Studies on the tissue activator of plasminogen. Distribution of activator and proteolytic activity in the subcellular fractions of rabbit kidney

1. On subcellular fractionation of rabbit kidney by differential and density-gradient centrifugation, a high proportion of the tissue activator of plasminogen activity was found to be particulate and displayed sedimentation properties associated with the lysosome-rich fraction as judged biochemically by the acid-phosphatase activity. 2. The activator activity is closely associated with a latent protease whose activity is enhanced in the presence of Triton X-100 or sodium deoxycholate in the neutral pH range. Besides hydrolysing casein this protease is also capable of attacking fibrinogen at pH7·4. 3. The pH optimum for activator activity and its inhibition by -hexanoic acid (-aminocaproic acid) point to its possible similarity to urokinase, an activator of plasminogen present in the urine of most mammals.     

Biosynthesis of sulfated glycoprotein-N-glycans present in recombinant human tissue plasminogen activator.

Recombinant human tissue plasminogen activator expressed in murine epithelial cells carries, in part, sulfated N-glycans, which are characterized by the presence of a NeuAc alpha 3[SO4-6]Gal unit. In order to study the biosynthesis of this novel structural element, corresponding sulfated asialooligosaccharide alditols were resialylated in vitro using a crude sialyltransferase preparation from murine liver which was shown to contain Gal beta 1,3(4)GlcNAc alpha 2,3-sialyltransferase activity. Products were analyzed for transfer of sialic acid residues by anion-exchange HPLC. The results demonstrated that resialylation of SO4-6Gal-residues did not occur. Therefore, it may be concluded that transfer of the sulfate group is the final step in the biosynthesis of this structural epitope.     

N-glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line

To probe the effects of N-glycosylation on the fibrin-dependent plasminogenolytic activity of tissue-type plasminogen activator (t-PA), we have expressed a human recombinant t-PA (rt-PA) gene in Chinese hamster ovary (CHO) cells and in a murine C127 cell line. The resulting rt-PA glycoproteins were isolated and their associated N-linked oligosaccharide structures determined by using a combination of high-resolution Bio-Gel P-4 gel filtration chromatography, sequential exoglycosidase digestion, and methylation analysis. The results show that CHO rt-PA is N-glycosylated differently from murine C127 derived rt-PA. Further, both rt-PA's are N-glycosylated differently from t-PA derived from a human colon fibroblast and the Bowes melanoma cell line (Parekh et al., 1989), confirming that N-glycosylation of the human t-PA polypeptide is cell-type-specific. Both CHO and murine rt-PA were fractionated on lysine-Sepharose chromatography. The N-glycosylation of the major forms was analyzed and their fibrin-dependent plasminogenolytic activity determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. The results suggest that the various forms of rt-PA differ from one another with respect to the kinetics of their fibrin-dependent activation of plasminogen. Together, these data support the notion (Wittwer et al., 1989) that N-glycosylation influences the fibrin-dependent catalytic activity of t-PA and that t-PA when expressed in different cell lines may consist of kinetically and structurally distinct glycoforms.     

Normal tissue plasminogen activator and plasminogen activator inhibitor activity in plasma from patients with type 1 diabetes mellitus.

The fibrinolytic system was investigated in 38 patients (21 males and 17 females) affected by type 1 diabetes mellitus (18 free from complications, 10 with retinopathy, and 10 with autonomic neuropathy) and in 8 healthy controls. Two separate fibrinolysis-stimulating tests were done: standardized venous occlusion and 1-desamino-8-D-arginine vasopressin infusion. Plasma tissue plasminogen activator antigen and activity and plasma plasminogen activator inhibitor activity were measured. All the patients were in good metabolic control (mean HbA1c 7.4%, range 6.1-8.0%). No significant differences were observed either between the diabetic patients and the control subjects, nor among the subgroups of diabetic patients. The fibrinolytic system is probably not involved in type 1 diabetes mellitus.     

Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator

Plasminogen activator inhibitor 1 (PAI-1) was purified from medium conditioned by cultured bovine aortic endothelial cells by successive chromatography on concanavalin A Sepharose, Sephacryl S-200, Blue B agarose, and Bio-Gel P-60. As shown previously for conditioned media (C. M. Hekman and D. J. Loskutoff (1985) J. Biol. Chem. 260, 11581-11587) the purified PAI-1 preparation contained latent inhibitory activity which could be stimulated 9.4-fold by sodium dodecyl sulfate and 45-fold by guanidine-HCl. The specific activity of the preparation following treatment with 0.1% sodium dodecyl sulfate was 2.5 X 10(3) IU/mg. The reaction between purified, guanidine-activated PAI-1 and both urokinase and tissue plasminogen activator (tPA) was studied. The second-order rate constants (pH 7.2, 35 degrees C) for the interaction between guanidine-activated PAI-1 and urokinase (UK), and one- and two-chain tPA are 1.6 X 10(8), 4.0 X 10(7), and 1.5 X 10(8) M-1 S-1, respectively. The presence of CNBr fibrinogen fragments had no affect on the rate constants of either one- or two-chain tPA. Steady-state kinetic analysis of the effect of PAI-1 on the rate of plasminogen activation revealed that the initial UK/PAI-1 interaction can be competed with plasminogen suggesting that the UK/PAI-1 interaction may involve a competitive type of inhibition. In contrast, the initial tPA/PAI-1 interaction can be competed only partially with plasminogen, suggesting that the tPA/PAI-1 interaction may involve a mixed type of inhibition. The results indicate that PAI-1 interacts more rapidly with UK and tPA than any PAI reported to date and suggest that PAI-1 is the primary physiological inhibitor of single-chain tPA. Moreover, the interaction of PAI-1 with tPA differs from its interaction with UK, and may involve two sites on the tPA molecule.     

Biphasic regulation of tissue plasminogen activator activity in ischemic rat brain and in cultured neural cells: essential role of astrocyte-derived plasminogen activator inhibitor-1

In brain, the serine protease tissue plasminogen activator (tPA) and its endogenous inhibitor plasminogen activator inhibitor-1 (PAI-1) have been implicated in the regulation of various neurophysiological and pathological responses. In this study, we investigated the differential role of neurons and astrocytes in the regulation of tPA/PAI-1 activity in ischemic brain. The activity of tPA peaked transiently and then decreased in cortex and striatum along with delayed induction of PAI-1 in the inflammatory stage after MCAO/reperfusion injury. In cultured primary cells, glutamate stimulation increased tPA activity in neurons but not in other cells such as microglia and astrocytes. With LPS stimulation, a model of neuroinflammatory insults, robust PAI-1 induction was observed in astrocytes but not in neurons and microglia. The upregulation of PAI-1 by LPS in astrocytes was also verified by RT-PCR analysis as well as PAI-1 promoter reporter assay. Lastly, we checked the effects of hypoxia on tPA/PAI-1 activity. Hypoxia increased tPA release from neurons without effects on microglia, while the activity of tPA in astrocyte was decreased consistent with increased PAI-1 activity in astrocyte. Taken together, the results from the present study suggest that neurons are the major source of tPA and that the glutamate-induced stimulated release is mainly governed by neurons in the acute phase. In contrast, the massive up-regulation of PAI-1 in astrocytes during subchronic and chronic inflammatory conditions, leads to decreased tPA activity in the later stages of MCAO. Differential regulation of tPA and PAI-1 in neurons, astrocytes and microglia suggest more attention is required to understand the role of local tPA activity in the vicinity of individual cell types.     

Metformin, but not thiazolidinediones, inhibits plasminogen activator inhibitor-1 production in human adipose tissue in vitro.

