Fibrinolytic behaviour of streptokinase-immobilized poly(methacrylic acid-g-ethylene oxide)

Streptokinase was immobilized on poly(methacrylic acid-g-ethylene oxide) surfaces by means of a modified coupling reagent method. The surfaces were activated with a carbodiimide to form an O-acyl isourea derivative, which, upon addition of the enzyme, condensed to form the corresponding amide. The quantity of enzyme immobilized on the surface increased as the reaction time with carbodiimide and streptokinase increased. The fibrinolytic activity of the immobilized enzyme systems was measured by a clot lysis assay. The dynamics of fibrin clot lysis was investigated for a period of up to 500 min. The lysis reaction mechanism was found to approximate a first-order fibrin degradation. In addition, the kinetic rate constant was found to increase with increasing immobilized enzyme content.     

A mathematical model of post-canalization thrombolysis

During the initial phase of lysis of an occlusive thrombus using lytic agents such as tissue plasminogen activator, blood flow through the centre of the clot is established (the process of recanalization). Following canalization, the clot remains on the vessel wall and further lysis is required. This paper develops a multi-species mathematical model to describe the bulk chemical reactions in the bloodstream and the convective and diffusive transport of chemical species to and from the clot surface in conditions following canalization. For the steady state case, the model indicates that the process of clot lysis following initial recanalization is dominated by surface chemical reactions and the bulk reactions play little role in the lytic process. Lytic rate is dependent on the clot geometry and flow conditions. The rate of clot dissolution is greatest at the upstream end of the clot and decreases steadily downstream due to lytic agent being removed from the flowing blood as it binds to the clot surface. This model may be further developed and used to simulate and compare different lytic regimes.     

Comparison of textilinin-1 with aprotinin as serine protease inhibitors and as antifibrinolytic agents

Textilinin-1 (Q8008) was isolated from the venom of the Pseudonaja textilis and has a 47% sequence identity to the antihaemorrhagic therapeutic agent aprotinin. When equimolar concentrations of enzyme and aprotinin were pre-incubated, plasmin was inhibited 100%, plasma kallikrein 58%, and tissue kallikrein 99%. Under the same conditions, textilinin-1 inhibited plasmin 98%, plasma kallikrein 16% and tissue kallikrein 17%. Whole blood clot lysis was inhibited strongly by both aprotinin and textilinin-1, as shown by thrombelastography. At 2 microM inhibitor lysis initiated by t-PA was greater than 99% inhibited by aprotinin (LY60 = 0.4 +/- 0.1) whereas textilinin-1, inhibited lysis by 91% (LY60 = 8.9 +/- 0.7). The same trend was found with the lysis of euglobulin fractions. From these data textilinin-1 appears to be a more specific plasmin inhibitor than aprotinin but aprotinin inhibits clot lysis to a greater extent.     

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.     

Assessing clot lysis strategies using a simplified mathematical model

This paper attempts to describe lysis of a clot by infusion of lytic agent using a simple geometrical approach, in which the rate of clot lysis is assumed proportional to the exposed surface of the clot and the concentration of lytic agent. Six simple realizations (a)-(f) of this basic model are developed which account for the dependence of clot lysis time on five different clot geometries. In all six cases the clot is initially described as a uniform cylinder which totally occludes a vessel. In model (a) lysis proceeds as an advancing front at the proximal face of the clot. In model (b) lysis proceeds radially outwards from the central axis of the vessel while in model (c) lysis occurs radially inwards from the surface adjacent the wall, of the cylinder. In models (d) and (e) it is assumed that the clot breaks into n uniform spherical and cylindrical fragments, respectively, while model (f) uses the spherical fragment model combined with a lytic agent concentration which decreases with time. The validity of the models was assessed using previously published data from 76 patients in whom lysis time and clot size were recorded. Least squares linear regression analysis based on the six model equations yielded highly significant correlation coefficients r² of 0.457, 0.412, 0.412, 0.495, 0.469, 0.663 for models (a)-(f), respectively. The results suggest that when a constant lytic agent concentration is assumed, no single geometry accounts for significantly more variation than any other, but that a combination of varying lytic agent concentration and clot geometry significantly influences clot lysis time and accounts for much of the observed variation.     

Mechanisms by which thrombolytic therapy results in nonuniform lysis and residual thrombus after reperfusion

A transport reaction model describing penetration of plasmin by diffusion and permeation into a dissolving fibrin gel was solved numerically to explore mechanisms that lead to the formation and growth of dissolution fingers through blood clots during thrombolytic therapy. Under conditions of fluid permeation driven by arterial pressures, small random spatial variations in the initial fibrin density within clots (±4 to 25% peak variations) were predicted by the simulation to result in dramatic dissolution fingers that grew in time. Within vitro experiments, video microscopy revealed that the shape of the proximal face of a fibrin gel, when deformed by pressure-driven permeation, led to lytic breakthrough in the center of the clot, consistent with model predictions of increased velocities in this region leading to cannulation. Computer simulation of lysis of fibrin retracted by platelets (where more permeable regions are expected in the middle of the clot due to retraction) predicted cannulation of the clot during thrombolysis. This residual, annular thrombus was predicted to lyse more slowly, because radial pressure gradients to drive inner clot permeation were quite small. In conjunction with kinetic models of systemic pharmacodynamics and plasminogen activation biochemistry, a two-dimensional transport-reaction model can facilitate the prediction of the time and causes of clot cannulation, poor reperfusion, and embolism during thrombolysis.     

