutes, suggesting that the inhibitory capacity was due to a shortened thrombin binding to fibrin rather than to a continued protection towards antithrombin. In plasmas from a cohort of 64 healthy subjects, application of Nb106 showed a consistent reducing effect on the parameters peak and ETP, with a moderate positive correlation with the fibrinogen level. Nb106 suppressed the thrombin generation amplitude, but not the kinetics in the presence of a panel of direct oral anticoagulants (DOAC). Strikingly, brightfield microscopy and SEM indicated that Nb106, but not control nanobodies, in triggered plasma, platelet-rich plasma and whole-blood, fully abrogated the formation of elongating fibrin fibers. In agreement with this, it strongly suppressed whole-blood clot retraction. Conclusion: Collectivity, these data point to the existence of two major pools of proteolytically active thrombin, both of which contributing to thrombin generation, that are formed upon plasma and blood coagulation. One pool of soluble thrombin steers the proteolytic coagulation cascade, while another pool is bound to growing fibrin fibers. The latter pool still cleaves the conventional AMC thrombin substrate, is temporarily protected against antithrombin, and is required for fibrin fiber extension and blood clotting and clot retraction. The Nb106, replacing thrombin from its binding site on fibrin, selectively annuls the second pool, thereby blocking the clotting process. The replacement ensures thrombin inactivation by antithrombin. Perspective: The novel anti-fibrin nanobody Syn-Nb-AF106 or a derivative has the potential to act as an alternative anticoagulant in thrombotic diseases with pathological fibrin formation.

Generation of iPS-derived endothelial cells and use in organoids vascularizationin

The use of human pluripotent stem cells (iPS) for in vitro disease modeling and clinical applications requires protocols that convert these cells into relevant adult cell types with an efficiency rate. The major challenge in the organoids formation is the vascularization of these. Here, we report the rapid and efficient differentiation of human iPS into mature vascular endothelial cells capable of forming vessels in collagen gels. iPS cells were committed to a mesodermal fate and subsequent exposure to VEGF resulted in the differentiation of endothelial cells. The protocol produced mature cells with efficiencies over 80% within six days. Upon purification to 99% via VECadherin as a surface marker, endothelial cells maintained their identity until passage 5, as assessed by marker gene expression and immunostainings (CD31, VECadherin, vWF). Functional tests have been carried out. The capability of endothelials cells to form capillaries in matrigel was tested and confirmed. IPS-derived endothelial cells are also able to capture oxidized LDL and form a tight monolayer that is destabilized by thrombin treatment. Tissue factor-induced thrombin generation in platelet free plasma is reduced in the presence of iPS-derived vascular endothelial cells showing an antithrombotic phenotype. Endothelial cells were cultivated in collagen gel during one week and form structured vessels with lumen. All together these results showed relevant in vitro functionality of iPS-derived vascular endothelial cells. Our results suggest that these endothelial cells could be used to vascularized organoids structure.

Atrial fibrillation and age synergistically increase clotting potential and promote atrial structural remodeling in goats

Abstract Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Netherlands Heart Foundation, European Union Background Age is a risk factor for atrial fibrillation (AF) as well as for stroke in patients with AF. However, the effect of ageing on AF mechanisms and on coagulation activity is not well understood. Purpose To evaluate the effect of age and AF on coagulation activity, AF characteristics, and atrial structural remodeling. Methods Four groups of female goats were investigated: Young sham (Y-Sh: sinus rhythm, &amp;lt;3 years old, n=9), Young AF (Y-AF: 4 weeks of AF, &amp;lt;3 years old, n=7), Old sham (O-Sh: sinus rhythm, &amp;gt;8 years old, n=6), and Old AF (O-AF, 4 weeks of AF, &amp;gt;8 years old, n=8). Groups were matched for body weight. AF was maintained using implantable pacemakers in both AF groups. Clotting potential, expressed as the ability of activated plasma to generate thrombin, was measured using tissue factor-induced thrombin generation assays at baseline and 4 weeks (final). A terminal experiment was performed with atrial contact mapping to study electrophysiological AF properties and to collect atrial tissue for histological analysis. Results Thrombin generation analysis showed that 4 weeks of AF induced a significant increase in clotting potential in old, but not in young goats. AF complexity and hemodynamics were not affected by age alone. AF, however, significantly increased the right atrial pressure in old (O-Sh: 6.4±1.9 vs. O-AF: 11.4±3.1 mmHg, p=0.02), but not in young goats. AF induced atrial myocyte hypertrophy and left atrial epicardial endomysial fibrosis in old goats, while this was not the case in young goats. Conclusion Four weeks of AF and advanced age synergistically enhanced coagulation potential and promoted atrial structural remodeling in goats. Complexity of atrial conduction was affected by AF, but not by age.

