Ambivalent roles of carboxypeptidase B in the lytic susceptibility of fibrin

Background

Removal of C-terminal lysine residues that are continuously exposed in lysing fibrin is an established anti-fibrinolytic mechanism dependent on the plasma carboxypeptidase TAFIa, which also removes arginines that are exposed at the time of fibrinogen clotting by thrombin.

Objective

To evaluate the impact of alterations in fibrin structure mediated by constitutive carboxypeptidase activity on the function of fibrin as a template for tissue plasminogen activator-(tPA) induced plasminogen activation and its susceptibility to digestion by plasmin.

Methods and results

We used the stable carboxypeptidase B (CPB), which shows the same substrate specificity as TAFIa. If 1.5 – 6 μM fibrinogen was clotted in the presence of 8 U/mL CPB, a denser fibrin network was formed with thinner fibers (the median fiber diameter decreased from 138 – 144 nm to 89 – 109 nm as established with scanning electron microscopy). If clotting was initiated in the presence of 5 – 10 μM arginine, a similar decrease in fiber diameter (82 -95 nm) was measured. The fine structure of arginine-treated fibrin enhanced plasminogen activation by tPA, but slowed down lysis monitored using fluorescent tPA and confocal laser microscopy. However, if lysis was initiated with plasmin in CPB-treated fibrin, the rate of dissolution increased to a degree corresponding to doubling of the plasmin concentration.

Conclusion

The present data evidence that CPB activity generates fine-mesh fibrin which is more difficult to lyse by tPA, but conversely, CPB and plasmin together can stimulate fibrinolysis, possibly by enhancing plasmin diffusion.     

Coagulofibrinolytic changes in patients with disseminated intravascular coagulation associated with post-cardiac arrest syndrome― Fibrinolytic shutdown and insufficient activation of fibrinolysis lead to organ dysfunction

Introduction

Post-cardiac arrest syndrome (PCAS) is often associated with disseminated intravascular coagulation (DIC), thus leading to the development of multiple organ dysfunction syndrome (MODS). The aim of this study was to examine the pathophysiological relationships between coagulation, fibrinolysis and fibrinolytic shutdown by evaluating the levels of coagulofibrinolytic markers, including soluble fibrin, thrombin-activatable fibrinolysis inhibitor (TAFI), tissue plasminogen activator-plasminogen activator inhibitor-1 complex (tPAIC), plasmin-alpha2 plasmin inhibitor complex (PPIC), neutrophil elastase and fibrin degradation product by neutrophil elastase (EXDP).

Materials and Methods

Fifty-two resuscitated patients were divided into two groups: 22 DIC and 30 non-DIC patients.

Results

The levels of soluble fibrin, PPIC, tPAIC, EXDP and neutrophil elastase in the DIC patients with PCAS were significantly higher than those observed in the non-DIC patients. The values of the tPAIC and JAAM DIC scores were found to be independent predictors of increased SOFA scores in the DIC patients. The MODS patients demonstrated significantly higher levels of soluble fibrin and tPAIC; however, the levels of TAFI and EXDP were identical between the patients with and without MODS. In addition, positive correlations were observed between the levels of tPAIC and EXDP in the patients with non-MODS; however, no correlations were observed between these markers in the MODS patients.

Conclusions

Thrombin activation and fibrinolytic shutdown play important roles in the development of organ dysfunction in PCAS patients. Neutrophil elastase-mediated fibrinolysis cannot overcome the fibrinolytic shutdown that occurs in DIC patients with PCAS, thus resulting in the development of MODS.     

Asymmetric dimethylarginine impairs fibrinolytic activity in human umbilical vein endothelial cells via p38 MAPK and NF-κB pathways

Introduction

Asymmetric dimethylarginine (ADMA) is a potent endogenous inhibitor of nitric oxide (NO) synthase. An increased synthesis and/or a reduced catabolism of ADMA might contribute to the onset and progression of thrombosis. The present study was designed to evaluate the effect of ADMA on fibrinolytic factors in endothelial cells, and to investigate the cellular mechanisms.

