Single-chain Urokinase-type Plasminogen Research Articles

A marine fibrinolytic compound FGFC1 stimulating enzymatic kinetic parameters of a reciprocal activation system based on a single chain urokinase-type plasminogen activator and plasminogen

Abstract A marine fibrinolytic compound FGFC1 enhancing fibrinolysis was obtained involving enzymatic kinetic parameters of a reciprocal activation system with a single chain urokinase type plasminogen activator and plasminogen. FGFC1, a kind of bisindole alkaloid from a metabolite of the rare marine fungi Starchbotrys longispora FG216, modulated enzymatic kinetic parameters including the fibrinolytic reaction rate and fibrin degradation characteristics. The enzymatic kinetics of fibrinolysis was described based on the enzymatic reaction of a chromogenic-substrate associated with p-nitroaniline (p-NA). While the single chain urokinase-type plasminogen activator (pro-uPA) activated plasminogen, kcat and kcat/Km increased significantly with an increase of FGFC1 concentration. Moreover, kcat and kcat/Km exhibited 26.5-fold and 22.8-fold enhanced activity at the concentration of 40 μg mL−1 of FGFC1, respectively. The results suggested that FGFC1 significantly improved the maximum catalytic efficiency and the total catalytic activity of fibrinolysis base on the reciprocal activation of pro-uPA and plasminogen. Km increased with increasing FGFC1 concentration, which indicated that FGFC1 slightly decreased the affinity activity of the pro-uPA and plasminogen versus the enzyme substrate. The marine bisindole alkaloid FGFC1 enhanced fibrinolysis, which was taken on enzymatic kinetic characteristics.

Thrombin–thrombomodulin inhibits prourokinase-mediated pleural mesothelial cell-dependent fibrinolysis

Fibrin deposition is a hallmark of pleural inflammation and loculation but understanding of mechanisms by which mesothelial cells regulate intrapleural fibrinolysins remains incomplete. We speculated that pleural mesothelial cells regulate local fibrinolytic capacity via processing of single chain urokinase type plasminogen activator (scuPA). Pretreatment of human pleural mesothelial (MeT-5A) cells with TGF-β or thrombin, either alone or in combination, inhibited urokinase (uPA)-mediated fibrinolysis by MeT-5A. Thrombin, unlike TGF-β, inhibited fibrinolysis without induction of PAI-1, suggesting that thrombin-mediated cleavage of scuPA inhibits the fibrinolytic capacity of MeT-5A cells. Thrombin cleaves both purified scuPA as well as that secreted by MeT-5A cells and cell surface thrombomodulin accelerates thrombin-mediated cleavage of scuPA to inhibit cellular fibrinolytic activity. Molecular dynamics analyses demonstrated that thrombin-cleaved scuPA (uPAt) do not acquire a catalytically active conformation and that secondary plasminogen binding sites of uPA implicated in plasminogen activation are distorted in uPAt, explaining, at least in part, why uPAt is a poor enzyme. uPAt was detectable in transudative and exudative pleural effusions from patients. Intrapleural administration of scuPA generated increased levels of uPAt in PF of rabbits with pleural injury and loculation induced by tetracycline in vivo. This pathway is operative in diverse forms of pleural injury, restricts the urokinase-dependent fibrinolytic capacity of pleural mesothelial cells and contributes to local control of fibrinolytic activity via processing of endogenous or exogenous scuPA within the pleural compartment.

Novel human plasma proteins, IHRP (acute phase protein) and PHBP (serine protease), which bind to glycosaminoglycans.

