Activatable Fibrinolysis Inhibitor Research Articles

Relative Inhibition of Thrombin Activatable Fibrinolytic Inhibitor by Newly Developed Thrombin Inhibitors: Impact on Bleeding and Antithrombotic Actions.

Thrombin activatable fibrinolytic inhibitor (TAFI) or carboxypeptidase U plays a key role in the fibrinolytic cascade. It acts as an inhibitor of fibrinolysis by cleaving the arginine and lysine amino acid residues from the carboxy terminus of fibrin. The cleavage then renders the fibrin resistant to digestion by plasmin, forming a clot that cannot be easily digested. This stabilized thrombus can then cause myocardial infarction, stroke or pulmonary embolism. It has been shown that individuals with acute coronary syndrome tend to have elevated TAFI, which may lead to further complications. Heparin and low molecular weight heparins (LMWHs) are the most common agents given to those at risk of thrombotic complications either during surgery, therapeutically or prophylacticly. Thrombin inhibitors are being developed to provide an alternative means of anticoagulation if a patient cannot be given heparin or LMWHs due to heparin induced thrombocytopenia (HIT) or some other condition. This study was done to determine the effect of different thrombin inhibitors on TAFI functionality and determine if there is a correlation to the thrombin inhibitors anti-IIa effects. Argatroban, one of the thrombin inhibitors used in this study, has been approved for use in patients who have a history of HIT. In addition to argatroban, the following thrombin inhibitors were tested: melagatran, angiomax, LU 208791, hirudin and dabigatran. All thrombin inhibitors were supplemented to normal human pooled plasma (NHP) in various concentrations (10 – 0.16 μg/ml). The functional TAFI levels were determined using a chromogenic substrate based method developed by Pentapharm Inc. (Basel, Switzerland), the Pefakit® TAFI. The anti-IIa (thrombin inhibition) was determined using a chromogenic substrate based method by American Diagnostica (Stamford, CN). All of the thrombin inhibitors were capable of inhibiting TAFI functionality; LU 208791 being the strongest inhibitor with an IC50 of 0.09 μg/ml while angiomax was the weakest with an IC50 of 6.6 μg/ml respectively. The other thrombin inhibitors showed IC50s ranging from 0.38 – 1.4 μg/ml. These results for the most part corresponded to the anti-IIa results. LU 208791 had the strongest anti-IIa with an IC50 of 0.39 μg/ml while angiomax had an IC50 >10 μg/ml. However, the IC50s for TAFI inhibition by argatroban and hirudin (1.3 and 1.2 μg/ml) were not significantly different, their IC50s for thrombin inhibition were (>10 and 3.0 μg/ml). This indicates that argatroban may inhibit TAFIa directly. The anti-IIa rank order was as follows: LU 208791 > dabigatran > hirudin > melagatran > angiomax = argatroban. These results indicate that the bleeding complications observed when using thrombin inhibitors may be due to the indiscriminate inhibition of TAFI yielding fibrin that is easily digested. It is vital that the balance between coagulation and fibrinolysis be maintained in patients on anticoagulants. When this balance is disrupted either bleeding or thrombosis can occur. This study indicates that TAFI may be the key to maintaining this balance. Too much TAFI inhibition may lead to bleeding while not enough inhibition may lead to thrombosis. Persistent inhibition of TAFI by thrombin inhibitors may compromise both their safety and efficacy.

Frequency of Anti-Erythropoietin Antibodies in Patients with End Stage Renal Disease Treated with Recombinant Erythropoietin.

End Stage Renal Disease (ESRD) manifests hematologic, cardiovascular, inflammatory, and immunologic dysfunctions. Erythropoietin deficiency is present in ESRD leading to anemic states. ESRD patients receive regular/repeated administration of commercially available recombinant erythropoietin (r-E) (Epogen® Procrit® Patients treated with r-E have been known to develop thrombotic complications during r-E therapy. Antibodies to erythropoietin are generated in some patients and lead to erythropoietic aplasia. The mechanisms of the anti-erythropoietin antibody induced aplasia and reported hypercoagulability state in patients treated with r-E are not fully understood. Anticoagulant drugs such as heparin are used to reduce the incidence of thrombotic complications during r-E therapy. To understand pathogenesis leading to hypercoagulable state and its potential link with r-E treatment, blood samples taken from a group of 60 patients with ESRD were drawn prior to initiation of antithrombotic therapy. These patients on periodic hemodialysis were administered r-E 6–8 weeks prior to blood drawn. Anti-erythropoietin antibody titer, anti-phospholipid antibody (APA) titer, CRP levels, fibrinopeptide-A (FPA), nitric oxide (NO) levels, asymmetric 1,3-dimethylarginine (ADMA) levels, and thrombin activatable fibrinolytic inhibitor (TAFI) levels were measured. Five of 60 patients (8.3%) showed higher titers of anti-erythropoietin antibodies. Three of 60 patients (5%) exhibited positive titers of anti-phospholipid antibodies. Marked elevation of the CRP, FPA, NO, ADMA, and TAFI levels were noted in comparison to match controls. Patients with positive levels of anti-erythropoietin antibody titer (3 to 5 folds) exhibited a simultaneous elevation of FPA, CRP and TAFI suggesting the potential role of anti-erythropoietin antibody in mediating these responses. These observations indicate that ESRD patients treated with r-E are at a higher risk of developing thrombotic complications. The upregulation of NO and ADMA in these patients is suggestive of ongoing inflammatory processes. Anticoagulants such as low molecular weight heparins may be useful in the management of these patients.

