Biochemical and Biological Properties of a Recombinant Tissue-Type Plasminogen Activator Derived from the Rat JMI-229 Cell Line

Abstract

SummaryRecombinant tissue-type plasminogen activator (rt-PA), produced by expression of the genomic t-PA DNA from the JMI-229 cell line, which is of rat origin, in the host cell line, was purified to homogeneity. JMI-229 rt-PA was obtained essentially as a single chain molecule which was quantitatively converted to a two-chain moiety by treatment with plasmin. The plasminogen activating potential of single chain JMI-229 rt-PA was 5-fold lower than that of commercially available human rt-PA (Actilyse®) in the absence of fibrin, but comparable in the presence of fibrin; it showed a concentration-dependent binding to fibrin, with a significantly more pronounced binding than Actilyse® at low fibrin concentration (85 ± 8% versus 20 ± 7% at 0.025 mg/ml fibrin; p = 0.004). In human plasma in the absence of fibrin, the concentrations of both single chain and two-chain JMI-229 rt-PA required to induce 50% fibrinogen degradation in 2 h, were about 15-fold higher than those of Actilyse®. Both single chain and two-chain forms of JMI-229 rt-PA and of Actilyse® induced a similar time- and concentration-dependent lysis of a 125I-fibrin-labeled plasma clot immersed in human plasma, in the absence of significant systemic fibrinolytic activation. Equally effective concentrations (causing 50% clot lysis in 2 h) were 0.11 or 0.10 pg/ml for single chain or two-chain JMI-229 rt-PA, as compared to 0.11 or 0.15 pg/ml for single chain or two-chain Actilyse®. Continuous infusion over 60 min of single chain JMI-229 rt-PA or Actilyse® in hamsters with a 125I-fibrin-labeled pulmonary embolus, revealed a very similar thrombolytic potency (clot lysis versus dose) and specific thrombolytic activity (clot lysis versus steady state plasma antigen level of t-PA). The initial plasma half-life following intravenous bolus injection of 0.10 mg/kg in hamsters was equally short for JMI-229 rt-PA or Actilyse® (1.2 or 1.4 min respectively).It is concluded that JMI-229 rt-PA has a higher fibrin-affinity and a higher fibrin-specificity in human plasma in the absence of fibrin than Actilyse®, but a comparable thrombolytic potency in a hamster pulmonary embolism model.

Vagus nerve stimulation inhibits activation of coagulation and fibrinolysis during endotoxemia in rats

“…Bound t-PA antigen was subsequently quantitated with biotinylated rabbit anti-rat t-PA antibodies, followed by avidin-peroxidase and tetramethylbenzimidine. As a standard recombinant rat JMI-229 t-PA was used [18]. PAI-1 activity was determined with an automated coagulation analyser (Behring Coagulation System) with reagents and protocols from Dade Behring.…”

Analysis of Tissue Plasminogen Activator Specificity Using Peptidyl Fluorogenic Substrates

“…Tissue-type plasminogen activator (tPA) is a serine protease which is produced by many cell lines (Booyse et al, 1981;Goldsmith et al, 1981;Dano et al, 1985) in a singlechain form (sc-tPA) (Rijken & Groeneveld, 1986) which can be converted rapidly to a two-chain form (tc-tPA) (Wallen et al, 1983;Bachmann & Kruithof, 1984;Rijken & Groeneveld, 1986;Andreasen et al, 1991). The amidolytic activity and efficiency toward various ligands by these two forms has been an object of substantial discussion (Rijken & Collen, 1981;Ranby, 1982;Rijken et al, 1982;Ichinose et al, 1984;Andreasen et al, 1984;Astedt et al, 1985;Kruithof et al, 1986;Tate et al, 1987;Petersen et al, 1988;Petersen, L., et al, 1990;Lijnen et al, 1992). Several assay methods have been described for tPA evaluation at its physiological concentration, including fibrinolytic, immunoradiometric, zymographic, and 125 I-fibrin microtiter well assays (Astrup & Stage, 1952;Astrup & Mullertz, 1952;Albrechtsen, 1957;Bachmann, 1987).…”

“…(Gyzander et al, 1984;Verheijen et al, 1985;Andreasen et al, 1991). For the evaluation of tPA at concentrations significantly higher than physiological, rapid and simple methods of direct chromogenic or fluorogenic assays have been used, employing low molecular weight synthetic substrates (Rijken et al, 1982;Wallen et al, 1982;Ichinose et al, 1984;Astedt et al, 1985;Tate et al, 1987;Petersen et al, 1988;Petersen, L., et al, 1990;Boose et al, 1989;Andreasen et al, 1991;Husain, 1991;Lijnen et al, 1992;Rydzewski & Castellino, 1993;Nieuwenhuizen et al, 1977Nieuwenhuizen et al, , 1978Petersen & Swenson, 1986). Substrate S-2288 (D-Ile-Pro-Arg-pNA) is the most commonly used in such assays.…”

1,25‐Dihydroxyvitamin D3 induction of the tissue‐type plasminogen activator gene is mediated through its multihormone‐responsive enhancer

“…Bands from autoradiographs were quantified on the PhosphorImager (Molecular Dynamics) and the values for the t‐PA transcript were normalized to the corresponding values of the 18S RNA. Rat t‐PA‐related antigen levels were determined in the conditioned medium by specific enzyme‐linked immunosorbent assays (ELISA) as described [23].…”