Impaired fibrinolysis as a risk factor for Budd-Chiari syndrome.

In Budd-Chiari syndrome (BCS), thrombosis develops in the hepatic veins or inferior vena cava. To study the relationship between hypofibrinolysis and BCS, we measured plasma levels of fibrinolysis proteins in 101 BCS patients and 101 healthy controls and performed a plasma-based clot lysis assay. In BCS patients, plasminogen activator inhibitor 1 (PAI-1) levels were significantly higher than in controls (median, 6.3 vs 1.4 IU/mL, P < .001). Thrombin-activatable fibrinolysis inhibitor and plasmin inhibitor levels were lower than in controls (13.8 vs 16.9 microg/mL and 0.91 vs 1.02 U/L, both P < .001). Median plasma clot lysis time (CLT) was 73.9 minutes in cases and 73.0 minutes in controls (P = .329). A subgroup of cases displayed clearly elevated CLTs. A CLT above the 90th or 95th percentile of controls was associated with an increased risk of BCS, with odds ratios of 2.4 (95% confidence interval, 1.1-5.5) and 3.4 (95% confidence interval, 1.2-9.7), respectively. In controls, only PAI-1 activity was significantly associated with CLT. Analysis of single nucleotide polymorphisms of fibrinolysis proteins revealed no significant differences between cases and controls. This case-control study provides the first evidence that an impaired fibrinolytic potential, at least partially caused by elevated PAI-1 levels, is related to the presence of BCS.

Generation and characterization of monoclonal antibodies against the N-terminus of alpha-2-antiplasmin.

Around 70% of circulating alpha-2-antiplasmin (α2AP), the main natural plasmin inhibitor, is N-terminally cleaved between residues Pro12 and Asn13 by antiplasmin-cleaving enzyme. This converts native Met-α2AP into the more potent fibrinolysis inhibitor Asn-α2AP. The Arg6Trp (R6W) polymorphism affects the N-terminal cleavage rate of Met-α2AP in a purified system, with ~8-fold faster conversion of Met(R6)-α2AP than Met(W6)-α2AP. To date, assays to determine N-terminally intact Met-α2AP in plasma have been limited to an ELISA that only measures Met(R6)-α2AP. The aim of this study was to generate and characterize monoclonal antibodies (mAbs) against Met(R6)-α2AP, Met(W6)-α2AP and all α2AP forms (total-α2AP) in order to develop specific Met(R6)-α2AP and Met(W6)-α2AP ELISAs. Recombinant Met(R6)-α2AP, Met(W6)-α2AP and Asn-α2AP were expressed in Drosophila S2 cells. Using hybridoma technology, a panel of 25 mAbs was generated against a mixture of recombinant Met(R6)-α2AP and Met(W6)-α2AP. All mAbs were evaluated for their specific reactivity using the three recombinant α2APs in one-site non-competitive ELISAs. Three mAbs were selected to develop sandwich-type ELISAs. MA-AP37E2 and MA-AP34C4 were selected for their specific reactivity against Met(R6)-α2AP and Met(W6)-α2AP, respectively, and used for coating. MA-AP15D7 was selected for its reactivity against total-α2AP and used for detection. With the novel ELISAs we determined Met(R6)-α2AP and Met(W6)-α2AP levels in plasma samples and we showed that Met(R6)-α2AP was converted faster into Asn-α2AP than Met(W6)-α2AP in a plasma milieu. In conclusion, we developed two specific ELISAs for Met(R6)-α2AP and Met(W6)-α2AP, respectively, in plasma. This will enable us to determine N-terminal heterogeneity of α2AP in plasma samples.

Circulating fibroblast activation protein activity and antigen levels correlate strongly when measured in liver disease and coronary heart disease.

BACKGROUND AND AIM: Circulating fibroblast activation protein (cFAP) is a constitutively active enzyme expressed by activated fibroblasts that has both dipeptidyl peptidase and endopeptidase activities. We aimed to assess the correlation between cFAP activity and antigen levels and to compare variations in levels. METHODS: In plasma of 465 control individuals, 368 patients with coronary heart disease (CHD) and 102 hepatitis C virus (HCV) infected patients with severe liver disease before and after liver transplant, cFAP activity levels were measured with a newly developed cFAP activity assay. In the same samples, cFAP antigen levels were measured using a commercially available cFAP ELISA. Correlation analyses between activity and antigen levels were performed by calculating Pearson's correlation coefficient (ρ). Additionally, normal ranges, determinants and differences between cohorts and between anticoagulants were investigated. RESULTS: cFAP activity and antigen levels significantly correlated in controls (ρ: 0.660, p<0.001) and in CHD patients (ρ: 0.709, p<0.001). cFAP activity and antigen levels in the HCV cohort were significantly lower in the samples taken after liver transplantation (p<0.001) and normalized toward levels of healthy individuals. Furthermore, cFAP activity and antigen levels were higher in men and significantly associated with body mass index. Also, cFAP activity and antigen levels were higher in EDTA plasma as compared to the levels in citrated plasma from the same healthy individuals. CONCLUSIONS: For analyzing cFAP levels, either activity levels or antigen levels can be measured to investigate differences between individuals. However, it is of importance that blood samples are collected in the same anticoagulant.

