Thrombin Activatable Fibrinolysis Inhibitor Activation Research Articles

Differential Modulation of Plasminogen Activator Mediated Thrombolysis By Recombinant Thrombomodulin and Activated Protein C

Introduction:Urokinase (UK) and tissue plasminogen activator (tPA) mediate thrombolytic actions by activating endogenous plasminogen. Thrombomodulin (TM) complexes with thrombin to activate Protein C and thrombin activatable fibrinolysis inhibitor (TAFI). Activated Protein C (APC) modulates coagulation by digesting factors V and VIII and activates fibrinolysis by decreasing PAI-1 functionality. Since the thrombin/thrombomodulin complex regulates both TAFI functionality and protein C activation, rTM modulates the fibrinolytic process by multiple mechanisms.Aim:To compare the effects of rTM and APC on urokinase and tPA mediated thrombolysis utilizing thromboelastography.Materials and methods:Native whole blood was activated using a diluted intrinsic activator (APTT reagent, Triniclot). The modulation of thrombolysis by tPA and UK (Abbott, Chicago, USA) was studied by supplementing these agents to whole blood and monitoring TEG profiles for 3 hours. To investigate effect of APC (Haematologic Technologies, VT, USA) and rTM (Asahi Kasai Pharma, Tokyo, Japan) these agents were also supplemented to the activated blood at a concentration range of 0.02 - 3.0 ug/ml. The modulation of tPA and UK induced thrombolysis by APC and rTM was studied in terms of thromboelastograph patterns. In addition, the effect of both APC and rTM on plasma based systems supplemented with tPA was also investigated.Results:In comparison to rTM, APC produced a stronger anticoagulant effect in terms of r time, k time, angle and MA. Fibrinolysis was assessed in terms of LY30 (%) and LY60 (%). At concentrations of up to 3.0ug/ml rTM and APC did not produce any direct fibrinolytic effects. APC also produced strong augmentation of the lytic of effects of tPA and urokinase in a concentration dependent fashion. rTM at lower concentrations produced stabilization of clot resisting fibrinolysis. In the plasma based system, APC exhibited a stronger interaction with tPA in comparison to rTM.Conclusion:These studies demonstrate the differential anticoagulant and procoagulant effects of APC and rTM. APC is a stronger anticoagulant than rTM and facilitates thrombolysis. rTM is a weaker anticoagulant and modulates clot stability branding it resistant to fibrinolysis. These observations suggest that while APC may impair hemostasis, rTM restores hemostasis via TAFI activation. The dual mode of action of the rTM may contribute to the regulation of fibrinolysis in facilitating hemostatic balance in both physiologic and pathologic states. [Display omitted] DisclosuresTanaka:Asahi Kasei Pharma America Corporation: Employment. Tsuruta:Asahi Kasei Pharma America Corporation: Employment.

Phosphinanes and Azaphosphinanes as Potent and Selective Inhibitors of Activated Thrombin-Activatable Fibrinolysis Inhibitor (TAFIa).

Selective and potent inhibitors of activated thrombin activatable fibrinolysis inhibitor (TAFIa) have the potential to increase endogenous and therapeutic fibrinolysis and to behave like profibrinolytic agents without the risk of major hemorrhage, since they do not interfere either with platelet activation or with coagulation during blood hemostasis. Therefore, TAFIa inhibitors could be used in at-risk patients for the treatment, prevention, and secondary prevention of stroke, venous thrombosis, and pulmonary embolisms. In this paper, we describe the design, the structure-activity relationship (SAR), and the synthesis of novel, potent, and selective phosphinanes and azaphosphinanes as TAFIa inhibitors. Several highly active azaphosphinanes display attractive properties suitable for further in vivo efficacy studies in thrombosis models.

Inside the Thrombus: Association of Hemostatic Parameters With Outcomes in Large Vessel Stroke Patients.

