Activatable Fibrinolysis Inhibitor Research Articles

Elevated plasma factor XI is predicts cardiovascular complications in patients with type 2 diabetes mellitus

Abstract Background Atherothrombotic complications are common in patients with type 2 diabetes mellitus (T2DM), reflecting a chronic prothrombotic state. Circulating factor XI (FXI) has been associated with a prothrombotic fibrin clot phenotype and predicted myocardial infarction, stroke and cardiovascular (CV) death in a number of diseases, including CAD. Its role in T2DM is largely unknown. Purpose The purpose of this study was to assess plasma FXI as a predictor of atherothrombotic events in T2DM patients. We also aimed to establish the determinants of elevated plasma FXI in this group of patients. Methods In a cohort of 156 patients aged 43–83 years, 56% male, with T2DM of a median duration time of five years, 64.7% with diagnosed CAD, we measured FXI along with a number of fibrin clot parameters, including turbidity, permeation, compaction, lysability, maximum concentration of D-dimer (D-D max), and rate of D-dimer release during tissue plasminogen activator-induced clot lysis. Patients were followed for a median time of 72 (68-74) months for a composite endpoint of nonfatal myocardial infarction (MI), nonfatal stroke, and cardiovascular (CV) death. Results Follow-up was complete for 133 (85.3%) patients. There were 21 (16%) cases of the composite endpoint, including one nonfatal MI, four nonfatal strokes and 16 cases of CV death. Patients with and without the composite endpoint shared a similar demographic and clinical profile with the exception of age and white blood cell count – patients with the endpoint were older (69.4 [8.4] vs. 65.4 [7.8] years, p=0.03) and had a higher leukocyte count (7.8 [1.9] vs. 7.0 [1.3], p=0.02) as compared with the remainder. FXI was elevated above the upper limit of 120% in 25 (18.8%) patients, including 16 (61.9%) with the composite endpoint vs. 12 (10.7%) without the composite endpoint, p<0.001. Individuals with the composite endpoint had higher baseline FXI concentrations as compared with the remainder (120.1±16.8 vs. 104.9±10.7%. p < 0.001), along with higher D-D max (4.1 [3.8-4.3] vs. 3.8 [3.6-4.1] mg/l, p = 0.006) and slightly longer lysis time (t50% 10.1 [9.8-10.9] vs. 9.8 [8.9-10.4] min, p = 0.03). On multivariable analysis, co-existing CAD, LDL cholesterol, and thrombin activatable fibrinolysis inhibitor activity were independent predictors of elevated FXI in T2DM patients. Conclusion Elevated plasma FXI in T2DM patients is associated with CAD, LDL cholesterol and TAFI, and predicts myocardial infarction, stroke, and CV death in long term follow-up. Our finding may have implications for risk stratification and suggests a role for FXI inhibitors in T2DM patients with a high risk of atherothrombotic complications.

RATIONALE FOR PRESCRIBING ANTICOAGULANT THERAPY IN THE COMPLEX TREATMENT OF PURULENT-NECROTIC COMPLICATIONS OF DIABETIC FOOT SYNDROME

