Activation with plasmin of tow-chain urokinase-type plasminogen activator derived from single-chain urokinase-type plasminogen activator by treatment with thrombin

Thrombin converts single-chain urokinase-type plasminogen activator (scu-PA) to an inactive two-chain derivative (thrombin-derived tcu-PA) by hydrolysis of the Arg-156--Phe-157 peptide bond. In the present study, we show that inactive thrombin-derived tcu-PA (specific activity 1000 IU/mg) can be converted with plasmin to active two-chain urokinase-type plasminogen activator (specific activity 43,000 IU/mg) by hydrolysis of the Lys-158--Ile-159 peptide bond. This conversion follows Michaelis-Menten kinetics with a Michaelis constant Km of 37 microM and a catalytic rate constant k2 of 0.013 s-1. The catalytic efficiency (k2/Km) for the activation of thrombin-derived tcu-PA by plasmin is about 500-fold lower than that for the conversion of intact scu-PA to tcu-PA. tcu-PA, generated by plasmin treatment of thrombin-derived tcu-PA, has similar properties to tcu-PA obtained by digestion of intact scu-PA with plasmin (plasmin-derived tcu-PA); its plasminogen activating potential and fibrinolytic activity in an in vitro plasma clot lysis system appear to be unaltered. These observations confirm that the structure of the NH2-terminal region of the B chain of u-PA is an important determinant for its enzymatic activity, whereas that of the COOH-terminal region of the A chain is not.     

Tissue plasminogen activator and plasmin independently decrease human neutrophil activation

Tissue-plasminogen activator (t-PA) and plasmin both decrease platelet aggregation, which may contribute to thrombolysis and tissue salvage. Since neutrophils may contribute to reperfusion injury, we examined the effects of t-PA and plasmin on human neutrophil function. t-PA (1 to 100 micrograms/ml) decreased f-MLP-induced chemotaxis and ionophore A23187-induced superoxide and LTB4 release in isolated neutrophils, and these effects were not blocked by the plasmin-inhibitor epsilon-aminocaproic acid (epsilon-ACA). On the other hand, plasmin (0.05 to 0.5 units/ml) also decreased these neutrophil functions but its effects were blocked in the presence of epsilon-ACA. Thus, while both t-PA and plasmin decrease neutrophil functions, effects of t-PA are independent of plasmin generation. Cumulative effects of t-PA and plasmin on neutrophil functions may relate to the overall efficacy of t-PA in thrombotic disorders.     

Anticoagulant low molecular weight heparin does not enhance the activation of plasminogen by tissue plasminogen activator

The activity of tissue plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) is stimulated by heparin. Heparin binds tightly to t-PA, u-PA, and plasminogen and decreases the usual stimulatory effect of fibrin on t-PA activity. In the present study we have found that low molecular weight heparin (LMW-heparin) preparations obtained by nitrous acid depolymerization or heparinase treatment of standard heparin have different properties with respect to their interaction with the fibrinolytic system. LMW-heparin prepared by either method does not stimulate plasmin formation by t-PA. However, these preparations of heparin still efficiently accelerate the inhibition of thrombin by antithrombin III. Binding data show that LMW-heparin does not bind t-PA and Glu-plasminogen and only binds very weakly to Lys-plasminogen. These results illustrate that it is possible to selectively destroy the fibrinolytic stimulating properties of heparin while leaving the classical anticoagulant characteristics intact.     

A study of tissue plasminogen activator forms from human small intestine

At least two forms of plasminogen activators which crossreacted with antiserum against tissue plasminogen activator (tPA) have been found in human small intestine homogenates. One of these activities has very slow mobility on Sephadex G-200 and is presumably a degraded form of tPA. The other moved very fast and was dispersed on gel filtration matrices, and probably represents aggregates of tPA with some other materials. Whereas 1 M NaCl, 1% Triton X-100 or 1 M potassium thiocyanate was unable to break up these aggregates, the high molecular weight components co-migrating with tPA could be separated from tPA by 4 M guanidine-HCl.     

