Substitution of Serine or Threonine at Position 473 of Tissue-type Plasminogen Activator Increases its Stability in Plasma

1996 ◽  
Vol 75 (01) ◽  
pp. 107-112 ◽  
Author(s):  
Murtaza Alibhai ◽  
Alice M Chow ◽  
Nicholas F Paoni
Keyword(s):  
Clearance Rate ◽  
Order Rate Constant ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Plasma Clot ◽  
Double Mutation ◽  
Lysis Activity ◽  
Inhibitor Resistance

SummaryInactivation by slow acting inhibitors in plasma is of little consequence for thrombolysis with wild type t-PA, since it is rapidly cleared from the blood stream and constantly replenished through infusion. However, it becomes increasingly important as the clearance rate of t-PA is reduced, through mutagenesis, to enable the molecule to be long acting and administered by a single bolus injection. The substitution of serine for alanine at position 473 substantially reduced the slow inactivation that occurs at pharmacological levels of t-PA in plasma. Approximately 70% of the activity of A473S remained after 4 h incubation in human plasma compared to approximately 25% for wild type t-PA. Wild type t-PA and A473S showed the same stability in alpha-2-antiplasmin depleted plasma, indicating that the resistance of A473S to inactivation is a result of reduced reactivity towards alpha-2-antiplasmin, the primary slow acting inhibitor of t-PA. The second order rate constant for the inactivation of A473S by purified alpha-2-antiplasmin was approximately 4 fold less than that of wild type t-PA, which is consistent with the results obtained in plasma. Substitution of threonine at position 473 also produced inhibitor resistance, but glycine did not. The substitution of charged or bulky residues at position 473 destroyed enzymatic activity. The mechanism of inhibitor resistance for A473S and A473T appears to be a reduced reactivity towards substrates with arginine at the PI position. The A473S mutation adds well to T103N, a mutation that causes an approximate 9 fold reduction in the clearance rate of t-PA. The double mutation variant, T103N, A473S had normal plasma clot lysis activity, and was stable in plasma over a 4 h incubation period at 37° C in vitro.

scholarly journals 3K3A-Activated Protein C Variant Does Not Interfere With the Plasma Clot Lysis Activity of Tenecteplase

Stroke ◽  
2020 ◽  
Vol 51 (7) ◽  
pp. 2236-2239
Author(s):  
Purba Mukherjee ◽  
Patrick Lyden ◽  
José A. Fernández ◽  
Thomas P. Davis ◽  
Kent E. Pryor ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Protein C ◽  
Activated Protein C ◽  
In Vitro Study ◽  
Tissue Type ◽  
Clot Lysis ◽  
Bolus Administration ◽  
Plasma Clot ◽  
Lysis Activity

Background and Purpose: A recombinant engineered variant of APC (activated protein C), 3K3A-APC, lacks anticoagulant properties (<10%) while preserving APCs anti-inflammatory, anti-apoptotic, and neuroprotective functions and is very promising in clinical trials for ischemic stroke. Therapeutic intervention with single bolus administration of the third-generation tPA (tissue-type plasminogen activator), tenecteplase, is anticipated to be widely adopted for treatment of acute ischemic stroke. 3K3A-APC is well-tolerated in stroke patients dosed with alteplase, and in vitro studies show 3K3A-APC does not interfere with alteplase-induced clot lysis. The purpose of this in vitro study was to assess the influence of 3K3A-APC on tenecteplase-induced clot lysis. Methods: Tenecteplase-mediated lysis of thrombin generated plasma clots of human normal pooled plasma was monitored in the presence of varying doses of 3K3A-APC. The effects on fibrinolysis by tenecteplase and alteplase were compared. Results: The presence of 3K3A-APC shortened the time for clot lysis induced by tenecteplase at very low levels but not at higher therapeutic concentrations of tenecteplase. Comparisons of alteplase-mediated clot lysis to tenecteplase clot lysis showed that both thrombolytic agents behaved similarly in the presence of 3K3A-APC. Conclusions: These results indicate that 3K3A-APC does not interfere with tenecteplase’s clot lysis function.

scholarly journals Biochemical and biologic properties of rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistant to plasminogen activator inhibitor-1

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 417-429
Author(s):  
XK Li ◽  
HR Lijnen ◽  
L Nelles ◽  
B Van Hoef ◽  
JM Stassen ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Plasma Clot ◽  
Type Plasminogen Activator ◽  
Dose Dependent

