scholarly journals The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1864-1872
Author(s):  
HR Lijnen ◽  
B Van Hoef ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Conformational Transition ◽  
Resistant Mutant ◽  
Fibrin Clot ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Plasma Clot ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

The relative contribution of several mechanisms to plasminogen activation and fibrin dissolution by urokinase-type plasminogen activator (u-PA) in vitro was quantitated. The activation of plasminogen by recombinant single chain u-PA (rscu-PA), by its two chain derivative (rtcu-PA) and by a plasmin-resistant mutant, rscu-PA- Glu158, obeys Michaelis-Menten kinetics with catalytic efficiencies of 0.00064, 0.046, and 0.00005 L/mumol.s for native plasminogen (Glu- plasminogen) and of 0.0061, 1.21, and 0.0004 L/mumol.s for partially degraded plasminogen (Lys-plasminogen). In a purified system consisting of a fibrin clot submerged in a plasminogen solution, the equi- effective doses (50% lysis in one hour) for rscu-PA, rtcu-PA, and rscu- PA-Glu158 were 16, 6.5, and 32,000 ng/mL for Glu-plasminogen and two- to fourfold lower for Lys-plasminogen. In a plasma milieu, 50% lysis in two hours was obtained for a plasma clot with 2.1 micrograms/mL rscu- PA, 0.5 micrograms/mL rtcu-PA, and greater than 200 micrograms/mL rscu- PA-Glu158 and for a purified fibrin clot with 1.3 micrograms/mL rscu-PA and 0.27 microgram/mL rtcu-PA. After predigestion of a purified fibrin clot with plasmin, the apparent potency of rscu-PA and rtcu-PA increased by 40% and 20%, respectively. In conclusion, rscu-PA has an intrinsic plasminogen activating potential that is only about 1% of that of rtcu-PA and that is 13 times higher than that of rscu-PA- Glu158. Conformational transition of Glu-plasminogen to Lys-plasminogen enhances its sensitivity to activation by all u-PA moieties ten- to 20- fold. Predigestion of fibrin clots with associated increased binding of plasminogen results in a minor apparent increase of the fibrinolytic potency of rscu-PA and rtcu-PA. The relative fibrinolytic potency of rtcu-PA is two to three orders of magnitude higher than that of rscu-PA- Glu158 but only two- to five-fold higher than that of rscu-PA, both in purified systems and in a plasma milieu. These results indicate that conversion of rscu-PA to rtcu-PA constitutes the primary mechanism of fibrin dissolution.

scholarly journals The mechanism of plasminogen activation and fibrin dissolution by single chain urokinase-type plasminogen activator in a plasma milieu in vitro

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1864-1872 ◽  
Author(s):  
HR Lijnen ◽  
B Van Hoef ◽  
F De Cock ◽  
D Collen
Keyword(s):  
Plasminogen Activator ◽  
Conformational Transition ◽  
Resistant Mutant ◽  
Fibrin Clot ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Plasma Clot ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract The relative contribution of several mechanisms to plasminogen activation and fibrin dissolution by urokinase-type plasminogen activator (u-PA) in vitro was quantitated. The activation of plasminogen by recombinant single chain u-PA (rscu-PA), by its two chain derivative (rtcu-PA) and by a plasmin-resistant mutant, rscu-PA- Glu158, obeys Michaelis-Menten kinetics with catalytic efficiencies of 0.00064, 0.046, and 0.00005 L/mumol.s for native plasminogen (Glu- plasminogen) and of 0.0061, 1.21, and 0.0004 L/mumol.s for partially degraded plasminogen (Lys-plasminogen). In a purified system consisting of a fibrin clot submerged in a plasminogen solution, the equi- effective doses (50% lysis in one hour) for rscu-PA, rtcu-PA, and rscu- PA-Glu158 were 16, 6.5, and 32,000 ng/mL for Glu-plasminogen and two- to fourfold lower for Lys-plasminogen. In a plasma milieu, 50% lysis in two hours was obtained for a plasma clot with 2.1 micrograms/mL rscu- PA, 0.5 micrograms/mL rtcu-PA, and greater than 200 micrograms/mL rscu- PA-Glu158 and for a purified fibrin clot with 1.3 micrograms/mL rscu-PA and 0.27 microgram/mL rtcu-PA. After predigestion of a purified fibrin clot with plasmin, the apparent potency of rscu-PA and rtcu-PA increased by 40% and 20%, respectively. In conclusion, rscu-PA has an intrinsic plasminogen activating potential that is only about 1% of that of rtcu-PA and that is 13 times higher than that of rscu-PA- Glu158. Conformational transition of Glu-plasminogen to Lys-plasminogen enhances its sensitivity to activation by all u-PA moieties ten- to 20- fold. Predigestion of fibrin clots with associated increased binding of plasminogen results in a minor apparent increase of the fibrinolytic potency of rscu-PA and rtcu-PA. The relative fibrinolytic potency of rtcu-PA is two to three orders of magnitude higher than that of rscu-PA- Glu158 but only two- to five-fold higher than that of rscu-PA, both in purified systems and in a plasma milieu. These results indicate that conversion of rscu-PA to rtcu-PA constitutes the primary mechanism of fibrin dissolution.

