PAI-1 Antigen Levels Research Articles

Antigen levels of urokinase type plasminogen activator and its inhibitors in primary breast cancer.

Primary Breast Cancer, Urokinase-Type Plasminogen Activator, Inhibitors The aim of the study was to monitor urokinase plasminogen activator antigen concentrations and its type 1 (PAI-1) and type 2 (PAI-2) inhibitors in histologically defined forms of primary breast cancer and a comparison with these antigens levels in normal tissue. Another goal was a search for a relationship/or its lack/between the occurrence of the new generation markers of neoplastic disease and a presence/or absence/of lymph node metastases. U-PA, PAI-1 and PAI-2 antigen levels were determined by ELISA tests in protein extracts of breast cancer tissues. Among the studied breast tumors 32 specimens were ductal carcinomas, 15 specimens were lobular carcinomas and the remaining 13 were other rare histological forms. In comparison to the obtained values of u-PA antigen levels in normal tissue, the values in neoplastic tissues were elevated several times: 11-fold, 6-fold and 15-fold in ductal c., lobular c. and other rare neoplasms. The values of PAI-1 antigen levels were about 20-fold higher for all studied, histologically defined primary breast cancers. The greatest differences of PAI-2 antigen levels growth was observed in histologically defined primary breast cancer forms. It was augmented 10-fold, 40-fold and 20-fold, respectively, for ductal carcinoma, lobular carcinoma and rare forms of neoplasms. In various forms of invasive breast cancer and those without lymph node metastases the content of u-PA, PAI-1 and PAI-2 were also significantly elevated. Among the new generation of independent markers of the neoplastic process, PAI-2 seems to be the most reliable marker for the identification of primary breast cancer. The goal of the present study was to evaluate a possible combined prognostic value of the three major components of the u-PA system (u-PA, PAI-1 and PAI-2) in patients with defined histopathological forms of primary breast cancer.

Prognostic significance of plasminogen activator inhibitor-1 in breast cancer, with special emphasis on locoregional recurrence-free survival

The independent prognostic value of protease uPA and its inhibitor PAI-1 for survival in breast cancer patients is firmly established. However, there is very little data on the prognostic value of serine proteases and their inhibitors for locoregional recurrence in breast cancer. The prognostic value of PAI-1 for local control in a group of 766 patients treated at our institute with either breast conserving treatment or modified radical mastectomy was evaluated. The locoregional recurrence-free survival (LRFS) of patients with PAI-1 values above the median value was significantly worse than that of patients with PAI-1 values below the median value (log-rank; p=0.0078). In multivariate analysis PAI-1 levels proved to be of independent statistical significance for LRFS (p=0.0401, relative risk 2.28, 95% confidence interval 1.04-5.02). The independent prognostic value of PAI-1 for metastasis-free survival and overall survival was also confirmed. In addition, our data suggest that PAI-1 antigen levels in tumor tissue might be of prognostic value for survival after locoregional recurrence (log-rank; p=0.0618). According to our findings, PAI-1 levels could be used as a biological marker that could facilitate the identifation of patients with a higher risk of local relapse already at the time of primary treatment. These patients should then be offered more aggressive treatment.

Prognostic Significance of Plasminogen Activator Inhibitor-1 in Breast Cancer, with Special Emphasis on Locoregional Recurrence-Free Survival

The independent prognostic value of protease uPA and its inhibitor PAI-1 for survival in breast cancer patients is firmly established. However, there is very little data on the prognostic value of serine proteases and their inhibitors for locoregional recurrence in breast cancer. The prognostic value of PAI-1 for local control in a group of 766 patients treated at our institute with either breast conserving treatment or modified radical mastectomy was evaluated. The locoregional recurrence-free survival (LRFS) of patients with PAI-1 values above the median value was significantly worse than that of patients with PAI-1 values below the median value (log-rank; p=0.0078). In multivariate analysis PAI-1 levels proved to be of independent statistical significance for LRFS (p=0.0401, relative risk 2.28, 95% confidence interval 1.04-5.02). The independent prognostic value of PAI-1 for metastasis-free survival and overall survival was also confirmed. In addition, our data suggest that PAI-1 antigen levels in tumor tissue might be of prognostic value for survival after locoregional recurrence (log-rank; p=0.0618). According to our findings, PAI-1 levels could be used as a biological marker that could facilitate the identifation of patients with a higher risk of local relapse already at the time of primary treatment. These patients should then be offered more aggressive treatment.