A mutant of recombinant tissue-type plasminogen activator (rt-PA), obtained by deletion of residues Lys296 to Gly302 [rt-PA del(K296- G302)], was previously shown to be resistant to inhibition by plasminogen activator inhibitor-1 (PAI-1) (Madison et al, Nature 339:721, 1989). This mutant was obtained by expression of its cDNA in Chinese hamster ovary cells and purification to homogeneity from conditioned cell culture medium. It was obtained as a single chain molecule with amidolytic activity, specific fibrinolytic activity, and binding to fibrin and lysine, which were comparable or somewhat lower than those of wild-type rt-PA obtained in the same expression system. The plasminogen-activating potential of rt-PA del(K296-G302) in the presence of CNBr-digested fibrinogen was about twofold lower than that of wild-type rt-PA. The inhibition rate of rt-PA del(K296-G302) by recombinant PAI-1 (rPAI-1) was more than 500-fold lower than that of wild-type rt-PA. In a human plasma milieu in vitro, rt-PA del(K296- G302) induced dose-dependent lysis of a 125I-fibrin-labeled plasma clot; equi-effective concentrations (causing 50% clot lysis in 2 hours) were 0.28 micrograms/mL and 0.36 micrograms/mL for mutant and wild-type rt-PA, respectively. In this system, addition of rPAI-1 to the plasma resulted in a concentration-dependent reduction of the fibrinolytic potency of rt-PA del(K296-G302) and of rt-PA; a 50% reduction required 2.4 micrograms/mL and 0.15 micrograms/mL rPAI-1, respectively. Continuous infusion of mutant or wild-type rt-PA over 60 minutes in hamsters with a 125I-labeled plasma clot in the pulmonary artery resulted in dose-dependent clot lysis, with a thrombolytic potency (percent clot lysis per milligram of compound administered per kilogram of body weight) and a specific thrombolytic activity (percent clot lysis per microgram per milliliter steady state rt-PA-related antigen level in plasma) that were not significantly different. Bolus injection in hamsters of 1 mg/kg rPAI-1 followed by bolus injection of 1 mg/kg rt- PA del(K296-G302) or wild-type rt-PA resulted in neutralization of the thrombolytic potency of wild-type rt-PA, while the mutant retained approximately half of its thrombolytic potency. These results indicate that rt-PA del(K296-G302), with a known resistance to inhibition by rPAI-1 in purified systems, maintains this property both in a plasma milieu in vitro and in an experimental animal model of thrombolysis in vivo.(ABSTRACT TRUNCATED AT 400 WORDS).

scholarly journals Biochemical and biologic properties of rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistant to plasminogen activator inhibitor-1

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 417-429 ◽  
Author(s):  
XK Li ◽  
HR Lijnen ◽  
L Nelles ◽  
B Van Hoef ◽  
JM Stassen ◽  
...  
Keyword(s):  
Plasminogen Activator ◽  
Plasminogen Activator Inhibitor ◽  
Tissue Type ◽  
Clot Lysis ◽  
Wild Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
Plasma Clot ◽  
Type Plasminogen Activator ◽  
Dose Dependent

