Synergistic Effect on Thrombolysis of Sequential Infusion of Tissue-Type Plasminogen Activator (t-PA) Single-Chain Urokinase-Type Plasminogen Activator (scu-PA) and Urokinase in the Rabbit Jugular Vein Thrombosis Model

1987 ◽  
Vol 58 (03) ◽  
pp. 943-946 ◽  
Author(s):  
D Collen ◽  
J M Stassen ◽  
F De Cock
Keyword(s):  
Plasminogen Activator ◽  
Jugular Vein ◽  
Blood Clot ◽  
Sequential Therapy ◽  
Quantitative Model ◽  
Tissue Type ◽  
Single Chain ◽  
Jugular Vein Thrombosis ◽  
Type Plasminogen Activator

SummaryIn a quantitative model of thrombolysis, consisting of rabbits with a 125T-fibrin labeled blood clot in the jugular vein, simultaneous intravenous infusion over 4 hours of t-PA and scu-PA or of t-PA and urokinase had a significantly greater (p <0.01) thrombolytic effect than could be anticipated on the basis of the added effects of each agent alone. In order to further investigate the mechanism of this in vivo synergism, recombinant t-PA (rt-PA) and scu-PA in synergistic amounts were infused: 1) simultaneously over 4 hours, 2) rt-PA over 1 hour, then 15 min later scu-PA over 2 hours and 3) scu-PA over 1 hour, then 15 min later rt-PA over 2 hours. Simultaneous infusion of 0.1 mg/kg rt-PA and 0.2 mg/kg scu-PA gave 48°2 percent thrombolysis (mean ° SEM, n = 5) and of 0.2 mg/kg rt-PA and 0.4 mg/kg scu-PA 67°5 percent (n = 5). When these infusions were given sequentially, rt-PA followed by scu-PA gave 32 °5 (n = 4) and 49 °8 (n = 4) percent lysis, but scu-PA followed by rt-PA yielded only 14° 1 (n = 4) and 21 ° 1 (n = 4) percent lysis, indicating that synergism occurs when rt-PA is followed by scu-PA but not when scu-PA is followed by rt-PA. In order to investigate the hypothesis that rt-PA predigests the clot resulting in more efficient plasminogen activation by scu-PA at the clot surface, partial thrombolysis was induced by injection of urokinase. Subsequent infusion of 0.4 mg/ kg of scu-PA did however not result in more thrombolysis than expected for additive effects. Infusion of 0.5 mg/kg of urokinase followed by 0.1 mg/kg rt-PA was not synergistic (24 °3 percent lysis, n = 3) whereas lysis by rt-PA followed by urokinase was 34°3 percent (p <0.1, n = 3).Sequential therapy with rt-PA followed by scu-PA might constitute an alternative to simultaneous infusion of synergistic thrombolytic agents.

ON THE MECHANISM OF THE IN VIVO SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR (t-PA) AND SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (scu-PA)

1987 ◽  
Author(s):  
J M Stassen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Rabbit Model ◽  
Sequential Therapy ◽  
Tissue Type ◽  
Single Chain ◽  
Molar Ratios ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

t-PA and scu-PA, in molar ratios between 1:4 and 4:1 do not act synergically in vitro (Thromb. Haemost. 56,35,1986) but display marked synergism in a rabbit model (Circulation 74, 838, 1986) and in man (Am. Heart J. 112, 1083, 1986). To investigate the mechanism of in vivo synergism in the rabbit model (J. Clin. Invest. 71, 368, 1983), t-PA and scu-PA were infused 1) simultaneously over 4 hrs, 2) t-PA over 1 hr, then 15 min later scu-PA over 2 hrs and 3) scu-PA over 1 hr, then 15 min later t-PA over 2 hrs.Significant synergism on thrombolysis is observed when t-PA and scu-PA are infused simultaneously or when t-PA is followed by scu-PA but not when scu-PA is followed by t-PA. These results suggest that low dose t-PA induces some plasminogen activation, sufficient to partially degrade fibrin, exposing COOH-terminal lysines with high affinity for plasminogen (Eur. J. Biochem. 140, 513, 1984). scu-PA might then activate surface-bound Glu-pla-minogen more efficiently.Sequential therapy with t-PA (or any other agent which "predigests" the thrombus), followed by scu-PA might constitute an alternative to simultaneous infusion of synergistic thrombolytic agents.

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

1992 ◽  
Vol 68 (03) ◽  
pp. 331-335 ◽  
Author(s):  
Giancarlo Agnelli ◽  
Claudia Pascucci ◽  
Mario Colucci ◽  
Giuseppe G Nenci ◽  
Antonio Mele ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Single Chain ◽  
Jugular Vein Thrombosis ◽  
Thrombolytic Activity ◽  
Thrombosis Model ◽  
Type Plasminogen Activator ◽  
Kringle 2 ◽  
Higher Doses

SummaryThe 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 <0.01). The thrombolytic activity of K2tu-PA was significantly higher than rt-PA at the two higher doses (p <0.01). Both K2tu-PA and rt-PA produced a statistically significant reduction of fibrinogen, α2-antiplasmin and plasminogen 3 h after the start of the infusions of the two higher doses. No statistically significant differences between K2tu-Pa and rt-PA were observed. Concomitant with the lower thrombolytic activity, the systemic proteolytic effects of FK2tu-PA and scu-PA were less pronounced. We conclude that the two HPAs we tested are effective thrombolytic agents. K2tu-PA deserves particular attention in future experiments.