Plasma Levels of Thrombin-Activatable Fibrinolysis Inhibitor in Primary and Secondary Thrombocytosis

An elevated platelet count is a common finding in both hospitalized and ambulatory patients. Thrombosis and bleeding complications are more frequently observed in patients with clonal thrombocytosis than secondary thrombocytosis. The aim of this study was to investigate the behaviors of thrombin-activatable fibrinolysis inhibitor (TAFI) activity, the inhibitor of fibrinolysis, and also prothrombin time (PT), active partial thromboplastin time, and D-dimer and fibrinogen levels in 21 patients affected with clonal thrombocytemia as compared with 21 patients with reactive thrombocytosis and 21 healthy controls. In the clonal thrombocytemia group, plasma levels of TAFI activity were significantly higher than in both the reactive thrombocytosis and the control group. Plasma levels of leukocyte and platelet counts were significantly higher in the clonal thrombocytemia group than in the other two groups and also higher in the reactive thrombocytosis group than in the control group, which was also significant. Fibrinogen and D-dimer levels were higher in patients than in the control group but showed no significant difference between the clonal and secondary thrombocytosis groups. Plasma levels of PT and aPTT were higher in secondary thrombocytosis group than the clonal thrombocytosis group. The results of this study showed for the first time that TAFI activity is increased in patients with clonal thrombocytosis. These increased levels in clonal thrombocytosis can be considered a factor to explain the thrombotic tendency in myeloproliferative disorders.

P-611 Thrombin activatable fibrinolytic inhibitor, fibrinogen andD-dimers in radically operated NSCLC

P-611 Thrombin activatable fibrinolytic inhibitor, fibrinogen andD-dimers in radically operated NSCLC

Enoxaparin (E) and warfarin (W) differentially regulate tissue factor (TF), tissue factor pathway inhibitor (TFPI) and thrombin activatable fibrinolytic inhibitor (TAFI) in cancer patients with thrombosis

2057 Background: Increased TF levels in malignancy produce consumption of endogenous TFPI, activate platelets and upregulate fibrinolytic deficit. Anticoagulants such as E and W have been used in the management of cancer associated thrombosis. Methods: To test the hypothesis that E and W differentially regulate TF mediated hypercoagulation in cancer, plasma samples were retrospectively analyzed from an open label, multidose, active comparator parallel design study in which all patients (n=110) were initially treated with E at 1–1.5 mg/kg sc for 5 days. These patients were further subdivided into two groups. The first group continued to receive E whereas the second group received W (INR target 2–3), for up to 12 weeks. Baseline blood samples (BL), 5 day post E (IPE) and 4–6 weeks sample from the E and W were analyzed for TF antigen, functional TFPI and antigen, functional TAFI and antigen and platelet marker CD 40L. Results: The initial treatment of all of patients with E at 1.0–1.5 mg/kg SC for 5 days res...

Renal transplant recipients with coronary artery disease exhibit impairment in fibrinolysis and structural changes in carotid arteries

Cardiovascular disease (CVD) is the main cause of mortality and morbidity among kidney transplant recipients (Tx). Intima-media thickness (IMT) of the common carotid artery is related to CVD. Hemostatic disturbances may contribute to the CVD pathogenesis in Tx. The aim of the study was to evaluate some hemostatic factors in Tx with and without coronary artery disease (CAD) and their correlations with IMT. Patients with CAD were significantly older, with thicker IMT, lower plasmin-antiplasmin complexes (PAP), higher fibrinogen, cholesterol, triglycerides, Thrombin-activatable fibrinolysis inhibitor (TAFI) antigen and activity, prolonged euglobulin clot lysis time when compared to those without CAD. Kidney transplant recipients have higher mean blood pressure, serum lipids, fibrinogen, TAFI antigen, TAFI activity, markers of coagulation and fibrinolysis, thicker IMT and lower PAP relative to healthy volunteers. In univariate analysis, IMT correlated significantly with age, time on dialysis prior to transplantation, PAP, fibrinogen, hematocrit, body mass index (BMI). Multiple regression analysis showed that only age, hematocrit, PAP, and time on dialysis prior to transplantation were positive independent predictors of IMT. Hypofibrinolysis may contribute to the arterial remodeling in Tx. Dialysis therapy before transplantation makes detrimental changes in arterial vasculature.