scholarly journals Comparative thrombolytic properties of single-chain forms of urokinase- type plasminogen activator

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 592-596
Author(s):  
DC Stump ◽  
JM Stassen ◽  
E Demarsin ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Molecular Forms ◽  
Clot Lysis ◽  
Single Chain ◽  
Plasma Clot ◽  
Thrombosis Model ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

The specific thrombolytic properties of urokinase and three molecular forms of single-chain urokinase-type plasminogen activator (scu-PA) were compared in a human plasma milieu in vitro and in an experimental thrombosis model in rabbits. These scu-PA molecules included Mr 54,000 scu-PA from human urine (urinary scu-PA), scu-PA from conditioned media of a human lung adenocarcinoma cell line (CALU-3,ATCC,HTB-55) (cellular scu-PA) and an Mr 32,000 proteolytic derivative of cellular scu-PA (scu- PA-32k). All four molecular forms induced significant lysis of a 125I- labeled human plasma clot immersed in citrated human plasma at concentrations between 50 and 200 IU/mL. None of the four showed absolute fibrin-specificity, but at equivalent lytic dose the three single-chain forms appeared to cause less fibrinogen degradation and alpha 2-antiplasmin consumption than two-chain urokinase. In addition, the fibrinolytic potential of the three single-chain forms was largely maintained during pre-incubation in plasma for up to 48 hours whereas that of urokinase was completely inhibited. Intravenous (IV) infusion of cellular scu-PA or scu-PA-32k into rabbits with a 125I-labeled thrombus in the jugular vein caused significant dose-dependent lysis at concentrations ranging from 8,700 to 35,000 and from 9,000 to 36,000 IU/kg respectively. Clot lysis was accompanied by minor alpha 2- antiplasmin consumption or fibrinogen breakdown. In contrast, urokinase induced lysis at doses between 20,000 and 200,000 IU/kg, but at higher doses was associated with significant systemic activation of the fibrinolytic system. It is concluded that scu-PA obtained from CALU-3 cell cultures has identical thrombolytic properties to that obtained from urine. In addition, the scu-PA-32k proteolytic derivative has the same fibrin-specific thrombolytic properties as the intact molecule. Cellular scu-PA and scu-PA-32k may therefore constitute more readily available alternatives for clot-selective thrombolytic therapy in man.

scholarly journals Comparative thrombolytic properties of single-chain forms of urokinase- type plasminogen activator