Materials and Methods

Human umbilical vein endothelial cells (HUVECs) were treated with different concentrations of ADMA for various periods; Then HUVECs were preincubated with NO precursor (L-arginine), MAPK inhibitors, or NF-κB inhibitor (PDTC) before ADMA treatment to repeat the experiment. Protein levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1), and NF-κB activity were measured by ELISA; mRNA levels of tPA and PAI-1 were assayed by qRT-PCR; The activation of MAPK was characterized by western blot analysis.

Results

(1) ADMA decreased tPA antigen levels in time- and concentration-dependent manners, with the maximum effect of 30 μmol/L ADMA for 48 h (control 109.01 ± 4.15 ng/ml vs ADMA 86.76 ± 5.95 ng/ml, p < 0.01); (2) 30 μmol/L ADMA elevated antigen levels of PAI-1 in a time-dependent manner, with the maximum effect of 30 μmol/L ADMA for 48 h (control 2721.12 ± 278.02 ng/ml vs ADMA 3435.78 ± 22.33 ng/ml, p < 0.05); (3) ADMA reduced tPA mRNA levels and increased PAI-1 mRNA levels; (4) L-arginine, SB203580 (p38 MAPK inhibitor) and PDTC attenuated the effects of ADMA on tPA and PAI-1 significantly. (5) ADMA induced a rapid phosphorylation of p38 MAPK, and stimulated NF-κB activity greatly.

Conclusions

ADMA may accelerate thrombosis development by impairing fibrinolytic activity in vascular via inhibiting nitric oxide production and then activating its downstream p38 MAPK and NF-κB pathways.     

Minimally oxidized low-density lipoprotein regulates hemostasis factors of brain capillary endothelial cells

Minimally oxidized low-density lipoprotein (MM-LDL) is a potent atherogenic lipoprotein. We analyzed the effects of MM-LDL on brain capillary endothelial expression of plasminogen activator inhibitor-1 (PAI-1), tissue-type plasminogen activator (tPA), and thrombomodulin (TM). Cultured bovine brain capillary endothelial cells (BEC) incubated with MM-LDL (25 microg/ml) for 24 h showed increased PAI-1 mRNA levels by approximately seven-fold, while tPA and TM mRNA levels were reduced by 84% and 75%, respectively. Moreover, PAI-1 protein levels increased two-fold (16.8+/-7.6 vs. 7.6+/-2.1 ng/ml, p<0.05), whereas tPA protein levels decreased by 45% (1.3+/-0.5 ng/ml vs. 2.3+/-0.7 ng/ml, p<0.05), and TM protein level decreased by 40%. Following incubation with MM-LDL, PAI-1 activity was increased 35% (18.4+/-5.0 vs. 24.8+/-5.2 AU/ml, p<0.05), while TM activity was decreased by 30%. MM-LDL therefore has substantial pro-thrombotic effects on brain capillary endothelial cells, reducing both endothelial fibrinolytic capacity (downregulating tPA while upregulating PAI-1) and anticoagulant function (downregulating TM). These results suggest that MM-LDL may contribute to thrombus formation in the brain.     

Residual vein thrombosis and onset of post-thrombotic syndrome: Influence of the 4G/5G polymorphism of plasminogen activator inhibitor-1 gene

Background

Plasminogen activator inhibitor-1 (PAI-1) is the most important inhibitor of plasminogen activator. The functional 4G/5G polymorphism of the gene coding for PAI-1 may affect PAI-1 plasmatic activity, influencing the imbalance between coagulation and fibrinolysis cascades.In this prospective cohort analytic study, we investigated the role of this single nucleotide polymorphism in the persistence of thrombotic lesion and the occurrence of post-thrombotic syndrome.