Protection of β2GPI Deficient Mice from Thrombosis May Reflect a Platelet Activation Defect

IntroductionAntiphospholipid syndrome (APS) is an autoimmune disorder caused by “antiphospholipid” antibodies (aPL) directed against β2-glycoprotein I (β2GPI). Although β2GPI is proposed to have both anti- and procoagulant properties in vitro, its physiological role in coagulation in vivo is not well understood. Previous studies have shown that β2GPI deficient mice display impaired thrombin generation but failed to demonstrate an anticoagulant role in vivo. Recent findings from our laboratory demonstrated that β2GPI-deficient mice (APOH -/-) developed by CRISPR/Cas9 had shown delayed time to thrombosis as evidenced by prolonged clotting times in carotid artery occlusion models. However, no mechanism was identified for the delayed time to thrombosis phenotype.MethodsVenous thrombosis by complete occlusion of the IVC was performed as described previously (Wrobleski et al., 2011). In brief, an abdominal incision was made on anesthetized mice to visualize IVC. The activated partial thromboplastin time (aPTT); prothrombin time (PT) and Tissue factor-induced thrombin generation time (TGT) were performed as described previously (Stavrou et al., 2014). Protein C activity was performed according to manufacturer's recommendations (Chromogenix Coamatic® Protein C, Diapharma). To assess platelet activation, platelets were isolated under resting conditions using retro-orbital blood and the final washed platelet pellet was resuspended in Tyrodes solution. Platelets were stimulated with 0.25 U/ml thrombin and incubated with 1 µL of CD62P-FITC or Oregon Green conjugated-fibrinogen for 30 minutes. Platelets were fixed with 100 μL 2% formalin, and quantification of platelet surface P-selectin and fibrinogen binding to activated platelet GPIIb/IIIa was performed by flow cytometry (Accuri Flow Cytometer, BD Biosciences). For mouse tail vein bleeding time assays, mice were anesthetized and the tail transected approximately 5 mm from the tip. The tail was then placed in a 50 ml falcon tube containing saline pre-warmed to 37°C. The time to cessation of bleeding was determined visually. All experiments were performed on 8-12 week old mice.ResultsaPTT and PT results for APOH +/+ and APOH -/- mice were 41.82 ± 1.174 and 41.38 ± 2.026 seconds, and 14.55 ± 2.262 and 11.30 ± 0.578 seconds, respectively (NS). After IVC ligation, thrombi in APOH -/- mice had a mean weight of 16.94 ± 1.782 mg compared to 27.69 ± 1.725 mg in APOH +/+ mice littermates (P = 0.0002, Figure A). Thrombin generation induced by either tissue factor or aPPT reagent showed peak thrombin generation at 15 min with no difference between APOH +/+ and APOH -/- mice. Likewise, no significant differences were observed in percent Protein C activity levels between APOH +/+ (8.058 ± 1.433) and APOH -/- (6.453 ± 0.924).Stimulation with 0.25 U/ml thrombin resulted in significantly greater expression of P-selectin) on platelets of APOH +/+ (161.3 ± 30.62 MFI) compared to those from APOH -/- mice (54.47 ± 9.721 MFI) (P= 0.0101;Figure 1B). Likewise, there was significantly greater fibrinogen binding to stimulated platelets from APOH +/+ (129.7 ± 32.21 MFI) compared to APOH-/- mice (35.30 ± 2.144 MFI) with P = 0.0111 (Figure 1C). Finally, consistent with platelet activation studies, tail vein bleeding times were mildly, but significantly prolonged in APOH -/- mice (192.3 ± 45.86 sec) compared to APOH+/+ mice (95.14 ± 9.582 sec) with P = 0..ConclusionThe effects of β2GPI, if any, on coagulation processes is controversial, and has not been thoroughly studied in β2GPI deficient animals. The results presented here suggest that the smaller thrombi that form in mice deficient in β2GPI following IVC occlusion may reflect a mild platelet function defect. This hypothesis is supported by diminished P-selectin expression and fibrinogen binding in response to 0.25 U/ml thrombin by platelets from β2GPI deficient mice compared to wild-type littermates. Moreover, tail vein bleeding times were mildly prolonged in β2GPI deficient mice. Taken together, these studies suggest that β2GPI may actually contribute to hemostasis by supporting platelet responses to low concentrations of thrombin. Additional studies are needed to characterize these effects in detail. [Display omitted] DisclosuresMcCrae: Sanofi, Novartis, Alexion, and Johnson & Johnson: Consultancy, Honoraria; Dova, Novartis, Rigel, and Sanofi Genzyme: Consultancy.

