Is exogenous tissue plasminogen activator necessary for antithrombotic efficacy of an inhibitor of thrombin activatable fibrinolysis inhibitor (TAFI) in rats?

INTRODUCTION: TAFI indirectly reduces the action of tPA on plasminogen. Whether exogenous tPA is necessary for TAFI inhibitor efficacy is unclear. Potato carboxypeptidase inhibitor (PCI), a TAFI inhibitor, has shown variable tPA dependence in rat models of arteriovenous shunt thrombosis (required) and microthrombosis (not required). This study was designed to further explore the importance of exogenous tPA in revealing PCI activity in rat models of venous and arterial thrombosis and provoked bleeding. METHODS: PCI was given as a bolus (5, 10 mg/kg) +/- infusion (5, 10 mg/kg/h) and with or without low dose tPA (5, 10, 25 microg/kg/min). In each instance tPA was adjusted to produce subthreshold thrombus reduction. Arterial thrombosis was induced by FeCl2; venous thrombosis by tissue factor or FeCl2. Bleeding was induced by kidney incision with PCI given (5 mg + 5 mg/kg/h) in the presence or absence of tPA (10, 150, 200 microg/kg/min). RESULTS: PCI was ineffective without exogenous tPA in all tested thrombosis models. With exogenous tPA, PCI decreased thrombus weight 85% in tissue factor thrombosis, 59% in FeCl2 thrombosis, and 46% in arterial thrombosis. PCI prolonged bleeding only when combined with a relatively high tPA dose (200 microg/kg/min) that increased bleeding alone. CONCLUSIONS: If the current results predict clinical efficacy, the need for exogenous tPA in combination with TAFI inhibition is a potential problem. However, in acute settings where intravenous fibrinolytics are administered, or indications in which tPA production increases, TAFI inhibitors may prove to be safe and moderately effective profibrinolytic agents.     

Thrombin activatable fibrinolysis inhibitor: at the nexus of fibrinolysis and inflammation

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.     

Prothrombotic alterations in plasma fibrin clot properties in thyroid disorders and their post-treatment modifications

Introduction

Available data on fibrin clot properties and fibrinolysis in hyperthyroidism and hypothyroidism are inconsistent. Our objective was to assess the impact of effective treatment of hyper- and hypothyroidism on fibrin clot characteristics.

Material and Methods

In a case-control study, ex vivo plasma fibrin clot permeability (K_s) and efficiency of fibrinolysis were assessed in 35 consecutive hyperthyroid and 35 hypothyroid subjects versus 30 controls. All measurements were performed before and after 3 months of thyroid function normalizing therapy.

Results

At baseline, hyperthyroid, but not hypothyroid, patients had lower K_s than controls (p < 0.0001). Hyperthyroid and hypothyroid groups compared with controls had prolonged clot lysis time (CLT), and lower rate of D-dimer release from clots (D-D_rate) (all p < 0.05). The regression analysis adjusted for fibrinogen showed that in hyperthyroid patients, pre-treatment thyroid stimulating hormone (TSH) independently predicted K_s, while thrombin activatable fibrinolysis inhibitor (TAFI) antigen predicted CLT. In hypothyroid individuals a similar regression model showed that TSH independently predicts CLT. After 3 months of thyroid function normalizing therapy, 32 (91.4%) hyperthyroid and 30 (85.7%) hypothyroid subjects achieved euthyroidism and had improved fibrin clot properties (all p < 0.05), with normalization of K_s in hyperthyroid and lysability in hypothyroid patients.

Conclusions

Both hyper- and mild-to-moderate hypothyroidism are associated with prothrombotic plasma fibrin clot phenotype and restoration of euthyroidism improves clot phenotype. Abnormal fibrin clot phenotype might contribute to thromboembolic risk in thyroid disease.     

Influence of exogenous and endogenous tissue-type plasminogen activator on the lysability of clots in a plasma milieu in vitro