Patients/Methods

In a group of 168 patients with post-surgical deep vein thrombosis of the legs, we analyzed the 4G/5G polymorphism in the promoter of PAI-1 gene and plasmatic PAI-1 activity.Enrolled patients were divided in two groups: patients with 4G/5G polymorphism and increased PAI-1 activity (n = 85) and patients without 4G/5G polymorphism and normal PAI-1 activity (n = 83). All patients were treated according to current protocols and re-examined after 3, 12 and 36 months in order to evaluate the persistence of thrombotic lesion and the occurrence of post-thrombotic syndrome.

Results

We found a significantly increased PAI activity in carrier of the 4G allele, who experienced much more frequently a persistence of thrombosis after 3, 12 and 36 months and/or the development of post-thrombosis syndrome, in spite of the anticoagulant treatment.

Conclusions

These data not only confirm the role played by PAI-1 activity and by the 4G/5G SNP of the PAI-1 gene, but also suggest that current therapeutic protocols, recommending the administration of low weight molecular heparin and oral anticoagulant for the treatment of deep vein thrombosis, could be non sufficient for patients genetically predisposed to a less efficient clot lysis.     

Decompressive craniectomy after intravenous tissue plasminogen activator administration for stroke

Objective

Intravenous tissue plasminogen activator (IV tPA) is an approved treatment for acute ischemic stroke. However, the effects of decompressive craniectomy (DC) after IV tPA administration for ischemic stroke are still largely unknown. The aim of this study was to investigate the safety and outcomes of DC after IV tPA administration.

Methods

We retrospectively reviewed patients who underwent DC for malignant hemispheric infarction. We compared 20 patients who underwent DC after IV tPA administration with another 20 patients who underwent DC without prior IV tPA administration.

Results

The patient characteristics did not differ between the DC patients with and without prior IV tPA administration. New intracranial bleeding or worsening of pre-existing ICH occurred in two patients (10%) in each group. Furthermore, the rates of an mRS score of 4–6, 5 or 6, and 6 did not differ significantly between the two groups.

Conclusion

DC may be a safe and useful surgical procedure for space-occupying edema after IV tPA administration for acute stroke.     

The alpha-2-antiplasmin Arg407Lys polymorphism is associated with Abdominal Aortic Aneurysm

Introduction

Abdominal Aortic Aneurysm (AAA) involves dilatation of the abdominal aorta, with a natural history of expansion and eventual rupture. We have previously shown that AAA patients form denser clots with smaller pores, which are more resistant to fibrinolysis. The aim of this study was to use functional polymorphisms of the fibrinolytic system to identify how changes to proteins involved in fibrinolysis may play a role in the development of AAA.

Methods

Caucasian subjects ≥ 55 years (602 AAA patients and 490 matched controls) were genotyped for four polymorphisms (α-2-antiplasmin α2AP Arg6Trp and Arg407Lys, Thrombin-activatable fibrinolysis inhibitor TAFI Thr325Ile and tissue plasminogen activator tPA 7351C → T). DNA was extracted from blood, and genotype identified using real time PCR. Fibrin clot structure was analysed by permeation and turbidity in a subset of patients and controls.

Results

Genotypes across the study population were in Hardy-Weinberg Equilibrium. The two α2AP polymorphisms, Arg6Trp and Arg407Lys were in linkage disequilibrium (P < 0.0001), and possession of a 407Lys allele negatively associated with AAA (odds ratio 0.833, CI95 0.7-0.991, P = 0.040). The TAFI Thr325Ile and the tPA 7351C → T polymorphisms were not associated with AAA. The α2AP 407Lys allele was not associated with in-vitro fibrinolysis times in plasma from patients with AAA.

Conclusion

Possession of the α2AP 407Lys allele was negatively associated with AAA, and thus changes in α2AP may affect aneurysm growth and development. These data indicate that the regulation of plasmin activity (through binding to α2AP), rather than plasmin generation (TAFI, tPA), may play a role in AAA.     