B-PO01-019 COAGULATION POTENTIAL, ELECTROPHYSIOLOGICAL CHARACTERISTICS AND STRUCTURAL REMODELING DUE TO ATRIAL FIBRILLATION IN YOUNG AND AGED GOATS

Age is a risk factor for atrial fibrillation (AF) as well as for stroke in patients with AF. The effect of ageing on AF mechanisms and on coagulation activity is not well understood. To evaluate the effect of age and AF on coagulation activity, AF characteristics, and structural remodeling. Four groups of goats were investigated: Young sham (Y-Sh: sinus rhythm, <3 years old, n=9), Young AF (Y-AF: 4 weeks of AF, <3 years old, n=7), Old sham (O-Sh: sinus rhythm, >8 years old, n=6), and Old AF (O-AF, 4 weeks of AF, >8 years old, n=8). Groups were matched for body weight. AF was maintained using implantable pacemakers in both AF groups. Clotting potential was measured using tissue factor-induced thrombin generation (TG) assays at baseline and 4 weeks (final). A terminal experiment was performed with atrial contact mapping to study electrophysiological AF characteristics and to collect atrial tissue for histological analysis. The clotting potential, expressed as the ability of activated plasma to generate thrombin, did not differ among groups. However, AF in old goats caused an increase in coagulation potential (thrombin nM) while this was not the case in young goats. Complexity of fibrillatory conduction (no. of waves) was higher in AF groups after 4 weeks as compared to Sham, but there was no effect of age. AF-related atrial myocyte hypertrophy occurred in old but not in young goats. Endomysial fibrosis was similar in all groups. Four weeks of AF caused an increase in clotting potential and myocyte size in old but not in young goats. Age did not cause fibrosis or increased AF complexity.

Supra-Physiological Concentrations of C1 Esterase Inhibitor Enhance Thrombin Generation in the Presence of Thrombomodulin

Introduction. Human C1-esterase inhibitor (C1INH) is a multifunctional plasma glycoprotein with a broad spectrum biological activities, best known for its role in regulation of the classical complement pathway, contact activation system and its essential roles in the coagulation and fibrinolytic cascades [1,2]. The main targets of C1INH are C1 esterase, Factors XIa and XIIa, kallikrein, thrombin and plasmin. Low levels of functional C1INH in plasma are considered to be the primary cause for hereditary angioedema (HAE) with symptoms of swelling in the face, throat, hands, feet and abdomen. HAE patients receive replacement therapy with plasma-derived C1INH products recently licensed in the US and EU for prophylactic and acute treatment of HAE [1]. Studies suggest that supra-physiological doses of C1INH can promote thrombotic events.. To date the use of C1INH at plasma concentrations exceeding normal have been proposed in a range of disease states including renal transplant, myocardial infarction and acute lung injury. However, the mechanism and concentration required to augment the risk of thromboembolic events remains unclear and therefore raises questions about the safety of high dose C1INH therapy. We evaluated all possible sites of the C1INH concentration-dependent interaction with the coagulation system and here we focus on a hypothesis that supra-physiological concentrations of C1INH alter the anticoagulant protein C system. To study the interactive role of C1INH and the protein C pathway we performed thrombin generation assays (TGA) on plasmas from both male and female healthy donors in the presence of supra-physiologic concentrations of C1INH and increasing concentrations of thrombomodulin (TM).Methods. Platelet poor plasma (PPP) was isolated by centrifugation from blood of 20 healthy donors and mixed with 16 IU/ml of C1INH or with the same volume of buffer. The used C1INH concentration was 16 times bigger than normal values. Concentrations of TM added to the plasma samples were equal to 3, 6, 9, 12 and 15 nM. 4.5 pm tissue factor-induced thrombin generation was performed by continuous measurement of thrombin proteolytic activity of thrombin-specific substrate Z-Gly-Gly-Arg-AMC added to PPP before induction of coagulation. Thrombin peak was calculated using Origin software. Wilcoxon and sign paired tests with P=0.05 were used to obtain statistics.Results and conclusions. C1INH significantly reduced thrombin peak up to 2 times in the absence and at the lowest concentrations of TM (3 and 6 nM). Addition of 9 nM TM caused disappearance of the difference between the samples with and without C1INH. Finally, in the presence of 12 and 15 nM of TM, C1INH significantly enhanced thrombin peak (Figure 1A, B). Figure 1B demonstrates that reducing C1INH concentration can bring the thrombin peak back to the values obtained without C1INH presence. These results indicate that presence of TM crucially change the action of C1INH on coagulation and explains how supra-physiological concentrations of coagulation inhibitor may cause thrombosis in certain clinical situations.Figure 1. A: Effect of elevation of C1INH concentration on thrombin peak. Statistics was obtained using Wilcoxon (*) and Sign (**) paired tests with P value equal to 0.05 from 20 healthy donors. B: Representative dependence of thrombin peak on TM concentration with and without the presence of 16 IU/ml C1INH. C: representative dependence of Thrombin peak on C1INH concentration in the presence of TM.

