Shinji Yoshitake

Antibody to Thrombin Receptor Inhibits Neointimal Smooth Muscle Cell Accumulation Without Causing Inhibition of Platelet Aggregation or Altering Hemostatic Parameters After Angioplasty in Rat

An antibody was raised in rabbits against SFFLRNPSEDTFEQF peptide, which is an NH2-terminal peptide of the thrombin-cleaved rat thrombin receptor. In vitro, the antibody inhibited rat smooth muscle cell proliferation but had no effect on rat platelet aggregation or clotting time. These data indicate that the antibody is a specific blocker of the thrombin receptor-signaling pathway in rat smooth muscle cells but does not work as a blocker in rat platelets, suggesting the existence of a second thrombin receptor in the platelets. Using an in vivo balloon catheter-induced injury model in rats, we examined the effect of the anti-rat thrombin receptor IgG on intimal smooth muscle cell accumulation 2 weeks after angioplasty. Analysis of the ratio of intimal to medial cross-sectional areas showed that injection of immune IgG resulted in 43.7% and 53.1% reduction (P<0.01) of neointimal smooth muscle cell accumulation compared with saline and nonimmune IgG treatment, respectively. Moreover, the injection of immune IgG caused a significant decrease of thrombin receptor mRNA expression and also 40.5% and 43.0% decreases (P<0.01) of the proliferating cell nuclear antigen (PCNA) index in the intima compared with the PCNA index after saline and nonimmune IgG treatment, respectively. The suppression of the PCNA index was also observed in the immune IgG-treated group at an early stage after angioplasty. These results suggest that thrombin receptor activation is involved in the proliferation and accumulation of neointimal smooth muscle cells induced by balloon injury.

Thrombolytic Properties of a Novel Modified Human Tissue-type Plasminogen Activator (e6010): A Bolus Injection of E6010 Has Equivalent Potency of Lysing Young and Aged Canine Coronary Thrombi

Summary: The thrombolytic properties of a novel modified human tissue plasminogen activator (E6010), in which cysteine 84 in the epidermal growth factor domain is replaced by serine and that has a prolonged biological half-life, were examined. The thrombolytic efficacies of E6010 and recombinant human tissue plasminogen activator (rt-PA) on the duration of coronary artery thrombus were evaluated in a canine model (123 anesthetized dogs) with copper coil–induced left anterior descending coronary artery thrombus. Thrombi established for periods of 1, 3, or 6 h, as documented by coronary arteriography, were employed. A single bolus i.v. injection of E6010 or rt-PA and an i.v. infusion of rt-PA over 60 min were compared (n = 6). Thrombolytic efficacy was evaluated by three criteria: time to reperfusion (TR), reperfusion rate at 60 min (RR), and reocclusion rate at 60 min after reperfusion (OR). With a bolus i.v. injection of E6010 at a dose of 0.2 mg/kg or an i.v. infusion of rt-PA at a dose of 0.6 mg/kg/h, these parameters were as follows: TR, 30.0 × 15.3 and 27.5 × 4.8 min; RR, 100 and 100%; OR, 17 and 33% for 1-h aged thrombi; TR, 30.0 × 9.5 and 35.0 × 8.2 min; RR, 83 and 50%; OR, 20 and 67% for 6-h aged thrombi. These data indicate that a bolus injection of E6010 is almost equally efficacious in lysing thrombi aged both 1 and 6 h. On the other hand, in the case of rt-PA, the thrombi aged 6 h were lysed significantly less than the thrombi aged 1 h. Plasma half-lives of E6010 were t1/2α, 4.8 × 0.95 (estimated by antigen level) and 3.0 × 0.78 min (estimated by activity), and t1/2β, 51 × 5.4 (antigen level) and 22 × 7.0 min (activity). The half-lives of rt-PA were t1/2α, 3.6 × 0.23 (antigen level) and 2.1 × 0.61 min (activity), and t1/2β, 36 × 2.3 (antigen level) and 7.0 × 3.5 min (activity). We conclude that a bolus injection of E6010 may have a more potent and longer-lasting effect than i.v.-infused rt-PA in clot lysis therapy.