A novel human plasma protein was found in the eluate from the dextran sulfate column, which was used for the treatment of the patients with hypercholesteremia to reduce plasma low density lipoprotein. The results of sequence analysis revealed that this protein was a homologue of heavy chains of inter-alpha-trypsin inhibitor (ITI) family, and it was termed IHRP (ITI family heavy chain related protein). IHRP was identified as an acute-phase protein in animals, and slightly increased concentrations in human plasmas were observed in the patients with inflammatory disorders. IHRP bound to actin and inhibited its polymerization, and IHRP suppressed the phagocytosis and chemotaxis of polymorphonuclear cells. These results suggest that IHRP may function as an anti-inflammatory protein. Plasma hyaluronan binding protein (PHBP) is a novel serine protease which was also found in human plasma. It is consisted of three epidermal growth factor domains, one kringle domain and one serine protease domain from its amino terminus. The amino acid sequence of PHBP is homologous to that of hepatocyte growth factor activator. Purified 75-kDa single chain pro-form of PHBP was auto-activated (auto-cleaved) to 50-kDa heavy chain and 25-kDa light chain, both of them are bridged by a disulfied bond. PHBP digested alpha-chain and beta-chain of fibrinogen to prevent coagulation and cleaved single chain urokinase type plasminogen activator (scuPA) to the active hetero dimer form (tcuPA). The auto-activation of PHBP was accelerated in the presence of dextran sulfate or phosphatidylethanolamine as well as factor XII of the coagulation system. C1 inhibitor of the complement system was identified as the main inhibitor of PHBP in human plasma. Partial hepatectomy and administration of carbon tetrachloride or galactosamine caused the conversion of pro-PHBP to the active form in mouse but administrations of turpentine and mercury chloride did not, suggesting the hepatic injury specific activation of PHBP. These results indicate that PHBP participates not only in the fibrinolytic system but also in the degradation cascade of extracellular matrix (ECM), i.e., PHBP activates scuPA to tcuPA, tcuPA activates matrix metalloproteases (MMPs) and activated MMPs degrade ECM for the tissue remodeling after hepatic injury.

New thrombolytic drugs.

Thrombolytic drugs reduce mortality from myocardial infarction and research is focused on newer drugs with improved clinical efficacy. The ideal thrombolytic drug should be effective in dissolving fresh and older thrombi, be rapid in its action with complete dissolution of the thrombus, be able to be given as a bolus and be safe without hypotensive or allergic or immunogenic reactions. The types of agents being developed fall into five broad categories: * mutants or variants of single chain urokinase type plasminogen activator; * mutants or variants of tissue type plasminogen activator; * recombinant chimaeric plasminogen activators; * conjugates of plasminogen activators and anti-fibrin monoclonal antibodies; * compounds derived from haemophagous animals. Within the mutant and variant groups there may be single point mutations which increase the half life or deletion of various amino acid sequences to increase resistance to plasma protease inhibitors or cause more selective binding to fibrin. The recombinant chimaeric plasminogen activators are designer drugs where the kringle regions, the growth hormone domain region, the finger domain region and the serine protease part of the molecules are all cleaved and recombined in various ways to improve potency. The conjugates of plasminogen activators and anti-fibrin and monoclonal antibodies improve the targeting of the agent to fibrin clot. Monoclonal antibodies are commonly used against the seven aminoterminal residues of the beta chain of fibrin. These are cross linked and bound to plasminogen activators. A variety of substances from haemophageous animals are currently being studied including salivary plasminogen activator from vampire bats, venom from southern copperhead snakes and staphylokinase from bacteria. Currently available thrombolytic agents have many limitations, but the novel thrombolytic agents have yet to be tested in clinical trials. With few exceptions, they all act via the plasminogen system and it may be in the future that combinations of anti-platelet and thrombolytic agents may prove to be more efficacious than thrombolytics and aspirin alone.

ＰＴＣＡカテーテルによって損傷させたイヌ冠動脈の血管狭窄ならびにヒト血管培養細胞に及ぼすｓｃｕ‐ＰＡとｔ‐ＰＡの影響

ＰＴＣＡカテーテルによって損傷させたイヌ冠動脈の血管狭窄ならびにヒト血管培養細胞に及ぼすｓｃｕ‐ＰＡとｔ‐ＰＡの影響

Functional properties of a recombinant chimeric protein with combined thrombin inhibitory and plasminogen-activating potential.