EVIDENCE FOR SYNERGISM BETWEEN TISSUE-TYPE PLASMINOGEN ACTIVATOR AND SINGLE CHAIN UROKINASE ON IN VITRO PLASMA CLOT LYSIS

1987 ◽  
Author(s):  
R S Rappaport ◽  
M R Blume ◽  
R L Vogel ◽  
M H Levner ◽  
P P Hung
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Molar Ratios ◽  
Type Plasminogen Activator

There is mounting evidence from animal models and the clinic that combination thrombolytic therapy with tissue-type plasminogen activator (tPA) and single chain urokinase (scuPA) is synergistic. Yet, efforts to demonstrate synergism between these two plasminogen activators in vitro have met with discordant results. Collen et al (Thromb. Haemostasis, 56:35, 1986) reported an absence of synergism between these two agents on clot lysis in an in vitro plasma milieu when they were evaluated at molar ratios of 1:4 (tPA:scuPA and vice versa). Gurewich and Pannell (Thromb. Res., 44:217, 1986), however, reported a synergistic effect on fibrin-specific clot lysis in vitro when the agents were combined in concentrations exceeding molar ratios of 1:4 (tPA:scuPA). Here, we present evidence that synergism between tPA and scuPA may be demonstrated in vitro provided that the molar ratio of tPA to scuPA exceeds 1:4 and that the concentration of clot bound or unbound tPA is minimized. In order to achieve this experimental condition, the standard in vitro plasma clot lysis assay was modified. Human plasma clots were incubated first for a short time in plasma containing varying amounts of tPA. After incubation, the clots were washed thoroughly and reimmersed in plasma alone or in plasma containing varying amounts of scuPA or tPA. Under these conditions, lysis proceeded at a greater rate and to a greater extent when tPA clots were immersed in plasma containing an appropriate amount of scuPA than when they were immersed in plasma alone or in plasma containing appropriate amounts of tPA. Lysis of untreated clots or clots exposed first to scuPA and then to plasma containing varying amounts of scuPA proceeded far less efficiently with a characteristic lag. The enhanced lysis produced by tPA and scuPA obeyed the classical definition of synergy: the same biological effect can be obtained with two drugs together at algebraic fractional combinations of less than 1 (Berenbaum, M.C., Clin. Exp. Immunol., 28:1-18, 1977). Thus, conditions that more closely mimic the in vivo situation resulting from a bolus injection of tPA followed by infusion with scuPA, may provide a system for duplication of in vivo synergism in. vi tro and investigation of the mechanism thereof.

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)

1993 ◽  
Vol 70 (02) ◽  
pp. 294-300 ◽  
Author(s):  
Giancarlo Agnelli ◽  
Claudia Pascucci ◽  
Giuseppe G Nenci ◽  
Antonio Mele ◽  
Rolf Bürgi ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Similar Degree ◽  
Single Chain ◽  
Jugular Vein Thrombosis ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Increased Risk ◽  
Plasma Half Life

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.

Potentiation by Heparin Fragments of Thrombolysis Induced with Human Tissue-Type Plasminogen Activator or Human Single-Chain Urokinase-Type Plasminogen Activator

1987 ◽  
Vol 58 (03) ◽  
pp. 947-950 ◽  
Author(s):  
J M Stassen ◽  
I Juhan-Vague ◽  
M C Alessi ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
San Francisco ◽  
Human Tissue ◽  
Factor Xa ◽  
Tissue Type ◽  
Single Chain ◽  
Jugular Vein Thrombosis ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryThe effect of heparin and of two low molecular weight (low M r) fractions of heparin on thrombolysis with recombinant human tissue-type plasminogen activator (rt-PA, Genentech Inc., So. San Francisco, CA) or human single chain urokinase-type plasminogen activator (scu-PA, Sandoz AG, Basle, Switzerland) was measured in a rabbit jugular vein thrombosis model. Four bolus injections of 200 anti-Factor Xa units/kg body weight of heparin (Liquemine, Hoffmann-La Roche, Basle, Switzerland), of 90 units/kg of CY 216 (Choay, Paris, France) or of 90 units/kg of CY 222 (Choay, Paris, France) were given intravenously, immediately after the start of the infusion of rt-PA or scu-PA and at hourly intervals during their intravenous infusion over 4 hours. The bolus injections resulted in anti-Factor Xa levels in plasma of 5.7 ± 1.2 units/ml just before the repeat bolus injections of heparin with corresponding values of 3.9 ± 0.2 units/ml for CY 216 and 1.6 ± 0.2 units/ml for CY 222.Thrombolysis with 0.25 mg/kg rt-PA was 36 ± 1 percent (n = 9) in the absence of anticoagulant, 40 ± 1 percent (n = 7, p <0.05) in the presence of heparin, 49 ± 5 percent (n = 7, p <0.02) with CY 216 and 62 ± 5 percent (n = 7, p <0.01) with CY 222. Thrombolysis with 0.5 mg/kg scu-PA was 23 ± 1 percent (n = 4) without heparin, and increased to 24 ± 1 percent (n = 4, p >0.1) with heparin, to 32 ± 2 percent (n = 4, p <0.01) with CY 216 and to 33 ± 3 percent (n = 4, p <0.01) with CY 222.It is concluded that, at these high doses, the two low M r heparin fractions CY 216 and CY 222, potentiate thrombolysis by rt-PA and scu-PA in this animal model.

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