Abstract. This article is based on the study of hemostasis indicators in 25 patients with diabetic foot syndrome. Diabetic foot syndrome (DFS) develops against the background of diabetic neuropathy, micro- and macroaniopathy, osteoarthropathy, which lead to frequent traumatization of the soft tissues of the foot, infection with the subsequent development of purulent-necrotic processes on the foot, and in the course of progression leads to high limb amputations. According to the international federation, which deals with the problem of diabetes mellitus (DM), the 21st century is considered a pandemic of DM. It should be noted that the number of patients with diabetes is increasing every year. Patients with diabetes are characterized by an atherothrombotic state caused by multiple disorders in the hemostasis system. Hemostatic disorders in patients with diabetes are complex and include activation of natural mechanisms of the coagulation system, inhibition of fibrinolytic activity, and various platelet dysfunctions.
 The purpose of the study is to substantiate the appointment of anticoagulation therapy in patients with SDS.
 Materials and methods. On the basis of KNP KMKL No. 6 in Kyiv in 2023, for a period of 6 months, 69 patients with SDS were treated. The state of indicators of the coagulation link of the hemostasis system in 49 people was studied. At the same time, 26 patients had purulent-necrotic complications of the neuroischemic form of SDS, 23 people were healthy (comparison group). The patients were divided according to the article and age, according to the duration of diabetes mellitus, and according to the stages according to Wagner. The following indicators were determined for patients: prothrombin index (PTI), plasma recalcification time, fibrinogen, B-naphthol test, ethanol test, Willebrand factor, endothelin-1, coagulation factor (VIII), fibronectin.
 The results of the study and their discussion: Based on the study, 61.5 % of patients with SDS are of working age. Of them, 57.7 % are men, 42.3 % are women. The largest number of patients with SDS is observed in the group of patients who have had diabetes for more than 10 years - 57.7 %, and the largest number of patients with stage III-IV according to Wagner - 88.5 %. In patients with diabetes with neuroischemic form of SDS, the PTI level is increased by 28.8 % and the prothrombin time is shortened by 21 %, which indicates an increase in the activity of external blood clotting mechanisms. An increase in fibrinogen by 29.7 % indicates an increase in the activity of coagulation hemostasis. Willebrand factor increased by 34.5 %, coagulation factor (VIII) increased by 20.3 %, fibronectin increased by 83.01 %, which indicates an increase in aggregation and procoagulant processes, which in turn increases thrombus formation.
 Conclusion. Thus, with diabetic foot syndrome, hypercoagulation is observed, which leads to an increase in the level of thrombus formation, which justifies the appointment of anticoagulant therapy in the complex treatment of diabetic foot syndrome.

Increased thrombin activatable fibrinolysis inhibitor activity is associated with hypofibrinolysis in dogs with sepsis.

Disorders of coagulation are well-recognized in dogs with sepsis, but data regarding fibrinolysis disorders are limited. We aimed to characterize fibrinolysis in dogs with sepsis compared to healthy controls. We hypothesized that dogs with sepsis would be hypofibrinolytic, and that hypofibrinolysis would be associated with non-survival. This was a prospective observational cohort study. We enrolled 20 client-owned dogs with sepsis admitted to the Cornell University Hospital for Animals and 20 healthy pet dogs. Coagulation and fibrinolytic pathway proteins including antiplasmin activity (AP), antithrombin activity (AT), thrombin activatable fibrinolysis inhibitor activity (TAFI), D-dimer concentration, fibrinogen concentration, and plasminogen activity were measured and compared between groups. Overall coagulation potential, overall fibrinolysis potential, and overall hemostatic potential were calculated from the curve of fibrin clot formation and lysis over time. Compared to healthy controls, dogs with sepsis had lower AT (P = 0.009), higher AP (P = 0.002), higher TAFI (P = 0.0385), and higher concentrations of fibrinogen (P < 0.0001) and D-dimer (P = 0.0001). Dogs with sepsis also had greater overall coagulation potential (P = 0.003), overall hemostatic potential (P = 0.0015), and lower overall fibrinolysis potential (P = 0.0004). The extent of fibrinolysis was significantly negatively correlated with TAFI. No significant differences were observed between survivors and non-survivors. Dogs with sepsis were hypercoagulable and hypofibrinolytic compared to healthy dogs, suggesting potential utility of thromboprophylaxis in this patient population. The association between high TAFI and low overall fibrinolysis potential might provide a potential mechanism for this hypofibrinolysis.

A missense mutation in lectin domain of thrombomodulin causing functional deficiency

Thrombomodulin (TM) functions in coagulation, fibrinolysis and inflammation by its cofactor activity for protein C, thrombin-activatable fibrinolysis inhibitor (TAFI) activation and high mobility group box 1 (HMGB1) degradation induced by thrombin. It has been widely reported that mutations in TM are related to thromboembolic diseases but hardly in lectin domain. Here we report our findings about the functional deficiencies in TM caused by substitution of aspartate with tyrosine at residue 126. Three patients suffering from recurrent thromboembolic diseases were identified with this mutation and their plasma soluble TM levels were decreased. Transfected cells expressing wild-type TM or the variant and corresponding proteins were used to examine TM functions in vitro. The cofactor activity of the mutant for protein C, TAFI activation was reduced to approximately 50% and 60% respectively. Loss in anti-inflammation due to weakened HMGB1 degradation was also observed. And the study with thrombosis models of mice suggested the decreased inhibition of thrombus development of the mutant. Together the results showed deleterious changes on TM function caused by this mutation, which may explain the thrombophilia tendency of the patients. This work provided supportive evidence that mutation in lectin domain of TM might be related to thrombotic diseases and may help us better understand the physiological roles of TM.