Plasma levels of soluble fibroblast activation protein in arterial thrombosis: determinants and cleavage of its substrate alpha-2-antiplasmin.

BACKGROUND: Fibroblast activation protein (FAP) is a transmembrane glycoprotein with dipeptidyl-peptidase and endopeptidase activities and circulates in blood in a truncated, soluble form (sFAP). Fibrinolysis inhibitor α2-antiplasmin (α2AP) has been described as a potential in vivo substrate of sFAP. We aimed to investigate sFAP levels and α2AP cleavage in young arterial thrombosis patients and in control individuals, study the correlation between sFAP levels and α2AP cleavage and investigate determinants of these variables. METHODS: sFAP levels and α2AP cleavage were determined by ELISA in the plasma samples of 391 coronary heart disease (CHD) patients, 221 ischemic stroke patients, 51 peripheral arterial disease patients and 501 control individuals. RESULTS: Median sFAP levels were similar in arterial thrombotic patients and in control individuals, but in CHD patients sFAP levels significantly increased with time (number of months) between the event and study inclusion (Spearman's rho: 0.209, p<0.001), indicating reduced sFAP levels at time of event. sFAP levels and percentage α2AP cleavage significantly correlated in controls and in patients. Furthermore, sex, use of oral contraceptives and hyperlipidemia were significant determinants of sFAP levels. CONCLUSIONS: sFAP levels were reduced in the CHD patient population, but only in the first months after the event, indicating that over time sFAP levels may normalize. The significant correlation between sFAP level and α2AP cleavage indicates that in vivo sFAP (at least partly) regulates cleavage of α2AP, irrespective of disease status. Differences in sFAP level due to sex, use of oral contraceptives and hyperlipidemia might suggest hormonal control of sFAP levels.

Biological variation in tPA-induced plasma clot lysis time.

Hypofibrinolysis is a risk factor for venous and arterial thrombosis, and can be assessed by using a turbidimetric tPA-induced clot lysis time (CLT) assay. Biological variation in clot lysis time may affect the interpretation and usefulness of CLT as a risk factor for thrombosis. Sufficient information about assay variation and biological variation in CLT is not yet available. Thus, this study aimed to determine the analytical, within-subject and between-subject variation in CLT. We collected blood samples from 40 healthy individuals throughout a period of one year (average 11.8 visits) and determined the CLT of each plasma sample in duplicate. The mean (± SD) CLT was 83.8 (± 11.1) minutes. The coefficients of variation for total variation, analytical variation, within-subject variation and between-subject variation were 13.4%, 2.6%, 8.2% and 10.2%, respectively. One measurement can estimate the CLT that does not deviate more than 20% from its true value. The contribution of analytical variation to the within-subject variation was 5.0%, the index of individuality was 0.84 and the reference change value was 23.8%. The CLT was longer in the morning compared to the afternoon and was slightly longer in older individuals (> 40 years) compared to younger (≤40 years) individuals. There was no seasonal variation in CLT and no association with air pollution. CLT correlated weakly with fibrinogen, C-reactive protein, prothrombin time and thrombin generation. This study provides insight into the biological variation of CLT, which can be used in future studies testing CLT as a potential risk factor for thrombosis.

The effect of ethanol and its metabolism on fibrinolysis.

The role of ethanol metabolism in possible haemostatic cardioprotective effects has not yet been determined. To this end, we investigated the effect of a moderate dose of ethanol (35 g) and its metabolism, on haemostatic variables over 14 hours (h). Eighteen Caucasian males participated in a placebo-controlled, randomised, cross-over study. Blood was collected prior to alcohol consumption, and at 10 time points for 14 h. Blood ethanol peaked at 1 h and was cleared after 8 h following ethanol consumption, significantly increasing plasma acetate (p=0.0028). Ethanol did not influence the coagulation factors significantly. PAI-1act increased (p<0.0001) and tPAact (p=0.047) decreased following alcohol consumption, reaching maximum (0.69 to 22.2 IU/ml) and minimum (0.88 to 0.33 IU/ml) levels at 5 h, respectively. Significantly increased plasma clot lysis times (46.8 to 67.6 minutes) and reduced global fibrinolytic capacity of whole blood, measured as D-dimer production during incubation of blood clots (2.26 to 0.29 µg/ml), were found at 5 h. Except for PAI-1act (borderline significance; p=0.05), there was no significant difference in the fibrinolytic markers between the two groups the following morning. Moderate ethanol consumption resulted in a significant temporary fibrinolysis inhibition. Any protective effects of moderate ethanol consumption on cardiovascular disease do not appear to be due to improvement in fibrinolytic potential within the first 14 h following consumption. The use of global fibrinolytic assays is recommended for determining the true effect of ethanol on fibrinolysis.