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Background: D-dimer (DD) is the most used fibrin-related marker and has been proposed, either alone or in combination with other variables, as prognostic factor in patients with sepsis. However, DD generation depends on both coagulation and fibrinolysis, meaning that it may give false negative results in conditions associated with marked fibrinolytic inhibition such as sepsis. In this study, we tested whether correction of DD for thrombin and plasmin generation could improve its prognostic significance in septic patients., Material and Methods: We performed a nested study in 269 septic patients from the ALBIOS trial. DD, prothrombin fragment 1+2 (F1+2) and plasmin-antiplasmin complex (PAP) were assayed at day 1. Corrected DD (DD corr ) was calculated by the formula DD×PAP/F1+2, such that the lower the DD corr the greater the imbalance in favour of fibrin formation over fibrin lysis, and vice-versa. Primary outcome was 90-day mortality., Results: DD corr showed a J-shaped relationship with mortality, which was highest in the first DD corr tertile (low fibrinolysis), intermediate in the 3 rd (high fibrinolysis), and lowest in the 2 nd (balanced fibrinolysis), suggesting an increased risk whenever the coagulation-fibrinolysis balance is tilted (p<0.0001). Neither DD, nor PAP or F1+2 showed a comparable association with mortality. DD corr was an independent prognostic factor in multivariable Cox models and significantly improved risk stratification (cNRI≥0.28). Finally, by combining DD corr and SOFA tertiles, we developed a score with high discriminatory power., Discussion: DD corr is a good marker of the in vivo coagulation-fibrinolysis balance and displays a prognostic value in sepsis much higher than DD.

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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Unlabelled: Essentials Plasma of factor XI-deficient patients (FXI-dp) displays enhanced fibrinolysis. We investigated the role of thrombin activatable fibrinolysis inhibitor (TAFI) in 18 FXI-dp. FXI-dp generated less activated TAFI (TAFIa) on clotting challenge and were resistant to TAFIa. TAFI activation and TAFIa resistance correlated with bleeding score and bleeding phenotype., Summary: Background Factor XI (FXI) deficiency, a rare disorder with unpredictable bleeding, has been associated with reduced fibrinolytic resistance as a result of abnormal fibrin density. Objective We investigated the involvement of thrombin-activatable fibrinolysis inhibitor (TAFI) in the increased lysability of FXI-deficient (FXI-def) clots and the role of thrombin. Patients/Methods Eighteen patients with FXI deficiency (1-58%) and 17 matched controls were investigated for fibrinolytic resistance to t-PA, thrombin generation, TAFI activation and response to TAFIa. Results When clotting was induced by 0.5 pm tissue factor (TF), FXI-def plasmas displayed less thrombin and TAFIa generation and shorter lysis time than controls. A 100-fold higher TF concentration (to bypass FXI) abolished the difference in thrombin generation but not in lysis time between patients and controls. Normalization of FXI levels by a FXI concentrate increased thrombin generation but had no effect on the lysis time of FXI-def plasma. Moreover, when clots were induced by purified thrombin and high concentrations of FXa inhibitor, FXI-def plasma still generated less TAFIa and displayed a shorter lysis time than controls. Finally, upon TAFIa addition, the lysis time of FXI-def plasma was prolonged significantly less than that of control plasma, suggesting a TAFIa resistance. TAFIa generation and TAFIa resistance were correlated with the bleeding score, displaying a considerable capacity to discriminate between patients with and without bleeding. Conclusions TAFI pathway impairment, largely caused by a hitherto unknown TAFIa resistance, appears to be one main cause of decreased fibrinolytic resistance in FXI deficiency and might be clinically useful for assessing the bleeding risk of FXI-def patients., (© 2016 International Society on Thrombosis and Haemostasis.)

Introduction: Most anticoagulants stimulate fibrinolysis in vitro through mechanisms dependent on and independent of thrombin activatable fibrinolysis inhibitor (TAFI). We evaluated the effect of dabigatran, rivaroxaban and apixaban treatment on plasma fibrinolysis in patients with non-valvular atrial fibrillation., Methods and Results: Patients treated with dabigatran etexilate (n=22), rivaroxaban (n=24) or apixaban (n=22) were studied. Plasma was obtained before (trough) and 2h after drug intake (peak). Fibrinolytic resistance of clots exposed to exogenous tissue plasminogen activator was significantly lower in peak than in trough samples and correlated with drug concentration only in dabigatran group. Moreover, fibrinolytic resistance at peak was lower in dabigatran than in rivaroxaban and apixaban groups. This difference disappeared if the TAFI pathway was inhibited. Thrombin generation and TAFI activation were markedly lower in peak than in trough samples in all three groups. However, TAFIa levels in trough and peak samples were significantly lower in dabigatran group than in rivaroxaban and apixaban groups. Circulating levels of prothrombin fragment F1+2 (reflecting in vivo thrombin generation) and plasmin-antiplasmin complex (reflecting plasmin generation) were not or barely influenced by drug levels in all groups., Conclusions: Our data suggest that dabigatran, contrary to rivaroxaban and apixaban, reduces fibrinolytic resistance by virtue of its greater impact on TAFI activation. The profibrinolytic effect of dabigatran may play a role locally, at sites of fibrin formation, by making the nascent thrombus more susceptible to plasminogen-dependent degradation., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Anticoagulants stimulate fibrinolysis in vitro, mainly by inhibiting thrombin-mediated TAFI activation. Surprisingly, however, direct thrombin inhibitors (DTIs) inhibit fibrinolysis and enhance thrombin generation in vitro when tested in the presence of high thrombomodulin (TM) concentrations. Because the paradoxical effect on thrombin generation was shown to be protein C (PC)-dependent, we investigated the role of PC in the antifibrinolytic effect of two DTIs, dabigatran and argatroban., Methods and Results: In the presence of 10 nM TM, both dabigatran (0.5 μM) and argatroban (1 μM) prolonged clot lysis time and enhanced thrombin generation. This notwithstanding, the DTIs inhibited thrombin-mediated TAFI activation, peak TAFIa activity being reduced by >60%. A specific feature of TAFI activation curve in the presence of DTIs was a much slower disappearance of TAFIa activity, which was likely the cause of fibrinolysis inhibition. The addition of an anti-PC antibody (αPC) nullified the paradoxical effect of DTIs on thrombin generation but influenced neither TAFI activation nor the fibrinolysis time., Conclusions: Our results suggest that the inhibition of PC activation by DTIs in the presence of TM, while enhancing thrombin generation, has no effect on thrombin-mediated TAFI activation. The inhibition of fibrinolysis by DTIs can be explained by the prolonged activation of TAFI resulting from the sustained release of thrombin from thrombin-DTI complex. While the clinical relevance of these findings needs to be investigated by in vivo studies, our data might help understanding the role of the different players in the regulation of thrombin generation, TAFI activation and fibrinolysis resistance., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Hypercoagulable conditions may determine a hypofibrinolytic state by increasing the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). Factor (F)IX-Padua is a mutated FIX with an eight-fold increased clotting activity and associates with a higher venous thrombotic risk. We evaluated the influence of FIX-Padua on TAFI-mediated regulation of fibrinolysis. A subject hemizygous for FIX-Padua, two family members (heterozygous and normal) and six healthy controls were studied. Clot lysis, TAFI activation and thrombin generation were evaluated in contact-inhibited plasma challenged with low concentrations of tissue factor. Fibrinolysis times were significantly longer in FIX-Padua carriers than controls. The difference disappeared when activated TAFI (TAFIa) was inhibited, when TAFI activation was avoided or when clotting was made independent of FIX. TAFIa generation was markedly enhanced in FIX-Padua carriers as compared to controls, and this could be explained by a greater thrombin generation in the former. Hyperactive FIX, but not wild-type FIX, enhanced fibrinolytic resistance also when the FXI-dependent positive feedback was blocked by a neutralising anti-FXI antibody. This thrombin-mediated, TAFI-dependent down-regulation of fibrinolysis provides new clues for explaining the heightened thrombotic risk in subjects carrying the FIX-Padua mutation.

