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High-frequency ultrasound has been shown to accelerate enzymatic fibrinolysis. One of the supposed mechanisms of this effect is the enhancement of mass transport by acoustic streaming, i.e., ultrasound-induced macroscopic flow around the clot. In this study, which is aimed at further elucidating the mechanisms of the acceleration of fibrinolysis by ultrasound, we investigated whether ultrasound would accelerate fibrinolysis if the flow around the thrombus is already present, as may occur in vivo. The effect of the ultrasound-induced temperature rise was also studied. In a model of a plasma clot submerged in plasma, containing tissue-type plasminogen activator, mild stirring of the outer plasma producing a shear rate of 40 seconds-1 at the surface of the clot resulted in a two-fold acceleration of lysis. A similar effect was obtained with ultrasound (1 MHz, 2 W/cm2). Furthermore, if ultrasound was applied together with stirring, only 30% acceleration by ultrasound was documented, fully attributable to the concomitant temperature rise. In a model with tissue-type plasminogen activator incorporated throughout a plasma clot, the effect of ultrasound (two-fold shortening of lysis time) was fully attributable to the concomitant temperature rise of a few degrees. We concluded that the acceleration of enzymatic plasma clot lysis by high-frequency ultrasound in the models we used can be largely explained by a combination of the effects of heating and acoustic streaming, equivalent to mild stirring. The thermal effects can hardly be utilized in vivo due to the danger of tissue overheat. The therapeutic advantage of transcutaneous high-frequency ultrasound as an adjunct to thrombolytic therapy may appear limited to the situations where there is no flow in the direct environment of the thrombus. Copyright (C) 2000 Elsevier Science Ltd. Chemicals/CAS: Tissue Plasminogen Activator, EC 3.4.21.68

SummaryFibrinolysis initially generates channels in an occluding thombus which results in blood flow through the thrombus. Since the impact of flow along the surface of a thrombus on thrombolysis has not been investigated in detail, we studied in vitro how such a flow affects lysis. Compacted and noncompacted plasma clots were used as model thrombi. With compacted clots, fibrin-specific lysis induced by alteplase in the outer plasma was accelerated about 2-fold by strong flow (arterial shear rate). Non-fibrin-specific lysis induced either by a high concentration of alteplase or by streptokinase was slow, was accompanied by rapid depletion of plasminogen in the outer plasma, and was only slightly accelerated by flow. With noncompacted clots, similar acceleration factors were documented, when mild flow (venous shear rate) was applied. Strong flow further accelerated fibrin-specific lysis, up to 10-fold as compared to lysis without flow, but paradoxically retarded non-fibrin-specific lysis. The data suggest that flow accelerates lysis by enhancing transport of plasminogen from the outer plasma to the surface of the clot. Both opposite effects of the strong flow were mediated by forceful intrusion of the outer plasma into the noncompacted clot due to flow irregularities. In the case of non-fibrin-specific lysis this resulted in the replacement of the plasminogen-containing milieu by plasminogen-depleted outer plasma in certain areas of the clot turning them into virtually unlysable fragments. This flow-enforced “plasminogen steal” may contribute to the relatively high percentage of incomplete thrombolysis (TIMI-2 grade flow) documented in a number of trials for non-fibrin-specific thrombolytic agents. In the case of fibrin-specific lysis, the effect of flow on the speed of fibrinolysis is always beneficial.

SummaryIn a number of cases, thrombolytic therapy fails to re-open occluded blood vessels, possibly due to the occurrence of thrombi resistant to lysis. We investigated in vitro how the lysis of hardly lysable model thrombi depends on the choice of the plasminogen activator (PA) and is accelerated by ultrasonic irradiation. Lysis of compacted crosslinked human plasma clots was measured after addition of nine different PAs to the surrounding plasma and the effect of 3 MHz ultrasound on the speed of lysis was assessed.Fibrin-specific PAs showed bell-shaped dose-response curves of varying width and height. PAs with improved fibrin-specificity (staphylokinase, the TNK variant of tissue-type PA [tPA], and the PA from the saliva of the Desmodus rotundus bat) induced rapid lysis in concentration ranges (80-, 260-, and 3,500-fold ranges, respectively) much wider than that for tPA (a 35-fold range). However, in terms of speed of lysis, these three PAs exceeded tPA only slightly. Reteplase and single-chain urokinase were comparable to tPA, whereas two-chain urokinase, anistreplase, and streptokinase were inferior to tPA. In the case of fibrin-specific PAs, ultrasonic treatment accelerated lysis about 1.5-fold. For streptokinase no acceleration was observed. The effect of ultrasound correlated with the presence of plasminogen in the outer plasma, suggesting that it was mediated by facilitating the transport of plasminogen to the surface of the clot.In conclusion, PAs with improved fibrin-specificity induce rapid lysis of plasminogen-poor compacted plasma clots in much wider concentration ranges than tPA. This offers a possibility of using single-or double-bolus administration regimens for such PAs. However, it is not likely that administration of these PAs will directly cause a dramatic increase in the rate of re-opening of the occluded arteries since they are only moderately superior to tPA in terms of maximal speed of lysis. Application of high-frequency ultrasound as an adjunct to thrombolytic therapy may increase the treatment efficiency, particularly in conjunction with fibrin-specific PAs.