Association Between Increased Homocysteine Levels and Impaired Fibrinolytic Potential: Potential Mechanism for Cardiovascular Risk

Elevated homocysteine levels increase cardiovascular risk although the mechanism is not well understood. Since thrombosis plays an important role in plaque development and acute coronary syndromes, hyperhomocysteinemia may increase risk by increasing the thrombotic potential. Hemostatic risk factors were measured in 3,216 individuals (1,451 men and 1,765 women) free of cardiovascular disease who participated in cycle 5 of the Framingham Offspring Study. An increase in homocysteine level was associated with a rise in plasminogen activator inhibitor (PAI-1), tissue plasminogen activator (TPA) antigen, von Willebrand factor and fibrinogen level. After regression analyses adjusting for covariates, there remained significant associations between homocysteine and PAI-1 and TPA antigen. Increasing homocysteine levels are associated with impaired fibrinolytic potential, as indicated by increased PAI-1 and TPA antigen levels. These data suggest that folic acid and other homocysteine lowering therapies may decrease cardiac events through a reduction in thrombotic tendency.

Relationships between Fibrinolytic and Inflammatory Parameters in Human Adipose Tissue: Strong Contribution of TNFα Receptors to PAI-1 Levels

Plasminogen activator inhibitor type 1 (PAI-1), a risk marker of atherosclerosis, is highly expressed in adipose tissue from obese subjects. PAI-1 is also considered as an acute phase protein. Recently, adipose tissue has been described as a source of inflammatory cytokines. Therefore, our aim was to study the relationships between PAI-1, and IL-6, TNF, TNF receptors (TNFRSF1s) and TGFbeta1, in plasma and adipose tissue from obese (n = 60) and lean (n = 28) subjects. Study has been extended to plasminogen activators (t-PA and u-PA). Compared to lean subjects, obese subjects exhibited higher plasma levels of all the studied parameters (except for TGFbeta1) whereas in adipose tissue only PAI-1, t-PA and TGFbeta antigen levels differed. In the obese population, plasma PAI-1 levels were weakly associated with circulating TNF, and this relationship disappeared after adjustment for plasma t-PA. Adipose tissue PAI-1 levels were positively associated with TNFRSF1s and TGFbeta, the strongest relationship being observed with TNFRSF1A, which explained 82% of PAI-1 variability. TNF and IL-6 were the main contributors to t-PA variability in plasma and in adipose tissue, respectively. Our results argue on the relevance of TNFRSF1s in the regulation of PAI-1 expression by adipose tissue. Association between t-PA, which is mainly produced by endothelial cells, and IL-6 or TNF suggest that inflammation might be involved in angiogenesis in adipose tissue.

Plasminogen Activator and Plasminogen Activator Inhibitor Gene Expression in Human Saphenous Vein Organ Culture

We describe the expression of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) in saphenous vein culture. Smooth muscle cells (SMC) are quiescent in fresh tissue, whereas these cells acquire a proliferating phenotype when venous segments are cultured in the presence of serum. t-PA and PAI-1 were localized immunohistochemically and quantified using biochemical techniques. t-PA and PAI-1 mRNA was quantified by reverse transcription polymerase chain reaction (RT-PCR) assay. Immunostaining showed an increase in the positivity of proliferating cell nuclear antigen (PCNA) from 10-day tissue culture. Tissue sections from fresh vein showed minimal t-PA and maximal PAI-1 immunostaining. In contrast, 10-day cultures showed an increase in t-PA and a decline in PAI-1 staining. Biochemical analysis revealed a 118% increase in t-PA and a 50% decrease in PAI-1 antigen levels from 10-day tissue cultures. RT-PCR demonstrated that the mRNA encoding t-PA increased, while PAI-1 decreased after 10 days of culture. In conclusion, venous culture showed an up-regulation of t-PA and a repression of PAI-1 gene expression during SMC proliferation in the vessel wall. The PAI-1 repression observed in venous culture is in contrast to the situation observed in human atheroma. A shift in the t-PA/PAI-1 balance may have a role in vascular remodeling.