Collagen deposition in myocardium after inhibition of fibrinolytic activity

Proper function of the heart directly depends on the protection of the myocardial architecture. The fibrinolytic system plays an important role(s) in the protection of myocardial architecture and aiding the pumping function of the heart. We investigated the effect of fibrinolytic system inhibition by tranexamic acid (TXA) on cat myocardium and collagen, which are important constituents of the extracellular matrix. Twenty-eight cats (seven per group) were used. Isotonic saline was administered to the control group (C) and TXA (200 mg/kg) diluted isotonic saline given to experimental groups 1 and 2 (E1 and E2). Experimental group 3 (E3) animals received 100 mg/kg TXA intravenously daily for 7 days. Bloods were drawn from groups C, E1, and E2, and fibrinolytic activity was determined by the euglobulin lysis time, fibrinogen degradation products and fibrin plate lytic area diameters. The group C and group E1 cats were sacrificed following the infusion. The group E2 and group E3 animals were sacrificed at 24 h and day 7, respectively. Light and electron microscopy, along with the collagen contents of the myocardium, were used to examine the myocardial tissues. Electron microscopic examination in groups E1 and E2 showed inter-myofibrillar edema, glycogen loss, mitochondrial swelling and disorganization of Z-bands, and a decrease of pinocytotic vesicles in capillary endothelial cells. In group E3, increases of collagen fibrils in intercellular areas and perivascular areas were noted. Biochemical analyses revealed a highly significant collagen accumulation in the myocardium in group E3 (P < 0.01) as compared with group C. These findings suggest that accumulation of the extracellular matrix containing collagen in the myocardium by fibrinolytic system inhibition may be responsible for the abnormal myocardial architecture, leading to cardiomyopathy and altered cardiac function .

A new global assay of coagulation and fibrinolysis

Introduction Global clotting assays may reflect an individual's net hemostatic balance and could contribute to prothrombotic and hemorrhagic risk assessment. In this research, a global assay that measures both coagulation and fibrinolytic capacities was developed and investigated. Materials and methods In the Clot Formation and Lysis (CloFAL) assay, a buffered reactant solution containing trace amounts of calcium, tissue factor, and tissue-type plasminogen activator is added to plasma samples on a 96-well microplate in an automated, thermoregulated (37 °C) spectrophotometer. Clot formation and lysis are monitored as continuous changes in absorbance over the course of 3 h. Measurements include maximum amplitude (MA), times to maximum absorbance ( T 1) and completion of the first phase of decline in absorbance ( T 2), and area under the curve (AUC), from which a coagulation index (CI) and various fibrinolytic indices (FI) may be calculated. Results and conclusions MA, T 1, and CI were principally influenced by fibrinogen and procoagulant factors. FI was found to be altered by inhibiting activation of plasminogen or thrombin activatable fibrinolytic inhibitor. Median CI was significantly decreased, while FI was markedly increased, in term neonates as compared to healthy adults (CI: 58% vs. 115%, FI: 210% vs. 90%; P<0.001 for each). By contrast, median CI was notably increased, and FI decreased, in healthy pregnant women when compared to adults (CI: 239% vs. 115%, FI: 59% vs. 90%; P<0.001 for each). The CloFAL global assay is analytically sensitive to several key components in the coagulation and fibrinolytic systems, as well as to physiologic alterations in hemostasis.

The profibrinolytic effect of plasma thrombomodulin in factor XI deficiency and its implications in hemostasis

Bleeding tendency in factor (F)XI deficiency may result from premature clot lysis due to insufficient thrombin activatable fibrinolysis inhibitor (TAFI) activation. Thrombomodulin (TM), upon binding to thrombin, is capable of modulating TAFI activation. In this study, we investigated the effects of plasma TM on fibrinolysis in FXI-deficient patients. A clot lysis assay showed the defective down-regulation of fibrinolysis in FXI-deficient patients as compared with normal controls. To evaluate the effects of plasma TM on fibrinolysis, a monoclonal anti-TM IgG was preincubated with plasma for 30 min. The presence of anti-TM IgG significantly prolonged the clot lysis times both in the FXI-deficient and normal plasma, indicating that plasma TM stimulated fibrinolysis. Furthermore, the presence of anti-TM IgG not only reduced protein C activation, but also increased thrombin generation and TAFI activation. The profibrinolytic effect of plasma TM was inhibited in the assay by including either a monoclonal anti-TAFI IgG or a specific TAFI inhibitor--carboxypeptidase inhibitor (CPI). Our results indicate that the impaired thrombin generation in FXI-deficient patients leads to the defective down-regulation of fibrinolysis, and that plasma TM stimulates fibrinolysis through APC pathway which inhibits TAFI activation. The profibrinolytic effect of plasma TM may contribute to the bleeding tendency observed in some FXI-deficient patients.

Expression of Urokinase-Type Plasminogen Activator in Platelets Decreases Oxygen-Induced Lung Injury in Mice.

