Single-chain Tissue Plasminogen Activator Research Articles

Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli.

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.

The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator

The structural events taking place during the reaction between PAI-1 (plasminogen-activator inhibitor 1) and the plasminogen activators sc-tPA (single-chain tissue plasminogen activator) and tc-tPA (two-chain tissue plasminogen activator) were studied. Complexes were formed by mixing sc-tPA or tc-tPA with PAI-1 in slight excess (on an activity basis). The complexes were purified from excess PAI-1 by affinity chromatography on fibrin-Sepharose. Examination of the purified complexes by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE) and N-terminal amino acid sequence analysis demonstrated that a stoichiometric 1:1 complex is formed between PAI-1 and both forms of tPA. Data obtained from both complexes revealed the amino acid sequences of the parent molecules and, in addition, a new sequence: Met-Ala-Pro-Glu-Glu-. This sequence is found in the C-terminal portion of the intact PAI-1 molecule and thus locates the reactive centre of PAI-1 to Arg346-Met347. The proteolytic activity of sc-tPA is demonstrated by its capacity to cleave the 'bait' peptide bond in PAI-1. The complexes were inactive and dissociated slowly at physiological pH and ionic strength, but rapidly in aq. NH3 (0.1 mol/l). Amidolytic tPA activity was generated on dissociation of the complexes, corresponding to 0.4 mol of tPA/mol of complex. SDS/PAGE of the dissociated complexes indicated a small decrease in the molecular mass of PAI-1, in agreement with proteolytic cleavage of the 'bait' peptide bond during complex-formation.

On the Properties of the International Standards for Tissue Plasminogen Activator

During the last four and a half years, several preparations of human single-chain tissue plasminogen activator (tPA) from melanoma cell cultures (1) were made. These preparations were analysed for homogeneity, protein content, fibrinolytic activity and amidolytic activity. Because of concern with increasing specific fibrinolytic activity, I investigated the effects of various solvents and the stability of the 2nd International Standard at room temperature

Inhibition by tranexamic acid of the conversion of single-chain tissue plasminogen activator to its two chain form by plasmin: The presence on tissue plasminogen activator of a site to bind with lysine binding sites of plasmin

The addition of tranexamic acid inhibited the conversion of single chain tissue plasminogen activator (sct-PA) to its two chain form (tct-PA) by plasmin. Increase in the concentrations of tranexamic acid resulted in more inhibition of the conversion, with 50 % inhibition being obtained at 0.251 mM of the concentration of tranexamic acid. The addition of the fragments of plasminogen such as kringle 1 to 3 (K1–3) and K4 resulted in the facilitation of the conversion of sct-PA to tct-PA by plasmin. The addition of tranexamic acid to the mixture of sct-PA and K4 inhibited the rate of the conversion of sct-PA by plasmin. These results suggest that binding of lysine binding sites of plasmin or plasminogen with sct-PA enhanced its conversion to tct-PA by plasmin, and that there is a site on a t-PA molecule to bind to plasminogen or plasmin.

Stimulation by fibrinogen of the amidolytic activity of single-chain tissue plasminogen activator

The amidolytic steady-state kinetic properties of a series of recombinant tissue plasminogen activators (rt-PA) have been examined in the presence and absence of the positive effectors fibrinogen (Fg) and native soluble (des-A)-fibrin (sFn). Two-chain (tc) native rt-PA displayed a K m value of 0.50 m m and a K cat value of 13.2 s −1 toward the substrate, HDIleProArg- p-nitroanilide (S2288) at 37 °. When these same assays were conducted in the presence of Fg or sFn, the K m and K cat values remained essentially the same. On the other hand, the activity of single-chain (sc) rt-PA was significantly increased in the presence of Fg or sFn, by approximately 3.4- to 4-fold, due to alterations in both the K m and K cat of the reaction. Similar results were obtained with rt-PA deletion variants, obtained by site-directed mutagenesis. With rt-PA domain-deletion derivatives, consisting of kringles 1 (K 1)2 (K 2)- protease (P), and K 2-P, the amidolytic activities of the scrt-PA preparations were significantly stimulated (2.0- to 2.5-fold) by Fg and sFn, a property not shared by the corresponding tcrt-PA. On the other hand, neither the single- nor two-chain derivatives of a deletion mutant containing only the finger (F)- growth factor (E)-P domains displayed stimulation by Fg or sFn, results suggestive of the importance of the K 2 region in the observed Fg- and Fn-induced stimulations of rt-PA amidolytic activity. With one strategically important derivative, a molecule containing the amino acid replacement, Cys 264 → Gly [(Cys 264 → Gly)-rt-PA], a change resulting in the loss of covalent attachment of the heavy and light chains of tcrt-PA, the amidolytic activities of neither the single-chain nor the two-chain form of the molecule were stimulated by the presence of the above two positive effectors. With the single-chain form of this same derivative, the K cat of the reaction was extremely low (1.5 s −1), but increased to approximately 50.5 s −1 for the two-chain form, this latter value being nearly 4-fold higher than that of any of the wild-type recombinant rt-PA preparations examined. This suggests that the latent heavy chain of rt-PA inhibits the amidolytic activity found in the trypsin-like P domain. However, with another variant rt-PA, containing a Arg 275 → Ser [(Arg 275 → Ser)-rt-PA)] substitution, a change that leads to a loss of ability of plasmin-like enzymes to catalyze conversion of scrt-PA to tcrt-PA, a much larger stimulation by Fg and sFn of its amidolytic activity toward S2288 occurred, due to an approximate 10-fold decrease in the K m of the reaction. These results indicate major differences in the enzymatic properties of scrt-PA and tcrt-PA, regarding their respective abilities to be stimulated in their amidolytic properties by Fg and sFn. Additionally, the capacity for scrt-PA to be enhanced in this manner is dependent on the integrity of the two covalent bonds i.e., Cys 264Cys 395 and Arg 275Ile 276, that stabilize the latent heavy and light chains of tPA. Finally, the nature of the amino acid residue located at the cleavage site of scrt-PA is of great importance to its ability to be stimulated by Fg and Fn.