The fibrinolytic potential of tissue-type plasminogen activator (t-PA) either incorporated in a clot (endogenous) or added to the surrounding plasma (exogenous), was studied in an in vitro system consisting of 125I-labeled human plasma clots (200 microliters) immersed in human plasma (2 ml). Clot lysis was measured as a function of endogenous t-PA concentration (in the absence of added exogenous t-PA), as a function of exogenous t-PA concentration (without added endogenous t-PA) and as a function of the same concentration of both endogenous and exogenous t-PA. Equivalent clot lysis was obtained with a 2 to 4 times lower concentration of endogenous t-PA as compared to exogenous t-PA, corresponding to a 20 to 40 times smaller total amount of endogenous versus exogenous t-PA. Fifty percent lysis in 5 hrs was obtained with about 5 IU/ml of endogenous t-PA or with 10 IU/ml of exogenous t-PA. The presence of both exogenous (10 IU/ml) and endogenous (5 IU/ml) t-PA resulted in 50 percent lysis in 1.5 hrs, indicating that t-PA incorporated in a thrombus contributes significantly to its lysis by exogenous t-PA. Similar results were obtained with plasma obtained after 10 min of venous occlusion in seven healthy subjects. Spontaneous clot lysis within 5 hrs was only observed with post-occlusion clots in pre- or post- occlusion plasma in two subjects in whom the t-PA level rose to 10-15 IU t-PA/ml. In the five other subjects with post-occlusion t-PA levels below 2 IU/ml, no clot lysis was observed within 24 hrs. The influence of the fast-acting inhibitor of t-PA on clot lysability by endogenous or exogenous t-PA was investigated by immersing clots prepared from normal or inhibitor-rich plasma (endogenous inhibitor) in normal or inhibitor-rich plasma (exogenous inhibitor). Exogenous t-PA inhibitor efficiently neutralizes clot lysis by both exogenous and endogenous t-PA. Endogenous t-PA inhibitor, however, efficiently neutralizes endogenous t-PA but has little influence on clot lysis by exogenous t-PA. These findings indicate that t-PA inhibitor is not concentrated into a clot and that t-PA inhibitor in plasma efficiently neutralizes t-PA incorporated in a clot. alpha 2-Antiplasmin depleted plasma clots were more susceptible to lysis by both endogenous and exogenous t-PA than normal clots.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of thrombin activatable fibrinolysis inhibitor by cysteine derivatives

Thrombin Activatable Fibrinolysis Inhibitor (TAFI) is a basic carboxypeptidase that functions as a fibrinolysis inhibitor through the cleavage of C-terminal lysine on partially degraded fibrin. Modulation of TAFI activity provides a potential therapy for thrombosis complications by potentiating fibrinolysis. In our study, we identified three novel TAFI inhibitors containing a cysteine backbone. Three cysteine derivatives, guanidinyl-L-cysteine, glycyl-L-cysteine, and glycyl-glycyl-L-cysteine were tested in TAFI substrate assays and showed K(app)(i)=0.08, 0.14, and 0.99 microM, respectively. Subsequent fibrinolysis assays confirmed their TAFI inhibitory activities. Guanidinyl-L-cysteine showed inhibitory activity in a human plasma clot lysis assay (IC(50)=9.4 microM). Identification of these cysteine derivatives represents an opportunity to develop potent and specific TAFI inhibitors.     

The role of thrombin-activatable fibrinolysis inhibitor in diabetic wound healing

Introduction

One of the major complications in patients with diabetes mellitus is impaired wound healing. The fibrinolytic system is involved in parts of the wound healing process and deficiency of thrombin-activatable fibrinolysis inhibitor (TAFI) results in delayed wound closure. Moreover, levels of TAFI are affected by diabetes mellitus. The aim of this study was to elucidate the effect of hyperglycaemia on TAFI and to determine the effect of deficiency of TAFI on wound healing under hyperglycaemic conditions.

Materials and methods

Hyperglycaemia was induced with streptozotocin (STZ) and used as a model for diabetes mellitus. TAFI plasma levels and TAFI gene expression in the liver were determined. Incisional and excisional wound healing were studied in non-treated and STZ-treated wild-type and TAFI-deficient mice. Wound closure was scored daily as open or closed.

Results

Mice treated with STZ showed hyperglycaemia, and TAFI plasma levels and TAFI gene expression were increased in diabetic mice. TAFI-deficient mice and diabetic wild-type and diabetic TAFI-deficient mice showed delayed wound healing of incisional wounds. No differences were observed between diabetic and non-diabetic TAFI-deficient mice and between diabetic wild-type and diabetic TAFI-deficient mice.

Conclusions

This study illustrated that TAFI was affected by hyperglycaemia and confirmed that TAFI is involved in wound healing. No additional effect was observed under hyperglycaemic conditions, indicating that deficiency of TAFI did not have an additive or synergistic effect in diabetic wound healing. Further research has to elucidate if TAFI and hyperglycemia affect wound healing via similar mechanisms.     