Comparative thrombolytic and immunogenic properties of staphylokinase and streptokinase

The comparative thrombolytic properties of natural (STAN) and recombinant (STAR) staphylokinase (STA) and of streptokinase were studied in hamsters with a pulmonary embolus consisting of a platelet-poor, platelet-rich (300000 platelets/μI), platelet-enriched (1500 000 platelets/ μl) or mechanically compressed human plasma clot. Intravenous infusion over 1 h in doses ranging between 0 and 0.50 mg/kg induced dose-dependent progressive clot lysis without systemic fibrinogen breakdown. The relative thrombolytic potencies, on a weight base, of the STA preparations versus streptokinase were comparable in the platelet-poor clot model (50% lysis requiring 13 μg/kg STA and 12 μg/kg streptokinase) and in the platelet-rich clot model (50% lysis with 28 μg/kg STA and with 32 μg/kg streptokinase), but 5-fold higher in the platelet-enriched clot model (50% lysis with 46 μg/kg STA and with 210 μ/kg streptokinase) and 2-fold higher in the mechanically compressed clot model (50% lysis with 36 μg/kg STA and with 71 μg/kg streptokinase).Dog plasma at baseline contained streptokinase-neutralising activity (neutralising 0.24±0.14 μg streptokinase per ml plasma in a human plasma-based clot lysis assay, mean ±SD) but apparently no STA-neutralising activity. Repeated administration of 40 μg/kg over 1 h of streptokinase at weekly intervals in 5 dogs, increased the streptokinase-neutralising titre to 1.1±0.98 μg/ml plasma after 2 weeks and to 2.9±2.5 μg/ml after 3 weeks (p=0.13 and 0.05 versus baseline, respectively), whereas the thrombolytic potency towards a ¹²⁵I-fibrin-labelled plasma clot inserted into an extracorporeal arteriovenous loop was reduced from 58±4% at baseline to 10±3% after 2 weeks and 4±1% after 3 weeks (p<0.001 vs baseline). Repeated weekly administration of an equipotent dose of STA (4 μg/kg) did not induce measurable STA-neutralising activity after 2 weeks and was associated with persistent, although somewhat reduced clot lysis (58 ± 4% lysis at baseline and 33±7% at 2 weeks, p=0.02). After 3 weeks however, STA-neutralising activity became detectable in plasma (0.34±0.30 μg/ml, p=0.07 vs baseline) and resistance to clot lysis became obvious (6±2% versus a baseline value of 58±4%, p<0.01).Thus, in the hamster model, STA has a thrombolytic potency towards platelet-poor and platelet-rich clots comparable to that of streptokinase, but it is relatively more efficient than streptokinase for the dissolution of platelet-enriched or retracted clots. Furthermore, STA might be less immunogenic than streptokinase as evidenced by less rapid induction of antibody formation and resistance to clot lysis upon repeated administration. Thrombolytic therapy of patients with acute myocardial infarction with STA thus might constitute a potential alternative to treatment with streptokinase, with a better efficacy towards platelet-rich arterial clots and possibly with reduced allergic side effects.     

Experimental thrombosis in rabbit marginal ear vein and evaluation of the thrombolytic action of longolytin

A model of venous thrombosis on the rabbit marginal ear vein was developed. It is based on occlusion of a segment of the left or right ear vein followed by injection of thrombin into this segment. Thrombolytic activity of longolytin applied externally onto the thrombotic venous segment was evaluated. The drug 2-fold shortened the time of clot lysis and 4–4.5-fold increased the rate of thrombolysis. The effect of longolytin was potentiated by heparin. The rate of lysis was higher in large thrombi and in the left vein (effect of asymmetry). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 143, No. 5, pp. 511–514, May, 2007     

A comparison of the rates of clot lysis in a plasma milieu induced by tissue plasminogen activator (t-PA) and rec-pro-urokinase: Evidence that t-PA has a more restricted mode of action