Kinetics of the inhibition of plasminogen activators by the plasminogen-activator inhibitor. Evidence for 'second-site' interactions.

The reactions between plasminogen-activator inhibitor (PAI) and different plasminogen activators were studied in the presence of chromogenic peptide substrates for the enzymes. Our findings suggest that the rate constants for the reactions of PAI with single-chain tissue plasminogen activator (tPA), two-chain tPA, high-Mr urokinase and low-Mr urokinase are high and quite similar (1.6 X 10(7)-3.9 X 10(7) M-1.s-1). A free active site in the enzymes seems to be necessary for their reaction with PAI. Amino acids with antifibrinolytic properties did not interfere with the reactions. However, di-isopropyl phosphorofluoridate-inactivated tPA inhibited the reaction between PAI and all plasminogen activators in a similar way. These findings clearly demonstrated that a 'second-site' interaction, in addition to that between the enzyme active site and the inhibitor 'bait' peptide bond, is of importance for the high reaction rate. The reaction rate between PAI and single-chain tPA in the presence of an activator substrate (D-Ile-Pro-Arg p-nitroanilide) was decreased in the presence of fibrin. Fibrin caused a decrease in the Km for the single-chain tPA-substrate reaction. As a consequence, the 'free' concentration of single-chain tPA in the system decreased in the presence of fibrin, affecting the reaction rate between PAI and single-chain tPA. The phenomenon might be of physiological relevance, in the sense that single-chain tPA bound to fibrin in the presence of plasminogen would be protected against inactivation by PAI.

Determination of plasminogen activator inhibitor in plasma using t-pa and a chromogenic single-point poly-D-lysine stimulated assay

A two stage method for determination of plasminogen activator inhibitor (PAI) activity in blood plasma is described. In the first stage, an excess of single-chain tissue plasminogen activator (t-PA) is added to plasma. In the second stage, the residual t-PA activity is determined with a plasminogen/chromogenic plasmin substrate assay utilizing poly-D-lysine as a t-PA stimulator. The method proved accurate since it correctly determined the PAI activity (range 0 to 110μ/mL) in citrated plasma samples with levels established by time course analysis of t-PA inhibition and by titration with t-PA. Furthermore, correlation was excellent (r=0.97) with the method of Chmielewska et al Thromb. Res. 31, 427–436, 1983. Plasma samples with increased platelet factor 4, indicative of platelet release, did not show increased levels of PAI activity.

172 Marked reduction in infarct size and recovery of ventricular function following experimental coronary thrombolysis by single chain tissue plasminogen activator in dogs

172 Marked reduction in infarct size and recovery of ventricular function following experimental coronary thrombolysis by single chain tissue plasminogen activator in dogs

Large-scale purification of human tissue-type plasminogen activator using monoclonal antibodies

Tissue plasminogen activtor produced by a human melanoma cell line (Bowes), was purified from large volumes of supernatant fluid using immunosorbent chromatography on monoclonal antibodies, followed by chromatography on lysine-Sepharose 4B and gel filtration on Sephadex G-150. Immunosorbents based on monoclonal antibodies were shown to be preferable to those based on polyclonal antibodies for several reasons: efficient binding and desorption, high yield along with a high degree of purification. The overall recovery was about 50%. A homogeneous sample of single-chain tissue plasminogen activator with a molecular weight of approx. 65 000 was obtained as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Amino acid sequence analysis revealed two N-terminals in equal yields, corresponding to the long and short tissue plasminogen activator structures previously reported by others. The pure preparations of tissue plasminogen activator showed specific activities of about 200 000 IU/mg protein.

イヌ大たい静脈血せんモデルにおける組織性プラスミノーゲンアクチベーターの効果

Tissue plasminogen activator (TPA) was purified either as a single chain or as a two chain form from the culture medium of a human melanoma cell line. The thrombolytic activity of TPA was investigated in beagle dogs with an experimental femoral vein thrombosis and compared with urokinase. The 125I-fibrinogen labeled thrombus was formed in the femoral vein, and the thrombolytic agents were infused over a 4 hour period. The degree of thrombolysis was measured as the difference between the injected and recovered 125I. In six control animals with a saline infusion, the degree of thrombolysis was 16.3±3.8 percent (mean±S. E. M.), in 5 dogs receiving 100, 000IU urokinase, 17.4±3.7 percent and in 4 dogs with 1, 000, 000IU urokinase, 40.6±4.8 percent. Infusion of 100, 000IU single chain TPA in 5 dogs resulted in 33.5±7.8 percent lysis and of 100, 000IU two chain TPA in 5 dogs in 60.1±10.8 percent. Infusion of 300, 000IU one chain TPA yielded 57.5 percecent lysis and of the same amount of two chain TPA 72.9 percent. Significant activation of plasminogen, consumption of α2-antiplasmin and fibrinogen breakdown was only observed in the animals receiving the high dosis of urokinase but not in the saline, TPA or the low dose urokinase groups. It is thus concluded that in this thrombosis model, human TPA has a higher specific thrombolytic effect than urokinase, without systemic fibrinolytic activation and fibrinogen breakdown.