The 4G/5G polymorphism of the type 1 plasminogen activator inhibitor gene and thrombosis in patients with antiphospholipid syndrome.

To investigate the relationship between the 4G/5G polymorphism of the type 1 plasminogen activator inhibitor (PAI-1) gene and thrombotic manifestations in patients with antiphospholipid syndrome (APS). We studied a total of 247 patients included in the following 4 groups: 70 patients with primary APS, 104 patients with systemic lupus erythematosus (40 with antiphospholipid antibodies [aPL] and clinical [secondary] APS, 13 with aPL but without clinical APS, and 51 with neither detectable aPL nor a history of thrombosis), 14 asymptomatic individuals with aPL, and 59 patients with thrombosis but without known thrombosis risk factors. A control group of 100 healthy individuals was also analyzed. PAI-1 4G/5G polymorphism was determined by polymerase chain reaction and endonuclease digestion. The allele frequency of 4G/5G in controls was 0.47/0.53. There were no differences in allele distribution among patient groups or between patients and controls. However, a higher frequency of the 4G allele was observed in APS patients with versus those without thrombosis (0.57 versus 0.39; P < 0.05) (odds ratio [OR] 2.83, 95% confidence interval [95% CI] 1.18-6.76). This higher frequency of the 4G allele was attributable to the higher frequency in patients with versus those without arterial thrombosis (0.64 versus 0.43; P < 0.01) (OR 5.96, 95% CI 1.67-21.32), while patients with venous thrombosis had an allele distribution similar to that of those without venous thrombosis (0.49 versus 0.50; P not significant). There was a trend toward higher PAI-1 antigen and activity levels in APS patients and controls with the 4G/4G genotype, but this did not reach statistical significance. The presence of the 4G allele of the 4G/5G polymorphism of the PAI-1 gene may be an additional risk factor for the development of arterial thrombosis in APS.

New ELISA for quantitation of human urokinase receptor (CD87) in cancer.

The serine protease urokinase-type plasminogen activator (uPA), its inhibitor (PAI-1), and its receptor (uPAR; CD87) facilitate cancer cell invasion and metastasis. Whereas uPA and PAI-1 antigen levels determined in tumor tissue extracts of breast cancer patients correlate with disease recurrence and overall survival, the prognostic relevance of uPAR is still a matter of debate. We established two new sandwich-type enzyme-linked immunosorbent assay (ELISA) formats (HU/IIIF10-ELISA and HU/HD13-ELISA) using the epitope-defined monoclonal antibody (mAb) IIIF10 or the conformation-dependent mAb HD13.1, a polyclonal chicken antibody (HU277), and recombinant soluble uPAR (CHO-suPAR) as the standard. The lower detection limit of the assays was at 0.16 ng/ml, with a linear dose-response up to 5 ng/ml of uPAR antigen. Both ELISA formats showed good reproducibility and recovery. The intra-assay and the inter-assay variation coefficients were respectively 4.3% and 11.7% (HU/IIIF10-ELISA) and 4.0% and 10.7% (HU/HD13-ELISA). The recovery rate of uPAR in cell lysates spiked with CHO-suPAR was above 82% and 88%, respectively. With these new ELISA formats, uPAR antigen content in breast cancer tissue extracts and tumor cell lysates was determined and compared to a commercially available ELISA (ADI-ELISA). By all of the three uPAR ELISA formats CHO-suPAR and uPAR present in lysates of non-malignant epithelial cells and stimulated monocytes were quantified with similar sensitivity. Interestingly, in breast cancer cell lines of epitheloid origin a higher uPAR antigen content was determined by the HU/IIIF10-ELISA than the HU/HD13- or ADI-ELISA formats. In lysates of fibroblastic breast cancer cell lines similar uPAR values were obtained with the HU/IIIF10- and ADI-ELISA formats, whereas with the HU/HD13-ELISA significantly lower uPAR concentrations were determined. The prognostic relevance of tumor uPAR antigen was evaluated in 199 primary breast cancer patients with a median follow-up of 24 months. uPAR antigen values above the cut-off of 3.33 ng/mg protein as determined by the HU/IIIF10-ELISA were significantly correlated with short disease-free survival (p=0.025). Results obtained by the other two ELISA formats (HU/HD13-ELISA and ADI-ELISA) were not associated with prognosis. Our findings stress the need of well-characterized antibodies, which detect both uPAR of non-malignant and tumor cells, in setting up a uPAR-ELISA useful for assessing breast cancer patient prognosis.