Effects of carvedilol or metoprolol on PAI-1, tPA-mass concentration or Von Willebrand factor in chronic heart failure - a COMET substudy

Introduction

In COMET (Carvedilol or Metoprolol European Trial), carvedilol reduced mortality compared with metoprolol in patients with chronic heart failure. We hypothesized that carvedilol might have greater effects on endothelial derived haemostatic factors than metoprolol. We aimed to study the effects of carvedilol or metoprolol on tissue plasminogen activator (tPA), its inhibitor PAI-1 and Von Willebrand factor (VWF) in patients with heart failure.

Material and Methods

We recruited 260 patients (134 on carvedilol, 126 on metoprolol), mean age 66 years and 84% of them men. Plasma mass concentrations of tPA and PAI-1and percent of VWF were measured at baseline and after one and two years of treatment.

Results

Plasma tPA, PAI-1 and VWF were similar between treatment groups at baseline and no significant differences between groups emerged after one or two years of treatment. In paired analyses in patients assigned to carvedilol, median PAI-1 level decreased from 37.2 to 32.1 µg/l at two years (p = 0.034) and of VWF decreased from baseline to one year (240 vs. 218%, p = 0.023) in patients assigned to carvedilol but were not reduced at any time in patients assigned to metoprolol. Plasma tPA increased over time in both treatment groups (p = 0.013 and 0.027 respectively).

Conclusion

We found no significant difference in the effects of carvedilol or metoprolol on tPA, PAI-1 and VWF. Comparison over time within treatment groups suggested that PAI-1 and VWF might have declined on carvedilol but not on metoprolol. Our hypothesis is not proved but this may reflect an inadequate sample size rather than lack of an effect.     

Long-term stability of fibrinolytic factors stored at -80 °C

Introduction

Blood samples in epidemiological studies are often stored for several years and analysed at different occasions. The reagent kits are continually modified for better precision and accuracy. Our hypothesis was that epidemiological studies are affected by long-term storage and/or modifications of reagent kits.

Materials and Methods

Plasma samples stored at -80 ^°C from two populations were used: A case-referent study with samples collected from 1985 to 2000 and analysed 2005 (n = 1598) were used to study influence of long-term storage. A cross-sectional study analysed 1990 (n = 1558) and re-analysed 2001 (n = 78) and 2005 (n = 828) was used to study influence of reagent kit modifications. Fibrinolytic analyses included immunoassays of tPA, PAI-1 and tPA-PAI-1 complex and chromogenic substrate assays of the activities of tPA and PAI-1.

Results

Long-term storage for a median time of 11.6 years (range 5 to 20) showed an effect of time on tPA antigen R² = 0.01, PAI-1 antigen R² = 0.01 and tPA-PAI-1 complex R² = 0.02. Modifications in reagent kits affected the levels of fibrinolytic factors; for tPA antigen the slope coefficients were between 0.72 and 0.95 (R² 0.47 - 0.75), whereas tPA activity showed an agreement with slope coefficients 1.06 to 1.09 (R² 0.67 - 0.93).

Conclusions

This study showed that long-term storage affects fibrinolytic variables to a negligible extent, but modifications in reagent kits introduced an element of bias. We conclude that analysis of samples on a single occasion is preferable to multiple occasions, as storage has negligible effect.     

Direct microscopic monitoring of initial and dynamic clot lysis using plasmin or rt-PA in an in vitro flow system

Introduction

Plasmin is a direct-acting thrombolytic agent with a favorable safety profile upon intra-arterial delivery in pre-clinical and phase I studies. However, the thrombolytic efficacy of plasmin, relative to that of rt-PA, remains to be established. We have compared the dynamics of clot lysis with plasmin or rt-PA in an in vitro perfusion system, in which thrombolytic agent is administered locally, allowed to induce lysis for short intervals, then washed with plasma in a re-circulation circuit.

Materials and Methods

Whole blood human clots were prepared in observation chambers, exposed to plasmin or rt-PA at equimolar concentrations (1.2/1.0, 1.8/1.5 and 2.4/2.0 mg/ml) for measured intervals of time, followed by perfusion with human plasma. Clot size was monitored by digital analysis of sequential photographs obtained through an optical microscope.