Background: Recently, anti-thrombin-activatable fibrinolysis inhibitor (TAFI) mAbs selectively inhibiting plasmin-mediated TAFI activation were shown to stimulate fibrinolysis in vitro and in vivo, suggesting, in contrast to other findings, that plasmin-mediated TAFI activation plays an important role in fibrinolysis regulation., Objective: To further characterize the effects of two plasmin-specific anti-TAFI mAbs (MA-TCK11A9 and MA-TCK26D6) on TAFI-dependent inhibition of fibrinolysis., Methods and Results: Both mAbs inhibited plasmin-mediated but not thrombin/thrombomodulin-mediated TAFI activation, whereas neither inhibited the cleavage of hippuryl-arginine by activated TAFI (TAFIa). They stimulated tissue-type plasminogen activator-induced fibrinolysis in different clot lysis models through a TAFI-dependent mechanism, especially in the presence of thrombomodulin (TM), a condition in which TAFI is largely activated by the thrombin-TM complex. In a fibrinolysis-based TAFIa activity assay, both mAbs inhibited TAFIa, whereas other mAbs targeting thrombin-TM-mediated TAFI activation did not. The inhibition of TAFIa activity, however, was substrate-specific, because neither mAb inhibited the cleavage of thrombin-activated osteopontin and C5a by TAFIa, thus sparing the anti-inflammatory activity of TAFIa., Conclusions: Our anti-TAFI mAbs, by selectively inhibiting TAFIa activity on fibrin, may represent the prototype of a new class of TAFI inhibitors with improved pharmacologic activity., (© 2013 International Society on Thrombosis and Haemostasis.)

Background: Severe clotting deficiencies are associated with enhanced in vitro fibrinolysis due to insufficient thrombin activatable fibrinolysis inhibitor (TAFI) activation. Because oral anticoagulant therapy (OAT) with warfarin causes a partial deficiency of vitamin K-dependent factors, its effect on clot lysability remains unclear., Objectives: To evaluate plasma and blood fibrinolytic capacity in patients under stable OAT (n = 221) as compared with controls (n = 132)., Methods: Fibrinolysis resistance of plasma (turbidimetry) and blood (thromboelastography) clots was calculated as the lysis time of tissue factor-induced clots exposed to 30 and 100 ng mL(-1) t-PA, respectively., Results: Plasma PAI-1 was similar in the two groups, whereas TAFI was slightly lower in patients. OAT plasma clots lysed faster than controls (P = 0.001). The addition of the TAFIa inhibitor PTCI reduced lysis time by 14% in OAT and 34% in controls, and the difference between the groups disappeared. Similar data were obtained with blood clots. Thrombin and TAFIa generation in OAT plasma amounted to roughly 50% of controls, supporting a reduced thrombin-dependent TAFI activation. Clot resistance of OAT plasma was normalized by Ba-citrate plasma eluate or prothrombin but not by BaSO(4) serum eluate, rFVIIa or FX. Surprisingly, circulating levels of TAFIa and its inactive derivative TAFIai were higher in warfarin patients (P < 0.0001) and correlated with plasmin-antiplasmin (P = 0.0001) but not with prothrombin F(1) (+) (2) ., Conclusions: OAT enhances both plasma and blood fibrinolysis by reducing thrombin-dependent TAFI activation, a phenomenon largely determined by low prothrombin levels. At variance with in vitro data, 'basal' in vivo TAFIa/ai levels seem related to plasmin rather than thrombin generation., (© 2012 International Society on Thrombosis and Haemostasis.)

Paediatric obesity, like adulthood obesity, is associated with an increase of fibrinolysis inhibitors. No study, however, has evaluated the impact of these changes on plasma fibrinolytic capacity. We investigated plasma fibrinolysis and the role therein of the fibrinolytic changes associated with obesity in 59 obese children (body mass index > 95th percentile) and 40 matched controls. Fibrinolysis was investigated by measuring 1) the plasma levels of relevant fibrinolytic factors; 2) the in vitro fibrinolytic capacity under different conditions, using a microplate plasma clot lysis assay; 3) the circulating levels of markers of clotting and fibrinolysis activation. Plasminogen activator inhibitor 1 (PAI-1), total thrombin activatable fibrinolysis inhibitor (TAFI) and fibrinogen levels were higher in obese children as compared to controls (p<0.01). Plasma clots from obese children lysed significantly slower than control clots when exposed to exogenous plasminogen activator, indicating a greater resistance to fibrinolysis. By the use of a selective inhibitor of activated TAFI and by regression analyses we found that fibrinolysis resistance in obese samples was attributable to PAI-1 increase and to enhanced TAFI activation. The ratio between the circulating levels of D-dimer and thrombin-antithrombin complex, a marker of in vivo fibrinolysis, was significantly lower in obese children, suggesting a reduced fibrinolytic efficiency. These data indicate that paediatric obesity is associated with a hypofibrinolytic state which might contribute to the increased thrombotic risk associated with this condition.