The kinetics of plasmin inhibition by aprotinin in vivo

INTRODUCTION: The purpose of this study was to estimate, in patients undergoing cardiopulmonary bypass (CPB), the in vivo rates of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) secretion, plasmin generation, fibrin degradation, and plasmin inhibition by aprotinin versus antiplasmin. MATERIALS AND METHODS: Estimates of in vivo rates were based on measured levels of tPA, PAI-1, antiplasmin, plasmin-antiplasmin complex (PAP), total aprotinin, plasmin-aprotinin complex and D-dimer, combined with a computer model of each patient's vascular system that continuously accounted for secretion, clearance, hemodilution, blood loss and transfusion. Plasmin regulation was studied in nine control patients undergoing CPB without aprotinin versus six patients treated with aprotinin. RESULTS: In controls, plasmin-antiplasmin levels rose from a baseline of 3.0+/-0.9 to a peak of 8.1+/-2.7 nmol/L after CPB due to an average 44-fold rise in the plasmin generation rate. This rise in plasmin generation during CPB lead to increased fibrin degradation causing D-dimer levels to increase from a baseline of 1.2+/-0.6 to a peak of 9.7+/-4.4 nmol/L due to an average 74-fold rise in the D-dimer generation rate. During CPB in the aprotinin group, plasmin-antiplasmin levels dropped, plasmin-aprotinin complex levels rose, while D-dimer levels remained unchanged from baseline. Compared to controls, the aprotinin group showed similar rates of plasmin generation during CPB, but an 11-fold faster plasmin inhibition rate and a 10-fold lower D-dimer generation rate. CONCLUSIONS: The rise in plasmin generation and fibrin degradation that occurs during standard CPB is suppressed by the addition of aprotinin, which returns the patient to near baseline fibrin degradation rates during CPB.     

The paradoxical antifibrinolytic effect of dabigatran and argatroban in the presence of soluble thrombomodulin is unrelated to protein C-dependent increase of thrombin generation

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.     

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.     

Tissue factor pathway inhibitor and thrombin-activatable carboxypeptidase B for prediction of early atherosclerosis in gouty arthritis

Background

Gouty arthritis (GA) is a chronic inflammatory arthritis in which both clinical and subclinical atherosclerosis are more frequent. The dynamic equilibrium between coagulation and fibrinolysis is impaired in inflammatory diseases. We determined TFPI and TAFI antigen levels in GA patients and evaluated their association with subclinical atherosclerosis.

Methods

We included 45 GA patients (41 males, 4 females; mean age: 51.6 years) and 25 asymptomatic hyperuricemic (AHU) subjects (19 males, 6 females; mean age: 48.1 years). Cardiovascular risk factors were determined. TAFI and TFPI levels were determined by ELISA. B-mode ultrasonography was used to detect subclinical atherosclerosis.

Results

Cardiovascular risk factors were similar in both groups. The carotid IMT was significantly higher in GA group than in AHU group (0.74 ± 0.23 mm vs. 0.61 ± 0.13 mm, p = 0.009). TFPI level was significantly higher in GA group than in AHU group (86.2 ± 48.9 ng/mL vs. 25.8 ± 21.4 ng/mL, p < 0.001); TAFI antigen was significantly higher in AHU group (22.6 ± 3.6 ng/mL vs. 25.7 ± 5.3 ng/mL, p = 0.006) than in GA patients. Atherosclerotic plaque formation was more frequent in GA group (p = 0.041). When GA patients with and without plaques were compared, the first group had significantly higher mean age (p = 0.01) and TFPI level (p = 0.028). TFPI level correlated with carotid IMT (r = 0.302; p = 0.028). Logistic regression analysis showed that age (OR: 1.236, 95%CI: 1.059-1.443, p = 0.007) and TFPI (OR: 1.031, 95%CI: 1.008-1.054, p = 0.008) were independent risk factors for the presence of plaques.

Conclusions

GA patients had more frequent subclinical atherosclerosis than subjects with AHU. Higher TFPI levels in GA patients –probably associated with enhanced endothelial damage- were related to subclinical atherosclerosis. Lower TAFI levels in GA pointed to impaired fibrinolysis.     

Co-administration of low molecular weight heparin enhances the profibrinolytic effect of warfarin through different mechanisms

Background and Objective

Treatment with vitamin K antagonists (VKA) reduces fibrinolytic resistance through the inhibition of thrombin-mediated activation of thrombin activatable fibrinolysis inhibitor (TAFI). Because low-molecular weight heparin (LMWH) is co-administered with VKA during initiation of anticoagulant treatment, we evaluated the effect of dual anticoagulation on fibrinolytic resistance.