CM-352 Efficacy in a Mouse Model of Anticoagulant-Associated Intracranial Hemorrhage.

Background  Intracranial hemorrhage (ICH) is one of the major devastating complications of anticoagulation. Matrix metalloproteinase (MMP) inhibition has been proposed as a novel pharmacological approach for ICH treatment. Objectives  We evaluated the effects of CM-352 (MMP-fibrinolysis inhibitor) in an experimental ICH model associated with oral anticoagulants as compared with clinically used prothrombin complex concentrate (PCC). Methods  ICH was induced by collagenase injection into the striatum of wild type (C57BL/6J) anticoagulated mice (warfarin or rivaroxaban) and Mmp10   −/− mice. Hematoma volume and neurological deficits were measured 24 hours later by diaminobenzidine staining and different behavioral tests. Circulating plasminogen activator inhibitor-1 (PAI-1) activity and interleukin-6 (IL-6) were measured in plasma samples and local inflammation was assessed by neutrophil infiltration. Finally, fibrinolytic effects of MMP-10 and rivaroxaban were evaluated by thromboelastometry and thrombin-activatable fibrinolysis inhibitor (TAFI) activation assays. Results  Only PCC reduced hemorrhage volume and improved functional outcome in warfarin-ICH, but both PCC and CM-352 treatments diminished hemorrhage volume (46%, p  < 0.01 and 64%, p  < 0.001, respectively) and ameliorated functional outcome in rivaroxaban-ICH. We further demonstrated that CM-352, but not PCC, decreased neutrophil infiltration in the hemorrhage area at 24 hours. The effect of CM-352 could be related to MMP-10 inhibition since Mmp10   −/− mice showed lower hemorrhage volume, better neurological score, reduced IL-6 levels and neutrophil infiltration, and increased PAI-1 after experimental ICH. Finally, we found that CM-352 reduced MMP-10 and rivaroxaban-related fibrinolytic effects in thromboelastometry and TAFI activation. Conclusion  CM-352 treatment, by diminishing MMPs and rivaroxaban-associated fibrinolytic effects, might be a novel antihemorrhagic strategy for rivaroxaban-associated ICH.

Вплив альбумінурії на стан плазмового гемостазу у хворих на гіпертонічну хворобу

В останні десятиріччя численні наукові роботи присвячено дослідженню клінічної та прогностичної значимості альбумінурії, методів її виявлення, а також лікувальних заходів, що направлені на боротьбу з даним патологічним станом. Метою роботи було встановити особливості змін плазмового гемостазу у хворих на гіпертонічну хворобу залежно від наявності альбумінурії. Матеріали і методи. Нами було обстежено 113 осіб: 14 практично здорових (1-ша група, контроль), 41 хворий на гіпертонічну хворобу (ГХ) II стадії із супутньою альбумінурією (2-га група), 58 хворих на ГХ II стадії без супутньої альбумінурії (3-тя група). Проводили лабораторні дослідження: активований частковий тромбопластиновий час, протромбіновий індекс, тромбіновий час, фібриноген, розчинні фібрин-мономерні комплекси (РФМК), час ХІІа-залежного фібринолізу, антитромбін ІІІ, протеїн С. Результати. Був виявлений підвищений вміст РФМК, який у 3-й групі хворих на 135 %, а в 2-й групі хворих — на 247 % перевищував конт­рольні значення. При цьому у хворих з альбумінурією він був набагато вищим навіть за показник у групі хворих на ГХ без альбумінурії — на 48 %. Час XII-а залежного фібринолізу як у 2-й, так і в 3-й групі був набагато більшим за норму. Виражені зміни відбувались з боку системи природних антикоагулянтів, що були статистично значущими по відношенню до групи контролю лише у групі хворих з альбумінурією, а у групі хворих без альбумінурії вони суттєво не відрізнялись від групи контролю. Висновки. У хворих на гіпертонічну хворобу незалежно від вираженості альбумінурії спостерігалось значне пригнічення фібринолітичної активності крові та активації останніх ланок згортання крові — фібриноутворення. Наявність альбумінурії асоціювалась з більш суттєвою активацією фібриноутворення та поєднувалась із пригніченням власної антикоагулянтної активності крові, про що свідчило зменшення вмісту антитромбіну III та протеїну С.

