Efficacy of ultrasound assisted catheter-directed thrombolysis compared to catheter-directed thrombolysis in vitro

Background Catheter-directed thrombolysis (CDT) is an effective and safe endovascular method used in critical limb ischemia and many other thromboembolic events. Ultrasound-assisted catheter-directed thrombolysis (US-CDT) is an emerging technique considered to accelerate thrombolysis and therefore is supposed to improve outcome. Purpose To evaluate the efficacy of US-CDT in comparison to standard CDT in vitro. Material and Methods A total of 69 sets of human venous blood were evaluated, each comprising a tube just treated with CDT, a tube treated with US-CDT, and a control tube. All tubes were kept under physiological conditions. Except for the controls, in all tubes 5 mg of tissue-type plasminogen activator was administered over the predetermined treatment interval. Thrombus mass was weighted at the end of the lysis intervals at 6 h or 24 h, respectively. Results CDT led to a mean thrombus reduction of 32% and ultrasound-assisted lysis led to a mean thrombus reduction of 41% ( P < 0.001 for both). Thrombus reduction was significantly higher after US-CDT compared to CDT ( P = 0.001). The better efficacy of US-CDT was mostly already apparent at early phases during thrombolysis and did further mildly increase over time (r = 0.24; P = 0.047). Conclusion In vitro US-CDT is significantly superior to standard CDT; this effect is apparent at an early timepoint of lysis and slightly further increases over time.

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Sonothrombolysis is effective with recombinant tissue-type plasminogen activator, but not with Abciximab

Thrombosis and Haemostasis ◽

10.1160/th09-03-0207 ◽

2009 ◽

Vol 102 (12) ◽

pp. 1274-1277 ◽

Cited By ~ 5

Author(s):

Stefan Ossadnik ◽

Heiko Hütten ◽

Günter Seidel ◽

Jürgen Eggers

Keyword(s):

Plasminogen Activator ◽

Venous Blood ◽

In Vitro Study ◽

Tissue Type ◽

Clot Lysis ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

The Us ◽

Vitro Model

SummaryTranscranial “diagnostic” ultrasound (US) has been shown to accelerate thrombolysis related to recombinant tissue-type plasminogen activator (rt-PA). In this in vitro study, we evaluated the potential of US to increase clot dissolution mediated by Abciximab (Abc) compared to rt-PA. The effect of 1.8-MHz pulsed wave (PW) Doppler US on dissolution of whole venous blood clots (WBC) and platelet-rich clots (PRC) treated with Abc and rt-PA was investigated in an in vitro model. Clot dissolution was measured by weight loss. Abc-related WBC dissolution was enhanced by additional US, but the effect was not any more detectable when the US was attenuated by a human temporal bone (US-tb). In PRC there was no additional effect of US on the Abc-related clot lysis. Rt-PA-related clot dissolution was increased by US in WBC and PRC as well, however, US-tb was only effective in WBC. The effect of insonation on WBC dissolution treated with the combination of Abc plus rt-PA was lower compared with those treated with rt-PA. In this in vitro experiment, the additional effect of “diagnostic” US in combination with Abc was only present in WBC and less strong than with rt-PA. The results do not support the use of Abc for sonothrombolysis targeting both, fibrin-rich and platelet-rich clots. In contrast, US when combined with rt-PA increases dissolution in both, WBC and PRC as well.

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A Monoclonal Antibody-Based Enzyme Immunoassay for Fibrin Degradation Products in Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1642777 ◽

1988 ◽

Vol 59 (02) ◽

pp. 310-315 ◽

Cited By ~ 64

Author(s):

P W Koppert ◽

E Hoegee-de Nobel ◽

W Nieuwenhuizen

Keyword(s):

Enzyme Immunoassay ◽

Degradation Products ◽

Blood Clot ◽

Tissue Type ◽

Fibrin Degradation Products ◽

Type Plasminogen Activator ◽

Strong Positive Reaction ◽

Sandwich Type ◽

Capture Antibody

SummaryWe have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes.* The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the capture antibody, binds both fibrinogen degradation products (FbgDP) and FbDP, but does not react with the parent fibrin(ogen) molecules. It has its epitope in the E-domain of the fibrinogen molecule on the Bβ-chain between amino acids 54-118. Antibody DD-13 was raised using D-dimer as antigen and is used as a tagging antibody, conjugated with horse-radish peroxidase. A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard. The EIA does virtually not detect FbgDP i. e. purified fragments X, Y, or FbgDP generated in vitro in plasma by streptokinase treatment. This indicates that the assay is specific for fibrin degradation products.We have successfully applied this assay to the plasma of patients with a variety of diseased states. In combination with the assay previously developed by us for FbgDP and for the total amount of FbgDP + FbDP (TDP) in plasma, we are now able to study the composition of TDP in patients plasma in terms of FbgDP and FbDP.

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Pharmacokinetics and Haemostatic Status During Consecutive Infusions of Recom binant Tissue-Type Plasminogen Activator in Patients with Acute Myocardial Infarction

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646622 ◽

1989 ◽

Vol 61 (03) ◽

pp. 497-501 ◽

Cited By ~ 47

Author(s):

E Seifried ◽

P Tanswell ◽

D Ellbrück ◽

W Haerer ◽

A Schmidt

Keyword(s):