Patients and Methods

Two groups of patients were studied: 1) patients on stable warfarin; 2) patients starting oral anticoagulant therapy, who were evaluated during dual anticoagulation and after enoxaparin withdrawal. Only samples with an INR between 2 and 3 were compared. The resistance of clots to t-PA-induced fibrinolysis was evaluated in blood and plasma by thromboelastography (TEG) and turbidimetry, respectively.

Results

In patients on dual anticoagulation, blood fibrinolysis time (TEG) was significantly shorter than in patients on warfarin alone and significantly correlated with LMWH level. The profibrinolytic effect was partly ascribable to a reduction of thrombin-dependent TAFI activation: 1) thrombin and TAFIa generation were significantly reduced by dual anticoagulation; 2) the addition of enoxaparin to warfarin-blood reduced TAFI-mediated fibrinolysis inhibition. Patients on dual anticoagulation also displayed a reduction in clot strength, a phenomenon known to reduce fibrinolytic resistance. The profibrinolytic effect of LMWH co-administration was not seen in plasma, likely because TAFIa generation was below the threshold required to inhibit fibrinolysis.

Conclusions

Co-administration of LMWH in patients under VKA reduces the fibrinolytic resistance of blood clots via TAFI-dependent and TAFI-independent mechanisms. Further studies are warranted to assess the clinical implications of these findings.     

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.     

Plasma TAFI and soluble CD40 ligand do not predict reperfusion following thrombolysis for acute myocardial infarction

INTRODUCTION: Thrombolytic therapy fails to achieve reperfusion in almost a third of patients with acute myocardial infarction. Thrombin activatable fibrinolysis inhibitor (TAFI) and soluble CD40 ligand (sCD40L) are novel endogenous fibrinolytic and atherothrombotic factors that determine clot stability. We investigated whether admission plasma thrombin activatable fibrinolysis inhibitor (TAFI) and soluble CD40 ligand (sCD40L) concentrations predicted reperfusion following thrombolytic therapy in patients with acute myocardial infarction. MATERIALS AND METHODS: Prior to administration of thrombolytic therapy, venous blood was collected from 110 patients presenting with acute ST segment elevation myocardial infarction and plasma assayed for tissue plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor type-1 antigen (PAI-1), TAFI antigen and activity, C-reactive protein (CRP) and sCD40L concentrations. Reperfusion was determined using continuous ST segment monitoring. RESULTS: Reperfusion occurred in 77 (70%) patients with a mean treatment to reperfusion time of 83 +/- 46 min. Peak creatine kinase was significantly lower in patients who reperfused (1578 +/- 1199 versus 2200 +/- 1744 U/L; P < 0.05) and correlated with time to reperfusion (r = 0.44 [95% CI: 0.23 - 0.61], P = 0.0001). There was a modest correlation between plasma TAFI antigen and activity (r = 0.3 [95% CI: 0.04 - 0.53]; P < 0.05). There were no significant associations between coronary reperfusion and plasma concentrations of t-PA, PAI-1, TAFI, CRP or sCD40L. CONCLUSIONS: Systemic plasma TAFI, sCD40L and CRP concentrations do not predict reperfusion in patients receiving thrombolytic therapy for acute ST elevation myocardial infarction.     

VEGF increases the fibrinolytic activity of endothelial cells within fibrin matrices: involvement of VEGFR-2, tissue type plasminogen activator and matrix metalloproteinases

Proteolysis of fibrin matrices by endothelial cells plays essential roles in the migratory and morphogenic differentiation processes underlying angiogenesis. Using an in vitro fibrinolysis model consisting of human umbilical vein endothelial cells (HUVECs) embedded in a three dimensional fibrin matrix, we show that VEGF, an angiogenic cytokine that plays a crucial role in the onset of angiogenesis, is a potent activator of HUVEC-mediated fibrinolysis. This VEGF-dependent fibrin degradation was completely abrogated by inhibitors of either the plasminogen activator/plasmin or matrix metalloproteinases (MMP) proteolytic systems, suggesting the involvement of both classes of proteases in fibrin degradation. Accordingly, VEGF-induced fibrinolysis correlated with an increase in the expression of tPA and of some MMPs, such as MT2-MMP and was completely blocked by a neutralizing antibody against tPA. Overall, these results indicate that efficient proteolysis of three dimensional fibrin matrices during VEGF-mediated angiogenesis involves a complex interplay between the MMP and plasmin-mediated proteolytic systems.     