Vascular and cellular proteolytic activity in mice with α2-antiplasmin gene inactivation

Objective: To study the role of α2-antiplasmin (α2-AP), the main physiological plasmin inhibitor, in controlling vascular and cellular proteolytic activity.Materials: Arteries, organs and cell cultures derived from α2-AP-deficient (α2-AP–/–) mice or from their wild-type littermates (α2-AP+/+).Results: In serum-free conditioned medium of α2-AP+/+or α2-AP–/–skin fibroblasts, the time course (0–72 h) of PAI-1 antigen and of t-PA or u-PA antigen and activity production was similar. Activation of proMMP-9 (gelatinase B) upon addition of plasmin(ogen) to serum-free conditioned medium of fibroblasts was consistently detectable with α2-AP–/–but not with α2-AP+/+cells. In aorta and femoral arterial extracts of α2-AP+/+or α2-AP–/–mice, t-PA and u-PA activity levels were comparable, and fibrin zymography with cryosections did not reveal significant differences in fibrinolytic activity. In liver or kidney extracts of α2-AP+/+or α2-AP–/–mice, t-PA, u-PA, PAI-1 and plasminogen antigen levels were comparable; t-PA or u-PA activity was not detected in liver extracts and was present at comparable levels in kidney extracts.Activation of plasminogen to plasmin in solution by cell-associated plasminogen activator, and activation of cell-bound plasminogen by tcu-PA was comparable for fibroblasts of both genotypes.Conclusions: α2-AP does not play a crucial role in controlling vascular or cellular proteolytic activity in mice.

Induction of type 1 plasminogen activator inhibitor in human liver ischemia and reperfusion

Background/Aims: The detailed role of type 1 plasminogen activator inhibitor (PAI-1) in liver with ischemia reperfusion injury remains unclear. The aim of the present study was to examine this mechanism, focusing on inflammatory cytokines. Methods: Eighteen patients who underwent partial hepatectomy (PH) for metastatic liver tumors using only Pringle's maneuver were enrolled in the study. Peripheral blood was taken perioperatively and PAI-1, tissue type plasminogen activator (tPA), plasma aspartate aminotransferase (AST) and cytokine levels were measured. Two liver specimens were taken from each patient, one before ischemia and the other after PH, for detection of mRNA of PAI-1, tPA, tumor growth factor (TGF)-β1, interleukin-1β and tumor necrosis factor-α. Immunohistochemistry and Western blotting were performed to detect PAI-1 protein in the liver. Results: The average plasma PAI-1 antigen level reached a maximum after the final clamp (121.6±5.9 ng/ml). The average PAI-1 level in hepatic veins (140.4±6.3 ng/ml) after the last Pringle's maneuver was higher than that in peripheral veins ( p<0.0001). A correlation was observed between the ratio of the corrected fluorescent activity of PAI-1 before and after ischemia and TGF-β1 mRNA levels in the liver after PH ( p=0.003). PAI-1 protein could be detected in parenchymal and non-parenchymal cells of the liver obtained after ischemia reperfusion injury. Conclusions: PAI-1 was produced in human livers after ischemia reperfusion injury provoked by Pringle's maneuver, giving valuable information regarding the degree of warm ischemic damage.

Abstracts of literature

Abstracts of literature

Long-term follow-up confirms prognostic impact of PAI-1 and cathepsin D and L in primary breast cancer.