Hyper-coagulability in obese patients accurately identified by combinations of global coagulation assay parameters

BackgroundRoutine laboratory methods are insensitive to hyper-coagulation, which may be detected by global hemostasis tests. Calibrated Automated Thrombogram (CAT) is a gold standard method to measure thrombin generation and coagulation potential. Thrombodynamics (TD) is a new global assay that monitors the spatio-temporal propagation of blood coagulation, separating initiation from amplification/propagation phases of coagulation and visualizing fibrin clot formation. AimWe investigated whether CAT and/or TD can identify hyper- and hypo-coagulable states in patients with well-characterized phenotypes and which parameters could be used as potential predictors of thrombotic risk. MethodsBlood was collected from: (1) forty healthy volunteers; (2) twelve obese patients scheduled for bariatric surgery (BMI ≥ 35 kg/m2); (3) nine patients under therapy with vitamin K-antagonists (median INR 2.7); (4) eight patients treated with low molecular weight heparins (anti-Xa activity between 0.5 and 0.7 U anti-Xa/ml); (5) ten patients with hemophilia A or B. Tissue factor induced thrombin generation was measured with CAT. Propagation of thrombin generation and clot growth from a tissue factor coated surface were monitored with TD. ResultsThrombin generation and fibrin clot formation parameters were significantly higher in obese patients compared to healthy volunteers and anticoagulated or hemophilic patients. ROC analysis of combined CAT or CAT/TD parameters (integrating thrombin generation and fibrin clot formation) demonstrated an excellent accuracy in detecting hyper-coagulability. ConclusionCombinations of CAT assay parameters and of parameters of thrombin generation burst with final fibrin clot properties allow recognizing accurately hyper-coagulable plasma and may represent predictive markers for thrombotic events.

Novel Thermostable Inhibitor of Contact Activation Tica Effectively Blocks Contact Activation in Low Tissue Factor Thrombin Generation

Background: Thrombin generation and other clotting assays suffer from a wide variation of pre-analytical variables. One of those pre-analytical variables is contact activation through blood withdrawal methods, different syringes, differences in blood coagulation tubes, blood transport and sample handling. It has been shown that the addition of contact activation inhibitors in low tissue factor activated thrombin generation leads to a correction of the, in these circumstances significant, increase in thrombin generation due to contact activation. We compare the novel 'thermostable inhibitor of contact activation' (TICA) to the current standard 'corn trypsin inhibitor' (CTI). Aim: Comparing the effectiveness of novel contact activation inhibitor TICA to the current standard CTI in low tissue factor-induced thrombin generation and recalcification in sodium citrate anticoagulated platelet poor plasma (PPP) and platelet rich plasma (PRP). Methods: We compared TICA, Corn trypsin inhibitor and plasma without contact activation inhibitors in low tissue factor PPP thrombin generation and in PRP recalcification thrombin generation, the latter the most sensitive condition for contact activation. In addition, we compared low tissue factor activated thrombin generation in plasma from severe hemophilia A patients with and without TICA during and after blood drawing. Thermostability - as a measure of shelf life - was measured and compared to CTI. Results: TICA is able to fully block contact activation in PRP recalcification experiments and is comparable to CTI in doing so. TICA significantly lowers low tissue factor induced thrombin generation by blocking contact activation. Pre-loading vacuum blood collection tubes with contact activation inhibitors is superior in inhibiting contact activation compared to addition of the inhibitor during the thrombin generation assay itself. TICA did not alter coagulation activity when added to FXIIa deficient plasma in thrombin generation. In contrast to CTI TICA is heat stable which will be of benefit to shelf life of pre-loaded blood drawing tubes. Conclusion: TICA is able to fully block contact activation and has several advantages over CTI. Disclosures No relevant conflicts of interest to declare.

In vitro effects of dalteparin on thrombin generation in canine plasma.

The calibrated automated thrombogram (CAT) is a functional thrombin generation (TG) assay that may provide a new approach for monitoring anticoagulant therapy in dogs. The effects of dalteparin on TG variables in dogs are unknown. Objectives were to establish normal TG variable ranges in dogs and measure the in vitro TG variables in canine pooled platelet-poor plasma (PPP) spiked with different dalteparin concentrations. In the first experiment, plasma samples from 25 adult healthy Beagle dogs and 11 client-owned healthy dogs of multiple breeds was measured individually for obtaining normal TG values. In the second experiment, separate pools of the remaining PPP from 24 of the 25 previous adult Beagles and from 45 different client-owned dogs were spiked with dalteparin at 9 concentrations with increasing anti-factor Xa (anti-FXa) activity. Activated partial thromboplastin time, tissue factor-induced TG, and anti-FXa activity were measured for each concentration. Concentration-response relationships were determined with ADAPT v.5, using various nonlinear regression models for stimulatory or inhibitory effects. Thrombin generation ranges of client-owned dogs and Beagles were equivalent only for time-to-peak (P < .05). In vitro dalteparin resulted in a concentration-dependent decrease in endogenous thrombin potential (ETP) in pooled PPP. The estimated dalteparin concentration that produced half the maximal inhibition of baseline ETP (IC50 ) was 0.289 U/mL. Thrombin generation and anti-FXa activity were more sensitive than APTT to detect the effects of dalteparin. The CAT assay can measure the effects of dalteparin in canine plasma, resulting in significant dose-dependent decreases in ETP, prompting further in vivo investigation.