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 592-596 ◽  
Author(s):  
DC Stump ◽  
JM Stassen ◽  
E Demarsin ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Molecular Forms ◽  
Clot Lysis ◽  
Single Chain ◽  
Plasma Clot ◽  
Thrombosis Model ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract The specific thrombolytic properties of urokinase and three molecular forms of single-chain urokinase-type plasminogen activator (scu-PA) were compared in a human plasma milieu in vitro and in an experimental thrombosis model in rabbits. These scu-PA molecules included Mr 54,000 scu-PA from human urine (urinary scu-PA), scu-PA from conditioned media of a human lung adenocarcinoma cell line (CALU-3,ATCC,HTB-55) (cellular scu-PA) and an Mr 32,000 proteolytic derivative of cellular scu-PA (scu- PA-32k). All four molecular forms induced significant lysis of a 125I- labeled human plasma clot immersed in citrated human plasma at concentrations between 50 and 200 IU/mL. None of the four showed absolute fibrin-specificity, but at equivalent lytic dose the three single-chain forms appeared to cause less fibrinogen degradation and alpha 2-antiplasmin consumption than two-chain urokinase. In addition, the fibrinolytic potential of the three single-chain forms was largely maintained during pre-incubation in plasma for up to 48 hours whereas that of urokinase was completely inhibited. Intravenous (IV) infusion of cellular scu-PA or scu-PA-32k into rabbits with a 125I-labeled thrombus in the jugular vein caused significant dose-dependent lysis at concentrations ranging from 8,700 to 35,000 and from 9,000 to 36,000 IU/kg respectively. Clot lysis was accompanied by minor alpha 2- antiplasmin consumption or fibrinogen breakdown. In contrast, urokinase induced lysis at doses between 20,000 and 200,000 IU/kg, but at higher doses was associated with significant systemic activation of the fibrinolytic system. It is concluded that scu-PA obtained from CALU-3 cell cultures has identical thrombolytic properties to that obtained from urine. In addition, the scu-PA-32k proteolytic derivative has the same fibrin-specific thrombolytic properties as the intact molecule. Cellular scu-PA and scu-PA-32k may therefore constitute more readily available alternatives for clot-selective thrombolytic therapy in man.

scholarly journals Effect of chemical conjugation of recombinant single-chain urokinase- type plasminogen activator with monoclonal antiplatelet antibodies on platelet aggregation and on plasma clot lysis in vitro and in vivo

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 1005-1018 ◽  
Author(s):  
M Dewerchin ◽  
HR Lijnen ◽  
JM Stassen ◽  
F De Cock ◽  
T Quertermous ◽  
...  
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Human Platelet ◽  
Single Chain ◽  
Plasma Clot ◽  
Antiplatelet Antibodies ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Induced Aggregation

Abstract The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein IIb/IIIa) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immunoadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiometry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL rscu-PA/MA-PMI-2 or 1.2 micrograms u- PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125I-fibrin- labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu- PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P less than .005) and fivefold to sevenfold higher than that of the control conjugate (P less than .01).

scholarly journals Effect of chemical conjugation of recombinant single-chain urokinase- type plasminogen activator with monoclonal antiplatelet antibodies on platelet aggregation and on plasma clot lysis in vitro and in vivo

Blood ◽  
1991 ◽  
Vol 78 (4) ◽  
pp. 1005-1018 ◽  
Author(s):  
M Dewerchin ◽  
HR Lijnen ◽  
JM Stassen ◽  
F De Cock ◽  
T Quertermous ◽  
...  
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Human Platelet ◽  
Single Chain ◽  
Plasma Clot ◽  
Antiplatelet Antibodies ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Induced Aggregation

The murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against ligand-induced binding sites [LIBS] on human platelet glycoprotein IIb/IIIa) were conjugated with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) using the cross-linking reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP). The conjugates (rscu-PA/MA-TSPI-1, rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, and rscu-PA/MA-LIBS-1), purified by immunoadsorption and gel filtration, were obtained with recoveries of 34% to 45%, with an average stoichiometry of 1.6 to 1.8 IgG molecules per rscu-PA molecule, and with unaltered specific activities and affinities. Preincubation of human platelet-rich plasma with rscu-PA/MA-PMI-2, rscu-PA/MA-PMI-1, or unconjugated rscu-PA resulted in partial inhibition of ADP-induced aggregation; 25% inhibition was obtained with 63 micrograms/mL rscu-PA and with 6 micrograms u-PA/mL rscu-PA/MA-PMI-2 or 1.2 micrograms u- PA/mL rscu-PA/MA-PMI-1. In an in vitro system composed of a 125I-fibrin- labeled platelet-rich human plasma clot immersed in normal human plasma, the conjugates had threefold to greater than 15-fold less fibrinolytic potency than unconjugated rscu-PA. The thrombolytic potency of rscu-PA/MA-PMI-1 and rscu-PA/MA-LIBS-1 was compared with that of rscu-PA and that of a control conjugate rscu-PA/MA-1C8 in a pulmonary embolism model in the hamster, using clots prepared from platelet-poor or platelet-rich human plasma. Lysis was measured 30 minutes after the end of a 60-minute intravenous infusion of the thrombolytic agents. rscu-PA, rscu-PA/MA-PMI-1, rscu-PA/MA-LIBS-1, as well as rscu-PA/MA-1C8 had comparable thrombolytic potencies (percent lysis per dose administered) towards platelet-poor human plasma clots. In contrast, the thrombolytic potency of rscu-PA/MA-PMI-1 and of rscu- PA/MA-LIBS-1 towards platelet-rich clots was 2.3- to 3-fold higher than that of rscu-PA (P less than .005) and fivefold to sevenfold higher than that of the control conjugate (P less than .01).