Results

Plasma perfusion after incubation with thrombolytic agent rapidly removed superficial clot fragments. This initial decrease in clot size was greater with plasmin than with rt-PA when tested at the highest concentrations of agent (0.63 ± 0.11 vs. 0.30 ± 0.11, p = 0.001 for clots with non-cross-linked fibrin and 0.53 ± 0.15 vs. 0.14 ± 0.15, p = 0.02, for clots with cross-linked-fibrin). Subsequent clot lysis during plasma flow was greater after prior incubation with rt-PA. Longer incubation times of plasmin resulted in larger portions of the clot being washed free. Repeated plasmin incubations and plasma perfusions of a clot successfully induced stepwise reductions in clot size.

Conclusions

Initial clot lysis is greater with direct exposure using plasmin than rt-PA. During washout and circulation with plasma, rt-PA induced continued clot lysis, while plasmin lysis was curtailed, presumably because of plasmin inhibition.     

Tissue plasminogen activator (tPA) activity is a novel and early marker of asymptomatic LEAD in type 2 diabetes

Introduction

Lower extremity arterial disease (LEAD) is often one of the first signs of a generalized atherosclerotic disease in type 1 and type 2 diabetic subjects.

Materials and methods

We studied 143 diabetic subjects at 30-70 years of age, M/F 69/74, 74 with type 1 and 69 with type 2 diabetes, without previously known or suspected lower extremity arterial disease. The relationship between early asymptomatic lower extremity arterial disease and blood levels of HbA1c, lipids and fibrinolysis markers (tPA-activity, tPA mass, PAI-1 activity, tPA-PAI-1 complex) was assessed. In parallel, a group with non-diabetic subjects (n = 80) was studied.

Results

35 (24%) diabetic subjects were classified as having sign(s) of LEAD, defined as having at least one reduced peripheral blood pressure measurement, 28% in type 1 vs 20% in type 2 diabetic subjects (p = NS). In univariate logistic regression analyses age, glycemic level (HbA1c), male gender (only in type 1 diabetic subjects), hypertension and tPA activity (only in type 2 diabetic subjects) were positively associated with LEAD. When markers of fibrinolysis were entered into a multivariate model adjusting for age, hypertension, and HbA1c, only tPA activity remained independently associated with LEAD (p = 0.01) and this was also found in type 2 diabetic subjects (p = 0.05). In type 1 diabetic subjects the increase in odds ratio was non-significant.

Conclusions

Tissue plasminogen activator (tPA) activity may be an independent and early marker for asymptomatic lower extremity arterial disease in diabetic subjects, particularly in type 2 diabetes. Thus an altered fibrinolytic activity could be an early marker of atherosclerosis development in the lower extremities but the cause-effect relationship remains unclear.     

In vitro effects of detergent sclerosants on fibrinolytic enzymes and inhibitors

Objective

To investigate the effects of Sodium Tetradecyl Sulphate (STS) and Polidocanol (POL) on fibrinolytic mechanisms.

Materials and methods

Measurements were done with serial dilutions of sclerosants in whole blood (WB), platelet rich (PRP) and platelet poor plasma (PPP). Control experiments were done in 5% bovine serum albumin (BSA), spiked with the enzyme/inhibitor. Plasminogen was measured with a chromogenic assay. Alpha-2-antiplasmin (AP) activity, plasmin-alpha-2-antiplasmin (PAP) complexes, plasminogen activator inhibitor-1 (PAI-1) activity, tissue plasminogen activator (t-PA) total antigen, t-PA activity, t-PA/PAI-1 complexes, thrombin activatable fibrinolysis inhibitor (TAFI) antigen and activated TAFI (TAFIa) were measured by ELISA.

