Thrombolytic and Haemorrhagic Effects of Bolus Doses of Tissue-Type Plasminogen Activator and a Hybrid Plasminogen Activator with Prolonged Plasma Half-Life (K2tu-PA: CGP 42935)

Abstract

SummaryK2tu-PA is a hybrid plasminogen activator linking the kringle 2 domain of tissue-type plasminogen activator (t-PA) to the catalytic protease domain of single-chain urokinase-type plasminogen activator (scu-PA). K2tu-PA, as t-PA has high affinity for fibrin and is activated by fibrin but has a longer plasma half-life (over 30 min). The aim of this study was to compare the effects of bolus doses of recombinant t-PA (rt-PA) and K2tu-PA, on: 1) lysis of preformed thrombi (fibrinolysis), 2) accretion of new fibrin on pre-existing thrombi during fibrinolysis (thrombus growth), 3) thrombolysis as assessed by reduction of thrombus weight and 4) systemic plasma proteolysis and blood loss from a standard wound. A jugular vein thrombosis model and an ear bleeding model were adopted in rabbits. Saline produced 11 ± 2% fibrinolysis. rt-PA, 0.2 mg/kg, 0.4 mg and 0.8 mg/kg produced 35 ± 4%, 54 ± 4% and 78 ± 6% fibrinolysis, respectively. K2tu-PA, at the same doses, produced 39 ± 5%, 57 ± 6% and 83 ± 6% fibrinolysis, respectively. Thus, no differences in the fibrinolytic activity of rt-PA and K2tu-PA were observed. Injection of saline was followed by an accretion of 56.4 ± 5.9 μg of radioactive new fibrin on the thrombi. The injection of the three increasing doses of rt-PA was followed by an accretion of 54.9 ± 5.3 μg, 49.1 ± 6.1 μg and 47.2 ± 4.8 μg. The injection of three increasing doses of K2tu-PA was followed by an accretion of 38.1 ± 3.4 μg, 29.6 ± 2.5 μg and 17.1 ± 3.4 μg. At each of the three doses, K2tu-PA was more effective than rt-PA in reducing the accretion of new fibrin on the thrombi (p <0.01) and, as a consequence, in reducing thrombus weight (p <0.01). The two lower doses of rt-PA and K2tu-PA did not produce systemic proteolysis and bleeding. The highest dose of K2tu-PA produced a statistically significant more intense systemic proteolysis and bleeding than the highest dose of rt-PA.This study demonstrates that bolus doses of K2tu-PA and rt-PA produce a similar degree of fibrinolysis. Due to its longer half-life K2tu-PA is more efficient than rt-PA in inhibiting accretion of new fibrin on the thrombi during thrombolysis so that the thrombus size is more efficiently reduced. As a consequence the concomitant use of heparin might not be necessary. The potential increased risk of bleeding with bolus of high doses of K2tu-PA has to be seen in view of the advantage of avoiding the concomitant use of heparin.

Clot penetration and fibrin binding of amediplase, a chimeric plasminogen activator (K2tu-PA)

“…The effect of the presence of kringle 2 on the fibrinolytic properties of K 2 tu-PA has been extensively described by Colucci et al (5). Animal studies in dogs (coronary thrombosis model) and rabbits (jugular vein thrombosis model and ear bleeding model) indicated that K 2 tu-PA has potent thrombolytic properties with a prolonged plasma half-live, which may allow administration via an intravenous bolus injection (6)(7)(8). After a dose finding study in patients with acute myocardial infarction (9), a phase II trial was performed in 241 patients with acute myocardial infarction, which showed a high TIMI grade 3 flow (58%) and TIMI grade 2 and 3 flow (76%) at 60 min (10).…”

Serine-proteases as plasminogen activators in terms of fibrinolysis

“…Amediplase (K2tu‐PA, CGP 42935, MEN 9036) consists of the kringle 2 domain of t‐PA (1–3 + 176–275) and the protease domain of single‐chain urokinase (159–411) [88] . The 356‐amino‐acid enzyme has a molecular weight of about 39.9 kDa and is produced in CHO cells [89] .…”

“…The 356‐amino‐acid enzyme has a molecular weight of about 39.9 kDa and is produced in CHO cells [89] . The half‐life after a bolus injection in rabbits was over 30 min [88] . A study that compared the in‐vitro clot penetration in relation to the fibrin binding of amediplase with alteplase showed 10 times less lysis activity in an internal clot lysis model (injection in the clot) but it was similar in an external model showing that amediplase binding to fibrin was significantly lower, but it had a better clot penetration [90] .…”

Amplification and expression of recombinant genes in serum‐independent Chinese hamster ovary cells

“…activator Alter activation in vitro with plasmin, no significant difference was seen in the specific enzymatic activity between purified SSF3-derived K2tu-PA and material derived from transfected standard CHO(dhfr-) cells [8][9][10]. However, 20-60% of the K2tu-PA from the conditioned medium of transfected CHO(dhfr ) cells (if cultured without addition of protease inhibitors) was found to be in the two-chain form and was already enzymatically active prior to deliberate activation with plasmin.…”

“…This protein is currently investigated in a clinical trial as treatment for acute myocardial infarction. It is composed of the Kringle-2 domain of human tissue-type plasminogen activator (t-PA) attached to the serine protease domain of the human urokinase-type plasminogen activator (u-PA) [7][8][9][10][11]. Like in the native plasminogen activators, proteolytic cleavage converts the otherwise inactive proenzyme K2tu-PA into an active plasrninogen activator.…”