Anticoagulant activity of oral rivaroxaban in healthy dogs

Rivaroxaban is an oral, direct factor Xa inhibitor used in human thrombotic disorders. In view of the in vitro concentration dependent anticoagulant effects of rivaroxaban in dogs, the time course of its anticoagulant effects was characterized in healthy dogs. Twenty-four healthy Beagles were randomized into three groups (n = 8 per group) and received orally either a placebo or 20 mg rivaroxaban once or twice at an 8 h interval. Fifteen blood samples were collected over a 30 h period, and blindly assayed for prothrombin time (PT), activated partial thromboplastin time (aPTT), tissue factor induced thrombin generation (TG) and anti-factor Xa activity. Thromboelastography (TEG) was evaluated at 0, 1, 4, 8 and 24 h. Peak/baseline anticoagulant effect ratios were analyzed with generalized linear models using β distributions and times to return to baseline with survival analyses (α = 0.05). Peak/baseline anticoagulant effect ratios of PT, aPTT, anti-factor Xa activity, TG and R (TEG) differed significantly between placebo and both rivaroxaban groups (P < 0.0001). The peak anticoagulant effect of rivaroxaban occurred 1.5 to 2 h after dosing. The median return to baseline occurred significantly sooner (P < 0.01) with 20 mg rivaroxaban administered once (7.9–18.7 h) versus twice (17.5–26.8 h). The inter-individual variability differed amongst assays, but overall was moderate to large. No adverse effects were recorded. Twice oral administration of 2 mg/kg rivaroxaban at an 8 h interval maintained 24 h anticoagulant activity, but larger studies are needed to establish guidelines for the use of rivaroxaban in dogs.

An in-vitro evaluation of direct thrombin inhibitor and factor Xa inhibitor on tissue factor-induced thrombin generation and platelet aggregation: a comparison of dabigatran and rivaroxaban.

Dabigatran and rivaroxaban may simultaneously inhibit coagulation and platelet activation. This study aimed to reveal the in-vitro effects of dabigatran and rivaroxaban on thrombin generation and platelet aggregation (PAg) derived via tissue factor (TF) pathway. Citrated blood was obtained from six healthy adults (26-60 years old) and pretreated with increasing concentrations of dabigatran or rivaroxaban. Plasmatic endogenous thrombin potential (ETP) was measured by the calibrated automated thrombogram method. The whole blood PAg was evaluated via a kinetic counting method. TF produced an ETP of 1904.69 ± 121.42 nmol min and a PAg of 78 ± 5%. Dabigatran and rivaroxaban concentration-dependently reduced ETP with half-maximal inhibitory concentrations of 460.1 ± 1.4 and 678.1 ± 1.4 nmol/l, and inhibited PAg with half-maximal inhibitory concentrations of 119.5 ± 1.5 and 77.5 ± 1.6 nmol, respectively. Dabigatran and rivaroxaban significantly inhibit TF-induced hypercoagulation and platelet activation in vitro in a concentration-dependent manner. Rivaroxaban displays stronger inhibition on thrombin generation and PAg than dabigatran.

Altered fibrinolysis in autosomal dominant thrombomodulin-associated coagulopathy

AbstractThrombomodulin-associated coagulopathy (TM-AC) is a newly recognized dominant bleeding disorder in which a p.Cys537Stop variant in the thrombomodulin (TM) gene THBD, results in high plasma TM levels and protein C-mediated suppression of thrombin generation. Thrombin in complex with TM also activates thrombin-activatable fibrinolysis inhibitor (TAFI). However, the effect of the high plasma TM on fibrinolysis in TM-AC is unknown. Plasma from TM-AC cases and high-TM model control samples spiked with recombinant soluble TM showed reduced tissue factor–induced thrombin generation. Lysis of plasma clots from TM-AC cases was significantly delayed compared with controls but was completely restored when TM/thrombin-mediated TAFI activation was inhibited. Clots formed in blood from TM-AC cases had the same viscoelastic strength as controls but also showed a TAFI-dependent delay in fibrinolysis. Delayed fibrinolysis was reproduced in high-TM model plasma and blood samples. Partial restoration of thrombin generation with recombinant activated factor VII or activated prothrombin complex concentrate did not alter the delayed fibrinolysis in high-TM model blood. Our finding of a previously unrecognized fibrinolytic phenotype indicates that bleeding in TM-AC has a complex pathogenesis and highlights the pivotal role of TM as a regulator of hemostasis.