Biguanides and thiazolidinediones (TZDs), which are primarily used as anti-diabetic drugs, are also associated with other beneficial effects on cardiovascular risk factors such as reduced plasma plasminogen activator inhibitor-1 (PAI-1) concentration in both diabetic and non-diabetic obese subjects. Since human adipose tissue is of importance for the production of PAI-1, the aim of the present study was to investigate the possible direct effects of these anti-diabetic agents on PAI-1 mRNA and secretion by human adipose tissue. Adipose tissue was obtained from biopsies taken from the subcutaneous abdominal depot. Adipose tissue fragments, isolated mature adipocytes, and preadipocytes were incubated in vitro with metformin and various TZDs. Metformin (0.1 - 10 mM) dose-dependently decreased PAI-1 production (and PAI-1 mRNA) under both basal (43 % inhibition at 10 mM, p < 0.05) and interleukin-1beta (IL-1beta)-stimulated conditions where the levels were inhibited by 47.8 % at 1 mM metformin (p < 0.05) and by 100 % at 10 mM (p < 0.01). None of the TZDs tested (PPAR-gamma agonists: troglitazone, pioglitazone, or ciglitazone) had any effects on PAI-1 production. Moreover, no effects on PAI-1 production were observed using various PPAR-alpha agonists such as 5, 8, 11, 14-eicosatetraynoic acid (ETYA), Wy14643 and fenofibrate. Our findings indicate no direct effects of TZDs on PAI-1 secretion, whereas metformin was able to directly inhibit PAI-1 production in human adipose tissue.     

In vivo and in vitro interaction of high and low molecular weight single-chain urokinase-type plasminogen activator with rat liver cells.

The plasma clearance and the interaction of high (HMW) and low (LMW) molecular weight single-chain urokinase-type plasminogen activator (scu-PA) with rat liver cells was determined. 125I-Labeled HMW- and LMW-scu-PA were rapidly cleared from plasma with a half-life of 0.45 min and a maximal liver uptake of 55% of the injected dose. Liver uptake of scu-PA was mediated by parenchymal cells. Excess of unlabeled HMW-scu-PA reduced the liver uptake of 125I-HMW-scu-PA strongly. In vivo liver degradation of scu-PA was reduced by inhibitors of the lysosomal pathway. A high affinity binding site (Kd 45 nM, Bmax 1.7 pmol/mg cell protein) for both HMW- and LMW-scu-PA was determined on isolated parenchymal liver cells. Cross-competition binding studies showed that LMW- and HMW-scu-PA bind to the same site. Tissue-type plasminogen activator, mannose- or galactose-terminated glycoproteins did not affect the scu-PA binding to parenchymal liver cells. It is concluded that LMW- and HMW-scu-PA are taken up in the liver by a common, newly identified recognition site on parenchymal liver cells and are subsequently degraded in the lysosomes. It is suggested that this site is important for the regulation of the turnover of scu-PA.     

Kinetic analysis of the interaction between plasminogen activator inhibitor-1 and tissue-type plasminogen activator.

The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-1 (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrin-bound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = 10(7) M-1.s-1) even in the presence of 2 microM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 +/- 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-1 a Ki value of 1.3 +/- 0.3 nM was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-1 and t-PA (Ki), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.     

Effects of N-glycosylation on in vitro activity of Bowes melanoma and human colon fibroblast derived tissue plasminogen activator

Tissue-type plasminogen activator (t-PA), when isolated from human colon fibroblast (hcf) cells, is N-glycosylated differently than when isolated from the Bowes melanoma (m) cell line (Parekh et al., 1988). Both hcf- and m-t-PA can be separated into type I t-PA (with three occupied N-glycosylation sequons, at Asn-117, -184, and -448) and type II t-PA (with two occupied sequons, at Asn-117 and -448). Oligosaccharide analysis of each of these types of t-PA indicates that hcf-t-PA and m-t-PA have no glycoforms in common, despite having the same primary amino acid sequence. We have therefore compared in vitro the enzymatic activities and fibrin binding of type I and type II hcf- and m-t-PA with those of aglycosyl t-PA isolated from tunicamycin-treated cells. Plasminogen activation kinetics were determined by using an indirect amidolytic assay with Glu-plasminogen and a chromogenic plasmin substrate. In the absence of stimulator, there was little difference in activity between type I and type II t-PA, but the activity of aglycosyl t-PA was 2-4-fold higher than that of the corresponding glycosylated t-PA. In the presence of a fibrinogen fragment stimulator, the Kcat value of type II t-PA was approximately 5-fold that of type I t-PA from the same cell line, while the Km values for activation of Glu-plasminogen were similar (0.13-0.18 microM). The stimulated activity of glycosyl t-PA was similar to that of type II t-PA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous fibrinolysis in whole human plasma. Identification of tissue activator-related protein as the major plasminogen activator causing spontaneous activity in vitro

