Fibrinolytic efficacy of Amediplase, Tenecteplase and scu-PA in different external plasma clot lysis models

2006 ◽  
Vol 96 (09) ◽  
pp. 325-330 ◽  
Author(s):  
Ana Guimarães ◽  
Marrie Barrett-Bergshoeff ◽  
Marco Criscuoli ◽  
Stefano Evangelista ◽  
Dingeman Rijken
Keyword(s):  
Thrombolytic Therapy ◽  
Thrombolytic Agent ◽  
Plasminogen Activators ◽  
Clot Lysis ◽  
Plasma Clot ◽  
Lysis Rate ◽  
Carboxypeptidase Inhibitor ◽  
Static Conditions

SummaryIn this study, the in-vitro fibrinolytic efficacy of Tenecteplase, Amediplase and scu-PA was investigated in different external lysis models by measuring the lysis of human plasma clots after the addition of the plasminogen activators (PAs) to the surrounding plasma. The effect ofTAFI was examined for each PA by neutralizing TAFI a with potato carboxypeptidase inhibitor (PCI). The lytic efficacy of Amediplase was lower than that of Tenecteplase at low PA concentrations but slightly higher at therapeutic concentrations. The activity of scu-PA was clearly lower than that of either Tenecteplase or Amediplase. The TAFI system inhibited external clot lysis mediated by all the PAs when thrombomodulin was present in the model. In the therapeutic range (5–10 µg/ml) however, the TAFIa effect was negligible for both Amediplase and Tenecteplase. At lower PA concentrations the effect of TAFI on Amediplase was slightly stronger than that on Tenecteplase. Under static conditions the lysis rates were lower than with stirring. The role of TAFI was similar under both conditions. In conclusion, at therapeutic concentrations Amediplase was slightly more active than Tenecteplase and scu-PA under all conditions used. Therefore, Amediplase might possibly be a more potent thrombolytic agent at these concentrations and increase the efficacy of thrombolysis. The potential of TAFI for inhibiting thrombolytic therapy is probably low. However in conditions where the local PA concentrations are sub-optimal TAFI might affect the lysis rate.

PLASMA-CLOT LYSIS INDUCED BY MONOCLONAL ANTIBODY AGAINST α2-PLASMIN INHIBITOR

1987 ◽  
Author(s):  
Y Sakata ◽  
J Mimuro ◽  
Y koike
Keyword(s):  
Clot Lysis ◽  
Blood Collection ◽  
Plasma Clot ◽  
Specific Patient ◽  
Lysis Time ◽  
Pi Deficiency ◽  
Dose Dependent ◽  
Plasmin Inhibitor