Abstract A mutant of recombinant tissue-type plasminogen activator (rt-PA), obtained by deletion of residues Lys296 to Gly302 [rt-PA del(K296- G302)], was previously shown to be resistant to inhibition by plasminogen activator inhibitor-1 (PAI-1) (Madison et al, Nature 339:721, 1989). This mutant was obtained by expression of its cDNA in Chinese hamster ovary cells and purification to homogeneity from conditioned cell culture medium. It was obtained as a single chain molecule with amidolytic activity, specific fibrinolytic activity, and binding to fibrin and lysine, which were comparable or somewhat lower than those of wild-type rt-PA obtained in the same expression system. The plasminogen-activating potential of rt-PA del(K296-G302) in the presence of CNBr-digested fibrinogen was about twofold lower than that of wild-type rt-PA. The inhibition rate of rt-PA del(K296-G302) by recombinant PAI-1 (rPAI-1) was more than 500-fold lower than that of wild-type rt-PA. In a human plasma milieu in vitro, rt-PA del(K296- G302) induced dose-dependent lysis of a 125I-fibrin-labeled plasma clot; equi-effective concentrations (causing 50% clot lysis in 2 hours) were 0.28 micrograms/mL and 0.36 micrograms/mL for mutant and wild-type rt-PA, respectively. In this system, addition of rPAI-1 to the plasma resulted in a concentration-dependent reduction of the fibrinolytic potency of rt-PA del(K296-G302) and of rt-PA; a 50% reduction required 2.4 micrograms/mL and 0.15 micrograms/mL rPAI-1, respectively. Continuous infusion of mutant or wild-type rt-PA over 60 minutes in hamsters with a 125I-labeled plasma clot in the pulmonary artery resulted in dose-dependent clot lysis, with a thrombolytic potency (percent clot lysis per milligram of compound administered per kilogram of body weight) and a specific thrombolytic activity (percent clot lysis per microgram per milliliter steady state rt-PA-related antigen level in plasma) that were not significantly different. Bolus injection in hamsters of 1 mg/kg rPAI-1 followed by bolus injection of 1 mg/kg rt- PA del(K296-G302) or wild-type rt-PA resulted in neutralization of the thrombolytic potency of wild-type rt-PA, while the mutant retained approximately half of its thrombolytic potency. These results indicate that rt-PA del(K296-G302), with a known resistance to inhibition by rPAI-1 in purified systems, maintains this property both in a plasma milieu in vitro and in an experimental animal model of thrombolysis in vivo.(ABSTRACT TRUNCATED AT 400 WORDS).

Recombinant Variants of Tissue-Type Plasminogen Activator Containing Amino Acid Substitutions in the Finger Domain

1992 ◽  
Vol 68 (06) ◽  
pp. 672-677 ◽  
Author(s):  
Hitoshi Yahara ◽  
Keiji Matsumoto ◽  
Hiroyuki Maruyama ◽  
Tetsuya Nagaoka ◽  
Yasuhiro Ikenaka ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Half Life ◽  
Tissue Type ◽  
Clot Lysis ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator

SummaryTissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7–9, 10–14, 15–19, 28–33, and 37–42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37–42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37–42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37–42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37–42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.

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.

scholarly journals A monoclonal antibody preventing binding of tissue-type plasminogen activator to fibrin: useful to monitor fibrinogen breakdown during t-PA infusion

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1482-1487 ◽  
Author(s):  
P Holvoet ◽  
HR Lijnen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activation ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Additional Support

Abstract One (MA-1C8) of 36 monoclonal antibodies obtained by fusion of P3X63- Ag8–6.5.3 myeloma cells with spleen cells of mice immunized with purified human tissue-type plasminogen activator (t-PA) blocked the activity of t-PA on fibrin plates but not on chromogenic substrates. MA- 1C8 at a concentration of 200 micrograms/mL inhibited plasma clot lysis and binding of t-PA to the clot. MA-1C8 had no influence on the activation of plasminogen by t-PA, which obeys Michaelis-Menten kinetics with Km = 105 mumol/L and kcat = 0.05 s-1; however, it abolished the influence of CNBr-digested fibrinogen on Km. These findings confirm that the stimulatory effect of fibrin on the activation of plasminogen by t-PA is mediated by binding of t-PA to fibrin and provide additional support for the kinetic model. Addition of t-PA to pooled fresh human plasma to a concentration of 5 micrograms/mL resulted in extensive fibrinogen breakdown after incubation for one hour at 37 degrees C or during storage at -20 degrees C for one day. In both instances, fibrinogen degradation was completely prevented by addition of MA-1C8 to a concentration of 200 micrograms/mL of plasma. MA-1C8 also effectively prevented in vitro fibrinogen degradation and in vitro plasminogen activation in plasma samples obtained during infusion of recombinant t-PA in patients with thromboembolic disease. Thus, MA-1C8 is a useful tool for discriminating between in vivo and in vitro fibrinolysis during thrombolytic therapy with t-PA.