Spontaneous fibrinolysis of plasma clots was studied by following the lysis of the clots formed in 125I-fibrinogen-supplemented citrated plasma. Lysis of the clots invariably follows sigmoidal kinetics with S50 (the time required for 50% clot lysis) ranging from 3.5 to 4.7 days in 8 samples of pooled blood bank plasma and in the majority of apparently healthy donor plasmas. The spontaneous lysis of factor XII-deficient and prekallikrein-deficient plasmas was found to be similar to that of normal plasma. Addition of ellagic acid or antibodies against kallikrein or urokinase to normal pooled plasma did not alter significantly its rate of spontaneous lysis. On the other hand the addition of antibody against tissue activator (t-PA) inhibited over 80% of the spontaneous fibrinolysis in a 7-day incubation period at 37 degrees C, and the clot visually persisted for more than a month. Therefore, the factor XII-dependent components and prourokinase/urokinase system do not contribute significantly in whole plasma fibrinolysis in vitro, while the t-PA-related protein appears to be the major plasminogen activator responsible for initiating spontaneous fibrinolysis in whole plasma. Exogenous addition of increasing amounts of purified HeLa cell t-PA to normal pooled plasma in the ng/ml range cause progressively faster clot lysis. By extrapolation, normal pooled plasma is found to contain endogenous tissue activator in an amount functionally equivalent to 2 ng/ml of purified 60-kDa t-PA. The molecular nature of the t-PA-related proteins in plasma was studied by zymographic and immunological methods. The major t-PA-related protein in plasma was found to have a molecular mass of 100 kDa as determined by zymography. By incubating purified HeLa 60-kDa t-PA with a t-PA-depleted plasma, the 100-kDa component can be generated in plasma, suggesting that the latter is formed as a result of the binding of 60-kDa t-PA to a binding protein in plasma.     

A novel sialylated N-acetylgalactosamine-containing oligosaccharide is the major complex-type structure present in Bowes melanoma tissue plasminogen activator.

We have employed fast atom bombardment mass spectrometry (FAB-MS) to screen the N-linked oligosaccharides of Bowes melanoma tissue plasminogen activator (mt-PA), and recombinant t-PAs produced by Chinese hamster ovary cells (rt-PA) and by a gene-enriched melanoma cell line (rmt-PA). These studies have confirmed the published structures for rt-PA, but are not in agreement with some of the structures reported for mt-PA. In the latter glycoprotein we have identified a novel structure as the major oligosaccharide attached to Asn-184 and Asn-448. This is a biantennary oligosaccharide consisting of a fucosylated trimannosyl core to which are attached two GalNAc(1----4)GlcNAc antennae, one of which carries a sialic acid linked at the 6-position of the GalNAc. Minor constituents are sialylated on both or neither antennae. The sialylated GalNAc moiety is unique in N-linked glycoproteins. The majority of complex structures in rmt-PA contain N-acetyllactosamine moieties at both the Asn-184 and Asn-448 sites with the novel oligosaccharide occurring as a minor component at the Asn-184 site. This study demonstrates the power of mass spectrometric strategies based on high-field two-sector FAB-MS for structure elucidations of natural and recombinant glycoproteins.     