A chimeric protein (rscu-PA-40-kDa/Hir), consisting of the C-terminal amino acids 53-65 of hirudin (Hir), fused via a 14-amino-acid linker sequence to the C-terminal of a 40-kDa fragment (Ser47-Leu411) of recombinant (r) single-chain (sc) urokinase-type plasminogen activator (rscu-PA), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. The thrombin inhibitory potential of purified rscu-PA-40-kDa/Hir was confirmed by complete inhibition of the coagulant activity of thrombin at 20-30-fold molar excess of the chimera, and by the resistance of rscu-PA-40-kDa/Hir to proteolytic cleavage by thrombin, rscu-PA-40-kDa/Hir prolonged the thrombin time of normal human plasma in a dose-dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin-mediated platelet aggregation (reduction of the apparent thrombin concentration to 50% with 40 nM chimeric protein). The chimera had a specific activity on fibrin films of 57,000 IU/mg as compared to 95,000 IU/mg for rscu-PA. The urokinase-like amidolytic activity of the single-chain protein was only 220 IU/mg but increased to 169,000 IU/mg after treatment with plasmin, which resulted in quantitative conversion to a two-chain (tc) derivative (rtcu-PA-40-kDa/Hir). Corresponding values for rscu-PA were 270 and 226,000 IU/mg. The catalytic efficiencies for plasmin-mediated conversion to two-chain molecules were comparable for rscu-PA-40-kDa/Hir and rscu-PA (0.63 and 0.65 microM-1.s-1, respectively). The plasminogen-activating potential of the single-chain chimera was comparable to that of rscu-PA; the catalytic efficiencies for plasminogen activation by their two-chain counterparts were also similar (0.55 and 0.73 microM-1.s-1, respectively). In 2 h, 50% lysis of 125I-fibrin-labeled clots prepared from platelet-poor human plasma and immersed in normal plasma was obtained with 1.3 micrograms/ml rscu-PA-40-kDa/Hir and with 0.67 micrograms/ml rscu-PA, with corresponding residual fibrinogen levels of 74% and 87%, respectively. In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal human plasma required 2.1 micrograms/ml rscu-PA, but 7.9 micrograms/ml rscu-PA-40-kDa/Hir. Thus, the chimera rscu-PA-40-kDa/Hir has maintained the specific fibrinolytic and plasminogen activating activity of rscu-PA as well as its fibrinolytic potency in plasma, whereas it displayed a similar or somewhat better fibrin specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Single chain urokinase type plasminogen activator (SCU-PA) complexes with and is inhibited by plasminogen activator inhibitor type 1 (PAI-1)

Single chain urokinase type plasminogen activator (SCU-PA) complexes with and is inhibited by plasminogen activator inhibitor type 1 (PAI-1)

Thrombolytic Activity of Two Chimeric Recombinant Plasminogen Activators (FK2tu-PA and K2tu-PA) in Rabbits

The aim of this study was to evaluate the thrombolytic activity of two hybrid plasminogen activators (HPAs) in a rabbit jugular vein thrombosis model. In the two HPAs the kringle-2 domain (K2tu-PA) or the finger and the kringle-2 domains (FK2tu-PA) of tissue-type plasminogen activator (t-PA) are linked to the catalytic protease domain of single chain urokinase type plasminogen activator (scu-PA). The two HPAs were compared with rt-PA and scu-PA on a weight/weight basis. K2tu-PA, FK2tu-PA, rt-PA and scu-PA were infused at doses of 0.4, 0.8 and 1.2 mg/kg over 3 h. Saline served as control. Saline produced 11 +/- 2% thrombolysis. The three doses of K2tu-PA led to 38 +/- 4%, 66 +/- 5% and 89 +/- 7% thrombolysis, respectively; the three doses of FK2tu-PA: 18 +/- 3%, 29 +/- 5% and 33 +/- 6%, respectively; the three doses of rt-PA 32 +/- 2%, 49 +/- 3% and 68 +/- 6%, respectively; the three doses of scu-PA 16 +/- 2%, 24 +/- 3% and 32 +/- 4%, respectively. K2tu-PA and rt-PA showed a statistically significant higher thrombolytic activity than FK2tu-PA and scu-PA at the three tested doses (p less than 0.01). The thrombolytic activity of K2tu-PA was significantly higher than rt-PA at the two higher doses (p less than 0.01). Both K2tu-PA and rt-PA produced a statistically significant reduction of fibrinogen, alpha 2-antiplasmin and plasminogen 3 h after the start of the infusions of the two higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of Intrinsic and Extrinsic Plasminogen upon the Lysis of Thrombi In Vitro