Background: Actual clinical management of ischemic stroke (IS) is based on restoring cerebral blood flow using tissue plasminogen activator (tPA) and/or endovascular treatment (EVT). Mechanical thrombectomy has permitted the analysis of thrombus structural and cellular classic components. Nevertheless, histological assessment of hemostatic parameters such as thrombin-activatable fibrinolysis inhibitor (TAFI) and matrix metalloproteinase 10 (MMP-10) remains unknown, although their presence could determine thrombus stability and its response to thrombolytic treatment, improving patient's outcome.Methods: We collected thrombi (n = 45) from large vessel occlusion (LVO) stroke patients (n = 53) and performed a histological analysis of different hemostatic parameters [TAFI, MMP-10, von Willebrand factor (VWF), and fibrin] and cellular components (erythrocytes, leukocytes, macrophages, lymphocytes, and platelets). Additionally, we evaluated the association of these parameters with plasma levels of MMP-10, TAFI and VWF activity and recorded clinical variables.Results: In this study, we report for the first time the presence of MMP-10 and TAFI in all thrombi collected from LVO patients. Both proteins were localized in regions of inflammatory cells, surrounded by erythrocyte and platelet-rich areas, and their content was significantly associated (r = 0.41, p < 0.01). Thrombus TAFI was lower in patients who died during the first 3 months after stroke onset [odds ratio (OR) (95%CI); 0.59 (0.36–0.98), p = 0.043]. Likewise, we observed that thrombus MMP-10 was inversely correlated with the amount of VWF (r = −0.30, p < 0.05). Besides, VWF was associated with the presence of leukocytes (r = 0.37, p < 0.05), platelets (r = 0.32, p < 0.05), and 3 months mortality [OR (95%CI); 4.5 (1.2–17.1), p = 0.029]. Finally, plasma levels of TAFI correlated with circulating and thrombus platelets, while plasma MMP-10 was associated with cardiovascular risk factors and functional dependence at 3 months.Conclusions: The present study suggests that the composition and distribution of thrombus hemostatic components might have clinical impact by influencing the response to pharmacological and mechanical therapies as well as guiding the development of new therapeutic strategies.

A prognostic model for thrombotic complications after pediatric cardiac surgery

Background. Cardiac surgery performed on pediatric patients with the use of artificial blood circulation (ABC) is accompanied by hemodilution, hypothermia and blood contact with artificial surfaces, as well as surgical trauma. All the above lead to endothelial cell injury, platelet aggregation and degranulation, activation of innate immunity, development of systemic inflammation and consumption of clotting, anti-coagulation and fibrinolytic factors, which is ultimately associated with the occurrence of thrombotic complications.Objective. The study aimed at developing a mathematical model for the prognosis of thrombotic complications in children which had undergone the ABC, based on assessment of their clinical and laboratory parameters.Methods. We have assessed clinical and laboratory data obtained from 153 children (newborn to 11 months 29 days of age) which had undergone cardiac surgery under conditions of ABC due to congenital heart defects (CHD). For all patients the general clinical and laboratory parameters: complete blood count, comprehensive metabolic panel, parameters of screening coagulogram, D-dimer concentration, von Willebrand factor activity, levels of antithrombin III, plasminogen, protein C and protein S, alpha-2-antiplasmin, thrombin activatable fibrinolysis inhibitor (TAFI) and fibrin-monomer have been assessed.Results. In 43 patients (28.1%) post-operative thromboses have been diagnosed. Examination of children revealed the presence of thrombosis of various localization including the intracardiac thrombi, ischemic cerebrovascular events, limb ischemia, etc. Based on logistic regression analysis, a model of development of thrombotic complications has been built which included 4 parameters: activity of lactate dehydrogenase (LDH), TAFI activity, von Willebrand factor activity and protein C activity. Model sensitivity was 95.3%, and its specificity — 96.4%.

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.

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

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.

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.