After long-term follow-up, the prognostic impact of the following proteolytic factors associated with tumor invasion and metastasis was evaluated in 276 primary breast cancer patients: uPA (urokinase-type plasminogen activator), PAI-1 (uPA inhibitor type 1), and cathepsins B, D and L. The median follow-up of patients still alive at the time of analysis was 109 months. To date 119 patients (43%) have relapsed and 117 (42%) have died. Antigen levels of uPA and PAI-1 were determined by ELISA in detergent extracts; cathepsin B, D, and L content was determined in cytosol fractions of the primary tumor: cathepsin D by ELSA and cathepsin B and L by ELISA. In multivariate analysis (Cox model) for disease-free survival (DFS), lymph node status (p < 0.001; RR = 3.8), cathepsin L (p < 0.001; RR = 2.6) and PAI-1 (p = 0.027; RR = 1.7) were significant factors in all patients. In addition to these factors, grading was significant for overall survival (OS). In another multivariate approach, CART (Classification And Regression Trees) analysis, lymph node status (p < 0.001) turned out to be the strongest discriminator for patients at high risk of relapse. In the node-negative patient subset, PAI-1 was the strongest risk group discriminator (p < 0.001): in this subset, patients with low levels of both PAI-1 and cathepsin D had a very low relapse rate of only 3.2% compared to 39% in the remaining node-negative patients. In node-positive patients cathepsin L gave the best risk group assessment (p = 0.001). In conclusion, tumor-associated PAI-1 and cathepsins D and L provide significant, statistically independent prognostic information for DFS and OS in primary breast cancer, even after a median follow-up period of almost 10 years.

Elevation of plasma fibrinogen in silent myocardial ischaemia.

High plasma levels of fibrinogen and plasminogen activator inhibitor (PAI-1) are reported to be correlated with coronary heart disease. Therefore the level of fibrinogen concentration in plasma was examined and verified for the possible correlation with the previously explored PAI-1 antigen and PAI-1 activity in the pathogenesis of premature atherosclerosis (Grzywacz et al., 1998, Blood Coagul Fibrinol. 9, 245-249). Examination included only men, aged 33-46 years, who were in a stable condition for at least six months after the acute event. They were divided into two subgroups: group A (n = 14) with and group B (n = 15) without ischaemic changes in 24 h Holter electrocardiogram. The number of involved vessels visible on the coronarography picture was similar in both groups. In the patients of group A the mean level of fibrinogen (3.92 vs 3.23 g/l, P < 0.05) was higher than in the controls (n = 15). No statistically differences were found between group B and control healthy subjects in any of the parameters measured. There were no correlation between fibrinogen concentration and PAI-1 antigen and activity levels, which were elevated in both groups of patients according to our previous study. Our results indicate that elevated levels of plasma fibrinogen and PAI-1 appeared in the group of patients with more severe disease, as revealed by silent myocardial ischaemia.

Larger and more invasive colorectal carcinoma contains larger amounts of plasminogen activator inhibitor type 1 and its relative ratio over urokinase receptor correlates well with tumor size

Considering recent findings that both urokinase plasminogen activator receptor (uPAR) and plasminogen activator inhibitors (PAIs) are involved in tumor growth through an urokinase-type plasminogen activator (uPA) activity-independent mechanism, the relation between the presence of these factors in tumor tissue and the clinicopathologic variables in colorectal carcinoma was reevaluated. In 100 colorectal carcinoma patients, antigen levels of u-PA, uPAR, and PAI-1 and PAI-2 were assayed in both tumor tissues and their normal counterparts. Plasma levels of soluble uPAR also were determined. All uPAR, uPA, PAI-1, and PAI-2 antigen levels in tumor tissue were significantly higher than those in normal tissue. Levels of both uPAR and PAI-1 were significantly higher (3.09 +/- 1.37 and 6.63 +/- 7.49, respectively) in large tumors (>/=50 mm in greatest dimension) than those in smaller tumors (< 50 mm) (2.50 +/- 1.07 and 2.72 +/- 2.70, respectively) (P < 0.05). Significant positive correlation coefficients (r) were obtained between tumor size and the calculated ratios of PAI-1/uPAR (r = 0.490; P < 0.0001) and PAI-1/uPA (r = 0. 469; P < 0.0001). In addition to liver metastases (P = 0.004) and lymph node involvement (P = 0.04), high levels of uPAR (P = 0.05) also were found to be of independent prognostic value by multivariate analysis. Higher expression of uPAR was related to poor prognosis of patients with colorectal carcinoma and excess amounts of PAI-1 over uPAR or uPAR-bound uPA appeared to play an important role in tumor progression.