A Monoclonal antibody (MCA) against α2-plasmin inhibitor α2-Pl) designated as JTP-1 inhibited antiplasmin act ivity and complex formation of a2-PI with plasmin. By using this MCA we tried to observe plasma-clot lysis (CL) in vitro and to estimate the level of total fibrinolytic capacity in plasma. As reported previously (Blood 55: U83, 1980) spontaneous CL is a striking feature of the plasma derived from a patient with α2-PI deficiency. We showed that this abnormally increased fibrinolysis was solely due to the deficiency of α2-PI. However, the contribution of plasminogen activator (PA) and its inhibitor to this specific patient' plasma -CL has been under discussion. Therefore, to test whether similar CL can be found in normal plasma without an addition of PA, plasma clots were made after incubation of plasma from normal volunteers containing 125i fibrinogen with various concentration of JTP-1, and fibrinolysis was measured by counting the soluble radioactivity. The addition of JTP-1 to plasma led to a dose-dependent enhancement of the soluble 125i fragment-release from the clot. However, JTP-1 had no effect on a2-PI-deficient plasma-CL. Other anti-a2-PI MCAs whose epitopes were not involved in the reactive site of a2-PI had no effect on CL and rabbit anti-mouse immunoglobulin IgG neutralized this JTP-l-inducing CL completely. Immunodepletion of tissue PA (tPA) or plasminogen from plasma decreased the rate of CL but that of prourokinase did not. To determine the role of PA inhibitor (PAl) released from platelets (pits) in the regulation of CL in vitro, plasma clots were made from pits poor plasma (PPP) and pits rich plasma (PRP), and CL was observed in the presence of JTP-1. There was almost no difference of the lysis time between PPP clot and PRP clot, although plasma clot from pregnant woman was lysed slowly. These results strongly suggest that endogenous t-PA in plasma is still functionally active after blood collection and CL is mainly prevented by a2-PI in vitro.-plasmin inhibitor (a2-Pl) designated as JTP-1 inhibited antiplasmin act ivity and complex formation of a2-PI with plasmin. By using this MCA we tried to observe plasma-clot lysis (CL) in vitro and to estimate the level of total fibrinolytic capacity in plasma. As reported previously (Blood 55: U83, 1980) spontaneous CL is a striking feature of the plasma derived from a patient with a2-PI deficiency. We showed that this abnormally increased fibrinolysis was solely due to the deficiency of a2-PI. However, the contribution of plasminogen activator (PA) and its inhibitor to this specific patient' plasma -CL has been under discussion. Therefore, to test whether similar CL can be found in normal plasma without an addition of PA, plasma clots were made after incubation of plasma from normal volunteers containing 125i fibrinogen with various concentration of JTP-1, and fibrinolysis was measured by counting the soluble radioactivity. The addition of JTP-1 to plasma led to a dose-dependent enhancement of the soluble 125i fragment-release from the clot. However, JTP-1 had no effect on a2-PI-deficient plasma-CL. Other anti-a2-PI MCAs whose epitopes were not involved in the reactive site of a2-PI had no effect on CL and rabbit anti-mouse immunoglobulin IgG neutralized this JTP-l-inducing CL completely. Immunodepletion of tissue PA (tPA) or plasminogen from plasma decreased the rate of CL but that of prourokinase did not. To determine the role of PA inhibitor (PAl) released from platelets (pits) in the regulation of CL in vitro, plasma clots were made from pits poor plasma (PPP) and pits rich plasma (PRP), and CL was observed in the presence of JTP-1. There was almost no difference of the lysis time between PPP clot and PRP clot, although plasma clot from pregnant woman was lysed slowly. These results strongly suggest that endogenous t-PA in plasma is still functionally active after blood collection and CL is mainly prevented by a2-PI in vitro.-plasmin inhibitor (a2-Pl) designated as JTP-1 inhibited antiplasmin act ivity and complex formation of a2-PI with plasmin. By using this MCA we tried to observe plasma-clot lysis (CL) in vitro and to estimate the level of total fibrinolytic capacity in plasma. As reported previously (Blood 55: U83, 1980) spontaneous CL is a striking feature of the plasma derived from a patient with α2-PI deficiency. We showed that this abnormally increased fibrinolysis was solely due to the deficiency of a2-PI. However, the contribution of plasminogen activator (PA) and its inhibitor to this specific patient' plasma -CL has been under discussion. Therefore, to test whether similar CL can be found in normal plasma without an addition of PA, plasma clots were made after incubation of plasma from normal volunteers containing 125i fibrinogen with various concentration of JTP-1, and fibrinolysis was measured by counting the soluble radioactivity. The addition of JTP-1 to plasma led to a dose-dependent enhancement of the soluble 125i fragment-release from the clot. However, JTP-1 had no effect on a2-PI-deficient plasma-CL. Other anti-a2-PI MCAs whose epitopes were not involved in the reactive site of a2-PI had no effect on CL and rabbit anti-mouse immunoglobulin IgG neutralized this JTP-l-inducing CL completely. Immunodepletion of tissue PA (tPA) or plasminogen from plasma decreased the rate of CL but that of prourokinase did not. To determine the role of PA inhibitor (PAl) released from platelets (pits) in the regulation of CL in vitro, plasma clots were made from pits poor plasma (PPP) and pits rich plasma (PRP), and CL was observed in the presence of JTP-1. There was almost no difference of the lysis time between PPP clot and PRP clot, although plasma clot from pregnant woman was lysed slowly. These results strongly suggest that endogenous t-PA in plasma is still functionally active after blood collection and CL is mainly prevented by α2-PI in vitro.