Results

At high concentrations (> 0.3%), STS destroyed plasminogen, PAI-1, t-PA/PAI-1 complexes and total t-PA antigen but increased t-PA activity. At low concentrations (< 0.3%), both agents reduced PAP complexes while increasing AP activity. Low concentration STS increased PAI-1 activity, t-PA/PAI-1 complexes, TAFI and TAFIa. Low concentration POL mildly increased the total t-PA antigen and TAFI.

Conclusion

At low concentrations, both agents demonstrated a prothrombotic, antifibrinolytic (increase in PAI-1, total t-PA antigen, AP, TAFI and TAFIa) activity. At high concentrations, STS demonstrated non-prothrombotic (destruction of PAI-1, t-PA/PAI-1 complexes), antifibrinolytic (destruction of plasminogen, increase in AP) activity while POL had minimal effect.     

Haemostatic factors in women with history of Preeclampsia

Objective

Evaluation of haemostatic parameters - Plasma tissue plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1) and fibrin fragment D-dimer several years after the end of pregnancy to investigate if they are modified in women with history of preeclampsia (PE). Study design: 65 healthy women with history of PE and 54 control women with previous normal pregnancy were enrolled in this study. Groups were matched for age, time period since delivery, smoking status and alcohol consumption. t-PA, PAI-1 and fibrin fragment D-dimer antigen levels were quantified using standards commercial ELISA methods. Plasma fibrinogen was measured using automated capillary zone electrophoresis.

Results

Systolic and diastolic blood pressures were higher in women with history of PE. Levels of t-PA, PAI-1 and fibrinogen were similar between groups as well as the t-PA/PAI-1 ratio. A significant increase in D-dimer levels was observed in women with history of PE.

Conclusion

The increase in D-dimer level suggests an abnormal haemostatic potential namely increased intravascular coagulation. This, together with the increased blood pressure, can reflect a tendency for an increased risk of cardiovascular/thrombotic events later in life.     

Simultaneous thrombin and plasmin generation capacities in normal and abnormal states of coagulation and fibrinolysis in children and adults

Introduction

Thrombin and plasmin are the key enzymes involved in coagulation and fibrinolysis, respectively. Plasma coagulative and fibrinolytic potentials in normal children and adults, and in representative pathologically altered hemostatic states, were evaluated via simultaneous assessment of thrombin and plasmin generation.

Materials and Methods

An assay of Simultaneous Thrombin and Plasmin generation (STP) was developed to measure thrombin and plasmin in plasma using individual fluorometric substrates. Coagulation is initiated with dilute tissue factor, phospholipid, and calcium in platelet-poor plasma; fibrinolysis is accelerated via tissue plasminogen activator (tPA). Abnormal states of hemostasis were investigated.

Results

STP assay reproducibility and normal adult and pediatric values for measured and calculated parameters have been established. Onset of both thrombin and plasmin generation was significantly delayed in children relative to adults (p < 0.001) and the maximum amplitudes of thrombin and plasmin generation were less in children than adults (p < 0.01). No significant differences were measured among pediatric age groups. The most profound impairments in thrombin generation were observed for extrinsic and common pathway factor deficiencies, with the exception of afibrinogenemia. Plasmin generation was severely impaired in deficiencies of fibrinogen and plasminogen as well as with decreased tPA reagent concentration and addition of aminocaproic acid. Plasmin generation was greatly enhanced by alpha-2-antiplasmin deficiency and excess tPA reagent.

Conclusion

Simultaneous assessment of thrombin and plasmin generation in plasma shows promise for affording an enhanced understanding of overall coagulative and fibrinolytic functions in physiological and pathologically altered states of hemostasis in children and adults.     