TAFI (thrombin activatable fibrinolysis inhibitor) is the precursor of a basic carboxypeptidase (TAFIa) with strong antifibrinolytic and anti-inflammatory activity. Compelling evidence indicates that thrombin, either alone or in complex with thrombomodulin, is the main physiological activator of TAFI. For this reason derangements of thrombin formation, whatever the cause, may influence the fibrinolytic process too. Experimental models of thrombosis suggest that TAFI may participate in thrombus development and persistence under certain circumstances. In several models of pharmacological thrombolysis, the administration of TAFI inhibitors along with the fibrinolytic agent leads to a marked improvement of thrombus lysis, underscoring the potential of TAFI inhibitors as adjuvants for thrombolytic therapy. The role of TAFI in inflammatory diseases is more complex as it may serve as a defense mechanism, exacerbate the disease, or have no influence, depending on the nature of the model and the role played by the mediators controlled by TAFIa. Finally, the numerous clinical studies in patients with thrombotic disease support the idea that increased levels of TAFI and/or the enhancement of TAFI activation may represent a new risk factor for venous and arterial thrombosis., (Copyright © 2011. Published by Elsevier Ltd.)

Background: The resistance of platelet-rich thrombi to fibrinolysis is generally attributed to clot retraction and platelet PAI-1 release. The role of TAFI in platelet-mediated resistance to lysis is unclear., Objective: We investigated the contribution of TAFI to the antifibrinolytic effect of platelets in whole blood by thromboelastography., Methods: Platelet-poor (PP-WB, < 40 × 10(3) μL(-1) ) and platelet-rich (PR-WB, > 400 × 10(3) μL(-1) ) blood samples were obtained from normal human blood (N-WB, 150-220 × 10(3) μL(-1) ). Clot lysis time was measured by thromboelastography in recalcified blood supplemented with t-PA (100 ng mL(-1) ) and tissue factor (1:1000 Recombiplastin)., Results: t-PA-induced lysis time increased in parallel with platelet concentration (up to 3-fold). Neutralization of TAFI, but not of PAI-1, shortened the lysis time by ∼ 50% in PR-WB and by < 10% in PP-WB. Accordingly, prothrombin F1+2 and TAFIa accumulation was greater in PR-WB than in PP-WB. A similar TAFI-dependent inhibition of fibrinolysis was observed when clot retraction was prevented by cytochalasin D or abciximab, or when platelet membranes were tested. Moreover, in blood with an intact contact system, platelet-mediated fibrinolysis resistance was attenuated by an anti-FXI but not by an anti F-XII antibody. Finally, platelets made the clots resistant to the profibrinolytic effect of heparin concentrations displaying a strong anticoagulant activity., Conclusions: Our data indicate that TAFI activation is one major mechanism whereby platelets make clots resistant to fibrinolysis and underscore the importance of TAFI inhibitors as new antithrombotic agents., (© 2010 International Society on Thrombosis and Haemostasis.)

Background: Anticoagulants are expected to promote fibrinolysis by counteracting the antifibrinolytic effects of thrombin, which include thrombin-activatable fibrinolysis inhibitor (TAFI) activation and clot structure enhancement. However, the efficiency of anticoagulants may vary remarkably, and the ability of direct thrombin inhibitors to facilitate clot lysis remains controversial., Objective: To evaluate the profibrinolytic effect of dabigatran, a new, direct thrombin inhibitor, using different in vitro models., Methods and Results: The resistance of tissue factor-induced plasma clots to fibrinolysis by exogenous tissue-type plasminogen activator (t-PA) (turbidimetric method) was reduced by dabigatran in a concentration-dependent manner, with > or = 50% shortening of lysis time at clinically relevant concentrations (1-2 microm). A similar effect was observed in the presence of low (0.1 and 1 nm) but not high (10 nm) concentrations of thrombomodulin. Acceleration of clot lysis by dabigatran was associated with a reduction in TAFI activation and thrombin generation, and was largely, although not completely, negated by an inhibitor of activated TAFI, potato tuber carboxypeptidase inhibitor. The assessment of the viscoelastic properties of clots showed that those generated in the presence of dabigatran were more permeable, were less rigid, and consisted of thicker fibers. The impact of these physical changes on fibrinolysis was investigated using a model under flow conditions, which demonstrated that dabigatran made the clots markedly more susceptible to flowing t-PA, by a mechanism that was largely TAFI-independent., Conclusions: Dabigatran, at clinically relevant concentrations, enhances the susceptibility of plasma clots to t-PA-induced lysis by reducing TAFI activation and by altering the clot structure. These mechanisms might contribute to the antithrombotic activity of the drug.