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

2004 ◽  
Vol 91 (01) ◽  
pp. 52-60 ◽  
Author(s):  
Marrie Barrett-Bergshoeff ◽  
A. F. H. Jie ◽  
Marco Criscuoli ◽  
Dmitri Sakharov ◽  
Dingeman Rijken
Keyword(s):  
Plasminogen Activator ◽  
Aminocaproic Acid ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Plasma System ◽  
Plasma Clot ◽  
Lysis Activity ◽  
Epsilon Aminocaproic Acid ◽  
Kringle 2

SummaryAmediplase (K2tu-PA) is a hybrid plasminogen activator, consisting of the kringle 2 domain of alteplase and the protease domain of urokinase. The objective of this study was to determine the in vitro clot penetration of amediplase in relation to its fibrin binding and to compare the properties with those of alteplase.The clot lysis activity of amediplase in internal clot lysis models (both purified system and plasma system) was about 10 times less than that of alteplase. The clot lysis activity of amediplase in an external clot lysis model (plasma system) was similar to that of alteplase at therapeutic concentrations around 1 µg/ml. The fibrin-clot binding properties of amediplase and alteplase were studied in a purified system as well as in a plasma system. In both systems amediplase bound to fibrin although to a significantly lower extent than alteplase. The binding of amediplase or alteplase did not increase during plasmin-mediated degradation of fibrin. The binding of amediplase was fully inhibited by epsilon-aminocaproic acid, indicating that the observed binding was specific and occurred via the lysine binding site in the kringle of amediplase. Clot penetration was studied during pressure-driven fluid permeation using syringes containing plasma clots. Amediplase was able to enter the clot without significant hindrance, while alteplase was concentrated on the top of the plasma clot and hardly entered into the inner parts of the clot. Diffusion-driven clot penetration was studied during clot lysis using confocal microscopy. Alteplase was detected on or close to the clot surface, while two-chain urokinase, which has no affinity to fibrin, was also detected deep inside the clot. Amediplase showed a penetration behaviour, which was distinct from that of alteplase and similar to that of two-chain urokinase.We concluded that the fibrin binding of amediplase is moderate and does not hinder clot penetration under permeationdriven or diffusion-driven transport conditions. Enhanced clot penetration, especially in large clots, could allow a more efficient lysis during thrombolytic therapy.

Biochemical, Thrombolytic and Pharmacokinetic Properties of rt-PA P47G, K49N, a Substitution Variant of Human Tissue-Type Plasminogen Activator

1992 ◽  
Vol 67 (04) ◽  
pp. 445-452
Author(s):  
L Nelles ◽  
X-K Li ◽  
I Vanlinthout ◽  
F De Cock ◽  
H R Lijnen ◽  
...  
Keyword(s):  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activation ◽  
Wild Type ◽  
Thrombolytic Activity ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Pharmacokinetic Properties

Summaryrt-PA P47G, K49N, a substitution variant of recombinant human tissue-type plasminogen activator (rt-PA), in which proline at position 47 and lysine at position 49 were replaced by glycine and asparagine respectively, was previously described by Ahem et al. (J Biol Chem 1990; 265: 5540—5) to have an extended in vivo half-life with unaltered in vitro fibrinolytic properties. Because this variant might possess an increased in vivo thrombolytic potency, we have constructed its cDNA, expressed it in Chinese hamster ovary cells and determined its biochemical, thrombolytic and pharmacokinetic properties relative to those of home-made rt-PA and of alteplase (Actilyse®).The specific fibrinolytic activities on fibrin plates were 160,000 ± 17,000, 210,000 ± 88,000 and 460,000 ± 72,000 IU/mg (mean ± SEM) for rt-PA P47G, K49N, rt-PA and alteplase, respectively, while the catalytic efficiencies for plasminogen activation (k 2 K m) in the absence of fibrin were comparable (1.1 to 1.7 × 10-3 μM-1s-1). Fibrin enhanced the rate of plasminogen activation by rt-PA P47G, K49N 100-fold and by both wild-type molecules 390-fold. Binding of the variant rt-PA to fibrin was significantly reduced, but its affinity for lysine-Sepharose was unaltered. In an in vitro clot lysis system, consisting of a radiolabeled human plasma clot submersed in plasma, 50% clot lysis in 2 h required 0.67 ± 0.14 pg/ml rt-PA P47G, K49N, 0.36 ± 0.01 pg/ml rt-PA and 0.17 ± 0.01 pg/ml alteplase, respectively (mean ± SEM; n = 3 or 4). At these doses residual fibrinogen levels at 2 h were in excess of 80%.The thrombolytic properties were examined in a hamster pulmonary embolism model. The thrombolytic potency, expressed as percent lysis per mg activator administered per kg body weight, was 160 ± 28 for rt-PA P47G, K49N, 150 ± 36 for wild-type rt-PA and 310 ± 42 for alteplase. The specific thrombolytic activity, expressed as percent lysis per pg steady-state t-PA-related antigen level per ml plasma, was 49 ± 18% for rt-PA P47G, K49N, 160 ± 49 for rt-PA and 500 ± 79 for alteplase. The clearance rates following bolus injection in hamsters were 0.18 ± 0.02, 1.9 ± 0.2 and 2.1 ± 0.1 ml/min respectively.Thus, the substitution of only two residues in wild-type rt-PA markedly reduces its clearance, but it also significantly alters its specific thrombolytic activity, resulting in a virtually unaltered thrombolytic potency.