Anti-inflammatory and anti-fibrinolytic effects of thrombomodulin alfa through carboxypeptidase B2 in the presence of thrombin

BackgroundThrombomodulin (TM) alfa, a recombinant human soluble TM, enhances activation of pro-carboxypeptidase B2 (pro-CPB2) by thrombin. Activated pro-CPB2 (CPB2) exerts anti-inflammatory and anti-fibrinolytic activities. Therefore, TM alfa may also have anti-inflammatory and anti-fibrinolytic effects through CPB2. However, these effects of TM alfa have not been elucidated. In the present study, we investigated the effects of TM alfa on inactivation of complement component C5a as an anti-inflammatory effect and prolongation of clot lysis time as an anti-fibrinolytic effect via CPB2 in vitro. MethodsCPB2 activity and tissue factor-induced thrombin generation was examined by a chromogenic assay. C5a inactivation was evaluated by C-terminal cleavage of C5a and inhibition of C5a-induced human neutrophil migration. Clot lysis time prolongation was examined by a tissue-type plasminogen activator-induced clot lysis assay. ResultsCPB2 activity in human plasma was increased by TM alfa and thrombin in a concentration-dependent manner. TM alfa inhibited tissue factor-induced thrombin generation and enhanced pro-CPB2 activation in human plasma simultaneously. The mass spectrum of C5a treated with TM alfa, thrombin, and pro-CPB2 was decreased at 156m/z, indicating that TM alfa enhanced the processing of C5a to C-terminal-cleaved C5a, an inactive form of C5a. C5a-induced human neutrophil migration was decreased after C5a treatment with TM alfa, thrombin, and pro-CPB2. TM alfa prolonged the clot lysis time in human plasma, and this effect was completely abolished by addition of a CPB2 inhibitor. ConclusionsTM alfa exerts anti-inflammatory and anti-fibrinolytic effects through CPB2 in the presence of thrombin in vitro.

PO-20 - Crosstalk between the lectin pathway and haemostasis in patients with pulmonary cancer

Recent research has focused on the complement system in cancer, including the lectin pathway of complement activation. Mannose-binding lectin (MBL), a key activator of the lectin pathway, can bind to tumor cell surfaces in vitro, and lectin pathway activation is increased in several types of cancer. The exact role of the complement system in cancer is currently discussed. However, one possible consequence of the increased complement activation could be contribution to the increased thrombosis risk which cancer patients experience. Proteins of the lectin pathway can activate coagulation and impair fibrinolysis in vitro, but the significance of this in a clinical setting is not well understood. We aim to investigate associations between lectin pathway and haemostatic activation in patients with lung cancer undergoing thoracoscopic surgery. Patients with lung cancer (n=60) eligible for thoracoscopic tumor resection were included as part of a randomized controlled trial, the COPPVATS project (EudraCT no: 2012-002409-23), conducted at the Department of Thoracic Surgery, Aarhus University Hospital. Exclusion criteria were arterial or venous thrombosis within the last three months, other systemic disease than cancer, and anticoagulant treatment prior to inclusion. Blood samples were obtained the day before surgery, perioperatively, and on the 1st and 2nd postoperative days. Laboratory analyses on the complement system include MBL, MBL-associated protease (MASP)-1 and -2, MBL-MASP complex, ficolin-1, -2, and -3 and complement factor C3b. Haemostasis was evaluated with routine coagulation parameters (INR, APTT, fibrinogen, fibrin d-dimer), platelet function, and tissue factor-induced thrombin generation. Recruitment of the study subjects is concluded, and laboratory work is in progress. The complement analyses and data processing will be performed during early spring 2016, so that results will be ready for presentation on the conference. The present project will provide new knowledge on lectin pathway activation and the pathogenesis of thrombosis in cancer patients. In the long term, this will help improve the individual risk assessment and lead to new studies on thromboprophylaxis and treatment in conditions with increased complement activation.