Background: To date, no study has tested the effect of different heparin dosages on the hemostatic changes during off-pump coronary artery bypass graft (OPCABG) surgery, and a wide variety of empirical anticoagulation protocols are being applied. We tested the effect of two different heparin dosages on the activation of the hemostatic system in patients undergoing OPCABG procedures., Methods: Forty-two patients eligible for OPCABG procedures were assigned in a randomized fashion to low-dose heparin (150 IU/kg) or high-dose heparin (300 IU/kg). Prothrombin fragment 1+2, plasmin/alpha(2)-plasmin inhibitor complex, D-dimer, soluble tissue factor, tissue factor pathway inhibitor, total thrombin activatable fibrinolysis inhibitor (TAFI), and activated TAFIa were assayed by specific enzyme-linked immunosorbent assays at six different timepoints, before, during, and after surgery. Platelet function was evaluated by means of an in vitro bleeding time test, platelet function analyzer-100., Results: The OPCABG surgery was accompanied by significant changes of all plasma biomarkers, indicative of systemic activation of coagulation and fibrinolysis. A significant increase in circulating TAFIa was detected perioperatively and postoperatively, and multiple regression analysis indicated that prothrombin F1+2 but not plasmin/alpha(2)-antiplasmin complex was independently associated with TAFIa level. Platelet function analyzer-100 values did not change significantly after OPCABG. All hemostatic changes were similar in the two heparin groups, even perioperatively, when the difference in anticoagulation was maximal., Conclusions: Both early and late hemostatic changes, including TAFI activation, are similarly affected in the low-dose and high-dose heparin groups, suggesting that the increase in heparin dosage is not accompanied by a better control of clotting activation during OPCABG surgery., (2010 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Thrombin is the main activator of the fibrinolysis inhibitor TAFI (thrombin activatable fibrinolysis inhibitor) and heightened clotting activation is believed to impair fibrinolysis through the increase of thrombin activatable fibrinolysis inhibitor activation. However, the enhancement of thrombin generation by soluble tissue factor was reported to have no effect on plasma fibrinolysis and it is not known whether the same is true for cell-associated tissue factor. The aim of this study was to evaluate the effect of tissue factor-expressing monocytes on plasma fibrinolysis in vitro., Design and Methods: Tissue factor expression by human blood mononuclear cells (MNC) and monocytes was induced by LPS stimulation. Fibrinolysis was spectrophotometrically evaluated by measuring the lysis time of plasma clots containing LPS-stimulated or control cells and a low concentration of exogenous tissue plasminogen activator., Results: LPS-stimulated MNC (LPS-MNC) prolonged fibrinolysis time as compared to unstimulated MNC (C-MNC) in contact-inhibited but not in normal citrated plasma. A significantly prolonged lysis time was observed using as few as 30 activated cells/microL. Fibrinolysis was also impaired when clots were generated on adherent LPS-stimulated monocytes. The antifibrinolytic effect of LPS-MNC or LPS-monocytes was abolished by an anti-tissue factor antibody, by an antibody preventing thrombin-mediated thrombin activatable fibrinolysis inhibitor activation, and by a TAFIa inhibitor (PTCI). Assays of thrombin and TAFIa in contact-inhibited plasma confirmed the greater generation of these enzymes in the presence of LPS-MNC. Finally, the profibrinolytic effect of unfractionated heparin and enoxaparin was markedly lower (approximately 50%) in the presence of LPS-MNC than in the presence of a thromboplastin preparation displaying an identical tissue factor activity., Conclusions: Our data indicate that LPS-stimulated monocytes inhibit fibrinolysis through a tissue factor-mediated enhancement of thrombin activatable fibrinolysis inhibitor activation and make clots resistant to the profibrinolytic activity of heparins, thus providing an additional mechanism whereby tissue factor-expressing monocytes/macrophages may favor fibrin accumulation and diminish the antithrombotic efficacy of heparins.

Background: Mild hyperhomocysteinemia (HHcy) has been shown to be associated with impaired fibrinolysis, but some aspects of this association are unclear., Objective: Using an in vitro model of physiological relevance, we investigated whether and how HHcy influences plasma fibrinolytic potential., Patients/methods: We studied 176 patients with previous venous thromboembolism, 58 with HHcy, 118 with normal total homocysteine (tHcy) levels (NHcy), at least 3 months after withdrawal of anticoagulant therapy. Plasma fibrinolytic potential was measured as the fibrinolysis time of tissue factor (TF)-induced clots exposed to 15 ng mL(-1) t-PA., Results: Fibrinolysis time was longer in HHcy than in NHcy patients, but this difference disappeared when the assay was performed in the presence of the TAFIa inhibitor, PTCI. Plasma levels of thrombin activatable fibrinolysis inhibitor (TAFI) and factor VIII (FVIII) were higher in HHcy than NHcy, whereas PAI-1, fibrinogen and endogenous thrombin potential were similar. Using multivariate analysis, plasma tHcy was identified as an independent predictor of fibrinolysis time. Experiments in which native fibrinogen was replaced by purified fibrinogen suggested that alterations of fibrinogen structure did not contribute appreciably to the hypofibrinolysis of HHcy plasma samples. The acute increase of tHcy either in vivo (after an oral methionine load) or in vitro (after incubation of normal plasma with 0.5 mm DL-Hcy) had no effects on fibrinolysis or TAFI levels., Conclusions: We describe an enhanced TAFI-related antifibrinolytic activity in mild HHcy, which might account for the reported heightened thrombosis risk; however, it is unknown whether HHcy is causally related to hypofibrinolysis or an associated bystander.

Introduction: TAFI (thrombin-activatable fibrinolysis inhibitor) is a potent anti-fibrinolytic and anti-inflammatory factor of liver origin. It is markedly reduced in liver cirrhosis but its effect on fibrinolysis remains controversial and no data are available on its prognostic value. We evaluated the relationship of TAFI level with plasma fibrinolysis and survival in cirrhotic patients., Patients and Methods: Sixty-five patients with liver cirrhosis were studied. TAFI antigen, plasma fibrinolysis and other laboratory variables were assayed at study entry and their association with mortality was assessed during a 3-year follow-up., Results: TAFI level and fibrinolysis time were markedly reduced in liver cirrhosis as compared to healthy subjects (p<0.0001) and TAFI deficiency was strongly correlated with fibrinolysis time (p=0.0002). TAFI level at entry, but not fibrinolysis time, was significantly lower in non-survivors (n=25) than in survivors (n=40, p=0.0001). By Cox regression analysis, after adjustment for possible confounding factors, TAFI, but not fibrinolysis time, was identified as an independent predictor of mortality. TAFI assay, moreover, showed a clinically relevant accuracy in assessing patients' survival (ROC curve analysis, p<0.0001) achieving a sensitivity of 92%, a specificity of 55%, and a negative predictive value of 91.7%., Conclusions: Our data indicate that TAFI deficiency in liver cirrhosis is associated with enhanced plasma fibrinolysis. Moreover, they suggest that TAFI, but not fibrinolysis time, is a strong predictor of survival and thus TAFI assay might prove useful to select candidates for liver transplantation.

Anticoagulants have been shown to stimulate fibrinolysis principally via inhibition of thrombin-mediated activation of TAFI (thrombin activatable fibrinolysis inhibitor). Their profibrinolytic effect, however, may vary according to their mechanism of action and to the clot composition. We compared the fibrinolytic activity of the direct thrombin inhibitor melagatran with that of unfractionated heparin in platelet-poor (PPP) and platelet-rich (PRP) models consisting of tissue-factor-induced clots exposed to exogenous t-PA (25 ng/ml). In the PPP clot model, both heparin (0.1-0.6 U/ml) and melagatran (20-320 ng/ml) caused a concentration-dependent shortening of lysis time. However, when drug profibrinolytic activity (lysis ratio) was expressed in function of the aPTT prolongation (aPTT ratio), melagatran was more efficient than heparin. In the PRP clot model, melagatran displayed a fibrinolytic activity fairly comparable to that observed in PPP whilst heparin caused a modest reduction of lysis time only at the highest concentrations. Assay of thrombin and TAFIa generation in defibrinated plasma showed that the presence of platelets markedly reduced the ability of heparin, but not that of melagatran, to inhibit the formation of these enzymes. Altogether these data indicate that melagatran is more efficient than heparin in promoting fibrinolysis, particularly in plateletrich clots, and may thus grant a greater antithrombotic activity by enhancing thrombus dissolution.