Plasma urokinase antigen and plasminogen activator inhibitor-1 antigen levels predict angiographic coronary restenosis.

The fibrinolytic system is intimately involved in several processes that contribute to restenosis, including clot dissolution, cell migration, and tissue remodeling. However, the role of the individual activators (urokinase [uPA] and tissue plasminogen [tPA] activators) and inhibitors (plasminogen activator inhibitor [PAI-1]) of the fibrinolytic system in maintaining patency after coronary artery angioplasty and stenting is unclear. We prospectively studied 159 patients with stable angina who underwent successful elective angioplasty (n=110) or stenting (n=49) of de novo native coronary artery lesions. Plasma samples were drawn at baseline (before angioplasty) and serially after angioplasty (immediately afterward and 6 hours, 24 hours, 3 days, 7 days, 1 month, 3 months, and 6 months afterward). Antigen and activity assays were performed for uPA, tPA, and PAI-1. Follow-up quantitative coronary angiography was performed in 92% of eligible patients. The overall angiographic restenosis rate (diameter stenosis >50%) was 31% (37% in PTCA patients, 17% in stented patients). At all time periods, including baseline, uPA antigen levels were significantly higher and PAI-1 antigen levels were significantly lower in patients with restenosis. Restenosis rates for patients in the upper tertile of baseline uPA antigen levels were 2-fold higher than for those in the lower 2 tertiles (46% versus 24% and 22%, respectively; P<0.004). In a stepwise regression multivariate analysis, obstruction diameter after the procedure and uPA antigen were significant predictors of follow-up diameter stenosis. Plasma uPA antigen levels and PAI-1 antigen levels identify patients at increased risk for restenosis after percutaneous coronary revascularization.

IL-1beta mediates induction of hepatic type 1 plasminogen activator inhibitor in response to local tissue injury.

Type 1 plasminogen activator inhibitor (PAI-1), a major physiological inhibitor of plasminogen activation, is an important component of the hepatic acute phase response. We studied the acute phase regulation of murine hepatic PAI-1 in response to systemic toxicity and local tissue injury in both wild-type mice and in mice in which the interleukin (IL)-1beta gene had been inactivated by gene targeting. Endotoxin induced plasma PAI-1 antigen levels and PAI-1 mRNA accumulation in liver to the same extent in both wild-type and IL-1beta-deficient mice. In contrast, turpentine increased plasma PAI-1 and hepatic PAI-1 mRNA accumulation in wild-type mice but not in IL-1beta-deficient mice. Intraperitoneal injection of murine IL-1beta rapidly increased plasma PAI-1 and hepatic PAI-1 mRNA in both wild-type and IL-1beta-deficient mice. These results suggest that IL-1beta is a critical inducer of hepatic PAI-1 gene expression during the acute phase response to local tissue injury. In situ hybridization studies revealed that hepatocytes are the cells primarily responsible for the hepatic expression of the PAI-1 gene induced by lipopolysaccharide and turpentine.

Relation between long-term steroid treatment after heart transplantation, hypofibrinolysis and myocardial microthrombi generation

