Cultured human mesangial cells produce both type 1 and type 2 plasminogen activator inhibitors.

Cultured human mesangial cells (HMC) derived from normal kidneys have been shown to synthesize tissue-type plasminogen activator (t-PA) and excess amounts of PA inhibitor type 1 (PAI-1). Conflicting results have been obtained concerning the production of urokinase-type PA (u-PA) and efforts to show PA inhibitor 2 (PAI-2) met with failure. We evaluated the fibrinolytic profile of cultured HMC lines obtained from 12 patients with renal carcinoma and one cadaveric kidney donor. Subconfluent cells (third passage) were incubated overnight in serum-free medium. t-PA, u-PA, PAI-1 and PAI-2 antigens were assayed by ELISA methods and PA and PAI activities by amidolytic methods both in conditioned medium (CM) and cell extracts (CE). Besides PAI-1, PAI-2 antigen was detected in all but one HMC lines. At variance with the former, which was largely released in the culture medium, PAI-2 was mainly cell-associated. t-PA antigen was found in all but two cell lines while u-PA antigen was detected in relatively high concentrations in 8 cell lines. PA activity, identified as u-PA by functional and immunological criteria, was measured in CM of six of the eight u-PA producing cell lines, whereas PAI activity was undetectable or very low in CM of all cell lines, suggesting that PAI-1 was largely inactive. Functional assays of cell extracts demonstrated the presence of PA activity, again identified as u-PA, only in samples (five lines) containing u-PA antigen in excess over PAI-2. PAI activity was found instead in the extracts in which the inhibitor was higher than the activator (six lines) and was identified as PAI-2, as it inhibited u-PA but not single-chain t-PA and was neutralized by a polyclonal anti-PAI-2 antibody. The heterogeneous fibrinolytic pattern of HMC lines was confirmed by mRNA analysis of three representative lines. Results were similar when HMC lines at passage five were used, except that the u-PA content was significantly reduced both in CM and CE. These findings indicate that the fibrinolytic profile of cultured HMC is more complex than previously reported. The production of large amounts of PAI-2 may represent an additional control mechanism of proteinase activity.

Enhanced endothelial tissue factor but normal thrombomodulin in endotoxin-treated rabbits.

Exposure of cultured endothelial cells to bacterial endotoxin induces an enhancement of cell procoagulant activity (PCA) and a simultaneous reduction of thrombomodulin activity (TM). We evaluated the effect of endotoxin on the expression of both endothelial PCA and TM in vivo, in rabbits. Animals were given a single i.v. injection of endotoxin (E. coli 0111:B4 LPS, W, 10-200 micrograms/kg); the thoracic aorta was harvested after 2 or 4 hours and placed in an ad hoc device to expose the endothelial surface only. Endotoxin treatment resulted in a dose-dependent increase of endothelial PCA (p < 0.001, at 100 micrograms/kg or more), which was totally dependent on factor VII and thus identified as tissue factor. In contrast, endothelial TM activity, as measured by the rate of thrombin-induced protein C activation, was similar in control and endotoxemic rabbits, even when the animals were given two injections (50 micrograms/kg, 24 h apart), or a continuous infusion (40 micrograms/kg/h during 4 hours) of endotoxin. To explore the effect of endotoxin on TM activity at the microcirculation level, we measured the extent of protein C activation in vivo, induced by a continuous infusion of low doses of thrombin (1 NIH U/kg/min for 60 min). Again, endotoxin administration was not associated with significant changes in TM-dependent protein C activation, as assessed by the anticoagulant activity present in a barium citrate plasma eluate obtained at the end of thrombin infusion. Although reduction of TM during persistent endotoxemia cannot be definitively excluded, our data support a major role of endothelial PCA in LPS-induced coagulative changes.

Properties of chimeric (tissue-type/urokinase-type) plasminogen activators obtained by fusion at the plasmin cleavage site.

Two hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 x 10(6) and 1.0 x 10(6) t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 microgram/ml for K2tu-PA and 1 microgram/ml for FK2tu-PA, as compared to 0.5 microgram/ml and > 2 micrograms/ml for t-PA and scu-PA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombin infusion in endotoxin-treated rabbits reduces the plasma levels of plasminogen activator inhibitor: evidence for a protein-C-mediated mechanism.

Plasminogen activator inhibitors (PAIs) play a pivotal role in the control of fibrinolysis. The mechanisms regulating the plasma levels of PAI(s) are still unknown. We report here that the infusion of bovine thrombin (1 U/kg/min, over 60 minutes) in rabbits treated with 0.5 microgram/kg endotoxin (to induce an increase in circulating fast-acting PAI) causes a marked reduction of PAI (50% of preinfusion value), as indicated by functional assay and reverse fibrin autography. Moreover, blood clots prepared from samples obtained after thrombin infusion lysed faster than preinfusion clots when exposed, in vitro, to tissue plasminogen activator. Donor-receiver transfusion experiments showed that the half-life of circulating PAI activity was shorter in thrombin-infused rabbits than in controls (4.1 minutes versus 7.4 minutes), suggesting an accelerated clearance. As expected, thrombin infusion resulted also in activation of protein C (PC). The following observations suggest a close relationship between PC activation and PAI reduction. (1) Infusion of thrombin in rabbits made deficient in vitamin K-dependent plasma proteins by warfarin treatment did not result in modification of PAI activity. (2) Treatment of the latter animals with a barium citrate eluate (PE) of rabbit plasma restored both the anticoagulant and profibrinolytic response to thrombin. (3) Short infusion of thrombin-activated PE (containing activated PC, PCa), but not of unactivated PE, induced both anticoagulation and reduction of PAI activity. In vitro, incubation of PAI-rich rabbit serum with thrombin-activated PE and phospholipids resulted in a progressive disappearance of PAI activity with a t1/2 of 30 minutes. However, this slow inactivation rate does not fully explain the results obtained in vivo. Our data suggest that thrombin infusion in rabbits causes a reduction of circulating PAI activity and that activation of PC is the intermediary mechanism involved in this phenomenon.

Impaired fibrinolysis in obstructive jaundice--evidence from clinical and experimental studies.

Microvascular thrombosis is considered an important pathogenetic factor in renal failure associated with obstructive jaundice but the mechanisms leading to fibrin deposition are still unknown. The plasma levels of plasminogen activator inhibitor (PAI) in 29 patients with obstructive jaundice were found significantly increased as compared to 20 nonjaundiced patients. Fibrin autography of plasma supplemented with tissue plasminogen activator (t-PA) revealed that in icteric samples most of the added activator migrated with an apparent Mr of 100 kDa, corresponding to t-PA-PAI complex, whereas in control samples virtually all t-PA migrated as free enzyme. PAI activity detected in icteric samples is similar to the endothelial type PAI since it is neutralized by a monoclonal antibody against PAI-1. Venous stasis in jaundiced patients was neither associated with an increase in blood fibrinolytic activity nor with a decrease in PAI activity. Immunologic assay showed that t-PA release was impaired in 3 out of 4 patients. In controls, venous occlusion induced an increase in both fibrinolytic activity and t-PA antigen and a reduction in PAI activity. Bile duct recanalization in jaundiced patients subjected to surgery was accompanied by a decrease in plasma PAI activity which paralleled the decrease in serum bilirubin levels. In nonjaundiced patients, surgical treatment did not cause significant changes in either parameter. Rabbits made icteric by bile duct ligation showed an early and progressive increase in plasma PAI activity indicating that obstructive jaundice itself causes the elevation of circulating PAI. it is concluded that obstructive jaundice is associated with a severe impairment of fibrinolysis which might contribute to microvascular thrombosis and renal failure.