Thrombomodulin alfa prevents oxaliplatin-induced neuropathic symptoms through activation of thrombin-activatable fibrinolysis inhibitor and protein C without affecting anti-tumor activity

Oxaliplatin, a platinum-based chemotherapeutic agent, is widely used to treat colorectal cancer, but it induces peripheral neuropathy as a serious dose-limiting side effect. Recently, thrombomodulin alfa, a recombinant human soluble thrombomodulin, was reported to prevent oxaliplatin-induced peripheral neuropathy in a clinical phase 2 study. Here we conducted preclinical pharmacology studies. Rats were given oxaliplatin (6 mg/kg) intravenously to induce mechanical hyperalgesia associated with peripheral neuropathy. Single intravenous administration of thrombomodulin alfa (0.1, 0.3, 1 mg/kg) dose dependently prevented the development of oxaliplatin-induced mechanical hyperalgesia, with no sex difference in the efficacy. The preventative effect of thrombomodulin alfa on mechanical hyperalgesia was attenuated by antithrombin or carboxypeptidase inhibitor. In addition, carboxypeptidase B, a homolog of activated thrombin-activatable fibrinolysis inhibitor (TAFI) and human-derived activated protein C, prevented mechanical hyperalgesia, whereas antithrombin or other anti-coagulants did not. These results suggest that thrombomodulin alfa prevents sensory symptoms of oxaliplatin-induced peripheral neuropathy through the activation of TAFI and protein C by modulating thrombin activity, but the effects are independent of an anticoagulant effect. On the other hand, thrombomodulin alfa did not affect the anti-cancer activity of oxaliplatin on human colon cancer cell lines or mice transplanted with HCT116 cells. These results indicate that thrombomodulin alfa prevents sensory symptoms of oxaliplatin-induced peripheral neuropathy without affecting the anti-tumor activity of oxaliplatin. Therefore, thrombomodulin alfa is a promising drug to prevent the symptoms of oxaliplatin-induced peripheral neuropathy.

Rosuvastatin use increases plasma fibrinolytic potential: a randomised clinical trial.

We conducted a study to assess the effect of rosuvastatin use on fibrinolysis in patients with previous venous thromboembolism (VTE). This was a post hoc analysis within the STAtins Reduce Thrombophilia (START) study (NCT01613794). Plasma fibrinolytic potential, fibrinogen, plasmin inhibitor, plasminogen activator inhibitor‐1 (PAI‐1) and thrombin‐activatable fibrinolysis inhibitor (TAFI) were measured before and after four weeks of rosuvastatin or no treatment in participants with prior confirmed VTE, after ending anticoagulant therapy. In the non‐rosuvastatin group (n = 121), plasma fibrinolytic potential and individual fibrinolysis parameters did not change at the end of the study versus the baseline, whereas in the rosuvastatin group (n = 126), plasma fibrinolytic potential increased: the mean clot lysis time decreased by 8·75 min (95% CI −13·8 to −3·72), and plasmin inhibitor levels and TAFI activity were lower at the end of the study (−0·05 U/ml; 95% CI −0·07 to −0·02 and −4·77%; 95% CI −6·81 to −2·73, respectively). PAI‐1 levels did not change and fibrinogen levels were 0·17 g/l (95% CI 0·04–0·29) higher. In participants with prior VTE, rosuvastatin use led to an increased fibrinolytic potential compared with non‐statin use. Our findings support the need for further studies on the possible role for statins in the secondary prevention of VTE.

The protein C activator AB002 rapidly interrupts thrombus development in baboons

AbstractAlthough thrombin is a key enzyme in the coagulation cascade and is required for both normal hemostasis and pathologic thrombogenesis, it also participates in its own negative feedback via activation of protein C, which downregulates thrombin generation by enzymatically inactivating factors Va and VIIIa. Our group and others have previously shown that thrombin's procoagulant and anticoagulant activities can be effectively disassociated to varying extents through site-directed mutagenesis. The thrombin mutant W215A/E217A (WE thrombin) has been one of the best characterized constructs with selective activity toward protein C. Although animal studies have demonstrated that WE thrombin acts as an anticoagulant through activated protein C (APC) generation, the observed limited systemic anticoagulation does not fully explain the antithrombotic potency of this or other thrombin mutants. AB002 (E-WE thrombin) is an investigational protein C activator thrombin analog in phase 2 clinical development (clinicaltrials.gov NCT03963895). Here, we demonstrate that this molecule is a potent enzyme that is able to rapidly interrupt arterial-type thrombus propagation at exceedingly low doses (<2 µg/kg, IV), yet without substantial systemic anticoagulation in baboons. We demonstrate that AB002 produces APC on platelet aggregates and competitively inhibits thrombin-activatable fibrinolysis inhibitor (carboxypeptidase B2) activation in vitro, which may contribute to the observed in vivo efficacy. We also describe its safety and activity in a phase 1 first-in-human clinical trial. Together, these results support further clinical evaluation of AB002 as a potentially safe and effective new approach for treating or preventing acute thrombotic and thromboembolic conditions. This trial was registered at www.clinicaltrials.gov as #NCT03453060.

Cardiac Myosin Promotes Thrombin Generation and Coagulation In Vitro and In Vivo.

Cardiac myosin (CM) is structurally similar to skeletal muscle myosin, which has procoagulant activity. Here, we evaluated CM's ex vivo, in vivo, and in vitro activities related to hemostasis and thrombosis. Approach and Results: Perfusion of fresh human blood over CM-coated surfaces caused thrombus formation and fibrin deposition. Addition of CM to blood passing over collagen-coated surfaces enhanced fibrin formation. In a murine ischemia/reperfusion injury model, exogenous CM, when administered intravenously, augmented myocardial infarction and troponin I release. In hemophilia A mice, intravenously administered CM reduced tail-cut-initiated bleeding. These data provide proof of concept for CM's in vivo procoagulant properties. In vitro studies clarified some mechanisms for CM's procoagulant properties. Thrombin generation assays showed that CM, like skeletal muscle myosin, enhanced thrombin generation in human platelet-rich and platelet-poor plasmas and also in mixtures of purified factors Xa, Va, and prothrombin. Binding studies showed that CM, like skeletal muscle myosin, directly binds factor Xa, supporting the concept that the CM surface is a site for prothrombinase assembly. In tPA (tissue-type plasminogen activator)-induced plasma clot lysis assays, CM was antifibrinolytic due to robust CM-dependent thrombin generation that enhanced activation of TAFI (thrombin activatable fibrinolysis inhibitor). CM in vitro is procoagulant and prothrombotic. CM in vivo can augment myocardial damage and can be prohemostatic in the presence of bleeding. CM's procoagulant and antifibrinolytic activities likely involve, at least in part, its ability to bind factor Xa and enhance thrombin generation. Future work is needed to clarify CM's pathophysiology and its mechanistic influences on hemostasis or thrombosis.

Thrombin activatable fibrinolysis inhibitor pathway alterations correlate with bleeding phenotype in patients with severe hemophilia A

BackgroundPatients with severe hemophilia A display varied bleeding phenotypes despite similar factor VIII (FVIII) activity levels. ObjectiveWe investigated different thrombin activatable fibrinolysis inhibitor (TAFI)‐related variables in patients with severe hemophilia A and their possible correlation with bleeding tendency. Patients/MethodsSixty‐one patients with severe hemophilia A (FVIII:C <1%], treated on demand, were included. Patients were categorized as mild, moderate, and severe bleeders according to number of bleeds per year (≤2, 3‐24, ≥25, respectively). Thirty healthy males served as controls. Clot lysis time was assessed by turbidimetric assay, TAFI activation by two‐stage functional assay, and response to TAFIa as the prolongation of fibrinolysis time upon addition of purified TAFIa. Circulating levels of activated TAFI (TAFIa/ai) were measured by specific enzyme‐linked immunosorbent assay. ResultsAs compared to controls, hemophilic patients displayed shorter lysis time, less TAFIa generation, and reduced response to TAFIa, but similar TAFIa/ai levels. Clot lysis time was similar in mild, moderate, and severe bleeders, whereas TAFIa generation and response to TAFIa decreased with the increase in bleeding tendency; moreover, circulating TAFIa/ai levels were highest in severe bleeders. Patients with markedly impaired TAFIa generation or TAFIa response (below median) displayed 3‐fold to 4‐fold higher bleeding rate and factor consumption than patients whose TAFI‐related values approached the control ones. ConclusionThe TAFI pathway impairment correlates with bleeding phenotype in severe hemophilia and may represent a promising tool to stratify the bleeding risk.

The role of haematological traits in risk of ischaemic stroke and its subtypes.

Thrombosis and platelet activation play a central role in stroke pathogenesis, and antiplatelet and anticoagulant therapies are central to stroke prevention. However, whether haematological traits contribute equally to all ischaemic stroke subtypes is uncertain. Furthermore, identification of associations with new traits may offer novel treatment opportunities. The aim of this research was to ascertain causal relationships between a wide range of haematological traits and ischaemic stroke and its subtypes. We obtained summary statistics from 27 published genome-wide association studies of haematological traits involving over 375 000 individuals, and genetic associations with stroke from the MEGASTROKE Consortium (n = 67 000 stroke cases). Using two-sample Mendelian randomization we analysed the association of genetically elevated levels of 36 blood cell traits (platelets, mature/immature red cells, and myeloid/lymphoid/compound white cells) and 49 haemostasis traits (including clotting cascade factors and markers of platelet function) with risk of developing ischaemic (AIS), cardioembolic (CES), large artery (LAS), and small vessel stroke (SVS). Several factors on the intrinsic clotting pathway were significantly associated (P < 3.85 × 10-4) with CES and LAS, but not with SVS (e.g. reduced factor VIII activity with AIS/CES/LAS; raised factor VIII antigen with AIS/CES; and increased factor XI activity with AIS/CES). On the common pathway, increased gamma (γ') fibrinogen was significantly associated with AIS/CES. Furthermore, elevated plateletcrit was significantly associated with AIS/CES, eosinophil percentage of white cells with LAS, and thrombin-activatable fibrinolysis inhibitor activation peptide antigen with AIS. We also conducted a follow-up analysis in UK Biobank, which showed that amongst individuals with atrial fibrillation, those with genetically lower levels of factor XI are at reduced risk of AIS compared to those with normal levels of factor XI. These results implicate components of the intrinsic and common pathways of the clotting cascade, as well as several other haematological traits, in the pathogenesis of CES and possibly LAS, but not SVS. The lack of associations with SVS suggests thrombosis may be less important for this stroke subtype. Plateletcrit and factor XI are potentially tractable new targets for secondary prevention of ischaemic stroke, while factor VIII and γ' fibrinogen require further population-based studies to ascertain their possible aetiological roles.

Similar Hemostatic Responses to Hypovolemia in Hemorrhage and Lbnp Reveals a Hyperfibrinolytic Subset of Baboons

Background: Lower body negative pressure (LBNP) has been established in human studies as an alternative method to blood removal (HEM) to study central hypovolemia. In a baboon model, LBNP also mimics the cardiovascular response of HEM. Hemostatic responses, including platelet activation, coagulation and fibrinolysis in response to LBNP and HEM in this baboon model are reported here. A previous meeting report from this study focused on hemostatic interactions; this report expands the assay of fibrinolytic proteins and includes detailed post-hoc analysis of interactions. Methods: Thirteen anesthetized baboons were exposed to progressive central hypovolemia by HEM and, four weeks later, by LBNP. Whole blood cell counts were determined; pro-coagulant and fibrinolytic activity was evaluated by plasma markers, thrombelastography, flow cytometry and platelet aggregometry at baseline (BL), intermediate hypovolemia (MID), presyncope or maximal hypovolemia (MAX) and after recovery (REC). Results: Baseline values for HEM (day 0) and LBNP (day 28) were indistinguishable for most analyzed parameters; however platelet number, maximal clotting (MA), Protein C, TAT, TAFI-activity, plasma renin, angiotensin and epinephrine were all significantly different (p&amp;lt;0.05). Central hypovolemia induced by both methods activated coagulation; TEG revealed decreased clotting time (R) and increased maximal clotting (MA) at MID, MAX and REC over BL. No differences during developing central hypovolemia were observed between HEM and LBNP. Platelets displayed activation markers on flow cytometry; absolute numbers and functional aggregometry were unchanged. At MAX hypovolemia, there was a modest hemodilution for HEM and a modest hemoconcentration for LBNP; red blood cell count decreased 5.8%±5.5%, p=0.0007 with HEM and increased 5.3±5.9%, p=0.0056 with LBNP. Coagulation factors were significantly increased with LBNP at MAX compared to HEM; fibrinolysis proteins were more varied. No hyper-fibrinolysis developed with progressive hypovolemia by either method; instead hyper-fibrinolysis already existed at BL with significant variance in TEG fibrinolysis at 30 min (LY30) values. Post hoc analysis separated study subjects into low lysis (LY30BL&amp;lt;2) or high lysis (LY30BL&amp;gt;2) whose fibrinolytic state matched at both HEM and LBNP BL. High lysis samples (n=8) showed a strong correlation (r=0.95; p&amp;lt;0.001) of tissue plasminogen activator (tPA) to LY30 at BL that was absent in low lysis samples (n=6). Thrombin activatable fibrinolysis inhibitor (TAFI) activity correlated with tPA levels for low lysis subjects (r=0.88) with no correlation for high lysis subjects (r=.08). Conclusions: Central hypovolemia resulted in activation of coagulation by either LBNP or HEM; thus LBNP is a powerful model for studying hemorrhage. This study also revealed a subset of baboons with baseline activation of fibrinolysis which was strongly coupled to tPA and uncoupled from TAFI activity. The methods demonstrated hemodilution with blood removal and hemoconcentration with LBNP, as has been observed in humans, but these modest changes had no significant effect on coagulation parameters. Unexpectedly, this study demonstrated that the effects of blood removal were sustained 28 days later on baseline parameters. Overall, these results in large primates suggest that baseline differences between apparently healthy and superficially homogeneous individuals may determine outcomes in studies of hemostasis. Disclosures No relevant conflicts of interest to declare.

Prevention of Bleeding-Induced Vascular Abnormalities in the Hemophilic Mouse Joint By Increasing TAFI Levels or Inhibiting uPA Activity