Kinetic analysis of the effects of heparin and lipoproteins on tissue plasminogen activator mediated plasminogen activation

Heparin sulfate and the less sulfated glycosaminoglycan heparan sulfate enhance human plasminogen (Pg) conversion to plasmin by tissue-type plasminogen activator (t-PA). Kinetic studies indicate that both heparin and heparan increase the kcat of t-PA-mediated Pg activation by 25- and 3.5-fold, respectively. The Km of plasmin formation is unaltered by the presence of either heparin or heparan. Both heparin and heparan stimulate the activity of t-PA by interacting with the finger domain of t-PA, with association constants of 1 microM and 200 nM, respectively. Additionally, the lipoproteins lipoprotein(a) [Lp(a)] and low-density lipoprotein (LDL) inhibit the heparin enhancement of Pg activation. Lp(a) is a competitive inhibitor and LDL is a mixed inhibitor of t-PA-mediated Pg activation, with inhibition constants of 30 and 70 nM, respectively. The inhibition constants correspond to physiologic concentrations of these lipoproteins. These data suggest that heparin, heparan, and lipoproteins may play an important in vivo role in regulating cell surface associated activation of the fibrinolytic system.     

Signal and propeptide processing of human tissue plasminogen activator: activity of a pro-tPA derivative

We have explored the heterogeneity in the proteolytic processing of the N-terminus of human tissue plasminogen activator. We demonstrate that normal propeptide processing occurs following Arg-4, preceding the sequence Gly-Ala-Arg-Ser+1. Generation of the previously designated Ser+1 occurs via secondary proteolysis following secretion. By site-directed mutagenesis, we have eliminated this cleavage site resulting in a derivative containing the propeptide sequence. N-terminal sequence analysis of this form indicated that signal peptide cleavage occurs following Ser-13. The pro-tPA derivative had near normal serine protease and plasminogen activating activities, and could be stimulated by fibrin. An additional derivative, containing the tribasic sequence from the human protein C propeptide preceding Ser+1, was secreted with full processing of the propeptide. Our data have defined the cleavages for the signal peptide and propeptide and demonstrate that a tribasic sequence can be used to eliminate N-terminal heterogeneity in this molecule. In addition, we demonstrate that, unlike several other serine proteases, a propeptide sequence does not alter the activity of this enzyme.     

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.     

Synergistic fibrinolysis: The combined effects of tissue plasminogen activator and recombinant staphylokinase in vitro

BackgroundMechanisms of fibrin-specificity of tissue plasminogen activator (tPA) and recombinant staphylokinase (STA) are different, therefore we studied in vitro the possibility of the synergy of their combined thrombolytic action.MethodsThrombolytic effects of tPA, STA and their combinations were measured by lysis rate of human plasma clot and side effects were evaluated by decreasing in fibrinogen, plasminogen and α₂-antiplasmin levels in the surrounding plasma at 37 °C in vitro.ResultsSTA and tPA induced dose- and time-dependent clot lysis: 50% lysis in 2 h was obtained with 30 nM tPA and 75 nM STA, respectively. At these concentrations, tPA produced greater degradation of plasma fibrinogen than STA. According to a mathematical analysis of dose–response curves by the isobole method, combinations of tPA and STA caused a considerable synergistic thrombolytic effect. The simultaneous and sequential combinations of tPA (< 4 nM) and STA (< 35 nM) induced a significant fibrin-specific synergistic thrombolysis, which was more pronounced in 2 h at simultaneous combinations than at sequential addition of STA after 30 min of tPA action. Simultaneous combination of 2.5 nM tPA and 15 nM STA showed a maximal 3-fold increase in thrombolytic effect compared to the expected total effect of the individual agents. Sequential combinations caused a lower depletion of plasma proteins compared to simultaneous combinations.ConclusionsThe simultaneous and sequential combinations of tPA and STA possessed synergistic fibrin-specific thrombolytic action on clot lysis in vitro.General significanceThe results show that combined thrombolysis may be more effective and safer than thrombolysis with each activator alone.