Plasminogen activator inhibitor-1 4G/5G polymorphism in breast cancer patients and its association with tissue PAI-1 levels and tumor severity

BACKGROUND: The plasminogen activator inhibitor type 1 (PAI-1) 4G/5G polymorphism may have significance for PAI-1 expression. High levels of PAI-1 in breast cancer patients are associated with a poor prognosis. In this study, we analyzed the influence of the PAI-1 4G/5G polymorphism on tissue PAI-1 levels and its association with tumor severity in women with breast cancer. MATERIAL AND METHODS: We studied 104 women with breast carcinoma (patient group) and 104 healthy age-matched women (control group). In patients and controls, the PAI-1 4G/5G polymorphism was determined by PCR amplification using allele-specific primers. In patients, PAI-1 levels were quantified in breast cancer tissue by using an ELISA. RESULTS: The frequency of the PAI-1 4G allele tended to be higher in patients than in controls (p=0.062). The presence of the 4G allele (4G/5G plus 4G/4G genotypes) was significantly higher among patients with histological grade 3 tumors than among those with grade 1 tumors (p=0.026). Furthermore, patients with the 4G/4G genotype had significantly higher tissue PAI-1 levels than those with the 5G/5G genotype. Moreover, tissue PAI-1 antigen levels were significantly and positively correlated with tumor severity (p=0.003) and tumor size (p=0.009). However, no significant differences in PAI-1 level were observed in relation to menopause, hormone receptor or nodal status. CONCLUSION: Tissue PAI-1 antigen levels and tumor severity seem to be associated with the PAI-1 4G/5G polymorphism. Further studies with a larger number of patients are needed to clarify the influence of this polymorphism in breast cancer.     

Altered fibrin clot properties are associated with residual vein obstruction: Effects of lipoprotein(a) and apolipoprotein(a) isoform

We tested the hypothesis that fibrin structure/function is unfavorably altered in patients with residual vein obstruction (RVO). Ex vivo plasma fibrin clot permeability, turbidimetry and efficiency of fibrinolysis were investigated in 86 patients with RVO following first-ever proximal deep vein thrombosis (DVT), and 86 DVT controls with no evidence of RVO. The RVO patients had 14.1% lower clot permeability (p = 0.011), 11.3% longer lysis time (p = 0.009) and 7.8% lower rate of D-dimer release from fibrin clots than controls (p = 0.022), with no differences related to thrombophilia, and duration or stability of anticoagulant therapy. RVO patients showed higher lipoprotein(a) (p = 0.014) with overrepresentation of smaller apolipoprotein(a) isoforms, corresponding approximately to 21 or fewer kringle IV type 2 repeats (p = 0.09), both associated with alterations to plasma fibrin clot characteristics. In conclusion, prothrombotic plasma fibrin clot phenotype related to elevated lipoprotein(a) with smaller apolipoprotein(a) isoforms might represent a novel risk factor for RVO.     

Importance of the interaction between plasminogen and fibrin for plasminogen activation by tissue-type plasminogen activator

The potentiating effect of fibrin monomer on plasminogen activation by tissue-type plasminogen activator is much more important with lys-plasminogen than with mini-plasminogen (which lacks the high affinity lysine-binding site important for binding to fibrin). Furthermore, this potentiating effect is totally abolished when lys-plasminogen is eluted from fibrin by the addition of 1 mM epsilon-amino caproic acid. Binding does however not seem to be the only condition required since it was found that fragment D is a much stronger potentiator of the activation of plasminogen by tissue-type plasminogen activator than fragment E although plasminogen binds to both fragment D and fragment E. Furthermore, fragment E has the same effect on the activation of lys-and mini-plasminogen by tissue-type plasminogen activator. Therefore, it is suggested that binding of plasminogen to fibrin involves a conformational change in the plasminogen molecule, facilitating its activation by tissue-type plasminogen activator.     

Alterations of profibrinolytic receptor annexin A2 in pre-eclampsia: a possible role in placental thrombin formation

Introduction

Deficient fibrinolytic activity due to alterations of profibrinolytic receptor annexin A2 has been shown to be related to increased thrombosis. We compared the expression of annexin A2 in pre-eclampsia with that in normal pregnancies and investigated its relationship to placental thrombin formation.

Materials and methods

Sixty patients with pre-eclampsia and 30 matched normal pregnant controls were included in the study. Expression of annexin A2 mRNA in placental tissues was analyzed using quantitative RT-PCR. Annexin A2 protein in placentas as well as blood was determined by western blot analysis and immunohistochemistry. Enzyme-linked immunosorbent assay (ELISA) was used to detect the presence of anti- annexin A2 antibodies in peripheral maternal blood.