TMEM16F is required for phosphatidylserine exposure and microparticle release in activated mouse platelets

Phosphatidylserine (PtdSer) exposure on the surface of activated platelets requires the action of a phospholipid scramblase(s), and serves as a scaffold for the assembly of the tenase and prothrombinase complexes involved in blood coagulation. Here, we found that the activation of mouse platelets with thrombin/collagen or Ca(2+) ionophore at 20 °C induces PtdSer exposure without compromising plasma membrane integrity. Among five transmembrane protein 16 (TMEM16) members that support Ca(2+)-dependent phospholipid scrambling, TMEM16F was the only one that showed high expression in mouse platelets. Platelets from platelet-specific TMEM16F-deficient mice exhibited defects in activation-induced PtdSer exposure and microparticle shedding, although α-granule and dense granule release remained intact. The rate of tissue factor-induced thrombin generation by TMEM16F-deficient platelets was severely reduced, whereas thrombin-induced clot retraction was unaffected. The imaging of laser-induced thrombus formation in whole animals showed that PtdSer exposure on aggregated platelets was TMEM16F-dependent in vivo. The phenotypes of the platelet-specific TMEM16F-null mice resemble those of patients with Scott syndrome, a mild bleeding disorder, indicating that these mice may provide a useful model for human Scott syndrome.

Thrombin generation induced by tissue factor plus ADP in human platelet rich plasma: A potential new measurement to assess the effect of the concomitant use of an oral factor Xa inhibitor edoxaban and P2Y12 receptor antagonists

IntroductionPatients with atrial fibrillation undergoing percutaneous coronary intervention may require combination therapy with anticoagulants and antiplatelet agents. The objectives of this study were to establish an assay which can evaluate the effects of both anticoagulants and P2Y12 receptor antagonists and determine the effects of edoxaban, a direct factor Xa inhibitor, and P2Y12 receptor antagonists (clopidogrel and ticagrelor) alone and when combined. Material and methodsHuman platelet-rich plasma (PRP) from healthy subjects was stimulated with adenosine diphosphate (ADP) plus tissue factor. Thrombin generation was measured by means of calibrated automated thrombography. ResultsCombination of 10μM ADP and low concentration (0.25 pM) tissue factor induced reproducible thrombin generation in human PRP. Edoxaban (40 and 80ng/mL), active metabolite of clopidogrel (AM-clopidogrel, 10 and 20μg/mL), and ticagrelor (3μg/mL) alone inhibited ADP plus tissue factor-induced thrombin generation. Edoxaban suppressed all 5 parameters (lag time, peak, time to peak, endogenous thrombin potential, and maximum rate), whereas AM-clopidogrel and ticagrelor inhibited 4 and 3 parameters, respectively. Concomitant treatment with edoxaban and AM-clopidogrel or ticagrelor produced an additive inhibition of thrombin generation compared to the single treatments. ConclusionsThe thrombin generation assay induced by ADP plus tissue factor can detect the activities of both edoxaban and P2Y12 receptor antagonists. Combination of edoxaban and a P2Y12 receptor antagonist shows additive inhibition. These results suggest that ADP plus tissue factor-induced thrombin generation may be a useful measurement to assess the combination effects of anticoagulants and P2Y12 receptor antagonists in a single assay.

Thrombin Generation and Bleeding in Hemophilia Inhibitor Patients during Immune Tolerance Induction

Background: Among the most serious complications of hemophilia A is anti-VIII inhibitor formation, which occurs in ~25% of patients, typically in childhood within the first 20 exposures. Morbidity is high, hospitalization is frequent, and healthcare cost is high. Thus, immune tolerance induction (ITI), a program of regular F.VIII therapy, is used to eradicate the inhibitor. A landmark study demonstrated ITI was equally effective with high-dose (HD) (200 IU/kg/day) or low-dose (LD) (50 IU/kg 3x/week) F.VIII, but found a bleeding rate 2-fold greater in the LD arm. As the inhibitor neutralizes F.VIII, no difference in bleeding was expected. We hypothesized that thrombin generation assay (TGA), a global measure of tissue factor-induced thrombin generation that predicts bleeding in hemophilia better than F.VIII, might clarify this question.Methods: Following IRB approval, TGA was performed on thawed, re-calcified CTI /citrate plasma samples from the ITI repository, using re-lipidated tissue factor and a BioTek Synergy 4 fluorescence plate reader. TGA parameters, i.e. peak thrombin (peak T), estimated thrombin potential (ETP), maximum rate (MaxR), lag time (LAG), were compared by dose group, and repeated after F.VIII spike to neutralize the inhibitor and enhance TGA sensitivity. Significance was calculated by fitting generalized estimating equations linear models to account for serial measurements, with log-transformed TGA as outcome and dose group as predictor. Wilcoxon rank sum test was used to compare bleeds by dose group. Spearman correlation was used to compare TGA and bleeds, and TGA and inhibitor titer. The first TGA sample per subject was used in the analysis; and, separately, all TGA samples per subject. Sensitivity analysis was performed by 95% bootstrap confidence intervals to account for multiple measurements per subject. Total F.VIII antibody (NNA+NA) was analyzed by fluorescent immunoassay (FLI) and compared with anti-VIII (BU/ml) by Pearson correlation.Results: Samples (n=83) from 31 subjects, 14 LD and 17 HD, were available. There was no difference in peak T, ETP, MaxR, or LAG between LD and HD; after VIII spike (Table); nor between non-tolerized (n=56) and tolerized (n=27) subjects, all p>.05. There was no correlation between TGA and bleeding, nor between TGA and anti-VIII titer, all p>.05. In 19 subjects in whom anti-VIII fell to <1.0 BU, including 12 (70.6%) HD and 7 (50.0%) LD, the NNA+NA prevalence was 17/19 (89.5%). Anti-VIII titer and total F.VIII antibody (NNA+NA) were highly correlated, r=.807, p<.001. [Display omitted] Discussion: Despite ITI, the majority of inhibitor patients have detectable non-neutralizing inhibitors. Further, TGA is detectable despite the presence of inhibitors, but does not predict bleeding by ITI dose arm, suggesting thrombin generated after F.VIII dosing may be too low or too short-lived to detect. Limitations include the small sample size and random post-F.VIII sampling. Future studies should evaluate TGA prospectively, early after dosing to determine if thrombin generation predicts bleeding in inhibitor patients. DisclosuresNo relevant conflicts of interest to declare.