We have previously shown that urokinase (uPA) can be ectopically expressed and stored in platelets in a transgenic mouse model and that such platelets can effectively prevent untoward thrombosis. One major advantage of this uPA delivery system is that there is no systemic fibrinolysis and the uPA is selectively released at sites of vascular injury. We have now tested whether such a delivery model may be of use in the care of patients with lung injury where inhibition of fibrinolytic activity and intra-alveolar fibrin deposition are of pathogenic importance. In this study, we evaluated (O2)-induced lung injury in transgenic mice expressing platelet uPA compared to wildtype (WT) littermate controls. Given that pulmonary sequestration of activated inflammatory cells, including neutrophils (PMN) and platelets, occurs relatively early in response to O2, and given the ability of the transgenic uPA platelets to enhance fibrin lysis, we hypothesized that hyperoxic-induced lung injury would be lessened in the transgenic animals. Lung injury was assessed by changes in bronchoalveolar lavage fluid (BAL) protein concentration. In this model, a significant increase in BAL protein concentration, indicative of injury to the alveolar capillary barrier, occurred in WT mice after 72 hr of >95% O2 exposure compared to controls [6.9 ± 0.9 vs. 0.14 ± 0.01 mg/ml (mean ± SEM)], respectively. BAL protein was significantly decreased in platelet uPA+ mice [1.5 ± 0.4 vs. 6.9 ± 0.9 mg/ml, n=6–8, p<0.05]. Immunohistochemical detection of fibrin in control and platelet uPA+ mice lungs using anti-murine fibrin(ogen) antibody showed that very little fibrin(ogen) was detected in control (0 hr) mice or WT mice exposed to >95% O2 for 24 hr. By 72 hr of O2 in WT mice, however, fibrin(ogen) immunoreactivity was seen within the alveolar space and diffusely throughout the interstitium. Fibrin clots within small blood vessels were also seen. In contrast, fibrin(ogen) immunoreactivity was greatly reduced in uPA+ mice. Moreover survival time of uPA+ mice was significantly longer than WT animals (127 ± 28 hr vs. 97 ± 14 hrs, respectively, n=12, p<0.003). Total white blood cell counts and % PMN in the BAL were significantly higher after 72 hr exposure to O2, suggesting that increased pulmonary inflammation is not necessarily associated with increased lung injury, a finding consistent with other studies showing that neutrophils do not play a significant role in the genesis of this lung injury. These data support the hypothesis that a decrease in lung fibrinolytic activity is a primary factor in the development of O2-induced lung injury and that platelet delivery of uPA may be an especially effective and safe way to prevent the development of such lung injury.

Evaluation of Physiologic and Pathologic Alterations of Coagulation and Fibrinolysis Using a New Global Assay.

BACKGROUND: The Clot Formation and Lysis (CloFAL) assay, modified from prior global assay methods by He et al., 1999, and Smith et al., 2003, involves activation of coagulation and fibrinolysis in platelet-poor plasma via addition of a physiologic reactant solution in a multi-well microassay plate. Continuous spectropohotometric data analysis allows measurement of kinetic absorbance changes of the plasma sample over three hours, which yields a unique clot formation and lysis curve. Using parameters of the curve, coagulation and fibrinolytic indices (CI and FI) are calculated relative to a simultaneously run pooled normal plasma standard.METHODS: Platelet-poor plasma obtained from pregnant women at term (n=24), neonatal cord blood (n=29), and healthy children (n=22) were analyzed using the CloFAL assay. Healthy adult (n=22) plasma samples, as well as those of individuals with factor deficiencies, were obtained commercially. Fibrinolytic alterations in vitro were also investigated. Intra-assay coefficients of variation (CVs) for normal controls ranged from 3–12% for all assay parameters, with inter-assay CVs of 5–15%.RESULTS: Representative CloFAL curves for healthy adults and children, pregnant women, and newborn infants are shown in Figure 1. Coagulation potential (measured by median CI) was significantly increased, while fibrinolytic capacity (measured by median FI) was markedly decreased, in pregnant women as compared to healthy adults (CI: 239% vs. 115%, FI: 59% vs. 95%; P<0.001 for each). By contrast, CI was decreased, and FI notably increased, in neonatal cords versus children, although the former comparison did not achieve statistical significance (CI: 58% vs. 69%, P=0.09; FI: 210% vs. 142%, P<0.001). The influence of deficiencies of coagulation factors and fibrinolytic regulators upon CloFAL parameters was also investigated. The greatest impact upon CI occurred with severe deficiency of fibrinogen or factors II, V, VII, VIII, IX, or X. Furthermore, CI was sensitive to deficiencies of factor VIII and fibrinogen in a concentration-dependent manner. In addition, FI was increased by PAI-1 deficiency and by inhibition of thrombin-activatable fibrinolysis inhibitor (TAFI) activation, and was zero in the setting of aminocaproic acid treatment. Further studies revealed that the influence of heparin concentrations of up to 2 U/mL in plasma was completely reversible by heparinase treatment of samples prior to assay.CONCLUSION: These results indicate that the CLoFAL assay is reproducible and analytically sensitive to known physiologic and pathologic alterations in coagulation and fibrinolysis. The application of this global assay to patients with a variety of disorders of thrombosis and hemostasis is currently ongoing. [Display omitted]

Differential down Regulation of Inflammatory Cytokines, CRP, CD 40-L, MCP-1 and NO Levels in Malignancy Associated Thrombosis by Extended Treatment of Enoxaparin and Warfarin.