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.

scholarly journals A monoclonal antibody specific for two-chain urokinase-type plasminogen activator. Application to the study of the mechanism of clot lysis with single-chain urokinase-type plasminogen activator in plasma

Blood ◽  
1990 ◽  
Vol 75 (9) ◽  
pp. 1794-1800 ◽  
Author(s):  
PJ Declerck ◽  
HR Lijnen ◽  
M Verstreken ◽  
H Moreau ◽  
D Collen
Keyword(s):  
Monoclonal Antibody ◽  
Human Plasma ◽  
Plasminogen Activator ◽  
Fibrin Clot ◽  
Clot Lysis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

Abstract A murine monoclonal antibody (MA-12E6A8) was raised against human urokinase-type plasminogen activator (u-PA), which, in an enzyme-linked immunosorbent assay (ELISA), reacted 15,000-fold better with recombinant two-chain u-PA (rtcu-PA) than with recombinant single-chain u-PA (rscu-PA). The antibody had no effect on the activity of rtcu-PA or on its inhibition by a chloromethylketone, but reduced the inhibition of rtcu-PA by recombinant plasminogen activator inhibitor-1 (rPAI-1) at least 10-fold. The dissociation constant of the rtcu-PA/MA- 12E6A8 complex was 7 nmol/L. An ELISA was developed using MA-12E6A8 as capture antibody and a horseradish peroxidase conjugated u-PA specific antibody for tagging. It recognized free and active site blocked rtcu- PA but not rtcu-PA in complex with rPAI-1 or with alpha 2-antiplasmin. This ELISA was used to monitor the generation of rtcu-PA during fibrin clot lysis with rscu-PA in human plasma. Addition of 5 micrograms/mL rscu-PA to 3 mL plasma containing a 0.2 mL 125I-fibrin labeled plasma clot caused 50% clot lysis in 62 +/- 13 minutes (mean +/- SD, n = 6), at which time 99 +/- 28 ng/mL rtcu-PA was detected but no fibrinogen breakdown had occurred. Fifty percent fibrinogen breakdown did occur only when rtcu-PA had reached a level of 1,000 +/- 270 ng/mL (at 150 +/- 21 minutes). rscu-PA, 2 micrograms/mL, induced 50% clot lysis in 160 +/- 41 minutes (n = 6); no fibrinogen degradation occurred within 4 hours and rtcu-PA levels did not exceed 80 ng/mL. In the absence of a fibrin clot, 5 micrograms/mL rscu-PA added to human plasma did not result in significant generation of rtcu-PA (less than 50 ng/mL after 4 hours) and no fibrinogen degradation was observed. These results indicate that clot lysis with rscu-PA in a plasma milieu does not require extensive systemic conversion of rscu-PA to rtcu-PA, and that fibrinogen degradation occurs secondarily to systemic conversion of rscu-PA to rtcu-PA.