Introduction: Helicobacter pylori and some of its virulence factors stimulate human blood mononuclear cells (MNC) in vitro to produce tissue factor (TF) and plasminogen activator inhibitor-2 (PAI-2). In this study we investigated the procoagulant-fibrinolytic potential of blood MNC in patients with H. pylori infection. In the same patients we also evaluated the coagulation-fibrinolysis profile in gastric tissue and in plasma., Methods and Results: The production of TF and PAI-2 was evaluated in 61 patients with dyspepsia, 31 positive and 30 negative for H. pylori infection. TF expressed by MNC and PAI-2 accumulation in cell culture medium after incubation for 20 h at 37 degrees C were significantly higher in H. pylori(+) than in H. pylori(-) patients and were significantly correlated. TF and PAI-2 content in extracts of gastric mucosa was similar in the two groups whereas lower levels of tissue plasminogen activator (t-PA) and thrombomodulin (TM) antigens were found in the antrum of H. pylori(+) patients. No difference between the groups was observed in plasma thrombus precursor protein, prothrombin fragment 1+2, D-dimer, t-PA, PAI-1, TM and thrombin activatable fibrinolysis inhibitor., Conclusions: H. pylori infection is associated with functional abnormalities of blood MNC resulting in the coordinate expression of TF and antifibrinolytic activity. Changes in cell coagulation-fibrinolysis balance may represent a link between H. pylori infection and ischemic heart disease.

The prothrombin gene mutation G20210A is a common risk factor for thrombosis and is associated with increased prothrombin levels. However, the mechanism whereby hyperprothrombinemia predisposes to thrombosis remains unclear. Because thrombin is the physiologic activator of TAFI (thrombin activatable fibrinolysis inhibitor), the precursor of an antifibrinolytic carboxypeptidase (TAFIa), we evaluated the influence of hyperprothrombinemia on fibrinolysis. Thirty-two heterozygous carriers of the G20210A mutation and 30 noncarriers were studied. Plasma fibrinolytic factors and TAFI levels were similar in the 2 groups. Mean lysis time of tissue factor-induced plasma clots exposed to 25 ng/mL exogenous tissue-type plasminogen activator (t-PA) was significantly longer in 20210A carriers than in control donors. This difference disappeared on addition of a specific inhibitor of TAFIa. Determination of thrombin and TAFIa activity, generated during clot lysis, revealed that G20210A mutation was associated with a significant enhancement of late thrombin formation and an increase in TAFI activation. Plasma prothrombin level was highly significantly correlated with both clot lysis time and TAFI activation. The addition of purified prothrombin, but not of factors X or VIIa, to normal plasma caused a concentration-dependent, TAFI-mediated inhibition of fibrinolysis. These findings provide a new mechanism that might contribute to the thrombotic risk in prothrombin 20210A carriers.

Background and Objectives: The case of a patient with thrombotic manifestations and severe activated protein C resistance due to an anti-factor V antibody has recently been described. Since activated protein C (APC) is also profibrinolytic we wanted to determine whether the presence of antibodies interfering with the anticoagulant activity of APC also inhibits its profibrinolytic effect., Design and Methods: Plasma clots were formed in the presence of tissue plasminogen activator, thrombin, phospholipids, Ca++, and various concentrations of APC, and the rate of lysis was monitored over time by the reduction in turbidity. Generation of endogenous thrombin and activation of thrombin activatable fibrinolysis inhibitor (TAFI) were also determined during fibrinolysis by clotting and spectrophotometric assays, respectively., Results: Addition of APC to the patient's plasma failed to stimulate fibrinolysis even at a concentration 4 times higher than that needed to produce the maximal effect in control plasma. Removal of IgG from the patient's plasma fully restored the fibrinolytic response to APC. Accordingly, addition of the patient's IgG to control plasma caused a concentration-dependent inhibition of APC-dependent fibrinolysis. The patient's IgG did not, however, inhibit the profibrinolytic effect of heparin. Determination of thrombin and activated TAFI generation during clot lysis showed that APC inhibited the generation of these enzymes by less than 20% in plasma supplemented with the patient's IgG as opposed to >80% in a control sample., Interpretation and Conclusions: Our data suggest that the anti-factor V antibody inhibits fibrinolysis by antagonizing the anticoagulant effect of APC thereby favoring thrombin generation and TAFI activation. Impaired fibrinolysis may represent an additional mechanism contributing to thrombosis in patients with severe APC resistance phenotype.

Hyperfibrinolysis is thought to contribute to bleeding associated with advanced cirrhosis. Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma precursor of a carboxypeptidase (TAFIa) with antifibrinolytic activity and was recently shown to be reduced in cirrhosis. In this study, we evaluated the influence of TAFI deficiency on in vitro fibrinolysis in cirrhotic patients. Fifty-three patients with cirrhosis and 43 healthy controls were studied. TAFI antigen was measured by enzyme-linked immunosorbent assay and TAFI activity by chromogenic assay. Fibrinolysis was evaluated as tissue plasminogen activator-induced plasma clot lysis time in the absence and in the presence of a specific inhibitor of TAFIa. TAFI antigen and activity levels were markedly reduced in cirrhotic patients (P <.0001). In these patients, the lysis time of plasma clots was shorter than in controls (median, interquartile range: 25 minutes, 21-36 minutes vs. 48 minutes, 40-60 minutes, respectively; P <.0001) and was poorly influenced by the TAFIa inhibitor. Accordingly, TAFIa and thrombin activity, generated in cirrhotic samples during clot lysis, were significantly lower than in control samples. Addition of purified TAFI to cirrhotic plasma prolonged the lysis time and enhanced the response to TAFIa inhibitor in a dose-dependent manner. In conclusion, our results indicate that in vitro plasma hyperfibrinolysis in cirrhosis is largely due to a defective TAFIa generation resulting from low TAFI levels and probably from impaired thrombin generation. Impairment of the antifibrinolytic TAFI pathway might contribute to bleeding associated with this disease.