Endogenous generation of inflammatory cytokines such as the C-reactive protein (CRP), tumor necorsis factor a (TNFα ), soluble CD 40-L (CD 40-L), monocyte chemotactic protein-1 (MCP-1) and enhanced formation of nitric oxide free radical (NO), significantly contribute to the pathogenesis of malignancy associated vascular and thrombotic disorders. An estimated 78 out of 1000 cancer patients develop symptomatic thrombosis (7.8%). Anticoagulant drugs such as the heparins and oral anticoagulants (warfarin) have been used in these patients for the overall management of cancer associated thrombotic and vascular events with improved clinical outcome. To further understand the pathogenesis of malignancy associated thrombosis and its modulation by a low molecular weight heparin (LMWH), enoxaparin (E) and warfarin (W), plasma samples were retrospectively analyzed from an open label, multidose, active comparator parallel design study in which all patients (n=110) were initially treated with enoxaparin at 1–1.5 mg/kg SC for 5 days. These patients were further subdivided into two groups. The first group continued to receive E whereas the second group received W (INR target 2–3), for up to 12 weeks. Baseline blood samples (BL), 5 day post enoxaparin (IPE) and 4–6 weeks samples from the E and W were analyzed for various inflammatory cytokines and NO. A highly sensitive CRP ELISA based assay (American Diagnostica, Stamford, CT), a sandwich based ELISA method for TNFα , CD 40-L and MCP-1 (R&D Systems, Minneapolis, MN) were used. In addition, tissue factor pathway inhibitor (TFPI) and thrombin activatable fibrinolytic inhibitor (TAFI) antigen levels were measured using commercially available ELISA kits (American Diagnostics, Stamford, CT and Hyphen Research, Paris, France), respectively. The results are summarized in the table below. The initial treatment of all of the cancer patients with thrombosis 1.0–1.5 mg/kg SC for 5 days resulted in a decrease of the circulating CRP, TNFα , CD 40-L and MCP-1 levels. NO levels were also dramatically reduced after this treatment. Increased levels of TFPI were also noted whereas, the TAFI antigen levels were reduced. Patients who were continued on E continued to exhibit reduced levels of most of these inflammatory markers and NO. However, patients treated with W exhibited a rebound increase in most of these markers and NO levels. At the 4–6 weeks time period in the W group the TFPI levels reverted back to normal levels. An increase in the TAFI levels were noted in this group. These results clearly indicate that various inflammatory markers and NO levels are upregulated in cancer patients with thrombosis. E is capable of down regulating these cytokines, however W treatment fails to sustain this down regulation.GroupCRP μg/mlTNF α(pg/ml)CD40-L(pg/ml)MCP-1 (pg/ml)NO M(μ)TFPI(ng/ml)TAFI( %NHP)BL15.1±1.237±101208±180540±6063±2374±1192±14IPE9.2±3.420±6590±125342±8222±8151±3476±124–6 weeks E7.8±2.819±2.3560±87290±7620.1±8168±2771±104–6 weeks W9.1±2.118±2.3690±79312±6525±1182±1596±15

Down Regulation of Inflammatory Markers after Treatment with Topically Administered Mucopolysaccharide Polysulfate.

Mucopolysaccharide polysulfate (MPS) represents a mammalian tracheal preparation, which mimics many of the properties of heparins including its anti-inflammatory actions. MPS is used topically for the treatment of inflammatory disorders. This study was designed to test the hypothesis that topical administration of MPS mobilizes many substances to mediate its therapeutic effects. A group of four non-human primates (Macaca Mulatta) were administered 4.5% MPS ointment in a dosage of 45 mg/kg, another four monkeys were administered placebo ointment at a dosage of 1.0 g/kg for 10 days. Citrated blood samples were drawn at baseline, 2, 4, 8 and 24 hours on days 1, 2, 5, 7 and 10. Markers of inflammation including thrombin activatable fibrinolytic inhibitor (TAFI), C-reactive protein (CRP), CD-40 Ligand (CD-40L), plasminogen activator inhibitor (PAI-1) and monocyte chemotactic protein (MCP-1) utilizing ELISA based methods, were used to measure the anti-inflammatory response. In the MPS treated group, a down regulation of all of these markers was observed over the 10 day period. CRP (baseline, 10.2 ±3.6; day 10, 5.1 ± 2.4 μg/ml), CD-40L (baseline, 298 ± 52; day 10, 138 ± 38 pg/ml) and MCP-1 (baseline, 350 ±68; day 10, 178 ± 42 pg/ml) showed a 50% decrease in the levels of these markers at day 10 in comparison to baseline. The PAI-1 (baseline, 65.1 ±21.2; day 10, 48.2 ±12.1 ng/ml) and TAFI (baseline, 138 ±25; day 10, 99 ± 12 % NHP) levels showed a slightly lesser effect. These results suggest that the topical administration of MPS leads to a decrease in the markers of inflammation that may result in promotion of the healing of inflammatory lesions.

Thrombin Activatable Fibrinolytic Inhibitor Modulation in Heparin Supplemented Plasma: Clinical Considerations.