Inhibition of the Activities of α2-Plasmin inhibitor (PI) and CI inhibitor (CI-Inh) by the Synthetic Fibrinolytic Agent, 3-Hydroxypropyl Flufenamide (Flu-HPA)

1979 ◽  
Author(s):  
L Miles ◽  
J Burnier ◽  
M Verlander ◽  
M Goodman ◽  
A Kleiss ◽  
...  
Keyword(s):  
Plasminogen Activators ◽  
Inhibitory Effect ◽  
Mechanisms Of Action ◽  
Flufenamic Acid ◽  
Clot Lysis ◽  
Factor Xiia ◽  
Dependent Inhibition ◽  
Normal Human ◽  
Plasmin Inhibitor

Flu-HPA is one of a series of flufenamic acid derivations that enhances plasminogen-dependent clot lysis in vitro. Studies of possible mechanisms of action of Flu-HPA were undertaken. The influence of Flu-HPA on the inhibition of purified plasmin by purified PI was studied. PI activity was assessed by its inhibition of the clevage of the tripeptide S-2251 (H-D-Val-Leu-Lys-pNA) by plasmin. Flu-HPA was dissolved in DMF or in methonol and preincubated with PI before addition of plasmin. At Flu-HPA concentrations greater than 1mM and up to 60mM, the inhibitory activity of PI was totally lost. The inhibitory effect of normal human plasma on plasmin was also completely abolished at concentrations of Flu-HPA between 2.5 and 40mM. The effect of Flu-HPA on the inhibition of purified plasma kallikrein by purified CI-Inh was also studied. CI-Inh activity was measured by its inhibition of cleavage of the tripeptide Bz-Pro-Phe-Arg-pNA by kallikrein. When Flu-HPA, dissolved in DMF or in methonol, was preincubated with CI-Inh, a concentration dependent inhibition of CI-Inh activity was observed. CI-Inh activity was abolished by concentrations of Flu-HPA greater than 1mM. Flu-HPA also inhibited the activity of CI-Inh on purified Factor XIIa. These observations suggest that this flufenamic acid derivative may enhance fibrinolysis not only by inhibiting PI activity but also by decreasing the inactivation of plasminogen activators by CI-Inh.

Thrombin activatable fibrinolysis inhibitor (TAFI) affects fibrinolysis in a plasminogen activator concentration-dependent manner

2004 ◽  
Vol 91 (03) ◽  
pp. 473-479 ◽  
Author(s):  
Ana Guimarães ◽  
Dingeman Rijken
Keyword(s):  
Plasminogen Activator ◽  
In Vitro Study ◽  
Inhibitory Effect ◽  
Retardation Factor ◽  
Clot Lysis ◽  
Dependent Manner ◽  
Plasma Clot ◽  
Concentration Dependent Manner ◽  
Plasmin Inhibitor

SummaryTAFIa was shown to attenuate fibrinolysis. In our in vitro study, we investigated how the inhibitory effect of TAFIa depended on the type and concentration of the plasminogen activator (PA). We measured PA-mediated lysis times of plasma clots under conditions of maximal TAFI activation by thrombin-thrombomodulin in the absence and presence of potato carboxypeptidase inhibitor. Seven different PAs were compared comprising both tPA-related (tPA, TNK-tPA, DSPA), bacterial PA-related (staphylokinase and APSAC) and urokinase-related (tcu-PA and k2tu-PA) PAs. The lysis times and the retardation factor were plotted against the PA concentration. The retardation factor plots were bell-shaped. At low PA concentrations, the retardation factor was low, probably due to the limited stability of TAFIa. At intermediate PA concentrations the retardation factor was maximal (3-6 depending on the PA), with TNK-tPA, APSAC and DSPA exhibiting the strongest effect. At high PA concentrations, the retardation factor was again low, possibly due to inactivation of TAFIa by plasmin or to a complete conversion of glu-plasminogen into lys-plasminogen. Using individual plasmas with a reduced plasmin inhibitor activity (plasmin inhibitor Enschede) the bell-shaped curve of the retardation factor shifted towards lower tPA and DSPA concentrations, but the height did not decrease. In conclusion, TAFIa delays the lysis of plasma clots mediated by all the plasminogen activators tested. This delay is dependent on the type and concentration of the plasminogen activator, but not on the fibrin specificity of the plasminogen activator. Furthermore, plasmin inhibitor does not play a significant role in the inhibition of plasma clot lysis by TAFI.