Biological and Thrombolytic Properties of Proenzyme and Active Forms of Human Urokinase – I. Fibrinolytic and Fibrinogenolytic Properties in Human Plasma In Vitro of Urokinases Obtained from Human Urine or by Recombinant DNA Technology

1984 ◽  
Vol 52 (01) ◽  
pp. 019-023 ◽  
Author(s):  
C Zamarron ◽  
H R Lijnen ◽  
B Van Hoef ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Recombinant Dna ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Clot Lysis ◽  
Recombinant Dna Technology ◽  
Plasma Clot ◽  
Type Plasminogen Activator ◽  
Intermediate Response

SummaryThe fibrinolytic and fibrinogenolytic properties of recombinant pro-urokinase (Rec-pro-UK) and recombinant urokinase (Rec- UK) obtained by expression of the human urokinase cDNA in E. coli, were compared with those of natural urokinase (Nat-UK) of urinary origin and of tissue-type plasminogen activator (t-PA) in a system, composed of a radioactive human plasma clot immersed in citrated human plasma.The specific fibrinolytic effects of Nat-UK, Rec-pro-UK and Rec-UK were very similar, causing significant clot lysis at concentrations of 100 IU/ml plasma or more. t-PA caused equivalent degrees of clot lysis at 10-fold lower concentrations.Activation of the fibrinolytic system in the plasma (fib- rinogenolysis), was not observed with t-PA in concentrations which induced complete clot lysis within 5 hr (20-30 IU/ml plasma). With Nat-UK and Rec-UK, all concentrations which caused significant clot lysis (100-200 IU/ml plasma) also caused extensive activation of the plasma fibrinolytic system. With Rec- pro-UK an intermediate response was obtained. The highest amounts required for complete clot lysis in 5 hr (200 IU/ml plasma) also caused significant fibrinogenolysis. At intermediate concentrations (50-100 IU/ml), however, significant clot lysis (40-80%) was observed without systemic fibrinolytic activation.

scholarly journals A monoclonal antibody preventing binding of tissue-type plasminogen activator to fibrin: useful to monitor fibrinogen breakdown during t-PA infusion

Blood ◽  
1986 ◽  
Vol 67 (5) ◽  
pp. 1482-1487
Author(s):  
P Holvoet ◽  
HR Lijnen ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Tissue Type ◽  
Clot Lysis ◽  
Plasminogen Activation ◽  
Plasma Clot ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Additional Support

One (MA-1C8) of 36 monoclonal antibodies obtained by fusion of P3X63- Ag8–6.5.3 myeloma cells with spleen cells of mice immunized with purified human tissue-type plasminogen activator (t-PA) blocked the activity of t-PA on fibrin plates but not on chromogenic substrates. MA- 1C8 at a concentration of 200 micrograms/mL inhibited plasma clot lysis and binding of t-PA to the clot. MA-1C8 had no influence on the activation of plasminogen by t-PA, which obeys Michaelis-Menten kinetics with Km = 105 mumol/L and kcat = 0.05 s-1; however, it abolished the influence of CNBr-digested fibrinogen on Km. These findings confirm that the stimulatory effect of fibrin on the activation of plasminogen by t-PA is mediated by binding of t-PA to fibrin and provide additional support for the kinetic model. Addition of t-PA to pooled fresh human plasma to a concentration of 5 micrograms/mL resulted in extensive fibrinogen breakdown after incubation for one hour at 37 degrees C or during storage at -20 degrees C for one day. In both instances, fibrinogen degradation was completely prevented by addition of MA-1C8 to a concentration of 200 micrograms/mL of plasma. MA-1C8 also effectively prevented in vitro fibrinogen degradation and in vitro plasminogen activation in plasma samples obtained during infusion of recombinant t-PA in patients with thromboembolic disease. Thus, MA-1C8 is a useful tool for discriminating between in vivo and in vitro fibrinolysis during thrombolytic therapy with t-PA.

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