Myocardial Infarction ◽

Acute Myocardial Infarction ◽

Plasminogen Activator ◽

Peak Plasma ◽

Peak Plasma Concentration ◽

Tissue Type ◽

Tissue Type Plasminogen Activator ◽

Precise Control ◽

Type Plasminogen Activator

SummaryPharmacokinetics and systemic effects of recombinant tissue type plasminogen activator (rt-PA) were determined during coronary thrombolysis in 12 acute myocardial infarction patients using a consecutive intravenous infusion regimen. Ten mg rt-PA were infused in 2 minutes resulting in a peak plasma concentration (mean ±SD) of 3310±950 ng/ml, followed by 50 mg in 1 h and 30 mg in 1.5 h yielding steady state plasma levels of. 2210±470 nglml and 930±200 ng/ml, respectively. All patients received intravenous heparin. Total clearance of rt-PA was 380±74 ml/min, t,½α was 3.6±0.9 min and t,½β was 16±5.4 min.After 90 min, in plasma samples containing anti-rt-PA-IgG to inhibit in vitro effects, fibrinogen was decreased to 54%, plasminogen to 52%, α2-antiplasmin to 25%, α2-macroglobulin to 90% and antithrombin III to 85% of initial values. Coagulation times were prolonged and fibrin D-dimer concentrations increased from 0.40 to 2.7 μg/ml. It is concluded that pharmacokinetics of rt-PA show low interpatient variability and that its short mean residence time in plasma allows precise control of therapy. Apart from its moderate effect on the haemostatic system, rt-PA appears to lyse a fibrin pool in addition to the coronary thrombus.

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Comparison of Specific Antibody, D-Phe-Pro-Arg-CH2Cl and Aprotinin for Prevention of In Vitro Effects of Recombinant Tissue-Type Plasminogen Activator on Haemostasis Parameters

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646016 ◽

1987 ◽

Vol 58 (03) ◽

pp. 921-926 ◽

Cited By ~ 21

Author(s):

E Seifried ◽

P Tanswell

Keyword(s):

Specific Antibody ◽

Plasma Concentrations ◽

Fibrinolytic Therapy ◽

Tissue Type ◽

Control Value ◽

Type Plasminogen Activator ◽

In Vitro Effects ◽

Fibrinolytic Parameters

SummaryIn vitro, concentration-dependent effects of rt-PA on a range of coagulation and fibrinolytic assays in thawed plasma samples were investigated. In absence of a fibrinolytic inhibitor, 2 μg rt-PA/ml blood (3.4 μg/ml plasma) caused prolongation of clotting time assays and decreases of plasminogen (to 44% of the control value), fibrinogen (to 27%), α2-antiplasmin (to 5%), FV (to 67%), FVIII (to 41%) and FXIII (to 16%).Of three inhibitors tested, a specific polyclonal anti-rt-PA antibody prevented interferences in all fibrinolytic and most clotting assays. D-Phe-Pro-Arg-CH2Cl (PPACK) enabled correct assays of fibrinogen and fibrinolytic parameters but interfered with coagulometric assays dependent on endogenous thrombin generation. Aprotinin was suitable only for a restricted range of both assay types.Most in vitro effects were observed only with rt-PA plasma concentrations in excess of therapeutic values. Nevertheless it is concluded that for clinical application, collection of blood samples on either specific antibody or PPACK is essential for a correct assessment of in vivo effects of rt-PA on the haemostatic system in patients undergoing fibrinolytic therapy.

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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648982 ◽

1994 ◽

Vol 72 (06) ◽

pp. 906-911 ◽

Cited By ~ 8

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.

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Interaction of Plasminogen Activators and Plasminogen with Heparin: Effect of Ionic Strength

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649685 ◽

1993 ◽

Vol 70 (05) ◽

pp. 867-872 ◽

Cited By ~ 21

Author(s):

Dingeman C Rijken ◽

Gerard A W de Munk ◽

Annie F H Jie

Keyword(s):

Molecular Weight ◽

Ionic Strength ◽

Plasminogen Activator ◽

Plasminogen Activators ◽

Fibrinolytic System ◽

Tissue Type ◽

Single Chain ◽

Physiological Conditions ◽

Amino Terminal ◽

Type Plasminogen Activator

SummaryIn order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparinagarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

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Behaviour and Quantitation of Extrinsic (Tissue-Type) Plasminogen Activator in Human Blood

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665244 ◽

1983 ◽

Vol 50 (02) ◽

pp. 518-523 ◽

Cited By ~ 16

Author(s):

C Kluft ◽

A F H Jie ◽

R A Allen

Keyword(s):

Plasminogen Activator ◽

Venous Occlusion ◽

Tissue Type ◽

Functional Assay ◽

Uterine Tissue ◽

Tissue Type Plasminogen Activator ◽

Type Plasminogen Activator ◽

Baseline Values

SummaryFunctional assay of extrinsic (tissue-type) plasminogen activator (EPA) in plasma on fibrin plates was evaluated. Using specific quenching antibodies, we demonstrated the method to be specific for EPA under all conditions tested. Contributions of urokinases and intrinsic activators were excluded. The quantity of EPA in blood samples, as compared with purified uterine tissue activator, shows 1 blood activator unit (BAU) to be comparable to 0.93 ng.The median values for EPA activity for healthy volunteers were: baseline, 1.9 BAU/ml (n = 123); diurnal, 5.5 BAU/ml (n = 12); DDAVP administration, 11.7 BAU/ml (n = 39); exhaustive exercise, 25 BAU/ml (n = 24); venous occlusion (15 min), 35 BAU/ml (n = 61). A large inter-individual variation in EPA activity was found, while individual baseline values tended to be constant for periods of weeks.In vitro in blood EPA activity shows a disappearance of 50% in about 90 min at 37° C; EPA activity in euglobulin fractions is stable for ≤2 hr at 37° C.A rapid decrease in EPA activity occurs in vivo, as noted after extracorporal circulation and exercise stimulation (t½ decay, 2-5 min).

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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

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661598 ◽

1986 ◽

Vol 56 (01) ◽

pp. 035-039 ◽

Cited By ~ 42

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.

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