Construction and expression of hybrid plasminogen activators prepared from tissue-type plasminogen activator and urokinase-type plasminogen activator genes

Recent data from several studies have suggested that the non-protease domains in tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) determine their biological specificities, including binding to fibrin clots and survival in the circulatory system (Van Zonneveld, A.-J., Veerman, H., and Pannekoek, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 4670-4674; Rijken, D. C., and Emeis, J. J. (1986) Biochem. J. 238, 643-646). Structural manipulations (e.g. deletions, additions, or substitutions) in these domains can thus be utilized to maximize the desired biological effects. Using recombinant DNA technology, we constructed a number of hybrid molecules from the t-PA and u-PA genes. In hybrid A, the epidermal growth factor and finger domains of t-PA (residues 1-91) were replaced by the epidermal growth factor and kringle of u-PA (residues 1-131). In hybrids B and C, the u-PA kringle (residues 50-131) was inserted either before (residue 92) or after (residue 261) the double-kringle region of t-PA. All these hybrid PAs containing three kringles were expressed in mouse fibroblast cells (C-127). The hybrid proteins were synthesized in predominantly a single-chain form with molecular weights of 70,000-80,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were enzymatically active as assayed by the fibrin-agar plate method. In vitro studies on the binding of hybrid PAs to fibrin showed that hybrid B, like t-PA, possesses affinity toward fibrin, while hybrid A shows lower binding. This suggests that the finger domain, which is not present in hybrid A, plays a role in conferring fibrin affinity to the hybrid PAs. The enzymatic activities of the hybrids were compared with that of recombinant t-PA (rt-PA) expressed in the same vector/host system and found to be similar in activity toward a chromogenic peptide substrate. In addition, plasminogen activation with all the hybrid-PAs, as with rt-PA, was stimulated by fibrin, with the order of activity being rt-PA greater than or equal to hybrid B greater than hybrid C greater than hybrid A. This study shows the feasibility of shuffling functional domain(s) of known specificity in plasminogen activators which may lead to the design of a superior thrombolytic agent.     

A factor from endothelium facilitates activation of plasminogen by tissue plasminogen activator

A component extracted from endothelium and partially purified has been found to have a capacity to enhance the rate of plasminogen activation by tissue-type plasminogen activator. The mechanism of action of this cofactor differs from that of others, such as fibrin.     

Purification and partial characterization of plasminogen activator from human uterine tissue.

A procedure was developed for the purification of a plasminogen activator from human uterine tissue. It involves six consecutive steps: (1) extraction of the plasminogen activator from delipidated uterine tissue with 0.3 M potassium acetate buffer, pH 4.2; (2) ammonium sulphate precipitation; (3) zinc chelate-agarose chromatography; (4) n-butyl-agarose chromatography; (5) concanavalin A-agarose chromatography; and (6) gel filtration on Sephadex G-150. The specific activity of the final plasminogen activator preparation was increased by a factor 4500 as compared with the crude extract. The purified plasminogen activator showed a strong tendency to adsorb to surfaces. This could be effectively prevented by Tween-80. The molecular weight of the plasminogen activator was 64 000 as estimated by gel filtration in 1.0 M NaCl and 69 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The plasminogen activator consisted of two chains (molecular weights 31 000 and 38 000) connected by disulphide bridges. The smallest chain contained the serine residue of the active site as deduced from the incorporation of the tritium label of [3H]diisopropylphosphofluoridate.     

Stimulation and desensitization of tissue plasminogen activator release from human endothelial cells

Tumor-promoting phorbol esters and histamine induce tissue plasminogen activator (tPA) release from human endothelial cells in a dose- and time-dependent manner. Phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu) increased tPA concentration in the culture medium by eight to 12 times after 24 h with half-maximal stimulation at 13 and 55 nM, respectively. Maximum release by histamine was only half that of the phorbol esters and required 18 microM for half-maximal response. Kinetics of enhanced release was similar with both types of agonists: a 4-h lag period followed by a period of rapid release (4 h in PMA-treated and 10 h in histamine-treated cultures) followed by a decline toward pretreatment rates. The PMA and histamine effects were additive while histamine and thrombin, which also stimulates tPA release in human endothelial cells, were no more effective together than they were alone. Exposure of the cells to PMA, PDBu, or phorbol 12,13-didecanoate caused a loss of responsiveness to second treatment of the homologous agent that was time- and dose-dependent, sustained, and specific to active tumor promoters (half-maximal desensitization = 52 nM PDBu). A partial desensitized state was also established by histamine which resulted in a 60% lower response to a second challenge dose. Histamine-induced desensitization did not interfere with the PMA response. However, PMA-induced desensitization caused a 75% loss of the histamine and a 67% loss of the thrombin effects. These studies indicate that tumor promoters are potent agonists of tPA release from human endothelial cells and establish a desensitized state to further stimulation. Treatment of these cells with histamine has similar effects which may be mediated at least in part by pathways common to phorbol ester stimulation.     