Intracoronary infusion of E6010 has more potent thrombolytic activity than tissue plasminogen activator (t-PA) in dogs : a higher plasma level of E6010 than t-PA causes potent thrombolytic activity

Summary: We examined the thrombolytic properties of a novel modified human tissue plasminogen activator (PA) (E6010), in which cysteine 84 is replaced by serine, and which has a prolonged biologic half-life (t½). We compared the thrombolytic efficacy of continuous intracoronary (i.e.) infusion of E6010 with that of recombinant human tissue PA (rt-PA) in a canine model with copper coil-induced 1-h-old coronary artery thrombi and also compared the relation between thrombolytic efficacy and plasma clearance represented by pharmacokinetic parameters of i.e.-infused E6010 and rt-PA. Sixty-minute E6010 and rt-PA i.e. infusions were compared. The thrombolytic effects of i.e.-infused E6010 and rt-PA, represented by time to reperfusion (TR), reperfusion rate at 60 min (RR), and reocclusion rates at 60 min after reperfusion (OR) were as follows. E6010: Dose 0.06, 0.15, 0.3 (mg/kg/h); TR 25 ± 10, 15 ± 10, 13 ± 5 (min); RR 100, 100, 100 (%); and OR 0, 0, 17 (%), respectively. Recombinant t-PA: Dose 0.06, 0.15, 0.3 (mg/kg/h); TR 47 ± 12, 18 ± 17, 14 ± 4 (min); RR 50, 75, 100 (%); and OR 100, 33, 33 (%), respectively. These findings indicate that E6010 has more potent thrombolytic activity than rt-PA. After the i.e. infusion of E6010 and rt-PA at doses of 0.3 mg/ kg/h, the i.e. and peripheral venous (intravenous, i.v.) plasma clearances, represented by the area under the plasma concentration-time curve (AUC), were assessed by antigen and by activity: E6010 assessed by antigen, i.e. 200.1 ± 88.6, i.v. 70.0 ± 39.9 (µg · min/ml); assessed by activity, i.e. 142.5 ± 93.3, i.v. 23.5 ± 15.3 (µg · min/ ml); and rt-PA assessed by antigen, i.e. 81.4 ± 47.4, i.v. 14.1 ± 7.1 (µg · min/ml); and assessed by activity, i.e. 55.1 ± 23.0, i.v. 9.4 ± 5.0 (µg · min/ml). We conclude that the higher plasma level of the active form of E6010, especially its i.e. level, was responsible for its enhanced thrombolytic effect

Immunogenic properties of structurally modified human tissue plasminogen activators in chimpanzees and mice

Immunogenic properties of second generation human tissue plasminogen activator (tPA) derivatives were examined in chimpanzee and mouse systems. Five species of modified tPAs (mtPAs) (designated 2660, 2663, 2810, 8000, and 9200), recombinant native tPA or bovine serum albumin (BSA) as a positive control were subcutaneously injected nine times at suitable intervals into chimpanzees, genetically the closest species to man. These animals were tested for antigen(Ag)-specific antibodies to the corresponding proteins by means of enzyme-linked immunosorbent assay and Western blot analysis. Neither 9200, one of the five mtPAs tested, nor tPA was immunogenic, although BSA and the other four mtPAs were immunogenic under these conditions. Thus, an antigenic determinant was not exposed by the modification on 9200 and this modified tPA is expected not to be immunogenic in humans. In the mouse studies, mice were immunized with mtPAs. Serum samples from these animals were tested for antibodies to the mtPAs which did not concomitantly recognize native tPA by immune adsorption of the antibodies to tPA. The amount of such antibodies after the elimination of native tPA-reactive antibodies was little or none when the serum samples from 9200 and from the other mtPAs, except 8000, were tested. Taking into consideration the results of the chimpanzee studies, it can be concluded that Ag-specific antibodies are dominantly produced to unchanged epitopes present in modified proteins in the mouse system, in which the native protein is immunogenic.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of biotinylated thrombin receptor peptide to cloned human thrombin receptor overexpressed in baby hamster kidney cells.

Baby hamster kidney (BHK) cells transfected with an expression vector for the human thrombin receptor, and then treated with basic fibroblast growth factor, were found to express specific and saturable binding sites for biotinylated thrombin receptor peptide (SFLLRNPNDKYEPF). Analysis of the binding to live BHK cells yielded an equilibrium dissociation constant (Kd) of 3.0 +/- 0.3 mu mol/l and a maximal binding capacity (Bmax) of 31.0 +/- 0.5 nmol/mg of protein. In competitive binding experiments, the thrombin receptor agonist peptide (SFLLRN), which is a strong inducer of human platelet aggregation, was the most potent competitor. In contrast, position 1 to 2 inverted peptides such as FSLLRNPNDKYEPF and FSLLRNP, which fail to induce for the platelet aggregation, were less potent. This simple and convenient binding assay system using the biotinylated thrombin receptor peptide as a labeled ligand and the cloned thrombin receptor overexpressed in BHK cells may be useful for exploring specific antagonists of the receptor.