Rate Of Clot Lysis Research Articles

Combination tissue plasminogen activator and ticlopidine therapy in a rabbit model of acute thromboembolic stroke

The successful application of thrombolytic therapy to acute myocardial infarction has prompted a reinvestigation of thrombolytic therapy for acute stroke. However, an examination of safety and efficacy of thrombolytic therapy in acute thromboembolic stroke has precluded the entry of patients taking either antiplatelet or anticoagulant therapy. It was therefore of interest, in an established rabbit model of thromboembolic stroke, to examine the use of tissue plasminogen activator therapy in combination with ticlopidine treatment. Following ticlopidine administration (10 mg kg-1, i.v., daily for 5 days), rabbits (n = 7) were embolized by injecting a tin-laden clot into the internal carotid artery with clot placement confirmed by x-ray. Three hours later, t-PA was initiated as a square-wave pulse (6.3 mg kg-1 total dose, given as a 20% bolus, with the remainder administered over 2 h as a continuous infusion). The protocol was continued for a total of 7 h following embolization. Complete clot lysis was demonstrated in 6 of 7 animals. Brain infarct size (triphenyltetrazolium chloride staining) was 36.0 +/- 12.9% hemisphere (mean +/- SEM). Both clot lysis rate and infarct size were very similar to that previously seen following administration of t-PA alone (58% and 31.6 +/- 6.4% hemisphere, respectively) but in marked contrast to previous results seen with intravenous aspirin (no clot lysis). These results suggest that antiplatelet agents used clinically for stroke prophylaxis (aspirin or ticlopidine) may influence the success rate of thrombolysis following initiation of thrombolytic therapy for acute thromboembolic stroke.

Optimizing Coronary Thrombolysis With IV Administration of Recombinant Tissue Plasminogen Activator: Single Bolus vs Double Bolus vs Front-loading

This study was designed to compare the efficacy of coronary thrombolysis obtained with i.v. administration of three dose regimens of recombinant tissue plasminogen activator (rtPA). Although many studies have confirmed the efficacy of thrombolytic therapy in treatment of acute myocardial infarction, few prospective studies have been designed to determine which dose regimen optimizes the rate of coronary thrombolysis. A canine model was used. Coronary thrombosis was induced by injection of radioactive, autologous blood clots through a catheter placed in the left anterior descending coronary artery. Subsequently, 15 dogs were randomized into 3 groups of 5 dogs each. In group 1 dogs, 1.25 mg/kg of rtPA was administered i.v. as a bolus; in the group 2 dogs, 1.25 mg/kg of rtPA was administered over 60 min. The administration was "front loaded" so that 15% was administered as a bolus, 60% over 30 min, and 25% over 30 min; in group 3, 1.25 mg/kg of rtPA was divided into two i.v. boluses and administered 15 min apart. Coronary thrombolysis was assessed with a gamma camera. Despite differences in rate of administration of rtPA, at 15, 30, and 90 min after onset of treatment, extent of clot lysis was similar between groups. These results indicate that despite differences in dose regimens, rates of thrombolysis are similar when i.v. rtPA is relatively rapidly administered. Further, the similar rates of clot lysis over time between groups suggest both an effective upper limit to the dose-thrombolytic rate relationship and relatively high, sustained steady-state plasma concentrations of rtPA.

Plasma carboxypeptidases as regulators of the plasminogen system.

Carboxy-terminal lysine residues on the surface of cells and fibrin bind plasminogen and control its activation. Since plasma contains basic carboxypeptidases, which remove carboxy-terminal lysines from protein substrates, we investigated if these enzymes are involved in the regulation of plasminogen binding sites. Plasma reduced plasminogen binding to cells, and this effect could be ascribed to the activity of the plasma carboxypeptidases. Purified carboxypeptidase N, which is constitutively active, and plasma carboxypeptidase B, which circulates as a zymogen, were both capable of significantly reducing plasminogen binding to cells. Dose titration experiments verified that plasma concentrations of either carboxypeptidase were sufficient to maximally affect plasminogen binding to cells. Furthermore, plasma carboxypeptidase B, but not carboxypeptidase N, reduced the rate of whole blood clot lysis induced by tissue-type plasminogen activator. These findings establish that plasma carboxypeptidases can modulate plasminogen binding to cells and control the rate of fibrinolysis. These functions delineate a novel role for the plasma carboxypeptidases in the regulation of the plasminogen system.

Comparison of streptokinase, urokinase, and recombinant tissue plasminogen activator in an in vitro model of venous thrombolysis

Presumed differences in the thrombolytic activity and fibrinolytic specificity of the three commonly used thrombolytic agents, streptokinase, urokinase, and recombinant tissue plasminogen activator (rt-PA), are based on clinical study results, where variability renders meaningful comparisons difficult. An in vitro model of catheter-directed venous thrombolysis was used to compare the three agents. Retracted iodine 125-radiolabeled clots that simulate those observed in the venous system were infused with thrombolytic agents at doses analogous to those used clinically. Perfusion with heparinized, whole human blood was undertaken for 60 minutes, measuring the efficacy of thrombolysis through serial quantification of radio tracer released into the circuit. Fibrinolytic specificity was determined by following decrements in perfusate fibrinogen concentration. Streptokinase was the agent associated with the slowest rate of clot lysis (p = 0.01 vs urokinase and rt-PA). Urokinase was associated with an intermediate rate of lysis but appeared to be the agent with the greatest degree of fibrinolytic specificity (p = 0.02 vs streptokinase, p = 0.05 vs rt-PA). Although rt-PA was associated with improved efficacy early in the perfusions, the differences between rt-PA and urokinase dissipated after 30 minutes. These laboratory observations suggest that urokinase may be the most appropriate agent for catheter-directed venous thrombolysis, offering an advantageous compromise between fibrinolytic specificity and thrombolytic speed.

Differential effects of staphylokinase, streptokinase and tissue-type plasminogen activator on the lysis of retracted human plasma clots and fibrinolytic plasma parameters in vitro

A novel plasma clot lysis system was used to compare the fibrinolytic characteristics of staphylokinase, streptokinase and tissue-type plasminogen activator. 125I-fibrinogen-labelled human plasma clots were formed on needles and mechanically compressed after spontaneous retraction. This model is relatively resistant to lysis and differentiates between fibrin-specific and non-fibrin-specific plasminogen activators. The novel plasminogen activator, recombinant staphylokinase, produced high rates of clot lysis without markedly influencing fibrinogen, plasminogen and alpha 2-antiplasmin in the plasma containing the clots. At equimolar concentrations, streptokinase markedly depleted these parameters in plasma despite low clot lysis rates. Tissue-type plasminogen activator showed relatively high lysis rates at low concentrations, but at higher concentrations, plasminogen depletion caused a decrease in clot lysis. Staphylokinase can be characterised as a fibrin-specific and plasminogen-saving fibrinolytic agent with a high clot lysis potential.

Intravenous Aspirin Causes a Paradoxical Attenuation of Cerebrovascular Thrombolysis

Aspirin treatment is recognized as an advantageous adjunct to thrombolytic agents in myocardial infarct patients. In this study we examined the effects of aspirin on the rate of clot lysis and on the frequency and extent of hemorrhagic transformations in rabbit models of embolic stroke. Rabbit models of ex vivo platelet aggregation and cutaneous template bleeding times were used to show the anticoagulant effects of aspirin in our experimental paradigm. We monitored tissue-type plasminogen activator (TPA)-induced clot lysis in two rabbit models of embolic stroke by (1) scintigraphically following the dissolution of a 99mTc-tagged clot or (2) using roentgenography to follow the disappearance of an Sn-tagged clot. In animals pretreated (18 hours) with a single administration of aspirin (1, 5, or 20 mg/kg IV) or 1 mg/kg per day for 3 days, the aggregation response of platelets to collagen (3.3 micrograms/mL) or arachidonic acid (0.5 mmol/L) was attenuated. High-dose aspirin also increased ear template bleeding time from 1.6 to 2.6 minutes. When aspirin (20 mg/kg) was administered 18 hours before embolism and subsequent lysis with TPA (0.3 mg/kg bolus; 3 mg/kg per hour IV), the pretreatment significantly antagonized the rate and extent of TPA-induced clot lysis by up to 70%. This was confirmed in a second embolic stroke model. The suppression of TPA-induced lysis was reversed by administration of the prostacyclin analogue iloprost (10 micrograms/kg per hour) directly into the cerebral circulation. We conclude that aspirin reduces the effects of TPA in embolic stroke models. This effect may be the result of a loss of endothelial prostacyclin production since the effect is reversed by iloprost.

An increase in low aortic pressure increases coronary artery flow and coronary thrombolysis induced by intravenous administrarion of recombinant tissue plasminogen activator

Purpose: Our study investigated the effects of an increase in aortic pressure, induced by norepinephrine (NE) administration on coronary artery flow in a clotted artery, and rate of coronary thrombolysis induced by intravenous (IV) administration of recombinant tissue plasminogen activator (rtPA). Methods: A canine model of coronary thrombosis, induced by intracoronary injection of radioactive autologous blood clots, was used to test the hypothesis that an increase in aortic blood pressure will increase coronary artery flow and the rate of clot lysis induced by IV administration of rtPA. Results: After clot injection, 11 dogs were phlebotomized to decrease systolic aortic pressure to 75 mm Hg. Subsequently, .25 mg/kg of rtPA was administered intravenously over two 15-minute intervals, one during hypotension, and the other after NE infusion had increased systolic blood pressure to 130 mm Hg. In six dogs the hypotensive condition was studied first, and in five dogs the NE-induced normotensive condition was studied first. In all dogs, coronary artery flow and the rate of clot lysis were significantly increased in the normotensive condition. Conclusions: These results indicate that an increase in a low coronary artery perfusion pressure may enhance coronary artery flow and the rate of thrombolysis.

Acute release of t-pa in rats and mice is inhibited by endotoxin and dexamethasone but is not affected by retinoic acid

To investigate the contribution of acute release of tissue type plasminogen activator (t-PA) on blood fibrinolytic activity, the ability to modulate the PAF-induced release of t-PA in perfused hindquarters of rats, mice, and hamsters was studied. Effects on t-PA release were compared with effects on ex vivo blood clot lysis. Animals were orally pretreated with dexamethasone (0.1–1 mg/kg) or retinoic acid (3 mg/kg) for a period of 5 days or intravenously with lipopolysaccharide (LPS: 0.03–50 μg/kg) for a period of 4 h. Maximum release of t-PA in response to PAF was 150, 40, and 6 U/kg in rats, mice, and hamsters, respectively, at concentrations of 2, 30, and 20 μM of PAF, respectively. Retinoic acid pretreatment had no effect on acute t-PA release in any of the species. Pretreatment with dexamethasone resulted in a dose-dependent decrease of the t-PA release in rats and mice to 25 ± 7% and 48 ± 6% (relative to vehicle treated animals), respectively at a dose of 1 mg/kg. LPS pretreatment resulted in about 45 % inhibition of t-PA release in both species at a dose of 50 μg/kg. No effects of dexamethasone or LPS were detected on acute release of t-PA in hamsters. In vivo acute release of t-PA in rats was inhibited by about 75% after dexamethasone pretreatment (I mg/kg) and by about 35% after LPS pretreatment (50 μg/kg). Pretreatment of rats with retinoic acid (3 mg/kg) had no effect on in vivo acute release of t-PA. LPS treatment resulted in complete inhibition of ex vivo measured blood clot lysis at doses of 0.3, 10, 0.1 μg/kg for rats, mice, and hamsters, respectively. Dexamethasone and retinoic acid affected the lysis rate of rat blood clots 1,2 but no effects on the lysis rates of murine or hamster blood clots were observed. It is concluded that in rats and mice but not in hamsters acute release of t-PA can be inhibited by pretreatment with dexamethasone or endotoxin. These effects do not parallel with effects on the rate of normal blood clot lysis.

Transglutaminase inhibition by 2-[(2-oxopropyl)thio]imidazolium derivatives: mechanism of factor XIIIa inactivation.

The physiologic role of several transglutaminases could be more precisely defined with the development of specific inhibitors for these enzymes. In addition, specific plasma transglutaminase (fXIIIa) inhibitors may have therapeutic utility in the treatment of thrombosis. For these purposes, the inactivation of fXIIIa and human erythrocyte transglutaminase (HET) by 2-[(2-oxopropyl)thio]imidazolium derivatives, which comprise a novel class of transglutaminase inactivators, was studied. As a specific example, 1,3,4,5-tetramethyl-2-[(2-oxopropyl)thio]imidazolium chloride (III) inactivated fXIIIa with an apparent second-order rate constant (specificity constant of inactivation) of 6.3 x 10(4) M-1 s-1, corresponding to a rate 4 x 10(7) times greater than its reaction rate with glutathione (GSH). The mechanism of fXIIIa inactivation by this class of compounds was investigated utilizing two [14C]-isotopic regioisomers of 1,3-dimethyl-2-[(2-oxopropyl)thio]imidazolium iodide (II). Structural analyses demonstrated that acetonylation of the active site cysteinyl residue of fXIIIa occurred along with the stoichiometric release of the complementary fragment of the inactivator as the corresponding thione. Kinetic analysis of the inactivation of fXIIIa by nonquarternary analogs of II and III indicated the formation of a reversible complex between the inactivator and fXIIIa prior to irreversible modification of the enzyme. At 1 mM, III displayed no detectable levels of inhibition or inactivation with several serine proteases and thiol reagent-sensitive enzymes. 2-[(2-Oxopropyl)thio]imidazolium derivatives and the related molecule 2-(1-acetonylthio)-5-methylthiazolo-[2,3]-1,3,4-thiadiazo lium perchlorate (I), when present at the time of clot formation at 1-10 microM, enhanced the rates of tissue plasminogen activator catalyzed clot lysis in vitro. These inactivators prevented the fXIIIa-catalyzed covalent incorporation of alpha 2-antiplasmin into the alpha chain of fibrin and the formation of high molecular weight fibrin alpha chain polymers, providing the basis for the observed enhancements in clot lysis rates.

Thrombolytic therapy for stroke

The physiology of thrombi and the pharmacology of thrombolytic drugs are under active study and improved regimens for the dosing of thrombolytic agents have been developed. In the setting of myocardial infarction, recently reported differences among thrombolytic agents have been slight, including the frequency of thrombolysis-associated hemorrhagic stroke following tissue plasminogen activator or streptokinase. In the setting of ischemic stroke, recanalization rates following intravenous tissue plasminogen activator have been modest and at least partly dependent on clot size. Conclusions regarding clinical benefit will depend on the results of multicenter randomized trials that should available in 1995. Studies of locally administered intra-arterial thrombolytic therapy demonstrate high rates of clot lysis, but clinical benefits have yet to be established. The results of randomized trials will be important in clarifying any cause-effect relationships between thrombolytic therapy and symptomatic and asymptomatic intracranial hemorrhage. Thrombolytic therapy in the study of subarachnoid hemorrhage is under active investigation.

Plasminogen acceleration of urokinase thrombolysis.

A relative deficiency of plasminogen within the thrombus may be the rate limiting factor in clot lysis. To investigate this hypothesis, we used an in vitro perfusion system and expanded polytetrafluoroethylene graft segments filled with radiolabeled human thrombus. Three groups of five perfusions were compared: (1) urokinase infusion (333 IU/min) into clots laced with buffer, (2) urokinase infusion (333 IU/min) into clots laced with plasminogen (44 CU), and (3) control, D5W infusion into clots laced with buffer. Two end points were measured over time: the amount of lysed thrombus and the flow through the graft. Urokinase infusion resulted in augmented flow through the graft when compared with control (p < 0.05). Lacing with plasminogen resulted in more rapid restoration of flow when compared with urokinase infusion alone (p < 0.05). Similarly, the rate of clot dissolution was significantly greater in plasminogen-laced thrombi (p < 0.05) when compared with the control and urokinase groups. Embolization of particles of thrombus was uniformly observed in the urokinase group, resulting in a temporary decrease in flow through the thrombosed graft. This event characteristically occurred after 60 minutes of infusion but was never seen in the urokinase/plasminogen treatment group. These results suggest that plasminogen supplementation of urokinase thrombolysis may result in significant clinical benefits with respect to the rate of clot lysis and the uniformity of clot dissolution with a lower likelihood of secondary embolization.

Mice release less tissue-type plasminogen activator (t-PA) and have more active clot stabilization by FXIII compared to rats which might explain their low rate of blood clot lysis

Fibrinolysis regulates the removal of blood clots from the bloodstream and is mediated by tissue-type plasminogen activator (t-PA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1). Rats and mice are commonly used animal species in pharmacological studies. However, very little is known about the fibrinolytic system of both species which prompted us to study the fibrinolytic system of rats and mice. At first we studied the ex vivo measured blood clot lysis. Blood clots prepared from 10% murine blood did not lyse spontaneously while blood clots prepared from 10% rat blood lysed for about 30% within 5 h. Addition of 2.5IU of human t-PA per ml diluted murine blood enhanced the lysis rate to a level comparable to rat blood. After i.v. administration of PAF, bradykinin, carbachol or adrenalin the lysis rate of both rat and murine blood clots was enhanced. The minimum dose of PAF, bradykinin, carbachol, or adrenalin which could induce a significant enhanced clot lysis was 0.06, 2.0, 0.8 and 0.8 μg/kg respectively for rats and 0.6, 2.0, 7.7 and 7.7 μg/kg respectively for mice. When no t-PA was added to mice blood clots these doses were not sufficient to induce a significant enhanced clot lysis; 3- to 10-fold higher doses were required. Addition of a FXIII inhibitor to diluted murine blood also increased the sensitivity for the used inducers. The doses which evoked an increased lysis rate under these conditions were 0.6, 0.6, 2.3 and 7.7 μg/kg respectively. No effects of FXIII inhibition were seen on lysis rates of rat blood clots. Secondly, acute t-PA release was studied in the perfused hindleg model with PAF, bradykinin, carbachol and adrenalin as inducers. Both rats and mice showed an acute t-PA release but differences in sensitivity were seen. The concentrations that induced half maximal release were 0.01, 1.2, 12 and 3 μM respectively for rats while for mice these values were 20, 3.7, 5.3 and 4.8 μM. The maximum amount of releasable t-PA in the perfusate was 26 U/kg for rats and 14 U/kg for mice measured using PAF and bradykinin respectively as inducer. Plasma PAI-1 levels were 1.7±0.2 U/ml and 3.7±0.3 U/ml for rats and mice respectively. Our results suggest that the high level of t-PA release and the lack of FXIII a mediated clot stabilization in rats contribute to the high rate of clot lysis compared to mice.

The Role of the Finger Domain of Tissue-type Plasminogen Activator (t-PA) and Plasminogen Activator Inhibitor 1 (PAI-1) in Initiation and Progression of Thrombolysis

We further investigated the role of the finger (F) and the kringle-2 (K2) domains of tissue-type plasminogen activator (t-PA) in fibrin-stimulated plasminogen activation. To that end, the action of purified (wt) t-PA or of variants lacking F (del.F) or K2 (del.K2) was assessed either in a static, human whole blood clot-lysis system or in whole blood thrombi generated in the "Chandler loop". In both clot-lysis systems, significant differences were observed for the initiation of thrombolysis with equimolar concentrations of the t-PA variants. A relatively minor "lag phase" occurred in thrombolysis mediated by wt t-PA, whereas a 6.4-fold and 1.6-fold extension is found for del.F and del.K2, respectively. We observed identical lag-times, characteristic for each t-PA variant, in platelet-rich heads and in platelet-poor tails of thrombi. Since plasminogen activator inhibitor 1 (PAI-1) is preferentially retained in the platelet-rich heads, we conclude that the inhibitor does not interfere with the initial stage of thrombolysis but exerts its action in later stages, resulting in a reduction of the rate of clot lysis. A complementation clot-lysis assay was devised to study a potential interplay of del.F and del.K2. Accordingly, clot lysis was determined with combinations of del.F and del.K2 that were inversely varied in relation to equipotent dosage to distinguish between additive, antagonistic or synergistic effects of these variants. The isobole for combinations of del.F and del.K2 shows an independent, additive action of del.F and del.K2 in clot lysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-linking of α2-antiplasmin to fibrin is a key factor in regulating blood clot lysis

The basal lysis rates ofex-vivoprepared blood clots from rats, mice, hamsters, dogs, and humans and levels of α2-antiplasmin (α2-AP) cross-linked to fibrin have been studied in the presence and absence of factor XIII (FXIII) inhibitors using a blood clot lysis assay and an α2-AP binding assay. Clots prepared from rat or human blood lysed spontaneously within 3-5h. Clots from hamster blood lysed completely within 30 min but clots prepared from murine or canine blood lysed only after addition of 1.0 IU human t-PA. To study the effect of activated FXIII (FXIII) inhibition on blood clot lysis the FXIII inhibitors L722151 and mono-dansylcadaverine (dansyl) were used. In the presence of the FXIII inhibitors human, murine, and canine blood clots showed increased lysis rates. The lysis rate of rat blood clots was not affected. Effects on hamster blood clots could not be detected because of the high basal lysis rate. In clots prepared from human, murine, or canine plasma about 20% of the plasma α2-AP concentration was cross-linked to fibrin. FXIII inhibitors inhibited crosslinking by more than 80%. No significant cross-linking of α2-AP could be detected in rat and hamster plasma clots. When 0.1 volume of human plasma was added to 0.9 volume of rat plasma the level of α2-AP cross-linking was equal to that in human plasma indicating that rat α2-AP can be cross-linked to human fibrin. The same effect was obtained with human FXIII deficient plasma but not with human fibrinogen deficient plasma. These experiments indicate that rat fibrinogen is not a substrate for the cross-linking of rat α2-AP catalysed by rat FXIII, Addition of human plasma to hamster plasma did not result in an increased level of α2-AP cross-linking which indicates that other factors are involved.

Cross-linking of α2-antiplasmin to fibrin is a key factor in regulating blood clot lysis

The basal lysis rates of ex-vivo prepared blood clots from rats, mice, hamsters, dogs, and humans and levels of alpha 2-antiplasmin (alpha 2-AP) cross-linked to fibrin have been studied in the presence and absence of factor XIII (FXIII) inhibitors using a blood clot lysis assay and an alpha 2-AP binding assay. Clots prepared from rat or human blood lysed spontaneously within 3-5 h. Clots from hamster blood lysed completely within 30 min but clots prepared from murine or canine blood lysed only after addition of 1.0 IU human t-PA. To study the effect of activated FXIII (FXIIIa) inhibition on blood clot lysis the FXIII inhibitors L722151 and mono-dansylcadaverine (dansyl) were used. In the presence of the FXIII inhibitors human, murine, and canine blood clots showed increased lysis rates. The lysis rate of rat blood clots was not affected. Effects on hamster blood clots could not be detected because of the high basal lysis rate. In clots prepared from human, murine, or canine plasma about 20% of the plasma alpha 2-AP concentration was cross-linked to fibrin. FXIII inhibitors inhibited cross-linking by more than 80%. No significant cross-linking of alpha 2-AP could be detected in rat and hamster plasma clots. When 0.1 volume of human plasma was added to 0.9 volume of rat plasma the level of alpha 2-AP cross-linking was equal to that in human plasma indicating that rat alpha 2-AP can be cross-linked to human fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of ultrasound-potentiated fibrinolysis in vitro

We have characterized the effects of ultrasound on fibrinolysis in vitro to investigate the mechanism of ultrasonic potentiation of fibrinolysis and to identify potentially useful ultrasound parameters for therapeutic application. Radiolabeled clots in thin walled tubes were exposed to ultrasound fields in a water bath at 37 degrees C, and lysis was measured by solubilization of radiolabel. Ultrasound accelerated lysis of plasma, whole blood, and purified fibrin clots mediated by recombinant tissue-type plasminogen activator (rt-PA), urokinase, or streptokinase, but ultrasound by itself caused no clot solubilization. The degree of ultrasonic potentiation was dependent on plasminogen activator concentration, increasing from 2.2-fold at a streptokinase concentration of 75 U/mL to 5.5-fold at 250 U/mL in a 1 MHz ultrasound field at 4 W/cm2. Ultrasound exposure resulted in heating due to absorption by the plastic tube, but the temperature increase was insufficient to account for the increase in clot lysis rate, indicating that the primary effect was nonthermal. Ultrasound did not accelerate hydrolysis of a peptide substrate by rt-PA and did not alter the rate of plasmic degradation of fibrinogen, indicating that the augmentation of enzymatic fibrinolysis required the presence of a fibrin gel. The acceleration of fibrinolysis by ultrasound was greater at higher intensities and duty cycles and was maximum at frequencies between 1 and 2.2 MHz, but decreased at 3.4 MHz. These findings suggest that ultrasound accelerates enzymatic fibrinolysis by increasing transport of reactants through a cavitation-related mechanism.

Characterization of Ultrasound-Potentiated Fibrinolysis In Vitro

AbstractWe have characterized the effects of ultrasound on fibrinolysis in vitro to investigate the mechanism of ultrasonic potentiation of fibrinolysis and to identify potentially useful ultrasound parameters for therapeutic application. Radiolabeled clots in thin walled tubes were exposed to ultrasound fields in a water bath at 37°C, and lysis was measured by solubilization of radiolabel. Ultrasound accelerated lysis of plasma, whole blood, and purified fibrin clots mediated by recombinant tissue-type plasminogen activator (rt-PA), urokinase, or streptokinase, but ultrasound by itself caused no clot solubilization. The degree of ultrasonic potentiation was dependent on plasminogen activator concentration, increasing from 2.2-fold at a streptokinase concentration of 75 U/mL to 5.5-fold at 250 U/mL in a 1 MHz ultrasound field at 4 W/cm2. Ultrasound exposure resulted in heating due to absorption by the plastic tube, but the temperature increase was insufficient to account for the increase in clot lysis rate, indicating that the primary effect was nonthermal. Ultrasound did not accelerate hydrolysis of a peptide substrate by rt-PA and did not alter the rate of plasmic degradation of fibrinogen, indicating that the augmentation of enzymatic fibrinolysis required the presence of a fibrin gel. The acceleration of fibrinolysis by ultrasound was greater at higher intensities and duty cycles and was maximum at frequencies between 1 and 2.2 MHz, but decreased at 3.4 MHz. These findings suggest that ultrasound accelerates enzymatic fibrinolysis by increasing transport of reactants through a cavitation-related mechanism.

Recombinant factor XIII supplemented clots resist lysis by plasmin and leucocyte elastase

We have examined the lysis of fibrin and plasma clots supplemented with supraphysiological concentrations of recombinant factor XIII(rFXIII) prior to clotting with thrombin. rFXIII supplements of up to 200 μg/mL reduced the lysis rate of fibrin clots by either plasmin or leucocyte elastase in a dose-dependent fashion. Plasma clots supplemented with rFXIII were larger and more resistant to plasmin lysis relative to controls. These effects were all greater when the clots were incubated for 20 h compared to 1 h. To determine whether these in vitro findings might have an in vivo correlate, rFXIII supplemented and unsupplemented fibrin clots labelled with 125I-fibrin were implanted into the peritoneum of mice and lysis followed by the appearance of 125I in the urine. Supplemented clots released significantly less 125I over the first 10 days post-surgery. We conclude that supraphysiological concentrations of rFXIII reduce lysis to both plasmin and leucocyte elastase in vitro and that supplemented clots show a similar resistance in vivo.

Thrombolytic profiles of clot-targeted plasminogen activators. Parameters determining potency and initial and maximal rates.

Targeting of plasminogen activators to the thrombus by means of fibrin-specific monoclonal antibodies may enhance their thrombolytic potency. The kinetics of clot binding of two human fibrin-specific monoclonal antibodies (MA-12B3 and MA-15C5) and of clot lysis with their chemical 1:1 stoichiometric complexes with recombinant single-chain urokinase-type plasminogen activator (rscu-PA) (rscu-PA/MA-12B3 and rscu-PA/MA-15C5) were determined in hamsters and rabbits. Thrombolytic potencies, maximal rates of clot lysis, and the duration of the lag phases before clot lysis of the antibody/rscu-PA conjugates were compared with those of rscu-PA and tissue-type plasminogen activator (rt-PA). Bolus injection of 7.5 micrograms of 125I-labeled antibody in rabbits with an extracorporeal arteriovenous loop containing a 0.3-mL human plasma clot produced clot-to-blood ratios of 6.6 +/- 1.0 (mean +/- SEM) for MA-12B3 and 1.1 +/- 0.15 for MA-15C5 (p < 0.001 versus MA-12B3) within 6 hours. Progressive digestion of the clot did not alter the binding of MA-12B3 but resulted in as much as a 10-fold increase of the binding of MA-15C5. The conjugates infused intravenously over 90 minutes in hamsters with a human plasma clot in the pulmonary artery produced dose-related in vivo clot lysis. Thrombolytic potencies (maximal slope of the percent lysis versus dose in milligrams of u-PA equivalent per kilogram body weight) were 2,500 +/- 440 for rscu-PA/MA-12B3, 3,600 +/- 640 for rscu-PA/MA-15C5 (p = NS vs. rscu-PA/MA-12B3), 60 +/- 8 for rscu-PA (p < 0.001 versus both conjugates), and 380 +/- 66 for rt-PA (p < 0.001 versus both conjugates). The plasma clearances of the conjugates were fourfold to sixfold slower than those of rscu-PA and rt-PA. Maximal rates of clot lysis, determined by continuous external radioisotope scanning over the thorax, were 0.90 +/- 0.13%, 0.91 +/- 0.17%, 0.84 +/- 0.12%, and 1.1 +/- 0.16% lysis per minute for rscu-PA/MA-12B3, rscu-PA/MA-15C5, rscu-PA, and rt-PA, respectively; these maximal rates were obtained with 0.016, 0.016, 1.0, and 0.25 mg/kg, respectively, and were associated with minimal lag phases of 18 +/- 3.2, 28 +/- 4.9, 34 +/- 3.7, and 25 +/- 3.9 minutes, respectively. The thrombolytic potency of the rscu-PA/antifibrin conjugates is determined by their clearance, as well as by rate and extent of initial binding to clots and by changes in binding during clot lysis. Clot targeting of rscu-PA with fibrin-specific antibodies increases its thrombolytic potency but does not alter the maximal rate or the minimal lag phase of clot lysis. These parameters appear to be independent of the nature of the plasminogen activator and of targeting.

Pharmacokinetic and Thrombolytic Properties of Chimeric Plasminogen Activators Consisting of a Single-Chain Fv Fragment of a Fibrin-Specific Antibody Fused to Single-Chain Urokinase

The pharmacokinetic and thrombolytic properties were determined of two recombinant single-chain chimeric plasminogen activators (PA) consisting of u-PA-33k, a low-molecular weight derivative of single-chain urokinase-type PA (scu-PA) comprising amino acids Ala132 through Leu411, and of either a single-chain variable region fragment (Fv) derived from the fibrin fragment D-dimer-specific monoclonal antibody MA-15C5 (K12G0S32) or of the deglycosylated single-chain Fv fragment obtained by substitution of Asn88 with Glu (K12G2S32). Following bolus injection in hamsters, clearances of recombinant scu-PA (rscu-PA) and of K12G0S32 were similar. In contrast, clearance of K12G2S32 was fourfold slower than that of rscu-PA. The thrombolytic potency (percent lysis per u-PA administered in milligrams per kilogram body weight) and specific thrombolytic activity (percent lysis per microgram per milliliter steady-state plasma u-PA antigen level) of these compounds were studied in hamsters with an experimental pulmonary embolus consisting of a human plasma clot injected via the jugular vein. The doses of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were sixfold and 11-fold lower than that of rscu-PA. The steady-state u-PA-related plasma antigen levels of K12G0S32 and K12G2S32 required to obtain maximal rate of clot lysis were 10-fold and fourfold lower than that of rscu-PA. Thus, targeting of K12G0S32 to the clot surface by means of its glycosylated Fv fragment results in a 10-fold increase of its specific thrombolytic activity and sixfold increase of its thrombolytic potency as compared with those of rscu-PA. Targeting of K12G2S32 to the clot surface by means of its deglycosylated Fv fragment results in only a twofold increase of its thrombolytic activity. However, its fourfold slower clearance, combined with its twofold higher specific thrombolytic activity, results in an 11-fold increase of its thrombolytic potency over that of rscu-PA. These findings indicate that the thrombolytic potency of chimeric antibody-targeted PA may be increased by increasing the specific thrombolytic activity, reducing the clearance, or both.