Background Thrombotic complications and transplant coronary artery disease are among the main causes of morbidity and mortality after heart transplantation. A thrombophilic state has been described in transplant recipients, and correlated to immunosuppressive therapy with Cyclosporine A or Azathioprine, whereas the prothrombotic effects of steroids, even though always given, have never been duly considered. A reduced fibrinolytic capacity due to high levels of PAI-1, the most important inhibitor of plasminogen activators, was suggested to play a role in the development of cardiovascular diseases and transplant coronary artery disease. A severe hypofibrinolytic state secondary to PAI-1 increase has been found in patients with Cushing’s disease, and in hypercorticism secondary to long-term steroid treatment after renal transplantation. Methods We evaluated plasma clotting and fibrinolytic behaviors in 2 groups of heart transplant patients treated with (26 cases) or without (23 cases) steroids together with Cyclosporine A and Azathioprine. Twenty-five healthy subjects were studied as normal controls. The following tests were assayed at least 1 year after transplantation: fibrinogen, factor VIII coagulant activity, von Willebrand factor antigen, Euglobulin Lysis Time, tissue plasminogen activator antigen and activity, PAI-1 antigen and activity. In addition, the presence of cardiac microthrombi was evaluated on 2 endomyocardial biopsy specimens obtained in each patient both on Day 7 after heart transplantation (first control) and usually 1 year or more later (last control). Results Plasma levels of fibrinogen, factor VIII and von Willebrand factor were significantly higher in both groups of patients than in normal controls. Fibrinolytic activity was significantly reduced in transplant patients treated with steroids, compared with steroid-free patients and normal controls. In steroid-treated heart transplant recipients, the hypofibrinolytic state was due to a significant and pathological increase in PAI-1 antigen and activity levels. The fibrinolytic impairment was more evident in patients transplanted for ischemic heart disease and treated with steroids than in patients with previous dilated cardiomyopathy and treated either with or without steroids. Myocardial microthrombi were found in 2/49 cases at the first biopsy control, and in 12/49 cases at the last biopsy control after transplantation. This different prevalence was statistically significant (χ 2 = 8.33, p = .003). Plasma PAI-1 activity was significantly higher and, as a consequence, Euglobulin Lysis Time was more prolonged in microthrombi-positive patients than in microthrombi-negative ones. Among the 12 transplant recipients who developed cardiac microthrombi, 7 patients were treated with steroids and showed higher PAI-1 levels and more reduced fibrinolytic activity than the 5 steroid-free patients. Conclusions Our data confirm the prothrombotic state induced by long-term steroid treatment, characterized by an increase in PAI-1 levels and secondary impairment of fibrinolytic capacity. In heart transplant patients, steroid-related hypofibrinolysis might constitute a further risk factor for transplant coronary artery disease.

Hemostatic variables as independent predictors for fetal growth retardation in preeclampsia

Preeclampsia is a major contributor to perinatal disease and fetal growth retardation (FGR). It has been suggested that increased intravascular coagulation, fibrin deposition in spiral arteries and hypoperfusion of the placenta are involved in these pregnancy complications. Multiple variables of the hemostatic system and lipid metabolism, as well as clinical features, were entered into univariate and multivariate models in order to examine the association with preeclampsia and FGR. Two hundred women with preeclampsia and 97 normotensive pregnant women were examined. Plasma levels of the thrombin-antithrombin complex (TAT), tissue factor pathway inhibitor free antigen (TFPI-Fag), protein S free antigen, plasminogen activator inhibitor type-1 (PAI-1) activity and serum levels of triglycerides were significantly increased, whereas plasma levels of antithrombin (AT), fibrinogen, C4b-binding protein (C4b-BP), PAI-2 antigen and serum HDL-cholesterol levels were decreased in the presence of preeclampsia. In the multivariate regression analysis, high TFPI-Fag plasma levels were associated with the presence of preeclampsia. The presence of FGR was in the univariate analysis associated with decreased PAI-1 activity and lower concentrations of fibrin, fibrinogen, factor VII antigen and PAI-2 antigen, as well as with evidence of macroscopic placental infarction. In a multivariate regression model, low maternal weight, placental infarction and low PAI-2 levels were predictors for low birth weight. In a logistic regression model, with the presence or absence of FGR as the dependent variable, male sex of the infant, placental infarction, low PAI-1 activity and factor VII antigen or PAI-2 antigen levels were independent predictors. Our results are consistent with activated coagulation in the placental vessels in preeclampsia. A low concentration of PAI-2 antigen in plasma emerged as the most consistent risk factor for preeclampsia and FGR.