Heparin has been proposed to enhance thrombolysis by inhibiting thrombin-dependent generation of activated TAFI (thrombin activatable fibrinolysis inhibitor), a carboxypeptidase that inhibits fibrinolysis. We evaluated the effect of heparin in an in vitro thrombolysis model consisting of a radiolabelled blood clot submerged in defibrinated plasma. Fibrinolysis was induced by adding t-PA (250 ng/ml) and calcium to the plasma bath. Control experiments indicated that thrombin generation induced by recalcification caused significant TAFI activation and inhibited clot lysis. Heparin (up to 1 U/ml), added to the plasma bath, failed to enhance clot lysis. Thrombin generation in the fluid phase was totally inhibited by heparin at concentrations > 0.5 U/ml. In contrast, thrombin generation on the clot surface was not inhibited by heparin (1 U/ml). TAFIa generation did occur in heparin-containing samples (1 U/ml) and amounted to about 10% of TAFIa formed in control samples. This low amount of TAFIa did exert antifibrinolytic activity as indicated by the observation that the addition of a specific TAFIa inhibitor (PTI) along with heparin enhanced clot lysis. Hirudin (10 micrograms/ml), at variance with heparin, inhibited clot-bound thrombin and enhanced clot lysis. These data show that heparin is unable to stimulate fibrinolysis through a TAFI-dependent mechanism, most likely because of its inefficiency in inhibiting thrombin generation on the clot surface. Moreover, they suggest that clot-bound thrombin plays a major role in TAFI-mediated inhibition of fibrinolysis through "localized" TAFIa generation.

TAFI (thrombin activatable fibrinolysis inhibitor) is a plasma procarboxypeptidase that upon activation inhibits the fibrinolytic process by removing the C-terminal lysines from partially degraded fibrin. The generation of activated TAFI (TAFIa) has been suggested to represent a mechanism of thrombus resistance to thrombolytic therapy. However, the ability of TAFI to inhibit fibrinolysis by pharmacological concentrations of t-PA has not been properly investigated. We used an in vitro model consisting of 125I-fibrin blood clots submerged in autologous defibrinated plasma. Upon addition of t-PA (125-5,000 ng/ml) and CaCl2 (25 mM), samples were incubated at 37 degrees C, and clot lysis was measured at intervals from the radioactivity released into solution. The role of TAFI was assessed either by neutralizing the generated TAFIa with the specific inhibitor PTI (50 microg/ml) or by enhancing TAFI activation through the addition of recombinant soluble thrombomodulin (solulin, 1 microg/ml). In our clot lysis model, activation of TAFI amounted to about 20% of inducible carboxypeptidase activity. Addition of PTI, however, produced a significant increase in the extent of lysis only at concentrations of t-PA equal to or lower than 250 ng/ml. When solulin was added to the plasma surrounding the clot, about 70% of TAFI was activated within 15 min. Under these conditions, inhibition of clot lysis was very marked in samples containing 125 or 250 ng/ml of t-PA, but negligible in those containing pharmacological concentrations of the activator (1,000 and 5,000 ng/ml). Additional experiments suggest that loss of fibrin-dependence by elevated concentrations of t-PA may be one of the mechanisms explaining the lack of effect of TAFIa. Our data indicate that, under our experimental conditions, clot lysis by pharmacological concentrations of t-PA is not influenced by TAFIa even after maximal activation of this procarboxy-peptidase.

Using an in vitro model of clot lysis, the individual response to a pharmacological concentration of recombinant tissue plasminogen activator (rt-PA) and the influence on this response of the physiological variations of blood parameters known to interfere with the fibrinolytic/thrombolytic process were investigated in 103 healthy donors. 125I-fibrin labelled blood clots were submersed in autologous plasma, supplemented with 500 ng/ml of rt-PA or solvent, and the degree of lysis was determined after 3 h of incubation at 37 degrees C. Baseline plasma levels of t-PA, plasminogen activator inhibitor 1 (PAI-1), plasminogen, alpha 2-antiplasmin, fibrinogen, lipoprotein (a), thrombomodulin and von Willebrand factor as well as platelet and leukocyte count and clot retraction were also determined in each donor. rt-PA-induced clot lysis varied over a wide range (28-75%) and was significantly related to endogenous t-PA, PAI-1, plasminogen (p < 0.001) and age (p < 0.01). Multivariate analysis indicated that both PAI-1 antigen and plasminogen independently predicted low response to rt-PA. Surprisingly, however, not only PAI-1 but also plasminogen was negatively correlated with rt-PA-induced clot lysis. The observation that neutralization of PAI-1 by specific antibodies, both in plasma and within the clot, did not potentiate clot lysis indicates that the inhibitor, including the platelet-derived form, is insufficient to attenuate the thrombolytic activity of a pharmacological concentration of rt-PA and that its elevation, similarly to the elevation of plasminogen, is not the cause of clot resistance but rather a coincident finding. It is concluded that the in vitro response of blood clots to rt-PA is poorly influenced by the physiological variations of the examined parameters and that factors other than those evaluated in this study interfere with clot dissolution by rt-PA. In vitro clot lysis test might help to identify patients who may be resistant to thrombolytic therapy.

Cultured human mesangial cells (HMC) derived from normal kidneys have been shown to synthesize tissue-type plasminogen activator (t-PA) and excess amounts of PA inhibitor type 1 (PAI-1). Conflicting results have been obtained concerning the production of urokinase-type PA (u-PA) and efforts to show PA inhibitor 2 (PAI-2) met with failure. We evaluated the fibrinolytic profile of cultured HMC lines obtained from 12 patients with renal carcinoma and one cadaveric kidney donor. Subconfluent cells (third passage) were incubated overnight in serum-free medium. t-PA, u-PA, PAI-1 and PAI-2 antigens were assayed by ELISA methods and PA and PAI activities by amidolytic methods both in conditioned medium (CM) and cell extracts (CE). Besides PAI-1, PAI-2 antigen was detected in all but one HMC lines. At variance with the former, which was largely released in the culture medium, PAI-2 was mainly cell-associated. t-PA antigen was found in all but two cell lines while u-PA antigen was detected in relatively high concentrations in 8 cell lines. PA activity, identified as u-PA by functional and immunological criteria, was measured in CM of six of the eight u-PA producing cell lines, whereas PAI activity was undetectable or very low in CM of all cell lines, suggesting that PAI-1 was largely inactive. Functional assays of cell extracts demonstrated the presence of PA activity, again identified as u-PA, only in samples (five lines) containing u-PA antigen in excess over PAI-2. PAI activity was found instead in the extracts in which the inhibitor was higher than the activator (six lines) and was identified as PAI-2, as it inhibited u-PA but not single-chain t-PA and was neutralized by a polyclonal anti-PAI-2 antibody. The heterogeneous fibrinolytic pattern of HMC lines was confirmed by mRNA analysis of three representative lines. Results were similar when HMC lines at passage five were used, except that the u-PA content was significantly reduced both in CM and CE. These findings indicate that the fibrinolytic profile of cultured HMC is more complex than previously reported. The production of large amounts of PAI-2 may represent an additional control mechanism of proteinase activity.