Changes in fibrinolysis following exercise above and below lactate threshold

This study sought to compare fibrinolytic responses to exercise above lactate threshold (LT) to longer-duration, equicaloric exercise below LT. Fifteen males performed cycle ergometer tests above (77% VO(2)peak) and below LT (41% VO(2)peak) to comparatively evaluate tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) responses. tPA activity significantly (P < 0.05) increased during the >LT test (pre-exercise = 1.57 +/- 0.44 IU ml(-1), post-exercise = 3.85 +/- 4.72 IU ml(-1)), but not the LT (pre-exercise = 8.32 +/- 4.48 ng ml(-1), post-exercise = 14.23 +/- 5.40 ng ml(-1)) and LT test. PAI-1 activity significantly (P < 0.05) decreased during both the >LT (pre-exercise = 15.00 +/- 2.73 AU ml(-1), post-exercise = 10.12 +/- 2.90 AU ml(-1)) and LT test. Our results suggest that exercise 相似文献   

Characterization of a small molecule PAI-1 inhibitor, ZK4044

Plasminogen activator inhibitor-1 (PAI-1) is a key negative regulator of the fibrinolytic system. In animal studies, inhibition of PAI-1 activity prevents arterial and venous thrombosis, indicating that PAI-1 inhibitors may be used as a new class of antithrombotics. In this study, we characterize a small molecule PAI-1 inhibitor, ZK4044, which was identified by high throughput screening and chemically optimized. In a chromogenic substrate-based urokinse (uPA)/PAI-1 assay and a tissue-type plasminogen activator (tPA)-mediated clot lysis assay, ZK4044 inhibited human PAI-1 activity with IC50 values of 644+/-255 and 100+/-90 nM, respectively. ZK4044 had no detectable inhibitory activity toward other serpins such as antithrombin III, alpha1-antitrypsin and alpha2-antiplasmin, indicating that ZK4044 is a specific PAI-1 inhibitor. ZK4044 was shown to bind directly to PAI-1 and prevent the binding of PAI-1 to tPA in a dose-dependent manner in surface plasmon resonance Biacore-based experiments. ZK4044 also prevented PAI-1/tPA complex formation, as analyzed by SDS/PAGE. ZK4044 had little effect on elastase-mediated cleavage of active PAI-1, indicating that the primary mode of action of ZK4044 is most likely to directly block the PAI-1/tPA interaction rather than to convert active PAI-1 to latent PAI-1. In the chromogenic substrate-based uPA/PAI-1 assay, ZK4044 was approximately 2-fold less potent against a mutant PAI-1 (14B-1), which contains four mutations at N150H, K154T, Q319L and M354I, compared with wild-type PAI-1, suggesting that the ZK4044 binding site on the surface of PAI-1 is close to these mutant residues. Together, our data show that ZK4044 represents a new class of small molecule PAI-1 inhibitors with anti-thrombotic potential.     

Impaired spontaneous thrombolytic activity measured by global thrombosis test in males with metabolic syndrome

Introduction

In patients with metabolic syndrome (MetS), activity of the fibrinolytic system is generally surmised to be decreased through increased plasminogen activator inhibitor-1 (PAI-1) generation. However, there have been no detailed reports describing whether the clot lysis activity is more dominant than increased clot formation activity for production of the thrombotic state in MetS.

Methods

The global thrombosis test (GTT) is a novel method designed to test both clot formation and clot lysis activities under physiological conditions by using non-anticoagulated blood samples in vitro. We used the GTT to examine the thrombotic or thrombolytic states in males with MetS.

Results

Lysis time, which reflects spontaneous clot lysis activity, was significantly longer in MetS subjects (median, 1494 s; range, 865-3596 s; n = 30) than in control subjects (median 1246 s; range, 667-2239 s; n = 53). There was no significant difference between the two groups in occlusion time, which reflects platelet function. The mean level of PAI-1 was significantly higher in MetS subjects than in controls (mean ± SE, 8.7 ± 1.1 and 5.0 ± 0.5 ng/mL, respectively). PAI-1 level and lysis time were significantly correlated (r = 0.400, P < 0.01).

Conclusion

These results suggest that male patients with MetS are more likely than controls to experience a thrombotic state through decreased fibrinolytic activity due to increased PAI-1 generation, and that the GTT is useful for evaluating fibrinolytic activity in vitro.     