Inhibitory effects of LDL-associated tissue factor pathway inhibitor

IntroductionTissue factor pathway inhibitor (TFPI) is present in plasma as full-length free TFPI (TFPIα) and as C-terminally degraded forms mainly associated with low-density lipoprotein particles (LDL-TFPI). Addition of TFPIα to plasma induces a prolongation of the clotting time when tested in a diluted prothrombin time (dPT) assay, whereas no prolongation is observed with LDL-TFPI or truncated recombinant TFPI (TFPI1–161). The aim was to further characterize kinetic properties of purified LDL-TFPI in thrombin generation and chromogenic activity assays. Materials and MethodsLDL-TFPI was purified from human plasma by sequential flotation ultracentrifugation. Thrombin generation was measured in human plasma or in FVIII-immunodepleted plasma using either 1 pM tissue factor and 4μM phospholipids or 0.5nM factor Xa (FXa) and 4μM phospholipids, respectively. ResultsTFPIα prolonged the lag-phase and decreased the thrombin peak in tissue factor-induced thrombin generation, whereas LDL-TFPI exclusively decreased the peak height of thrombin without effecting the lag phase. Steady-state and transient kinetics showed that LDL-TFPI was a more potent inhibitor of FXa than TFPIα and TFPI1–161, indicating that FXa inhibition was not rate determining for the lag phase, whereas it appeared to affect thrombin generation during the propagation phase. This was supported by FXa-induced thrombin generation showing that LDL-TFPI, compared with TFPIα, more actively decreased the peak height. ConclusionsOur results suggest that LDL-TFPI affects thrombin generation during the propagation phase, and is kinetically different from TFPI1–161. It may therefore play a more prominent physiological role in vivo than hereto anticipated from dPT measurements.

Rivaroxaban demonstrates in vitro anticoagulant effects in canine plasma

Rivaroxaban is an oral direct factor X inhibitor used in human thrombotic disorders and its oral administration makes it an attractive potential anticoagulant for dogs. The objective of this study was to evaluate the in vitro anticoagulant effect of rivaroxaban on canine pooled platelet-poor plasma (PPP). Pooled PPP was collected from 20 healthy adult Beagle dogs. Aliquots of pooled citrated PPP were treated in vitro with DMSO solutions of rivaroxaban (98% purity) to obtain 19 final concentrations ranging from 0 to 1000mg/L of drug. Samples were immediately submitted for the following coagulation assays: prothrombin time (PT), activated partial thromboplastin time (aPTT), tissue factor-induced thrombin generation and anti-factor Xa activity. Concentration–effect data were analyzed with various nonlinear regression models for stimulatory or inhibitory effects.Rivaroxaban caused a concentration-dependent prolongation of all coagulation parameters. Rivaroxaban concentration for 50% baseline inhibition of the propagation phase of thrombin (rate index) was 0.024mg/L, and for 50% baseline inhibition of the optical density in the anti-factor Xa activity assay was 0.053mg/L. At these concentrations, PT and aPTT remained within the reference range. Two-fold prolongation from baseline of PT and aPTT was achieved with higher concentrations, i.e. 1.24 and 1.69mg/L, respectively. Thrombin generation was completely suppressed by concentrations ⩾0.8mg/L. In conclusion, rivaroxaban showed an in vitro concentration-dependent anticoagulant effect on canine plasma. Thrombin generation and anti-factor Xa activity were more sensitive and accurate than PT and aPTT in detecting the anticoagulant effect of rivaroxaban.