In patients with colorectal cancer, profound alterations of the plasminogen activator system have been described at the tumor level, but conflicting results have been obtained for fibrinolytic parameters in plasma. Components of the fibrinolytic system, including tissue-type and urokinase-type plasminogen activators and their inhibitors type 1 and 2, were measured in tissue and/or plasma from 41 patients with colorectal cancer and in 40 controls. Procoagulant activity of freshly isolated mononuclear cells (basal activity) and the procoagulant activity and fibrinolytic proteins produced by the cells after incubation for 18 h without exogenous stimulation were also evaluated. Malignant tissue extracts had significantly higher levels of urokinase-type plasminogen activator and plasminogen activator inhibitor-1, but lower levels of tissue-type plasminogen activator than normal mucosa. Plasminogen activator inhibitor-1 alone was higher in advanced (Dukes' stages C + D) than limited (B) tumors. Plasminogen activator inhibitor-2 was not different in malignant tissue and normal mucosa. Plasma levels of plasminogen activator inhibitor-1 antigen were significantly increased in cancer patients compared with controls, but there were no differences in tissue-type and urokinase-type plasminogen activator, in plasminogen activator inhibitor-2, and D-dimer levels. Intra-patient analysis revealed no significant correlation between tumor and plasma levels of plasminogen activators or type 1 inhibitor. Tissue-type plasminogen activator, but not the urokinase type or inhibitor type 1, was higher in venous than in arterial blood collected at the tumor site during surgery. Basal procoagulant activity of mononuclear cells and the procoagulant activity and inhibitor type-2 produced by the cells after short-term culture were comparable in patients and controls. These findings indicate that, at least in our patients with colorectal cancer, the profound changes occurring at tumor level are barely detectable in the blood. Thus, the clinical relevance of plasma fibrinolytic parameters, especially urokinase-type plasminogen activator antigen, as tumor markers in colorectal cancer remains to be established.

Blood clotting activation and fibrin deposition are common findings in lymphoma patients. We evaluated the capacity of peripheral blood mononuclear cells to produce procoagulant activity (PCA) and plasminogen activator inhibitor (PAI) in 12 children with newly diagnosed lymphoma (8 non-Hodgkin's, 4 Hodgkin's) and in 12 matched healthy donors. In the same subjects we also measured plasma antigen levels of tissue-type PA (t-PA), urokinase-type PA (u-PA), PAI-1, PAI-2, and D-dimer. PCA generated by mononuclear cells after incubation for 20 h at 37 degrees C was significantly higher in patients than in controls (p = 0.027). In all samples it was identified as tissue factor by functional criteria (dependence on factor VII). Moreover, culture medium obtained from patients' mononuclear cells after incubation for 20 h at 37 degrees C contained significantly higher amounts of PAI activity and PAI-2 antigen than control samples (p < 0.001). Plasma PAI-1 and t-PA antigens were significantly augmented in patients (p < 0.005), the mean increase of PAI-I being about 5 times higher than that of t-PA. Plasma levels of D-dimer were markedly increased in the patient's group (p < 0.001), whereas u-PA and PAI-2 antigens did not differ from controls. It is suggested that monocytes from lymphoma patients are endowed with functional abnormalities leading to the simultaneous expression of tissue factor and antifibrinolytic activity. These abnormalities, coupled with a reduced plasma fibrinolytic potential, could play an important pathogenetic role in blood clotting activation and fibrin deposition associated with lymphoma.

Glomerular fibrin deposition may result from local activation of blood coagulation and/or impaired removal by the fibrinolytic system. We evaluated the fibrinolytic activity of glomeruli isolated from rabbits infused for 5 h with tumor necrosis factor (TNF) at the doses of 0.8 micrograms/kg/h (n = 5) and 8 micrograms/kg/h (n = 3) or with endotoxin (8 micrograms/kg/h, n = 7). Animals infused with vehicle (n = 11) served as control group. Plasminogen activator (PA) activity of glomerular extracts from rabbits infused with 8 micrograms/kg/h of TNF or endotoxin was significantly lower than that of samples from control animals (p = 0.013 and p = 0.003, respectively). At the dose of 0.8 micrograms/kg/h, TNF caused a reduction in glomerular PA activity which, however, did not reach statistical significance. Neither plasminogen-independent activity nor PA inhibitor activity was detected in glomerular extracts. Fibrin autography of control extracts revealed the presence of two main fibrinolytic activities comigrating with purified urokinase-type and tissue-type PAs. In treated samples, the bands corresponding to free PAs were markedly reduced, with no evidence of enzyme-inhibitor complex formation. Local reduction of fibrinolytic capacity may contribute to intraglomerular fibrin deposition during endotoxemia or in renal inflammatory diseases associated with TNF production.

Over the last few years, evidence has accumulated that the pathogenetic mechanism of disseminated intravascular coagulation encountered in patients with infectious diseases is extraordinarily complex and involves multiple interactions between the microorganism itself and/or a number of mediators, both microorganism derived and host manufactured, and multifunctional cellular systems, namely endothelial cells and mononuclear phagocytes. In particular, infectious agents and mediators shift the coagulation-fibrinolysis equilibrium of these cells towards fibrin formation and accumulation, via enhancement of procoagulant properties and reduction of both anticoagulant and fibrinolytic capacities. New insights into the pathogenetic mechanism may have important implications for the management of infected patients with disseminated intravascular coagulation.