scholarly journals A bleeding disorder due to deficiency of alpha 2-antiplasmin

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1246-1251 ◽  
Author(s):  
LA Miles ◽  
EF Plow ◽  
KJ Donnelly ◽  
C Hougie ◽  
JH Griffin
Keyword(s):  
System Analysis ◽  
Bleeding Disorder ◽  
Fibrinolytic System ◽  
In Vitro Assays ◽  
Clot Lysis ◽  
Plasma Clot ◽  
Normal Limits ◽  
Normal Plasma ◽  
Bleeding Episodes

Abstract A deficiency of alpha 2-antiplasmin has been identified in a female patient with severe and frequent bleeding episodes. Routine coagulation and platelet assays of the patient's plasma were within normal limits. However, abnormally rapid whole blood or dilute plasma clot lysis times and an abnormal FXIII test in which clots were lysed in the presence of urea or saline suggested an abnormal fibrinolytic system. Analysis of alpha 2-antiplasmin levels by radioimmunoassay revealed less than 1.0 microgram/ml alpha 2-antiplasmin. Functional assays indicated an alpha 2-antiplasmin level less than or equal to 10% of normal. Addition of purified alpha 2-antiplasmin to the patient's plasma restored its ability to inhibit plasmin in in vitro assays, and mixtures of patient plasma with normal plasma did not interfere with the antiplasmin activity of the normal plasma. Whereas normal platelets contain 68 ng alpha 2-antiplasmin/10(9) platelets, platelets from the patient contained 30% of the normal level of antigen. Analysis of alpha 2- antiplasmin functional and antigenic levels in the plasma of both parents and four siblings of the propositus provided evidence consistent with an autosomal mechanism of inheritance of alpha 2- antiplasmin deficiency. One sibling appeared to be homozygous and three siblings and the parents were heterozygous for the deficiency. Two heterozygotes had positive bleeding histories. The association of a bleeding disorder with a deficiency of alpha 2-antiplasmin emphasizes that lack of regulation of the fibrinolytic system can result in a hemostatic dysfunction.

Role of Erythropoietin in Megakaryocyte Colony Formation in Man

1979 ◽  
Author(s):  
W. Vainehenker ◽  
J. Breton-Gorius
Keyword(s):  
Bone Marrow ◽  
Linear Relationship ◽  
Colony Formation ◽  
Colony Stimulating Factor ◽  
Plasma Clot ◽  
In Vitro Technique ◽  
One Step ◽  
Stimulating Factor

We have recently realized megakaryocyte (MK) colony formation in culture from blood and bone marrow progenitors using the plasma clot technique. In this study, the MK stimulating factor was an erythropoietin (Epo) either a poorly purified one(step III from anaemic sheep serum, a crude serum from anaemic mice, an urinary human Epo) or a highly purified one (GOLDWASSER). Similar results were obtained with all these Epo. A linear relationship was found between the number of colonies and seeded cells. However with less than 5.105 plated cells from the blood, no MK colonies were obtained, although erythroid colonies could be grown. In contrast, without Epo, spontaneous colonies could be observed which represented 1/5 th of the maximum plating efficiency , in these eases no erythroid colonies were present. These data suggest that Epo itself acts an a MK colony stimulating factor; but is not the only factor involved in the formation of MK colonies. This in vitro technique will be useful of in determining the factors regulating megakaryocytopoiesis.