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.

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.

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.

Коагуляційні властивості cистеми зсідання крові при гострій тромбоемболії легеневої артерії та їх зміни на тлі проведеної системної тромболітичної терапії в пацієнтів з різним ризиком госпітальної летальності

Мета роботи – оцінити динаміку змін коагуляційних властивостей крові в пацієнтів з гострою тромбоемболією легеневої артерії (ТЕЛА) до і після проведення системної тромболітичної терапії (ТЛТ), порівнюючи групи з високим і проміжним-високим ризиком.Матеріали і методи. Проведене ініціативне контрольоване проспективне нерандомізоване дослідження результатів лікування 45 пацієнтів з первинним епізодом гострої ТЕЛА, яким здійснено ТЛТ у відділенні інтенсивної терапії. Досліджувану вибірку становили 29 (64 %) чоловіків і 16 (36 %) жінок віком у середньому (55,6±13,6) року. Відповідно до рекомендацій Європейського товариства кардіологів (2014) проведена стратифікація на нестабільних пацієнтів з високим ризиком з гіпотонією або епізодом синкопе (1-ша група, 28 (62 %) осіб) і пацієнтів з нормальним артеріальним тиском з проміжним-високим ризиком (2-га група, 17 (38 %) осіб) і значним двобічним емболічним навантаженням, верифікованим мультидетекторною спіральною комп’ютерною томографією в ангіорежимі (U1-2=2,2; р=0,33), ознаками дисфункції правого шлуночка і позитивними біомаркерами пошкодження міокарда. Ризик 30-денної летальності за PESI відповідає V (ІV) класу: в 1-й групі – (152±19) балів, у 2-й – (138,0±9,7) бала (p1-2&lt;0,01).Результати та обговорення. В обох групах виявлена подібна (р1-2&gt;0,25) первинна прокоагуляційна спрямованість змін за показниками тромбіноутворення обох ланок активації гемокоагуляції: протромбіновий час збільшений до 19,8 [16; 23] с, протромбіновий індекс – до 96,1 [86; 106] %. Зменшення активованого часткового тромбопластинового часу (АЧТЧ) до 23,5 [21; 24] с підтвердило визначену спрямованість гемостазу шляхом контактної коагуляції. Виявлено збільшений вміст основного коагуляційного субстрату фібриногену – до 4,3 [4,1; 4,5] г/л (р1-2=0,25) і маркерів тромбінемії: розчинного фібрину – до 17,0 [16; 18] мг, D-димеру – до 5214 [3605; 5643] нг/мл. Початково пригнічена системна фібринолітична активність – значення спонтанного фібринолізу зменшені до 9,5  [6,0; 12,2] %, власна ретракція – до 31,9  [26,1; 36,1] %. На 5-ту добу після проведеної ТЛТ на тлі базисної терапії динаміка виявилася насамперед підвищенням (Z=5,62; р&lt;0,00001) значень АЧТЧ до (46,1±6,0) с (р1-2=0,36), протромбінового часу – до 22,9 (18–26) с, рівень фібриногену зменшився до 3,5 г/л. Незважаючи на сприятливі зміни, залишалися збільшеними маркери тромбінемії – значення фібрину зменшилися (Z=3,03; р&lt;0,001) до 13,7 мг, але перевищували верхню межу референтного діапазону в обох групах (р1-2=0,21). Збільшилися значення спонтанного фібринолізу до 11,9 % [9,9; 12,4], власної ретракції (Z=2,64; р&lt;0,01) – до 32,0 % [27,9; 33,0], при цьому залишаючись значно зниженими за референтні, що засвідчує збереження високого ризику рецидиву тромбоемболічних подій.Висновки. У пацієнтів з гострою ТЕЛА, незалежно від наявності високого або проміжного-високого ризику, за даними коагулограми спостерігається однаковий за силою та спрямованістю прокоагулянтний стан гемостазу, пригнічення фібринолітичної активності, зменшення щільності згустка in vitro. На 5-ту добу після ТЛТ на тлі базисної антикоагулянтної терапії: попри певний рівень «терапевтичної» антикоагуляції, зберігається достатньо високий рівень маркерів тромбінемії, пригнічення фібринолізу та ретракції. Наявність однакових за силою та спрямованістю коагуляційних змін, особливо виснаження фібринолітичних механізмів гемостазу, позитивний клінічний вплив ТЛТ у групі з проміжним-високим ризиком засвідчують доцільність проведення ТЛТ у цій групі хворих.