Fibrin deposition in kidney is a common event in some forms of human and experimental glomerulonephritis, and is thought to result from local activation of blood coagulation and/or impaired removal by the fibrinolytic system. We studied the urinary procoagulant and fibrinolytic activities in 46 patients with renal disease (26 with IgA nephritis, 13 with other forms of glomerulonephritis and 7 with non-inflammatory kidney disease) and in 15 matched healthy subjects, as possible indicators of the coagulation-fibrinolysis balance in kidney. Procoagulant activity was slightly but not significantly increased in patients with serum creatinine levels higher than 1.5 mg/dl (group II) as compared with patients with normal creatinine (group I) and controls. It was identified as tissue factor by biological criteria (dependence on factor VII). Fibrinolysis studies showed that both plasminogen activator activity and urokinase antigen were significantly lower in group II than in group I patients and controls (P less than 0.0005). Reduced fibrinolytic activity in patients' urine was due to decreased excretion of urokinase since no inhibitor was detected by both fibrin autography and functional assay. No differences were found between patients and controls in plasma fibrinolytic activity, plasminogen activator inhibitor, and procoagulant activity of blood monocytes. The urinary changes in severe renal disease may reflect an unbalance of the coagulation-fibrinolysis equilibrium in kidney and might be of pathogenetic and clinical relevance.

Treatment of acute lymphoblastic leukaemia (ALL) with L-asparaginase (L-asp) may be associated with thrombotic complications, but the pathogenetic mechanisms of thrombus formation and persistence remain unclear. We studied the procoagulant activity (PCA) of peripheral blood mononuclear cells and some components of the plasma fibrinolytic system in 10 children with ALL undergoing remission induction therapy which includes L-asp. Mononuclear cells obtained 14 days after starting L-asp treatment generated significantly higher amounts of PCA (identified as tissue factor) than cells isolated before the first dose of L-asp and 7 days after the cessation of L-asp administration (p less than 0.01). Augmented PCA coincided with an increase in the plasma D-dimer. The plasma levels of type 1 plasminogen activator inhibitor were found significantly elevated during L-asp therapy (p less than 0.05), whereas plasminogen levels were markedly decreased (p less than 0.05). These findings suggest that, during the course of L-asp treatment, the coagulation-fibrinolysis balance is shifted towards promotion of fibrin formation and deposition. Although it remains to be conclusively established whether L-asp per se or the concurrent administration of multiple chemotherapeutic agents is responsible for these changes, the latter could contribute to the thrombotic complications associated with remission induction therapy for ALL.

The influence of endotoxin-induced elevated plasma levels of the fast-acting inhibitor of plasminogen activator (PA-inhibitor) on thrombolysis was investigated in rabbits with a jugular vein thrombus. Infusion of human tissue-type plasminogen activator (t-PA) produced similar degrees of thrombolysis in control and endotoxin-treated rabbits, although no free t-PA could be demonstrated in plasma of endotoxin-treated animals. Infusion of t-PA in an extracorporeal arteriovenous shunt resulted in loss of thrombolytic activity in endotoxin-treated animals but not in control animals. Blood clots superfused in vitro with mixtures of t-PA and normal plasma lysed in contrast to clots superfused with t-PA and PA-inhibitor-rich plasma. However, addition of rabbit lung slices to the plasma surrounding the blood clot, reversed the inhibition of thrombolysis by PA-inhibitor-rich plasma. This indicates that tissue-derived factor(s) are involved in the regulation of in vivo thrombolysis. These hypothetical factor(s) are, however, very unstable in plasma, which has thus far precluded their further characterization.

Protein C is a circulating proenzyme which, upon activation, exerts a potent anticoagulant activity. Infusion of activated bovine protein C into dogs is accompanied by an increase of circulating tissue plasminogen activator (PA) activity. However, the evidence that human protein C shares a similar profibrinolytic capacity is still lacking. Therefore, we investigated the profibrinolytic properties of human protein C in squirrel monkeys (Samiri sciureus). Injection of activated human protein C resulted in prolongation of the activated partial thromboplastin time but was not associated with increased fibrinolytic activity of blood. Similarly, activation of endogenous protein C (up to 20-30%) by infusion of thrombin-thrombomodulin complex markedly reduced blood coagulability without being accompanied by an increase of circulating PA activity. The in vivo-generated anticoagulant activity was identified as activated protein C by the following observations. It was neutralized by rabbit anti-human protein C-IgG, was slowly inhibited by plasma but not by anti-thrombin III, was adsorbable on barium citrate, and expressed amidolytic activity. Activation of protein C appeared to be selective since other parameters such as thrombin time, platelet count, fibrinogen, and factor V levels were unaffected by thrombin-thrombomodulin infusion. Infusion of human plasma derived from whole blood incubated in vitro with human activated protein C also did not induce a fibrinolytic response, suggesting that no second messengers with PA-releasing activity were being generated in blood. It is concluded that in a primate, neither the administration of activated human protein C nor the activation of endogenous protein C are associated with an increase of fibrinolytic activity. These findings question the role of this enzyme in the regulation of PA release in man.

Microvascular thrombosis is considered an important pathogenetic factor in renal failure associated with obstructive jaundice but the mechanisms leading to fibrin deposition are still unknown. The plasma levels of plasminogen activator inhibitor (PAI) in 29 patients with obstructive jaundice were found significantly increased as compared to 20 nonjaundiced patients. Fibrin autography of plasma supplemented with tissue plasminogen activator (t-PA) revealed that in icteric samples most of the added activator migrated with an apparent Mr of 100 kDa, corresponding to t-PA-PAI complex, whereas in control samples virtually all t-PA migrated as free enzyme. PAI activity detected in icteric samples is similar to the endothelial type PAI since it is neutralized by a monoclonal antibody against PAI-1. Venous stasis in jaundiced patients was neither associated with an increase in blood fibrinolytic activity nor with a decrease in PAI activity. Immunologic assay showed that t-PA release was impaired in 3 out of 4 patients. In controls, venous occlusion induced an increase in both fibrinolytic activity and t-PA antigen and a reduction in PAI activity. Bile duct recanalization in jaundiced patients subjected to surgery was accompanied by a decrease in plasma PAI activity which paralleled the decrease in serum bilirubin levels. In nonjaundiced patients, surgical treatment did not cause significant changes in either parameter. Rabbits made icteric by bile duct ligation showed an early and progressive increase in plasma PAI activity indicating that obstructive jaundice itself causes the elevation of circulating PAI. it is concluded that obstructive jaundice is associated with a severe impairment of fibrinolysis which might contribute to microvascular thrombosis and renal failure.