Ambient temperature affects thrombotic potential at rest and following exercise

Introduction

During exercise, ischemic risk increases, possibly due to changes in coagulation and fibrinolytic activity. Previous research suggests ambient temperature affects resting thrombotic potential, but the effect of heat and cold on hemostasis during exercise is unknown. The purpose of this study was to assess changes in coagulation and fibrinolysis during maximal exercise in hot and cold temperatures, and to compare those responses to exercise under temperate conditions.

Materials & Methods

Fifteen healthy men completed maximal exercise tests in hot (30 °C), temperate (20 °C) and cold (5° - 8 °C) temperatures. Blood samples were obtained before and immediately after exercise and analyzed for concentrations of thrombin-antithrombin III (TAT), active tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). Results were analyzed by ANOVA.

Results

A main effect of time was observed for TAT (temperate = 1.71 ± 0.82 - 2.61 ± 0.43 ng/ml, hot = 1.81 ± 0.73 - 2.62 ± 0.67 ng/ml, cold = 2.33 ± 0.65 - 2.89 ± 0.81 ng/ml, PRE to POST, respectively) and tPA activity (temperate = 0.72 ± 0.44 - 2.71 ± 0.55 IU/ml, hot = 0.72 ± 0.38 - 2.64 ± 0.61 IU/ml, cold = 0.86 ± 0.45 - 2.65 ± 0.77 IU/ml, PRE to POST, respectively). A trend was observed for the PAI-1 response to exercise (temperate = 14.5 ± 23.7 - 12.3 ± 20.2 IU/ml, hot = 15.1 ± 26.5 - 10.0 ± 15.1 IU/ml, cold = 10.5 ± 10.4 - 7.9 ± 9.7 IU/ml, PRE to POST, respectively, p = 0.08). TAT concentrations were significantly higher in cold compared to temperate and hot conditions.

Conclusion

Coagulation potential is elevated during exposure to cold temperatures. These data suggest that risk of an ischemic event may be elevated in the cold.     

Sustained high plasma plasminogen activator inhibitor-1 levels are associated with severity and mortality in septic patients

Background

Higher plasma plasminogen activator inhibitor-1 (PAI-1) levels have been reported in septic patients. However, some questions remain unanswered, such as whether there is an association between plasma PAI-1 levels and sepsis severity and mortality, and inflammation state during the first week.

Methods

Multicenter, observational and prospective study carried out in six Spanish Intensive Care Units of 260 patients with severe sepsis. Circulating levels of PAI-1 and tumour necrosis factor (TNF)-α were measured at day 1, 4 and 8. End-point was 30-day mortality.

Results

Nonsurviving septic patients (n = 89) presented higher PAI-1 levels than surviving (n = 171) at day 1 (58.4 (33.3-83.8) vs 36.5 (21.1-62.5) ng/mL; p < 0.001), 4 (34.0 (14.7-53.3) vs 16.2 (10.2-27.4) ng/mL; p < 0.001) and 8 (30.6 (16.2-47.8) vs 18.9 (10.4-29.5) ng/mL; p = 0.004). We found a positive correlation of PAI-1 levels with SOFA, lactic acid, aPTT, INR and TNF-α, and negative with platelet count at day 1, 4 and 8. Logistic regression analyses showed that PAI-1 levels at day 1 (p < 0.001), 4 (p < 0.001) and 8 (p = 0.001) were associated with 30-day mortality. On ROC curve analysis to predict 30- day survival, the area under the curve of PAI-1 levels at day 1, 4 and 8 were 0.65 (95% CI = 0.58-0.72; p < 0.001), 0.69 (95% CI = 0.60-0.78; p < 0.001) and 0.65 (95% CI = 0.54-0.75; p = 0.005) respectively.

Conclusions

The most interesting findings of our study, to our knowledge the largest series reporting PAI-1 levels during follow-up in septic patients, were that plasma PAI-1 levels during the first week were associated with inflammation, severity and mortality.