Molecular Conversions of Recombinant Staphylokinase During Plasminogen Activation in Purified Systems and in Human Plasma

1993 ◽  
Vol 70 (03) ◽  
pp. 495-499 ◽  
Author(s):  
S Ueshima ◽  
K Silence ◽  
D Collen ◽  
H R Lijnen
Keyword(s):  
Amino Acid ◽  
Human Plasma ◽  
Catalytic Amount ◽  
Clot Lysis ◽  
Lag Phase ◽  
Plasminogen Activation ◽  
Plasma Clot ◽  
Acid Protein ◽  
Human Plasminogen

SummaryRecombinant staphylokinase (STAR) is produced as a 136 amino acid protein with NH2-terminal sequence Ser-Ser-Ser (mature STAR, HMW-STAR), which may be converted to lower molecular weight forms (LMW-STAR) by removal of the first six residues (yielding STAR-Δ6 with NH2-terminal Gly-Lys-Tyr-) or the first ten residues (yielding STAR-Δ10 with NH2-terminal Lys-Gly-Asp-). In the present study the occurrence and effects of these conversions during plasminogen activation by HMW-STAR were studied in purified systems and in human plasma.In stoichiometric mixtures of HMW-STAR and native human plasminogen (Glu-plasminogen), rapid and quantitative conversion of HMW-STAR to LMW-STAR occurred, concomitant with exposure of the active site in the plasmin-STAR complex. NH2-terminal amino acid sequence analysis revealed the sequence Lys-Gly-Asp- in addition to the known sequences of the Lys-plasmin chains, identifying STAR-Δ10 as the derivative generated from HMW-STAR. In mixtures of catalytic amount of HMW-STAR and human plasminogen, plasmin generation occurred progressively, following an initial lag phase, during which HMW-STAR was converted to LMW-STAR. Plasmin-mediated conversion of HMW-STAR to LMW-STAR obeyed Michaelis-Menten kinetics with K m = 3.6 μM and k 2 = 0.38 s−1. The specific clot lysis activities of HMW-STAR (122,000 ± 8,000 units/mg) and LMW-STAR (129,000 ± 8,000 units/mg) were indistinguishable.In an in vitro system consisting of a 60 μl plasma clot submerged in 250 μl plasma, 80% clot lysis within 1 h was obtained with 70 nM HMW-STAR. This was associated with fibrinogen depletion and conversion of 20% of the HMW-STAR to LMW-STAR. Addition of 100 nM HMW-STAR to human plasma in the absence of a clot did not induce significant fibrinogen breakdown (≥ 90% residual fibrinogen after 2 h), and was not associated with significant coversion to LMW-STAR (≤10% within 2 h). With 400 nM HMW-STAR, fibrinogen depletion in plasma occurred within 1 h, and 80% conversion to LMW-STAR was observed. Thus, at fibrinolytically active concentrations which do not cause fibrinogen breakdown, no significant conversion of HMW-STAR to LMW-STAR occurs in human plasma in the absence of a clot.These findings indicate that the conversion of HMW-STAR to LMW-STAR may occur in association with clot lysis, but is not required to induce it.

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.

Proteins of the Fibrinolytic System in Human Thrombi

1996 ◽  
Vol 75 (01) ◽  
pp. 127-133 ◽  
Author(s):  
Linda A Robbie ◽  
Bruce Bennett ◽  
Alison M Croll ◽  
Paul A J Brown ◽  
Nuala A Booth
Keyword(s):  
Immunohistochemical Staining ◽  
Strong Association ◽  
Plasminogen Activators ◽  
High Plasma ◽  
Clot Lysis ◽  
High Plasma Concentration ◽  
Quantitative Immunoassay ◽  
High Concentrations ◽  
Pai 1

SummaryThe proteins of fibrinolysis have been quantified in human thrombi, to assess the balance between plasminogen activators and their major inhibitor PAI-1. The relative roles of PAI-1 and α2-AP were also examined since we have previously shown that both platelet PAI-1 and plasma α2-AP are important determinants of clot lysis in vitro. Extracts and sections were prepared from human thrombi for quantitative immunoassay and immunohistochemical staining respectively. PAI-1 and α2-AP were present at high concentrations. Levels of t-PA and t-PA-PAI-1 complex were relatively low. Staining confirmed the presence of abundant PAI-1, associated primarily with platelet material within the thrombus and also with fibrin. Staining for α2-AP was also intense and demonstrated strong association with fibrin. The α2-AP concentration was similar to its high plasma concentration, whereas PAI-1 levels were up to 30 times greater than that in circulating blood, suggesting that active recruitment of platelets contributes to the high PAI-1 concentration in thrombi.

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