Synthesis of recombinant human single-chain urokinase-type plasminogen activator variants resistant to plasmin and thrombin

Single-chain urokinase-type plasminogen activator (scu-PA), a potential therapeutic reagent for thrombosis, is activated in plasma by plasmin. The activated enzyme is further digested by plasmin to generate low-molecular-weight urokinase (LMW-UK), which has no affinity for fibrin. To circumvent this dual effect of plasmin, we synthesized in Escherichia coli a variant of scu-PA, which is not converted to LMW-UK on treatment with plasmin. In another variant, the activation cleavage site was modified such that activation by plasmin was slowed down and that inactivation by thrombin was greatly diminished. The combination of these variants may be applicable as an effective thrombolytic reagent for clinical use.     

A functionally active heavy chain derived from human high molecular weight urokinase

Human high molecular weight urokinase, a plasminogen activator, when minimally reduced with 0.01 M 2-mercaptoethanol for 10 h at pH 8.0 and 25 degrees C and then carboxymethylated with sodium iodoacetate, gave two chains, a functionally active heavy chain with about 80% of the original activity and a light chain. These two chains were found to be linked by a single interchain disulfide bond. The functionally active heavy chain can be isolated by an affinity chromatography method with [N alpha-(epsilon-aminocaproyl)-DL-homoarginine hexylester]-Sepharose. The light chain, which has no enzyme activity, is not adsorbed to the affinity matrix, whereas the active heavy chain was adsorbed and subsequently eluted. The active heavy chain was further purified by gel filtration on Sephadex G-100. This preparation was found to be homogeneous by both analytical and sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. The molecular weight of the active heavy chain was determined to be 33,000 by Sephadex G-100 gel filtration and 31,000 by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis. Its specific activity, with L-pyroglutamyl-glycyl-L-arginine-p-nitroanilide, was determined to be 208,000 IU/mg of protein. Approximately 87% active sites were found by p-nitrophenyl-p'-guanidino-benzoate titration with a molar activity of 7.41 X 10(9) IU/mmol of active site. The active heavy chain when compared to low molecular weight urokinase has a similar molecular weight, specific activity, and amino acid composition. The NH2-terminal residue found in the active heavy chain was lysine which was the same as that found in low molecular weight urokinase, whereas the NH2-terminal residues found in high molecular weight urokinase were serine and lysine. Serine is the NH2-terminal residue of the light chain of high molecular weight urokinase. The steady state kinetic parameters of activation of human Glu-plasminogen by the active heavy chain were also similar to low molecular weight urokinase, as were the amidase parameters of these enzymes. The Michaelis constants of activation (Kplg) were 2.11 and 2.21 microM, respectively; the catalytic rate constants of activation (kplg) were 51.7 and 44.1 min-1, respectively, with second order rate constants, kplg/Kplg of 24.5 and 20.2 microM-1 min-1, respectively.     