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.

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.

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.

Regulation of Coagulation by Polyphosphate

Polyphosphate (polyP) is a biomolecule comprised of repeating units of inorganic phosphate residues linked by energy-rich phosphoanhydride bonds. PolyP is highly conserved throughout evolution and plays a vital role in the survival of prokaryotes and single-cell eukaryotes1 by functioning in processes such as motility, virulence and stress. Its role in mammalian systems was less well defined, however, identification of polyP in the dense granules of human platelets2 prompted analysis of the function of polyP in hemostasis. Stimulation of platelets by conventional agonists such as ADP, thrombin and collagen induces release of polyP, and promotes coagulation by acting at several points in the cascade3. These include 1) providing a surface for activation of the contact pathway protein factor XII, 2) augmenting conversion of factor V to factor Va which participates in the prothrombinase complex and 3) accelerating thrombin-mediated activation of factor XI4. The net outcome is a reduction in the lag time to thrombin formation thereby driving clot formation. The effect of polyP on thrombin generation has down-stream repercussions in terms of clot breakdown, as it augments the early activation of thrombin activatable fibrinolysis inhibitor (TAFI). There has been a degree of uncertainty and debate over whether polyP is capable of reaching adequate concentrations within the plasma milieu to elicit these profound effects on haemostasis. However, the observations that polyP can bind directly to fibrin5, allowing it to be incorporated into the forming clot, localise the biomolecule to its site of action. In addition, binding alters the structure of the fibrin clot altering its susceptibility to degradation by fibrinolytic enzymes6. These observations led to the hypothesis that polyP operates as a timed switch, being released from platelets upon activation thereby allowing rapid repair of an injury while simultaneously preventing clot breakdown until sufficient wound healing has occurred3. The ability of polyP to promote FXII-dependent coagulation has been reported in several studies but has been contested, as shorter soluble polymers, such as those found in platelets, are relatively inefficient in this process. Recent work from our group7 and others8 documents the retention of polyP on the surface of degranulated platelets. This pool of polyP is of longer chain length than the soluble secreted form and is complexed to divalent metal ions8 thereby augmenting its ability to stimulate factor XII activation. These studies highlight the potential that polyP may exert its biological cofactor activity when anchored to a surface such as platelets, fibrin and potentially other blood cells. Many questions remain as to how this polymer regulates its plethora of functions and whether the binding surface impacts on the target reaction and the half-life of the polymer. However, taken together these in vitro and in vivo studies have delineated a role for polyP in hemostasis and highlight the requirement for more efficient tools to quantify this interesting biomolecule in subsets of patients. References Kornberg A. Inorganic polyphosphate: a molecule of many functions. Prog Mol Subcell Biol. 1999;23:1-18. Ruiz FA, Lea CR, Oldfield E, Docampo R. Human platelet dense granules contain polyphosphate and are similar to acidocalcisomes of bacteria and unicellular eukaryotes. J Biol Chem. 2004;279(43):44250-44257. Smith SA, Mutch NJ, Baskar D, Rohloff P, Docampo R, Morrissey JH. Polyphosphate modulates blood coagulation and fibrinolysis. Proc Natl Acad Sci U S A. 2006;103(4):903-908. Choi SH, Smith SA, Morrissey JH. Polyphosphate is a cofactor for the activation of factor XI by thrombin. Blood. 2011;118(26):6963-6970. Whyte CS, Chernysh IN, Domingues MM, Connell S, Weisel JW, Ariens RA, Mutch NJ. Polyphosphate delays fibrin polymerisation and alters the mechanical properties of the fibrin network. Thromb Haemost. 2016;116(5):897-903. Mutch NJ, Engel R, Uitte de Willige S, Philippou H, Ariens RA. Polyphosphate modifies the fibrin network and down-regulates fibrinolysis by attenuating binding of tPA and plasminogen to fibrin. Blood. 2010;115(19):3980-3988. Mitchell JL, Lionikiene AS, Georgiev G, Klemmer A, Brain C, Kim PY, Mutch NJ. Polyphosphate colocalizes with factor XII on platelet-bound fibrin and augments its plasminogen activator activity. Blood. 2016;128(24):2834-2845. Verhoef JJ, Barendrecht AD, Nickel KF, Dijkxhoorn K, Kenne E, Labberton L, McCarty OJ, Schiffelers R, Heijnen HF, Hendrickx AP, Schellekens H, Fens MH, de Maat S, Renne T, Maas C. Polyphosphate nanoparticles on the platelet surface trigger contact system activation. Blood. 2017;129(12):1707-1717. Disclosures No relevant conflicts of interest to declare.