Binding of components of the fibrinolytic system to fibrin is important for the regulation of fibrinolysis. In this study, decomposition of the fibrin network and binding of plasminogen and plasminogen activators (PAs) to fibrin during lysis of a plasma clot were investigated with confocal microscopy using fluorescein-labeled preparations of fibrinogen, plasminogen, tissue-type PA (t-PA), and two-chain urokinase-type PA (tcu-PA). Lysis induced by PAs present throughout the plasma clot was accompanied by a gradual loss of fibrin content of fibers and by accumulation of plasminogen onto the fibers. Two sequential phases could be distinguished: a phase of prelysis, during which the fibrin network remained immobile, and a phase of final lysis, during which fibers moved with a tendency to shrink and eventually disappeared. The two phases occurred simultaneously but in different locations when lysis was induced by PAs present in the plasma surrounding the clot. The zone of final lysis was located within a 5-8-μm superficial layer, where fibers were mobile, and surface-associated fibrin agglomerates appeared. Plasminogen accumulated in these agglomerates up to 30-fold as compared with its concentration in the outer plasma. t-PA was also highly concentrated in the agglomerates, and tcu-PA bound to them slightly. The zone of prelysis, where plasminogen was moderately accumulated on the immobile fibers, was located deeper in the clot. This zone was much thinner in the case of t-PA-induced lysis than in the case of tcu-PA-induced lysis, reflecting the difference in penetration of the two PAs into the clot. We conclude that under conditions of diffusional transport of fibrinolytic enzymes from outside a plasma clot, extensive lysis is spatially restricted to a zone not exceeding 5-8 μm from the clot surface. In this zone the structure of the fibrin network undergoes significant changes, and strikingly high accumulation of fibrinolytic components takes place.

Background Binding of plasminogen to partially degraded fibrin is an important step in fibrinolysis, influencing its rate and fibrin specificity. Little is known about the spatial distribution of plasminogen and of plasminogen-binding sites inside thrombi during lysis. In the present study, we investigated this problem, which is important for a better understanding of the local regulation of fibrinolysis and the rate-limiting factors of therapeutic thrombolysis. Methods and Results An experimental system was used that allowed continuous visualization and quantification by fluorescence microscopy of the spatial distribution of fluorescein-labeled plasminogen inside and outside model thrombi. Strong superficial accumulation of plasminogen was observed during lysis of a plasma clot induced by tissue-type or urokinase-type plasminogen activators in the surrounding plasma. A distinctly visible plasminogen-accumulating shell moved continuously with the reducing surface of the clot. The accumulation decreased in conditions of exhaustive activation of plasminogen in the outer plasma. It was found in a purified system that a thin superficial layer (≈50 μm wide) of a plasmin-treated fibrin clot exposes about 2.5 plasminogen-binding sites per fibrin monomer with a K d of 2.2 μmol/L. At a physiological concentration of plasminogen (1.5 μmol/L) in the outer medium, plasminogen was concentrated about 10-fold in this layer. The binding was dose-dependently inhibited by ε-aminocaproic acid. Conclusions We conclude that the generation of potent surface-associated plasminogen-binding sites during thrombolysis results in a strikingly high plasminogen concentration at the dynamically changing surface of a lysing clot. The necessity of a continuous plasminogen supply from the plasma supports the use of fibrin-specific and plasminogen-sparing agents for thrombolytic therapy.

The precursor of plasma carboxypeptidase B (pCPB) also known as thrombin-activable fibrinolysis inhibitor can be converted by thrombin to an active enzyme capable of eliminating C-terminal Lys- and Arg-residues from proteins. The activation is about 1000-fold more efficient in the presence of thrombomodulin (TM). We investigated the antifibrinolytic potency of maximally activated pCPB in plasma and explored the antifibrinolytic mechanism of pCPB. During clotting of plasma in the presence of 3.3 NIH units/ml thrombin and 1 microg/ml soluble TM, more than 80% pro-pCPB was converted into the active form causing an increase of plasma carboxypeptidase activity from 100 units/liter (constitutive activity ascribed to plasma carboxypeptidase N) to 430 units/liter as measured with furoylacroleyl-alanyl-arginine substrate. Under these conditions, lysis of a plasma clot induced by a range of tissue-type plasminogen activator (t-PA) concentrations (0.2-2 microg/ml) was retarded more than 4-fold. A considerable retardation of fibrinolysis was observed upon addition of as little as 12 ng/ml soluble TM, a concentration comparable with physiological concentrations of soluble TM in human plasma. The presence of Ca2+ appeared to be a critical requirement for effective activation of pro-pCPB by thrombin-TM in plasma. Plasminogen-binding sites (C-terminal lysines) on the surface of a plasmin-treated fibrin clot were eliminated within 1-3 min by plasma with maximally activated pCPB, as studied in a recently described model involving fluorescence microscopy. Confocal fluorescence microscopy showed that in the absence of TM plasminogen strongly accumulated on fibrin fibers during t-PA-induced lysis of a plasma clot. In the presence of TM (and a concomitant pro-pCPB activation), lysis was slow and was not accompanied by accumulation of plasminogen on the fibers. In conclusion, generation of active pCPB during clotting of plasma in the presence of Ca2+ and TM leads to a retardation of plasma clot lysis in a wide range of t-PA concentrations, from low to therapeutic, and to a fast elimination of plasminogen-binding sites on partially degraded fibrin. This is a likely mechanism for the antifibrinolytic effect of active pCPB.