Phospholipid-associated annexin A2-S100A10 heterotetramer and its subunits: characterization of the interaction with tissue plasminogen activator,plasminogen, and plasmin

Annexin A2 (p36) is a highly alpha-helical molecule that consists of two opposing sides, a convex side that contains the phospholipid-binding sites and a concave side, which faces the extracellular milieu and contains multiple ligand-binding sites. The amino-terminal region of annexin A2 extends along the concave side of the protein and contains the binding site for the S100A10 (p11) subunit. The interaction of these subunits results in the formation of the heterotetrameric form of the protein, annexin A2-S100A10 heterotetramer (AIIt). To simulate the orientation of AIIt on the plasma membrane we bound AIIt to a phospholipid bilayer that was immobilized on a BIAcore biosensor chip. Surface plasmon resonance was used to observe in real time the molecular interactions between phospholipid-associated AIIt or its annexin A2 subunit and the ligands, tissue-type plasminogen activator (t-PA), plasminogen, and plasmin. AIIt bound t-PA (Kd = 0.68 microm), plasminogen (Kd = 0.11 microm), and plasmin (Kd = 75 nm) with moderate affinity. Contrary to previous reports, the phospholipid-associated annexin A2 subunit failed to bind t-PA or plasminogen but bound plasmin (Kd = 0.78 microm). The S100A10 subunit bound t-PA (Kd = 0.45 microm), plasminogen (Kd = 1.81 microm), and plasmin (Kd = 0.36 microm). Removal of the carboxyl-terminal lysines from the S100A10 subunit attenuated t-PA and plasminogen binding to AIIt. These results show that the carboxyl-terminal lysines of S100A10 form t-PA and plasminogen-binding sites. In contrast, annexin A2 and S100A10 contain distinct binding sites for plasmin.     

Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector

A baculovirus expression vector was constructed with the tissue plasminogen activator (TPA) cDNA under the control of the viral polyhedrin promoter. After infection of insect cells with the recombinant baculovirus, active TPA was secreted into the medium in which these cells were grown. TPA was isolated from the conditioned media using metal chelate affinity chromatography followed by immunoaffinity purification using mouse monoclonal anti-human TPA coupled to Sepharose. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions and sequence analysis of recombinant human TPA have revealed a two-chain form of the enzyme. The N-terminal amino acid was identified to be serine, indicating that it was processed at its N-terminus by the insect cell culture in a manner similar to that observed for mammalian cells. The relative specific activity of recombinant TPA from insect cells is comparable to that of Bowes melanoma TPA standard. Its activity is stimulated in the presence of fibrinogen fragments, but by a factor about 2.3-fold lower than the Bowes melanoma TPA. The apparent molecular weight of recombinant TPA from insect cells was about 60K by fibrin agar activity gels, suggesting less complex glycosylation than recombinant TPA from mammalian cells.     

The genetic relationships between the kringle domains of human plasminogen,prothrombin, tissue plasminogen activator,urokinase, and coagulation factor XII

Summary A computer-based statistical evaluation of the optimal alignments of the kringle domains of human plasminogen, human prothrombin, human tissue plasminogen activator, human urokinase, and human coagulation Factor XIIa, as well as the putative kringle of human haptoglobin, has been performed. A variety of different alignments has been examined and scores calculated in terms of the number of standard deviations (SD) of a given match from randomness. With the exception of human haptoglobin, it was found that very high alignment scores (8.9–23.0 SD from randomness) were obtained between each of the kringles, with the kringle 1 and kringle 5 regions of human plasminogen displaying the highest similarity, and the S kringle of human prothrombin and the human Factor XII kringle showing the least similarity. The relationships obtained were employed to construct an evolutionary tree for the kringles. The predicted alignments have also allowed nucleotide mutations in these regions to be evaluated more accurately. For those regions for which nucleotide sequences are known, we have employed the maximal alignments from the protein sequences to assess nucleotide sequence similarities. It was found that a range of approximately 40–55% of the nucleotide bases were placed at identical positions in the kringles, with the highest number found in the alignment of the two kringles of human tissue plasminogen activator and the lowest number in the alignment of the S kringle of prothrombin with the second kringle of tissue plasminogen activator. From both protein and nucleotide alignments, we conclude that haptoglobin is not statistically homologous to any other kringle.Secondary structural comparisons of the kringle regions have been predicted by a combination of the Burgess and Chou-Fasman methods. In general, the kringles display a very high number of -turns, and very low -helical contents. From analysis of the predicted structures in relationship to the functional properties of these domains, it appears as though many of their functional differences can be related to possible conformational alterations resulting from amino acid substitutions in the kringles.     

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)