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.

Rivaroxaban and apixaban induce clotting factor Xa fibrinolytic activity

When bound to an anionic phospholipid-containing membrane, activated clotting factorX (FXa) is sequentially cleaved by plasmin from the intact form, FXaα, to FXaβ and then to Xa33/13. Tissue-type plasminogen activator (t-PA) produces plasmin and is the initiator of fibrinolysis. Both FXaβ and Xa33/13 enhance t-PA-mediated plasminogen activation. Although stable in experiments using purified proteins, Xa33/13 rapidly loses t-PA cofactor function in plasma. Bypassing this inhibition, covalent modification of the FXaα active site prevents Xa33/13 formation by plasmin, and the persistent FXaβ enhances plasma fibrinolysis. As the direct oral anticoagulants (DOACs) rivaroxaban and apixaban bind to the FXa active site, we hypothesizedthat they similarly modulate FXa fibrinolytic function. DOAC effects on fibrinolysis and the t-PA cofactor function of FXa were studied in patient plasma, normal pooled plasma and purified protein experiments by the use of light scattering, chromogenic assays, and immunoblots. The plasma of patients taking rivaroxaban showed enhanced fibrinolysis correlating with FXaβ. In normal pooled plasma, the addition of rivaroxaban or apixaban also shortened fibrinolysis times. This was related to the cleavage product, FXaβ, which increased plasmin production by t-PA. It was confirmed that these results were not caused by DOACs affecting activated FXIII-mediated fibrin crosslinking, clot ultrastructure and thrombin-activatable fibrinolysis inhibitor activation in plasma. The current study suggests a previously unknown effect of DOACs on FXa in addition to their well-documented anticoagulant role. By enabling the t-PA cofactor function of FXaβ in plasma, DOACs also enhance fibrinolysis. This effect may broaden their therapeutic indications.

Impaired fibrinolysis in degenerative mitral and aortic valve stenosis

Valvular heart disease is associated with an increased thromboembolic risk. Impaired fibrinolysis was reported in severe aortic stenosis (AS). Little is known about fibrinolysis in mitral stenosis (MS). We sought to compare fibrinolysis impairment in AS and MS. We studied 121 individuals scheduled for elective aortic valve (AV) or mitral valve (MV) surgery for AS (n = 76) or MS (n = 45), in order to compare fibrinolysis impairment. Fibrinolytic capacity was assessed by determination of clot lysis time (t50%) and fibrinolysis inhibitors, including plasma plasminogen activator inhibitor-1 (PAI-1) antigen (PAI-1:Ag) and activity, thrombin-activatable fibrinolysis inhibitor (TAFI) antigen and activity. Prolonged t50% (+ 29%), elevated TAFI activity (+ 12%), TAFI:Ag (+ 21%), and PAI-1:Ag (+ 84%) were observed in patients with MS, compared with those with AS. t50% Correlated with mean and maximal MV gradients (r = 0.43, p < 0.0001 and r = 0.39, p < 0.0001, respectively), but not with AV gradients. Mean and maximal MV gradients correlated with TAFI activity and PAI:Ag. Patients with permanent atrial fibrillation (AF; 35 with MS and 5 with AS) had longer t50% (by 22%, p = 0.0002) and higher PAI-1:Ag (by 74%, p < 0.0001) than the remainder. In the whole group, postoperative drainage volumes correlated inversely with PAI-1:Ag (r = − 0.22, p = 0.02). MS is associated with more pronounced impairment of global fibrinolytic capacity than AS at the stage of surgical intervention, which is in part driven by AF. Our findings suggest that hypofibrinolysis might be implicated in the progression of MS and its thromboembolic complications.