Thrombin Stimulation of Platelets Induces Plasminogen Activation Mediated by Endogenous Urokinase-Type Plasminogen Activator

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3579-3586 ◽  
Author(s):  
Catherine Lenich ◽  
Jian-Ning Liu ◽  
Victor Gurewich
Keyword(s):  
Plasminogen Activator ◽  
Gene Knockout ◽  
Direct Action ◽  
Plasminogen Activation ◽  
Single Chain ◽  
Platelet Surface ◽  
Physiologic Mechanism ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator ◽  
Gene Knockout Mice

Abstract Gene knockout mice studies indicate that urokinase-type plasminogen activator (u-PA) is importantly involved in fibrinolysis, but its physiologic mechanism of action remains poorly understood. We postulated that platelets may be involved in this mechanism, as they carry a novel receptor for u-PA and a portion of the single-chain u-PA (scu-PA) intrinsic to blood is tightly associated with platelets. Therefore, plasminogen activation by platelet-associated u-PA was studied. When washed platelets were incubated with plasminogen, no plasmin was generated as detected by plasmin synthetic substrate (S2403) hydrolysis; however, after the addition of thrombin, but not other agonists, platelet-dependent plasminogen activation occurred. Plasminogen activation was surface-related, being inhibited by blocking platelet fibrinogen receptors or by preventing plasminogen binding to the thrombin-activated platelet surface. U-PA was identified as the only plasminogen activator responsible and enrichment of platelets with exogenous scu-PA significantly augmented plasminogen activation. These findings appeared paradoxical because thrombin inactivates scu-PA. Indeed, zymograms showed inactivation of scu-PA during the first hour of incubation with even the lowest dose of thrombin used (1 u/mL). However, this was followed by a thrombin dose-dependent (1 to 10 u/mL) partial return of u-PA activity. Reactivation of u-PA was not due to the direct action of thrombin, but required platelets and was found to be related to a platelet lysosomal thiol protease, consistent with cathepsin C. In conclusion, a new pathway of plasminogen activation by platelet-associated endogenous or exogenous scu-PA was demonstrated, which is specifically triggered by thrombin activation of platelets. These findings may help explain u-PA–mediated physiological fibrinolysis and have implications for therapeutic thrombolysis with scu-PA.

Role of α2-Antiplasmin in Fibrin-Specific Clot Lysis with Single-Chain Urokinase-Type Plasminogen Activator in Human Plasma

1991 ◽  
Vol 65 (04) ◽  
pp. 394-398 ◽  
Author(s):  
P J Declerck ◽  
H R Lijnen ◽  
M Verstreken ◽  
D Collen
Keyword(s):  
Human Plasma ◽  
Plasminogen Activator ◽  
Clot Lysis ◽  
Minor Role ◽  
Single Chain ◽  
Plasma Clot ◽  
Normal Plasma ◽  
Type Plasminogen Activator ◽  
Urokinase Type Plasminogen Activator

SummaryThe role of plasma α2-antiplasmin (α2-AP) in the fibrinspecificity of clot lysis by recombinant single-chain urokinase-type plasminogen activator (rscu-PA) and in the conversion of rscu-PA to its two-chain derivative (rtcu-PA, urokinase) was investigated in an in vitro human plasma clot lysis system. Fifty % lysis in 2 h of a 0.1 ml 125l-fibrin labeled human plasma clot immersed in 0.5 ml normal human plasma was obtained with 1.4 ± 0.15 µg/ml rscu-PA (mean ± SD, n = 8). This was associated with degradation of 23 ± 7% of fibrinogen and generation of 0.20 ± 0.09 µg/ml rtcu-PA. In α2-AP-depleted plasrna 50% clot lysis in 2 h required 2-fold less rscu-PA which was associated with 3-fold more extensive fibrinogen degradation and 2-fold more rtcu-PA generation. Fifty % lysis in? h, of a 0.1 ml α2-AP-depleted plasma clot, subriersed in 0.5 ml normal plasma, was obtained with 0.80 ± 0.05 µg/ml rscu-PA (n = 3, p <0.001 vs normal clot) and was associated with 17 ± 6% fibrinogen breakdown (p : 0.22 vs normal clot) and 0.08 ± 0.02 µg/ml rtcu-PA generation (p < 0.05 vs normal clot). In α2-AP-depleted plasma the equipotent rscu-PA concentration was 4-fold lower than in normal plasma and was associated with 3-fold more fibrinogen degradation and a similar extent of rtcu-PA generationThus, α2-AP in plasma contributes significantly to the fibrinspecificity of rscu-PA, primarily via prevention of conversion in plasma of rscu-PA to rtcu-PA. Clot associated α2-AP increases the resistance of the clot to lysis with rscu-PA, but plays an only minor role in the fibrin-specificity of clot lysis in normal plasma.

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