Results

The expression of annexin A2 mRNA in placentas was significantly decreased in pre-eclampsia compared to normal pregnancies. Levels of annexin A2 protein in placentas as well as in peripheral maternal blood were also significantly reduced in patients with pre-eclampsia compared with healthy pregnant women. High titers of anti- annexin A2 antibodies were more frequently detected in pre-eclampsia group. Decreased levels of annexin A2 expression and the presence of anti-annexin A2 antibodies were associated with increased placental thrombosis.

Conclusion

The down regulation of annexin A2 expression and the presence of anti-annexin A2 antibodies were correlated to PE and may increase placental thrombin formation.     

Association between genotype and plasma levels of thrombin-activated fibrinolysis inhibitor (TAFI) in the development of preeclampsia

Introduction

The objective was to evaluate if thrombin-activated fibrinolysis inhibitor (TAFI) polymorphisms (G505A, C1040T, and G-438A), and TAFIa plasma levels are associated with preeclampsia.

Materials and Methods

In a case-control study design, we evaluated preeclampsia patients and women with uncomplicated pregnancies. The TAFI polymorphisms were determined by real-time PCR method, and TAFIa plasma levels were established with a chromogenic assay.

Results

We included 87 women in each group. The TAFIa levels in the preeclampsia group were 20.4 μg/mL (CI 95% 17.3-23.5), while in the control group, they were significantly lower: 13.3 μg/mL (12.0-14.5, p 0.003). There were no differences in the genotype distribution or allelic frequency of TAFI polymorphisms between the two groups. In preeclampsia patients and controls heterozygous for the G505A polymorphism, the TAFIa values were 22.8 (16.7-28.9 μg/mL) and 13.2 (11.3-15.0 μg/mL, p 0.019), respectively. In G505A homozygous polymorphism the TAFIa values were 25.7 (18.7-32.6 μg/mL) and 13.5 (1.6-21.9 μg/mL, p 0.041), respectively. In the C1040T and G-438A TAFI wild type polymorphisms, the TAFIa values were 18.3 (12.5-23.9 μg/mL) and 11.5 (9.9-35.0, p 0.033), and 19.4 (10.9-27.9 μg/mL) and 12.5 (10.8-14.2 μg/mL, p 0.006), respectively, without differences in other genotypes.

Conclusions

Preeclampsia by itself may be responsible for the increase in TAFIa values rather than the presence of polymorphisms.     

Reduced plasma fibrin clot permeability and susceptibility to fibrinolysis are associated with increased intima-media thickness in patients with primary antiphospholipid syndrome

Introduction

Formation of denser fibrin networks displaying impaired lysability has been reported in subjects at an increased risk of atherosclerosis. Given recent data on prothrombotic fibrin clot phenotype reported in patients with antiphospholipid syndrome (APS), we tested the hypothesis that altered fibrin clot properties are associated with increased intima-media thickness (IMT) observed in PAPS.

Materials and methods

We studied 30 consecutive patients with PAPS and 30 controls matched for age, sex and the type of previous thromboembolism. We assessed plasma fibrin clot permeability (K_s) and clot lysis time (CLT) with their potential determinants. The IMT was measured in 3 segments of the carotid arteries.

Results

Patients with APS had 15.2% lower K_s (p = 0.002) and 9.7% prolonged CLT (p = 0.039) compared with controls. The IMT in the APS group was greater in the common carotid artery (5.7%; p = 0.002), at the bifurcation (17.46%; p < 0.001), and the internal artery (9.26%; p = 0.015). Patients with triple positivity in the antiphospholipid antibody profile (n = 9; 30%) had lower K_s and greater IMT (both, p < 0.05), compared with those with single positivity (n = 13; 43.3%). Multivariate analysis adjusted for potential confounders showed that in APS patients, oxidized low-density lipoproteins (p = 0.019) were the only independent predictor of K_s, while thrombin activatable fibrinolysis inhibitor activity (p < 0.001) predicted CLT. Plasminogen activator inhibitor-1 (PAI-1) was found to be the independent predictor of the IMT in the common carotid artery (p = 0.004), and in the internal carotid artery (p < 0.001).

Conclusions

Reduced K_s and susceptibility to lysis are associated with greater IMT in PAPS, which might contribute to the early atherosclerosis in this disease.