Platelet Drop and Fibrinolytic Shutdown in Patients With Sepsis.

Thrombocytopenia is the most common hemostatic disorder during sepsis and is associated with high mortality. We examined whether fibrinolytic changes precede incident thrombocytopenia and predict outcome in patients with severe sepsis. Nested study from the multicenter, randomized, controlled trial on the efficacy of albumin replacement in severe sepsis or septic shock (the Albumin Italian Outcome Sepsis trial). Forty ICUs in Italy. Three groups of patients were selected: 1) patients with platelet count less than or equal to 50 × 10/L at study entry (n = 85); 2) patients with baseline platelet count greater than or equal to 100 × 10/L who developed thrombocytopenia (≤ 50 × 10/L) within 28 days (n = 100); 3) patients with platelet count always more than or equal to 100 × 10/L (n = 95). Fibrinolytic variables, including fibrinolysis inhibitors and in vivo markers of plasmin generation, were measured on day 1. Patients with early thrombocytopenia (group 1) and those who developed it later (group 2) had similar illness severity and 90-day mortality, whereas patients without thrombocytopenia (group 3) had milder disease and lower mortality. Fibrinolysis was markedly (and similarly) depressed in groups 1 and 2 as compared with group 3. Major fibrinolytic changes included increased levels of plasminogen activator inhibitor 1 and extensive activation/consumption of thrombin activatable fibrinolysis inhibitor. Most fibrinolytic variables were significantly associated with mortality in univariate models. However, only thrombin activatable fibrinolysis inhibitor level and in vivo markers of fibrinolysis activation, namely plasmin-antiplasmin complex, and D-dimer, were independently associated with mortality after adjustment for Simplified Acute Physiology Score-II score, sex, and platelet count. Furthermore, the coexistence of impaired fibrinolysis and low platelets was associated with an even greater mortality. Impaired fibrinolysis, mainly driven by plasminogen activator inhibitor-1 increase and thrombin activatable fibrinolysis inhibitor activation, is an early manifestation of sepsis and may precede the development of thrombocytopenia. Thrombin activatable fibrinolysis inhibitor level, in particular, proved to be an independent predictor of mortality, which may improve risk stratification of patients with severe sepsis.

Grape intake reduces thrombin generation and enhances plasma fibrinolysis. Potential role of circulating procoagulant microparticles

Phytochemicals contained in grapes down-regulate several prothrombotic pathways in vitro. We evaluated the effect of grape consumption on coagulation and fibrinolysis in healthy volunteers. Thirty subjects were enrolled: 20 were given grape (5 g/kg body weight/day for 3 weeks), while 10 served as controls. Blood samples were taken at baseline (T0), at the end of the grape diet (T1) and after 4-week wash-out (T2). Grape intake caused a significant decrease of the procoagulant and inflammatory responses of whole blood and/or mononuclear cells to bacterial lipopolysaccharide at both T1 and T2. At plasma level, grape diet decreased thrombin generation at T1 and T2, largely through a reduction in the number and/or activity of procoagulant microparticles. This anticoagulant effect resulted in the formation of clots that were more susceptible to fibrinolysis, mainly because of a lesser activation of thrombin activatable fibrinolysis inhibitor. No difference in any variables was detected in controls at the time points considered. In conclusion, chronic grape consumption induces sustained anticoagulant and profibrinolytic effects with potential benefits for human health.