Thrombin activatable fibrinolytic inhibitor (TAFI) is a procarboxypeptidase that cleaves the arginine and lysine amino acid residues off the carboxyl terminus of fibrin strands rendering fibrin resistant to the digestive actions of plasmin. Anticoagulant drugs such as the heparins and antithrombin drugs (hirudin, angiomax, argatroban and ximelagatran) by virtue of their inhibition of thrombin and its complex formation with thrombomodulin can down regulate the functionality of TAFI. It is now known that there are several TAFI polymorphisms, but the exact function of each form of TAFI is unclear at this time. Heparin-antithrombin complex produces strong inhibition of thrombin and thrombin-thrombomodulin complex and thereby reduces the conversion of TAFI to TAFIa. However, despite anticoagulation some of the individuals do not exhibit decreased TAFI functionality. In order to determine if a population of normal healthy volunteers exhibits differences in TAFI functionality upon anticoagulation with heparin, plasma samples were divided into two aliquots; saline was added to one set and 2.5 U heparin was added to the second set. This resulted in a final concentration of 0.25 U of heparin, which mimics therapeutic levels. Functional TAFI levels were determined using a synthetic substrate based method from Pentapharm Inc. (Basel, Switzerland), functional tissue factor pathway inhibitor (TFPI) levels and anti-Xa levels were obtained using a chromogenic-based method from American Diagnostica (Stamford, CT). TAFI levels in the saline supplemented samples were 89.9 ± 16.0% (range; 58.9 – 126.7%) normal human pooled plasma (NHP). After heparin supplementation, TAFI levels were reduced to 45.6 ± 16.1% NHP (range: 24.2 – 139.8% NHP). Upon further analysis it was determined that 45 (33%) individuals showed TAFI levels > 50% NHP (62.7 ± 14.8; range: 51.5 – 139.8% NHP) and 16 (11.8%) showed TAFI levels > 65% NHP (74.0 ± 17.8; range: 65.1 – 139.8% NHP). The anti-Xa level in the heparin supplemented samples was 0.181 ± 0.179 U (range: 0 – 0.61 U/ml) of heparin showing that the samples were anticoagulated. Comparing the TAFI levels obtained from the individuals that showed higher than expected levels to the anti-Xa showed that although TAFI levels were higher, the anti-Xa levels showed that the individuals were adequately anticoagulated. Interestingly functional TFPI levels did not differ in the heparin supplemented (1.35 ± 0.4 U/ml) and controls (1.73 ± 0.6 U/ml). These studies suggest that normal individuals, and different patient populations, may respond differently to anticoagulation using heparin, as functional TAFI levels may remain normal even after heparinization. This may be due to TAFI polymorphism in some individuals that allow the activation of TAFI to TAFIa even though only a small amount of thrombin is present. It is also plausible that this form of TAFI is more readily activated by plasmin or that the TAFIa itself may be more efficient in cleaving the arginine and lysine amino acid residues from fibrin. This suggests that patients undergoing surgical procedures where anticoagulation with heparin is necessary may be at risk of a fibrinolytic deficit if their functional TAFI levels remain high. This may predispose these patients to impaired fibrinolysis leading to further complications.

More Information on Patients with Factor XI Deficiency

It has been called to our attention that our recent Clinical Concepts and Commentary article, “Current Concepts of Hemostasis: Implications for Therapy,” contains a statement that implies that postoperative bleeding in patients with factor XI deficiency is usually mild.1We wish to clarify this implication. Patients with factor XI deficiency over a lifetime are mild bleeders who do not usually experience the chronic, crippling hemarthrosis or other severe bleeding episodes so typical of severe classic hemophilia or hemophilia B. Nonsurgical bleeding episodes in factor XI–deficient patients are usually mild over a lifetime. We wish to make it clear, however, that it is quite possible for patients with factor XI deficiency undergoing surgery to bleed severely unless they are pretreated. The replacement therapy for factor XI deficiency in the United States usually consists of plasma replacement therapy before an operation. More recently, recombinant factor VIIa has been used for factor XI deficiency, and it has been found to be effective, although it is not yet approved by the US Food and Drug Administration for this indication.2One of the readers of the journal pointed out to one of us via e-mail that patients with factor XI deficiency undergoing cardiopulmonary bypass may bleed excessively. We agree with this statement, and at our institution, such patients would be treated, preoperatively and postoperatively, with recombinant factor VIIa or with plasma replacement therapy, which may require plasma exchange transfusions to increase factor XI to 50% or greater. Factor XI concentrates are available in Europe but not in the United States, and these concentrates have occasionally been associated with thrombotic side effects. In summary, we wish to emphasize that even though patients with factor XI deficiency usually have mild bleeding episodes over a lifetime, this does not mean that they may not experience extensive hemorrhage after severe trauma or surgery. Although some patients with factor XI deficiency do not bleed after operative procedures, a family history of bleeding after surgery is suggestive that relatives of such patients will also bleed during surgery.It has been recommended that replacement therapy for factor XI deficiency also include the use of antifibrinolytic agents such as tranexamic acid because it seems that one function of factor XI is to boost thrombin generation to the extent that the “thrombin activatable fibrinolytic inhibitor” (TAFI) can be activated.3Adding tranexamic acid enhances the antifibrinolytic effect.We are grateful to the reader who called this potential interpretation of factor XI deficiency to our attention.* The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina. hrr@med.unc.edu