Hemophilic arthropathy is characterized by vascular abnormalities in the joint soft tissue that may cause rebleeding and progressive joint destruction. Defective thrombin generation in hemophilia leads to impaired activation of Thrombin Activatable Fibrinolysis Inhibitor (TAFI). Activated TAFI (TAFIa) is a carboxypeptidase that cleaves C-terminal Lysine from fibrin or cell plasminogen receptors, thereby diminishing fibrinolysis or pericellular plasmin generation. In addition, TAFIa inactivates pro-inflammatory (i.e. C3a, C5a, bradykinin) and anti-angiogenic (i.e. VEGF-A, SDF-1α) substrates. We have recently shown that TAFI-gene deficiency causes vascular abnormalities in the mouse joints that phenocopy those in hemophilic arthropathy, either after joint bleeding or spontaneously with aging. Based on these mechanistic insights, we evaluated treatment with TAFI for its ability to mitigate bleeding-induced vascular abnormalities and associated bleeding susceptibility in the FVIII KO mouse joint. Knee joint bleeding in FVIII KO mice was induced by a subpatellar puncture injury that was standardized to cause severe bleeding as defined by a hematocrit (Hct) drop from 47 ± 2 % at baseline (BL) to 29 ± 11 % two days after injury. The vascular abnormalities formed in the hemophilic mouse joint 2 weeks after injury capture those in patients with hemophilic arthropathy: i.e. increased vascular permeability and abnormal vessel enlargement. Hydrodynamic gene delivery was used in mice for continuous treatment with human wild type TAFI (hTAFI-WT). To compensate for lower TAFI activation in hemophilia, two TAFI mutants were tested with extended enzymatic stability: hTAFI-CYQ (8-fold increased) and hTAFI-CIIYQ (180-fold increased). The TAFI constructs had comparable antigen expression profiles (reaching a similar level as in human plasma at day 2 and gradually returning to BL by week 2). Treatment efficacy to reduce vascular permeability, however, differed according to TAFIa's enzymatic stability in FVIII KO mice 2 weeks after injury. Without treatment, Evans blue-BSA extravasation in the joints was increased (1.8-fold compared to the contralateral joint; p= 0.0001 vs BL). hTAFI-WT overexpression initiated 2 days after injury had no effect, whereas hTAFI-CYQ and hTAFI-CIIYQ overexpression reduced the vascular permeability to 1.5-fold (n= 10; p= 0.10 vs untreated) and 1.3-fold (n= 8; p= 0.0032 vs untreated), respectively, indicating that hTAFI-CIIYQ was most effective. Histology at week 2 confirmed that hTAFI-CIIYQ reduced the number of excessively enlarged CD31+ vessels (with ≥ 20 µm diameter) by 3-fold (n= 6; p= 0.0011 vs untreated). To determine whether the protective effect of TAFI overexpression on bleeding-induced vascular abnormalities was associated with inhibition of the plasminogen activation system, continuous administration of tranexamic acid (TXA; 50 mg/ml in drinking water) or antibody-mediated blockage of uPA's proteolytic activity (mU1; 60 mg/kg; 2x weekly) was initiated in FVIII KO mice 2 days after injury. Histology at week 2 showed mU1, but not TXA, reduced vessel enlargement to the same extent as hTAFI-CIIYQ. Finally, the joint bleeding model was adopted to appreciate functional consequences of hypervascularity to changes in bleeding susceptibility. Aged TAFI KO mice (8 months) were used for their spontaneously (3-fold) increased number of enlarged vessels (≥ 40 µm diameter; p= 0.0076) in the joint that correlates with Power Doppler (PD) signal. A mild injury was optimized to cause minimal bleeding and showed that PD signals in joints of aged TAFI KO mice correlated negatively with the Hct 2 days after mild injury with injection of a FVIII inhibitor (r= -0.53, p= 0.024; n= 18), supporting hypervascularity as a major contributor to hemophilic joint bleeding. Furthermore, the same mild injury caused minimal bleeding in naive FVIII KO mice with low BL vascularity (median Hct 46 % vs 48 %; p= 0.81 vs BL; n= 7), but excessive bleeding in FVIII KO mice injured 2 weeks previously with vascular abnormalities (median Hct 38 %; p= 0.021 vs BL; n= 7). hTAFI-CIIYQ effectively reduced bleeding severity upon re-injury (median Hct 47 %; p&amp;gt; 0.99 vs BL; n=5). Our data provide an experimental link between vascular abnormalities and bleeding susceptibility in hemophilic joints and conclude that the vascular abnormalities were alleviated by increasing TAFI levels or inhibiting uPA activity. Disclosures von Drygalski: University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy; Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder. Mosnier:The Scripps Research Institute: Membership on an entity's Board of Directors or advisory committees, Patents &amp; Royalties.

TAFI deficiency causes maladaptive vascular remodeling after hemophilic joint bleeding.

Excessive vascular remodeling is characteristic of hemophilic arthropathy (HA) and may contribute to joint bleeding and the progression of HA. Mechanisms for pathological vascular remodeling after hemophilic joint bleeding are unknown. In hemophilia, activation of thrombin-activatable fibrinolysis inhibitor (TAFI) is impaired, which contributes to joint bleeding and may also underlie the aberrant vascular remodeling. Here, hemophilia A (factor VIII-deficient; FVIII-deficient) mice or TAFI-deficient mice with transient (antibody-induced) hemophilia A were used to determine the role of FVIII and TAFI in vascular remodeling after joint bleeding. Excessive vascular remodeling and vessel enlargement persisted in FVIII-deficient and TAFI-deficient mice, but not in transient hemophilia WT mice, after similar joint bleeding. TAFI-overexpression in FVIII-deficient mice prevented abnormal vessel enlargement and vascular leakage. Age-related vascular changes were observed with FVIII or TAFI deficiency and correlated positively with bleeding severity after injury, supporting increased vascularity as a major contributor to joint bleeding. Antibody-mediated inhibition of uPA also prevented abnormal vascular remodeling, suggesting that TAFI's protective effects include inhibition of uPA-mediated plasminogen activation. In conclusion, the functional TAFI deficiency in hemophilia drives maladaptive vascular remodeling in the joints after bleeding. These mechanistic insights allow targeted development of potentially new strategies to normalize vascularity and control rebleeding in HA.