Defective TAFI Activation in Hemophilia Worsens Joint Bleeding

Background: Joint bleeds are common in hemophilia A or B and cause hemophilic arthropathy. It is clinically well recognized that patients with acquired hemophilia generally do not suffer from joint bleeding, but the molecular mechanisms responsible for the difference in joint bleeding tendency between acquired and congenital hemophilia are ill defined. FVIII deficiency causes defective thrombin generation, impaired coagulation, and increased fibrinolysis. The latter is caused by impaired activation of thrombin activatable fibrinolysis inhibitor (TAFI). Our previous plasma-based analyses showed that clotting and thrombin generation were readily inhibited by an anti-FVIII antibody, whereas a 10-fold higher antibody concentration was required to inhibit thrombin-mediated TAFI activation. We hypothesize that residual TAFI activation occurring in acquired hemophilia, but not in congenital hemophilia, protects against joint bleeding. Here, we determine whether TAFI activation prevents joint bleeding in a mouse model of acquired hemophilia.Methods and results: A transient (anti-FVIII) acquired hemophilia A model was set up to compare joint bleeding in wild type (WT) vs. TAFI-/- mice. Joint bleeding was induced by a subpatellar needle puncture in the right knee. This model caused considerable joint bleeding in FVIII-/- mice as evidenced by the decreased hematocrit (Hct) 2 days post injury (D2 Hct) (D2 Hct= 29 ± 11 % (n= 9) vs. baseline Hct (46 ± 2 %); p< 0.0001). A single injection of the FVIII inhibiting antibody (GMA-8015; 0.25 mg/kg) in WT mice caused acquired hemophilia for up to 72 hours as evident from increased tail bleeding similar to that observed in FVIII-/- mice. Consistent with clinical findings, only minimal joint bleeding was observed in inhibitor-treated WT mice (D2 Hct= 44 ± 4 % (n= 15) for BALB/c and 40 ± 4 % (n= 17) for C57Bl/6J). Significant joint bleeding (D2 Hct= 36 ± 9% (n= 12) for C57Bl/6J; p< 0.05) could be induced by a higher dose of inhibitor (1 mg/kg), however bleeding remained considerably less severe than that observed in FVIII-/-mice.In vitro, the FVIII inhibitor readily inhibited thrombin generation but was relatively ineffective in inhibiting TAFI activation. Therefore, we tested our hypothesis that continued TAFI activation prevented severe joint bleeding in the inhibitor-treated WT mice. Indeed, administration of the FVIII inhibitor (0.25 mg/kg) in TAFI-/-mice resulted in excessive joint bleeding (D2 Hct= 25 ± 8 %; n= 14; p< 0.0001). Similarly, joint bleeding in WT mice was increased significantly when the FVIII inhibitor was co-administered with an inhibitory antibody against TAFI (D2 Hct= 34 ± 7 %; n= 13; p< 0.01). In contrast, TAFI deficiency did not increase tail bleeding with or without FVIII inhibitor, as determined by acute blood loss, 24-hour mortality, and Hct of the survivors at 24 hours post tail resection. These data clearly demonstrate that different vascular beds empower different mechanisms to curb bleeding and suggest that the protective effects of TAFI are specifically relevant for the vascular beds of the synovial joint.Activated TAFI (TAFIa) conveys multiple functions, including anti-fibrinolytic effects and numerous anti-inflammatory activities. Interestingly, tranexamic acid (TXA), a Lys analogue and potent anti-fibrinolytic agent, added at 50 mg/ml to the drinking water, did not reduce joint bleeding in FVIII-/- mice or TAFI-/- mice with the FVIII inhibitor, whereas TXA did correct tail bleeding in these mice. This suggests that the protective effects of TAFI on joint bleeding were independent of its anti-fibrinolytic effects and may result from its anti-inflammatory activities. This is supported by histological analysis at day 7 showing increased stromal proliferation and inflammatory cell recruitment in the joints of TAFI-/-mice.Conclusions:TAFI activation is impaired in congenital hemophilia but not in acquired hemophilia. Abrogation of TAFIa activity, either genetically or pharmaceutically, increased joint bleeding in mice with acquired hemophilia, indicating that TAFI may be responsible for the difference in joint bleeding tendency between acquired and congenital hemophilia. Protective effects of TAFI were vascular bed specific and independent of its anti-fibrinolytic effects, suggesting that one or more of TAFIa's other substrates promote hemophilic joint bleeding. Disclosuresvon Drygalski:Novo Nordisk: Consultancy, Honoraria, Speakers Bureau; CSL-Behring: Consultancy, Honoraria, Speakers Bureau; Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; Baxalta/Shire: Consultancy, Honoraria, Speakers Bureau. Mosnier:The Scripps Research Institute: Patents & Royalties; Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Bayer: Honoraria, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau.