Fibrin-specificity of a Plasminogen Activator Affects the Efficiency of Fibrinolysis and Responsiveness to Ultrasound: Comparison of Nine Plasminogen Activators In Vitro

1999 ◽  
Vol 81 (04) ◽  
pp. 605-612 ◽  
Author(s):  
Dmitry V. Sakharov ◽  
Marrie Barrett-Bergshoeff ◽  
Rob T. Hekkenberg ◽  
Dingeman C. Rijken
Keyword(s):  
Thrombolytic Therapy ◽  
Plasminogen Activator ◽  
Tissue Type ◽  
Bolus Administration ◽  
High Frequency Ultrasound ◽  
Single Chain ◽  
Response Curves ◽  
Maximal Speed ◽  
Frequency Ultrasound

SummaryIn a number of cases, thrombolytic therapy fails to re-open occluded blood vessels, possibly due to the occurrence of thrombi resistant to lysis. We investigated in vitro how the lysis of hardly lysable model thrombi depends on the choice of the plasminogen activator (PA) and is accelerated by ultrasonic irradiation. Lysis of compacted crosslinked human plasma clots was measured after addition of nine different PAs to the surrounding plasma and the effect of 3 MHz ultrasound on the speed of lysis was assessed.Fibrin-specific PAs showed bell-shaped dose-response curves of varying width and height. PAs with improved fibrin-specificity (staphylokinase, the TNK variant of tissue-type PA [tPA], and the PA from the saliva of the Desmodus rotundus bat) induced rapid lysis in concentration ranges (80-, 260-, and 3,500-fold ranges, respectively) much wider than that for tPA (a 35-fold range). However, in terms of speed of lysis, these three PAs exceeded tPA only slightly. Reteplase and single-chain urokinase were comparable to tPA, whereas two-chain urokinase, anistreplase, and streptokinase were inferior to tPA. In the case of fibrin-specific PAs, ultrasonic treatment accelerated lysis about 1.5-fold. For streptokinase no acceleration was observed. The effect of ultrasound correlated with the presence of plasminogen in the outer plasma, suggesting that it was mediated by facilitating the transport of plasminogen to the surface of the clot.In conclusion, PAs with improved fibrin-specificity induce rapid lysis of plasminogen-poor compacted plasma clots in much wider concentration ranges than tPA. This offers a possibility of using single-or double-bolus administration regimens for such PAs. However, it is not likely that administration of these PAs will directly cause a dramatic increase in the rate of re-opening of the occluded arteries since they are only moderately superior to tPA in terms of maximal speed of lysis. Application of high-frequency ultrasound as an adjunct to thrombolytic therapy may increase the treatment efficiency, particularly in conjunction with fibrin-specific PAs.

Influence of Intrinsic and Extrinsic Plasminogen upon the Lysis of Thrombi In Vitro

1991 ◽  
Vol 66 (06) ◽  
pp. 672-677 ◽  
Author(s):  
N Nishino ◽  
V V Kakkar ◽  
M F Scully
Keyword(s):  
Plasminogen Activator ◽  
Dose Response ◽  
Tissue Type ◽  
Single Chain ◽  
Response Curves ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Dose Response Curves

SummaryWhen the rate of lysis of artificial thrombi (prepared from plasma or whole blood) was expressed according to the concentration of tissue type plasminogen activator (t-PA) or single chain urokinase type plasminogen activator (sc-uPA) then bell-shaped dose response curves were obtained, low rates being observed at concentrations of activator greater than 500 units/ml. Bell-shaped dose response curves were not observed for rate of lysis of artificial thrombi over the concentrations of streptokinase tested (SK) or for the lysis of plasma gel clots by any of the activators tested. Further investigation indicated that the preponderant mechanism for dissolution of thrombi at 500 units/ml of t-PA was by activation of the plasminogen within the thrombus (intrinsic) since the plasminogen present in the plasma perfusing the thrombus (extrinsic) rapidly became depleted. On the other hand, at 50 units/ml t-PA the lysis was observed to be due preponderantly to the action of plasmin arising from extrinsic rather than intrinsic plasminogen. If "plasminogen enriched" thrombi were prepared in the presence of Lys plasminogen (Lys-Plg) faster rates of lysis occurred and bell-shaped biometric curves were not observed.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

Absence of Synergism Between Tissue-Type Plasminogen Activator (t-PA), Single-Chain UrokinaseType Plasminogen Activator (scu-PA) and Urokinase on Clot Lysis in a Plasma Milieu In Vitro

1986 ◽  
Vol 56 (01) ◽  
pp. 035-039 ◽  
Author(s):  
D Collen ◽  
F De Cock ◽  
E Demarsin ◽  
H R Lijnen ◽  
D C Stump
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Dose Response ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryA potential synergic effect of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (scuPA) or urokinase on clot lysis was investigated in a whole human plasma system in vitro. The system consisted of a human plasma clot labeled with 125I-fibrinogen, immersed in titrated whole human plasma, to which the thrombolytic agents were added. Clot lysis was quantitated by measurement of released 125I, and activation of the fibrinolytic system in the surrounding plasma by measurements of fibrinogen and α2-antiplasmin.t-PA, scu-PA and urokinase induced a dose-dependent and time-dependent clot lysis; 50 percent lysis after 2 h was obtained with 5 nM t-PA, 20 nM scu-PA and 12 nM urokinase. At these concentrations no significant activation of the fibrinolytic system in the plasma was observed with t-PA and scu-PA, whereas urokinase caused significant α2-antiplasmin consumption and concomitant fibrinogen degradation. The shape of the dose-response curves was different; t-PA and urokinase showed a log linear dose-response whereas that of scu-PA was sigmoidal.

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.

ANTITHROMBOTIC EFFECTS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR AT PHYSIOLOGICAL BLOOD LEVELS

1987 ◽  
Author(s):  
W Witt ◽  
B Baldus ◽  
P Donner
Keyword(s):  
Plasminogen Activator ◽  
Plasma Levels ◽  
Dose Range ◽  
Tissue Type ◽  
Clot Lysis ◽  
Blood Levels ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

Effective thrombolysis in human patients and experimental animals by tissue-type plasminogen activator (t-PA) usually requires t-PA plasma levels in the microgram range. Compared to that physiological plasma levels of t-PA are about 100 - 1000 times lower. To investigate the effects of t-PA at physiological blood levels rat studies were performed in vitro and in vivo employing highly purified recombinant single-chain t-PA (sct-PA: 500,000 IU/mg).t-PA activity in rat whole blood as assessed by dilute blood clot-lysis time (DBC-LT) was increased by addition of sct-PA as low as 3 ng/ml (20 % decrease in DBC-LT). Injection of brady-kinin 10, 100 and 1000 μg/kg i.v. shortened DBC-LT to 54, 23, and 10 % of controls corresponding to the effect of about 10, 30, and 100 ng/ml sct-PA added in vitro. Infusion of sct-PA 15 - 450 μg/kg/h i.v. shortened DBC-LT ex vivo dose-dependently by 20 - 90 % at steady state levels (n = 5). In the same dose range sct-PA inhibited thrombus formation along a silk thread introduced into an arteriovenous shunt in anaesthetized rats. The reduction in thrombus dry weight was dose-dependent amounting to 33 - 67 % of preapplication values (n = 5 - 8) at 15 - 450 μg/kg/h i.v. sct-PA. Already 50 μg/kg/h sct-PA corresponding to a sct-PA activity of about 15 ng/ml displayed a significant (a = 0.05) effect in this model.The results of this study suggest that t-PA present at physiological resting or activation (bradykinin) levels during acute clot formation may have potent antithrombotic efficacy. This study provides further evidence for the importance of a balance coagulation-fibrinolysis which can be influenced on both sides towards thrombophilia as well as to achieve antithrombotic therapy, e.g. by elevating plasma fibrinolytic activity with low-dose t-PA treatment or with drugs which stimulate the endogenous fibrinolytic potential.

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.

Characterization of Domain Deletion and/or Duplication Mutants of a Recombinant Chimera of Tissue-Type Plasminogen Activator and Urokinase-Type Plasminogen Activator (rt-PA/u-PAI)

1990 ◽  
Vol 64 (01) ◽  
pp. 053-060 ◽  
Author(s):  
L Nelles ◽  
H R Lijnen ◽  
A Van Nuffelen ◽  
E Demarsin ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Catalytic Domain ◽  
Tissue Type ◽  
Clot Lysis ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Specific Activities

SummaryChimeric molecules comprising the A-chain of tissue-type plasminogen activator (t-PA) and the catalytic domain of urokinase-type plasminogen activator (u-PA) have intact enzymatic characteristics of u-PA, but only partial fibrin-binding properties of t-PA (Nelles et al., J Biol Chem 1987; 262: 10855–62). The following domain deletion and/or duplication mutants of such a t-PA/u-PA chimera were constructed, purified and charactertzed: rt-PA-ΔFE∇/u-PA, with deletion of the finger-like (F) and epidermal growth factor-like (E) domains, rt-PA-ΔK1∇K2/u-PA, with kringle 1 (K1) replaced by a second copy of kringle 2 (K2), and rt-PA-ΔFEK1∇K2/u-PA, with F and E domain deletions in rt-PAΔK1∇K2/u-PA.The specific activities on fibrin plates of the single-chain (sc) chimeras ranged between 68,000 IU/mg for rt-PA-ΔK1∇K2/scu-PA and 200,000 IU/mg for rt-PA-ΔFEK1∇K2/scu-PA, as compared to L20,000 IU/mg for rscu-PA. The specific activities of their plasmin-generated two-chain (tc) derivatives ranged between 120,000 IU/mg for rt-PA-ΔK1∇K2/tcu-PA and 240,000 IU/mg for rt-PA-ΔFEK1∇K2/tcu-PA, as compared to 100,000 IU/mg for rtcu-PA. All two-chain chimeras activated plasminogen following Michaelis-Menten kinetics, with catalytic efficiencies between 0.072 μM−1s−1 for rt-PA-ΔK1∇K2/tcu-pA and 0.081 pM−1 s−1 for rt-PA-ΔFEK1∇K2/tcu-PA, as compared to 0.088 μM−1 s−1 for rtcu-PA. CNBr-digested fibrinogen enhanced the initial rate of plasminogen activation by a factor 2.2 to 6.2, as compared to 4.9 for rtcu-PA. The fibrin-affinity of the chimeras decreased in the order rt-PA > rt-PA-ΔK1∇K2/u-PA > u-PA and that for lysine in the order rt-PA-ΔFEK1∇K2/u-PA > > t-PA/u-PA ⩽ st-PA > rt-PA-ΔFE/u-PA > u-PA. All single-chain plasminogen activators caused a time and concentration-dependent clot lysis in an in vitro plasma clot lysis system, with equi-effective doses (causin g 50% clot lysis in 2 h) ranging between 0.53 and 0.90 μg/ml, as compared to 1 .7 μg/ml for rscu-PA, and were associated with comparable residual fibrinogen levels of approximately 80%.Thus, substitution of K1 by a second copy of K2 in the chimeric protein t-PA/u-PA enhances the affinity for both fibrin and lysine significantly and improves the fibrinolytic potency in an in vitro clot lysis system marginally.

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