Tissue Factor Pathway Inhibitor and Thrombin Activatable Fibrinolytic Inhibitor Plasma Levels following Burnand Septic Injuries in Rats

Burn and septic injuries induce profound changes in coagulation status. This study examined the changes in plasma tissue factor pathway inhibitor (TFPI) and thrombin activatable fibrinolytic inhibitor (TAFI) levels in a rat model of burn and septic injuries. Rats underwent 30% TBSA cutaneous scald burn injury and septic insult was induced by caecal ligation and puncture (CLP). CLP was superimposed on burn injury to mimic the clinical model of sepsis complicating burn injury. Rats were pretreated with Cprofloxacin orally to colonize their gut with Enterococcus faecalis. TFPI and TAFI plasma levels were measured using functional activity assay kit with a chromogenic method at 24 and 72 hours following the injuries. TFPI levels decreased significantly at 24 hours in burn, CLP, and burn+CLP groups, followed by incomplete rebound recovery at 72 hours in all three groups. On the other hand, TAFI levels increased significantly at 24- and 72-hour time points in all three groups. These results suggest that burn, septic, and their combined injuries perturb coagulation cascade and thrombotic process toward the procoagulant pathway by impairing fibrinolysis.

Role of PAI-1 and TAFI in the mediation of fibrinolytic deficit in cancer patients

9727 Background: Cancer associated thrombosis is known to have a multi-factorial pathogenesis involving up regulation of coagulation factors, down regulation of inhibitors and endothelial dysfunction. More recently, cancer patients have been shown to exhibit fibrinolytic deficit with impaired clot lysis. This has been mostly attributed to an increase in the circulating levels of plasminogen activator inhibitor 1 (PAI-1). Thrombin activatable fibrinolytic inhibitor (TAFI) has recently been identified as an additional mediator of fibrinolytic deficit that transforms fibrin to a plamsin resistant variant. Methods: To decipher the role of TAFI in this process, plasma samples obtained from 170 cancer patients with symptomatic thrombosis were analyzed for PAI-1, TAFI functional (F) and TAFI immunologic (I). An ELISA method was used for PAI-1 (Stago, Stamford, CT) and TAFI-I (American Diagnostic, Greenwich, CT), while a functional method was used for TAFI-F (Pentapharm, Ltd., Basel Switerland) Results: Baseline samples exhibited a marked increase in PAI-1 levels (140 ± 21) compared to normal (94 ± 14). The TAFI-I levels ranged from 56 –183 %NHP (123 ± 28 %NHP) which was higher than normal (85.1 ± 14 %NHP; n= 82). Interestingly, TAFI measured by functional levels were disproportionately lower (85 ± 26 %NHP) suggesting the consumption of this protease in cancer patients. In contrast to the TAFI I/F ratio approximately 1 for normal individuals, this ratio for cancer patients was 1.6 ± 0.1 (5.6 –0.7). When the PAI-1 levels were compared with F and I TAFI levels, no correlation was evident. This suggests independent regulation of these two proteins in cancer. The increased PAI-1 and TAFI-I levels were independent of the fibrinogen levels. Conclusions: The results clearly suggest that thrombotic complications in cancer patients may be partly due to the fibrinolytic deficit, which is modulated by an up regulation of both PAI-1 and TAFI. Furthermore, the two regulators of fibrinolysis mediate their effects independently. Since the TAFI-I levels are disproportionately high, this enzyme may complex with endogenous proteins modulating its functionality. No significant financial relationships to disclose.

Erythropoietin-induced thrombosis as a result of increased inflammation and thrombin activatable fibrinolytic inhibitor.