Evaluation of a bioimmuno assay for t-PA activity and its relation to PAI-1 activity and antigen levels

A chromogenic bioimmunoassay method for determination of t-PA activities has been evaluated on plasmas from healthy individuals and from thromboembolic patients. The assay consists of an initial binding and washing step, whereby plasma t-PA is bound to a specific monoclonal t-PA antibody, which is coated on to microplate wells, followed by a chromogenic plasmin generation step, using the chromogenic plasmin substrate S-2403. Through collection of blood at pH 6 and through maintaining this pH during the binding step, in vitro inhibition of t-PA by PAI-1 is prevented and the resulting t-PA activity reflects the condition in plasma at the time of blood sampling. Through parallel incubation of plasma at pH 8, an estimation of the PAI-1 activity is obtained from the ratio of t-PA activities at pH 6 and 8. A median t-PA activity of 0.46 IU/mL (range 0.05-1.3, n = 69) was obtained for healthy individuals, whereas analysis of plasma from thromboembolic patients resulted in a median value of 0.31 IU/mL (range 0.04-1.5, n = 107). These results were in striking contrast to those obtained from 40 individuals from each group with parallel blood sampling in neutral citrate and incubation at pH 8, where the corresponding activities were 0.03 IU/mL (range 0-0.5) and 0.014 IU/mL (range 0-0.13) respectively, illustrating the severe losses of t- PA activity obtained in vitro unless precautions to avoid inhibition by PAI- 1 are undertaken. Furthermore, the results Showed an inverse correlation of the t-PA activity with PAI-1 activity and antigen levels (r = 0.74-0.91). Elevated concentrations of t-PA antigen in combination with elevated levels of PAI-1 consistently resulted in low t-PA activities. PAI-1 antigen concentrations above 70 ng/mL were always connected with t-PA activities below 0.2 IU/mL and with BIA t-PA ratios below 0.4. Prevention of t-PA inhibition by PAI-1 at pH 8 through addition of the monoclonal PAI-1 antibody MAI-12 resulted in equal t-PA activities as obtained from incubation at pH 6. Altogether, the results suggest that the BIA t-PA assay performed on plasma samples collected in acid medium provides a convenient tool for determination of basal steady state t-PA activities.

Tissue-Type Plasminogen Activator (tPA) and Its Inhibitor (PAI-1) in Patients Treated with Continuous Ambulatory Peritoneal Dialysis

Aim: To establish whether the values of two key enzymes of fibrinolysis, tissue-type plasminogen activator (tPA) and its inhibitor (PAI-1), differ between patients treated with continuous ambulatory peritoneal dialysis (CAPD) and healthy volunteers and whether plasma and dialysate tPA and PAI-1 values vary during one exchange of dialysis solution. Methods: A total of 11 patients with chronic renal failure, treated with CAPD during the peritoneal equilibration test (in addition with blood sampling at time 0), and a control group of 11 healthy volunteers were examined. To identify the factors involved in the changes in tPA and PAI-1, 9 CAPD patients were subsequently monitored, in a crossover manner, during dialysis with solutions of 1.36 and 3.86% dextrose and off dialysis. Results: Compared with healthy individuals, CAPD-treated patients showed a significantly lower tPA activity (0.39 vs. 0.81 IU/ml, p < 0.05). Changes in plasma fibrinolysis during one exchange of dialysis solution were characterized mainly by a decrease in PAI-1 concentrations and activities caused by the circadian rhythm of fibrinolysis. To explain, in the crossover part of the study, the values of plasma PAI-1 antigen at time 0 (07.00 h) and at time 2 (09.00 h) were 9.4 versus 6.5 ng/ml with the 1.36% solution (p < 0.05), 8.2 versus 4.9 with the 3.86% solution (p < 0.05), and 14.1 versus 9.1 ng/ml off dialysis (p < 0.01). Compared to baseline (0 ng/ml with 1.36 as well as 3.86% solutions), the levels of PAI-1 antigen in dialysis solution rose, apparently due to local production in the peritoneal cavity, to 0.5 ng/ml (p < 0.05) with the 1.36% solution, to 0.7 (p < 0.05) with the 3.86% solution after a 2-hour dwell time, and to 1.6 (p < 0.05) and 1.3 ng/ml (p < 0.05) after a 4-hour dwell time, respectively. Hence, the different dextrose levels in the dialysis solutions had no effect on the monitored parameters of fibrinolysis. Conclusion: The lower activity of plasma tPA, and the increase in PAI-1 levels in dialysis solutions may contribute to the development of thromboses in CAPD patients and to fibrin formation on the peritoneal surface with consequences such as peritoneal fibrosis.