When the rate of lysis of artificial thrombi (prepared from plasma or whole blood) was expressed according to the concentration of tissue type plasminogen activator (t-PA) or single chain urokinase type plasminogen activator (sc-uPA) then bell-shaped dose response curves were obtained, low rates being observed at concentrations of activator greater than 500 units/ml. Bell-shaped dose response curves were not observed for rate of lysis of artificial thrombi over the concentrations of streptokinase tested (SK) or for the lysis of plasma gel clots by any of the activators tested. Further investigation indicated that the preponderant mechanism for dissolution of thrombi at 500 units/ml of t-PA was by activation of the plasminogen within the thrombus (intrinsic) since the plasminogen present in the plasma perfusing the thrombus (extrinsic) rapidly became depleted. On the other hand, at 50 units/ml t-PA the lysis was observed to be due preponderantly to the action of plasmin arising from extrinsic rather than intrinsic plasminogen. If "plasminogen enriched" thrombi were prepared in the presence of Lys plasminogen (Lys-Plg) faster rates of lysis occurred and bell-shaped biometric curves were not observed.

Novel Expression of Chimeric Plasminogen Activators in Insect Cells

The isolated protease domain of single chain urokinase–type plasminogen activator (scu–PA) preferentially activates fibrin–associated plasminogen in plasma, yet scu–PA binds poorly to forming fibrin clots. We are investigating the effect of combining this protease domain with protein domains that are reported to be involved in the binding of other plasma proteins to forming fibrin clots; the chimeric proteins consist of the protease domain and either the plasmin kringle 1 domain or the tissue plasminogen activator kringle 2 domain. Rapid construction of expression vectors for these chimeras was possible because much of the coding region required was synthesized either by polymerase chain reaction using RNA as the initial template or by the ligation of multiple long synthetic oligonucleotides. We also used an artificial signal peptide and baculovirus expression vectors to direct the secretion of these chimeras from insect cells. The signal peptide was designed by selecting those amino acid residues that occur more frequently than expected (based on amino acid composition) at positions −13 to +2 relative to the signal peptidase cleavage site in eukaryotic proteins. The chimeric proteins expressed by insect cells were enzymatically active, but their binding to forming fibrin clots was similar to that of the poorly binding scu–PA. In an in vitro clot lysis system, these chimeras displayed a specificity for fibrin–associated plasminogen greater than the specificity of two chain urokinase–type plasminogen activator and similar to the specificity of tissue plasminogen activator and scu–PA.

Vascular origin determines plasminogen activator expression in human endothelial cells: Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator

Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney.Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/105 cells/24 h and 50.42 ng/105 cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/105 cells/24 h and 2.17 ng/105 cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.

Characterization of commercially available plasminogen preparations in vitro: purity and reactivity.

Three different commercially available plasminogen preparations (Immuno-, Kabi-, and Behring-plasminogens) were examined regarding purity and reactivity to different activators (high molecular weight [HMW] or low molecular weight [LMW] two-chain urokinase type plasminogen activator [tcu-PA], single chain urokinase type plasminogen activator [scu-PA], tissue type plasminogen activator [t-PA], and streptokinase [SK]). The Immuno-preparation was a Lys-plasminogen, commercially available for therapeutical use, whereas the research reagents for KabiVitrum and Behringwerke were Glu-plasminogen. Activity data provided by the manufacturers correlated well with our findings. Also a good correlation of reactivity to activators measured with a chromogenic substrate and on fibrin plates could be observed. The Immuno-plasminogen showed a minimum contamination with plasmin which has to be taken into consideration for the interpretation for its apparently higher activation by plasminogen activators compared to the plasmin-free plasminogens. Further in vitro and in vivo research has to be performed to find out criteria for a practicable scheme of administration of fibrinolytic agents for therapeutical thrombolysis.