Chronic inflammation is a major cause of morbidity and mortality in end-stage renal disease. The associated anemia in these patients due to renal cortical atrophy and erythropoietin deficiency is treated with recombinant erythropoietin. Recent reports suggest a growing incidence of symptomatic venous thrombosis in cancer patients treated with recombinant erythropoietin. Several investigators have reported on different mechanisms of thrombosis in these patients. We hypothesize that thrombosis in patients with end-stage renal disease due to increased expression of C-reactive protein (CRP) as a result of chronic inflammation promotes the release of thrombin activatable fibrinolytic inhibitor causing fibrinolytic deficit and eventually thrombosis. Furthermore, because endothelial nitric oxide is responsible for the maintenance of the normal vascular function, the decreased levels of nitric oxide in chronic inflammation cause endothelial damage and result in thrombosis. To test this hypothesis, blood samples were collected from 106 patients (49 male and 57 female, aged 59.8+/-15.7 years) with end-stage renal disease undergoing hemodialysis and treated with recombinant erythropoietin at a mean dose of 201.8 U/kg/week. Blood samples were drawn in 5-mL tubes containing 3.2% sodium citrate just before the hemodialysis procedure. These blood samples were immediately centrifuged to obtain platelet-poor plasma, which was aliquoted and frozen at -70 degrees C until further analysis. Erytropoietin antibodies were measured using an anti-EPO enzyme-linked immunosorbent assay (ELISA) method developed in our laboratory. Nitric oxide was measured using a NO analyzer (Sievers 280I, Ionics, Boulder, CO). Plasma CRP levels were measured with a highly sensitive ELISA method IMUNOCLONE CRP ELISA (American Diagnostica, Greenwich, CT). TAFI antigen levels in plasma were analyzed with an IMUCLONE TAFI ELISA kit (American Diagnostica, Greenwich, CT). TAFI functional activity was assayed with an ACTICHROME TAFI activity kit. The measured levels of nitric oxide, CRP, TAFI antigen, and TAFI functional were 37.36+/-36.8 (normal value, 37.49+/-18.96; range, 19.3-102 microM), 12.27+/-10.6 (normal value, < 1 microg/mL), 146.9+/-28.4% NHP (normal, 100% NHP), and 102.55+/-37% NHP (normal range, 22.3-165.7; mean, 89.5% NHP), respectively. The erythropoietin antibody was detected in 9.4% of the patient group. While 20% of the erythropoietin antibody-positive and 27.1% of the erythropoietin antibody-negative patients experienced chest pain, thrombotic events developed in 9.4% of the erythropoietin antibody-negative patients. These data provide the rationale for a novel mechanism of thrombosis through increased activity of CRP, nitric oxide, and TAFI, leading to fibrinolytic deficit and thrombosis in patients treated with erythropoietin.

Thrombin activatable fibrinolysis inhibitor: not just an inhibitor of fibrinolysis.

To review the activation of thrombin activatable fibrinolysis inhibitor (TAFI) and activity of activated TAFI (TAFIa) as it relates to the regulation of both fibrinolytic and proinflammatory substances. Published articles and reviews (from PubMed, published between 1962 and 2003) on experimental studies of coagulation, fibrinolysis, and inflammation. The principal physiologic role of TAFI is still a matter of debate. Although TAFI activation can result from proteolysis by a number of proteases, the most likely physiologic activators are thrombin (in complex with the cofactor thrombomodulin) and plasmin (in complex with polysaccharide cofactors). The activated enzyme, TAFIa, displays carboxypeptidase B-like activity and probably regulates both fibrinolysis and inflammation in response to injury and infection. At present, there is limited understanding of the role that TAFI plays in the interrelationships between coagulation, fibrinolysis, and inflammation. Although the potential therapeutic value of TAFIa inhibition/TAFI activation awaits further investigation, the data gathered to date suggest that, like activated protein C, TAFIa may play a pivotal role in regulating the crosstalk between coagulation, fibrinolysis, and inflammation.

Effect of atorvastatin on different fibrinolyis mechanisms in hypercholesterolemic subjects

Background: Hydroxymethyl-glutaryl-CoA-reductase inhibitors (statins) reduce cardiovascular events by cholesterol lowering as well as non-lipid related actions. Among them, the modulation of fibrinolysis could play a relevant role in vascular protection. Atorvastatin is able of reducing platelet activity and thrombin generation before low-density lipoprotein cholesterol (LDL-C) decrease in hypercholesterolemic subjects in which coagulation and fibrinolysis are linked by the activation of thrombin activable fibrinolysis inhibitor (TAFI). The aim of our study was to evaluate whether atorvastatin could modulate fibrinolysis by interactions with endothelial mechanisms and thrombin generation. Methods: Forty-four pure hypercholesterolemic subjects (26 M, 18 F, mean age 52.7±13.7, LDL-C 194.8±9.3t mg/dl) were evaluated for plasmin–antiplasmin complexes (PAP), tissue-plasminogen acivator (t-PA) and its inhibitor (PAI-1) (ELISA), TAFI activity (HPLC), platelet P-selectin (P-sel) (cytofluorymetric detection), platelet-dependent thrombin generation (PDTG, coagulative-chromogenic method) and lipid profile at baseline and after 7, 14, 28 and 90 days of atorvastatin (10 mg/die) treatment. Results: PAP were significantly reduced at baseline in hypercholesterolemic versus control subjects ( P<0.05) and were related to P-sel ( P<0.01), PDTG ( P<0.01) and its inhibitor (PAI-1) after venous occlusion (VO) ( P<0.05). Atorvastatin induced a significant increase of PAP at T 2 related to modifications of P-sel ( P<0.01) and PDTG ( P<0.01) before significant LDL-C reduction ( P=0.132). PAI-1 was significantly changed at T 3 with relation to LDL-C ( P<0.01), Von Willebrand factor (VWF) ( P<0.01) and sE-sel ( P<0.05). Conclusions: The profibrinolytic activity of atorvastatin in hypercholesterolemic subjects is related, initially, to the positive effects exerted on platelet function and thrombin generation which can modulate fibrinolysis by TAFI activity.