Tissue plasminogen activator (t-PA) and pro-urokinase (pro-UK) were previously found to have distinct and complementary mechanisms of action in a plasma milieu. The t-PA activated primarily plasminogen bound to intact fibrin (internal lysine-bound), whereas pro-UK activated plasminogen bound to partially degraded fibrin (C-terminal lysine-bound). These observations therefore suggested that each of these activators was restricted in its fibrin-dependent lytic effect. At least this is true at activator concentrations where activation of plasminogen is strictly fibrin dependent; at some high concentration all plasminogen is activated including free plasminogen in the ambient plasma. However, since some pro-UK is converted to UK at the plasmin rich fibrin surface during the course of lysis and since UK is a non-selective plasminogen activator, the pro-UK/UK system should not be similarly restricted. The present study was therefore designed to examine whether this is the case by determining the highest rates of lysis achievable at the threshold of specificity for t-PA and for pro-UK/UK respectively. Maximal rates of clot lysis by t-PA (0.1–5 μg/ml) and by rec-pro-UK (0.5–5 μg/ml) were determined and correlated with the degree of preservation of fibrinogen. The highest rate of clot lysis induced by a fibrin-specific (<10% fibrinogen degradation) dose of rec-pro-UK was 80%/h. A similar rate could not be induced by t-PA without causing >80% fibrinogen degradation. The highest fibrin-specific rate of lysis achievable with t-PA was only 42%/h. When a small amount of t-PA was added at intervals during clot lysis induced by rec-pro-UK, the t-PA significantly foreshortened the lag phase, but it did not enhance the maximal rate of clot lysis. By contrast, when rec-pro-UK was added during the course of clot lysis induced by a large, but fibrin-specific amount of t-PA, rec-pro-UK accelerated the rate of lysis thereby demonstrating the presence of some fibrin-bound plasminogen not activated by t-PA. The study indicates that in a plasma milieu t-PA is more restricted in its action and activates only about half the fibrin-bound plasminogen which is activated by rec-pro-UK/UK.     

Characterisation of rt-PA I276G,a recombinant human tissue-type plasminogen activator mutant with altered plasmin cleavage site

rt-PA I276G, a mutant of recombinant human tissue-type plasminogen activator (rt-PA) with altered plasmin cleavage site, was obtained by site-directed mutagenesis of Ile²⁷⁶ to Gly. rt-PA I276G, purified to homogeneity from the conditioned medium of transfected Chinese hamster ovary cells, was obtained as a single chain molecule, which was quantitatively converted to a two chain moiety by cleavage of the Lys²⁷⁷-Gly²⁷⁸ peptide bond with plasmin. The specific activities on fibrin films of the single chain and two chain forms of rt-PA I276G were 8900 and 15000 IU/mg respectively, as compared to 210000 and 130000 IU/mg respectively for the single chain and two chain forms of wild-type rt-PA obtained in the same expression system. The amidolytic activity of the rt-PA I276G moieties was 3- to 5-fold lower and their catalytic efficiency for plasminogen activation 20- to 50-fold lower than those of the wild-type rt-PA moieties. Both single chain and two chain rt-PA I276G induced concentration-dependent lysis of a ¹²⁵I-fibrin labelled plasma clot submersed in human plasma; equi-effective concentrations (causing 50% clot lysis in 2 h) were 0.55 and 1.40 μg/ml respectively, as compared to 0.36 and 0.60 μg/ml for single chain or two chain wild-type rt-PA respectively. Continuous infusion over 60 min of single chain rt-PA 1276G or wild-type rt-PA in hamsters with a pulmonary embolus, revealed an approximately 2-fold lower thrombolytic potency (clot lysis versus dose) for the mutant, but a comparable specific thrombolytic activity (clot lysis versus steady state plasma antigen level). It is concluded that replacement of Ile²⁷⁶ by Gly in single chain rt-PA significantly reduces the intrinsic enzymatic activity in purified systems. In a plasma milieu in the presence of fibrin the fibrinolytic potential of the mutant is, however, only 2-fold lower than that of wild-type rt-PA obtained in the same expression system.     

Hageman factor-independent fibrinolytic pathway

The lysis of clots formed by the addition of thrombin to dilute whole plasma containing ¹²⁵I-labelled fibrinogen was examined in kinetic assays which assessed clot size and release of radiolabel into the lysate. Neither platelets, immunoglobulin, nor Hageman factor was essential for clot lysis in this system. Clot lysis was normal in plasma from patients genetically deficient in either C2 or C6 but abnormal in the plasma of two patients with acquired C3 deficiency. Plateletdeficient plasma depleted of C3 by cobra venom factor or adsorbed with zymosan did not sustain clot lysis. These findings indicate the existence of a humoral fibrinolytic pathway in whole human plasma dependent upon C3 but independent of Hageman factor.     

Plasmin-catalysed cleavage of single chain tissue-type plasminogen activator in fibrin clots

The activity of single chain t-PA in the presence of fibrin was investigated and compared to that of its twochain counterpart. A plasmin resistant t-PA analogue in which Arg-275 is replaced by Gly was included in this study in order to avoid the complications caused by concomitant two chain generation during plasminogen activation experiments. Substantial plasminogen activation of single chain t-PA was observed during fibrin clot dissolution, still the clot lysis activity of two chain t-PA was found to be about 20% higher than that of the single chain form. Plasmin-catalysed cleavage of single chain t 25I-t-PA was studied in the presence and absence of fibrin. This reaction was not enhanced by fibrin, rather a small inhibition was observed. In addition to the primary cleavage site at Arg-275 Ile-276 secondary plasmin-catalysed cleavage resulting in a 30 000 Da fragment was observed. Appearance of this fragment took place roughly at the same time as a decline in clot lysis activity (but not in amidolytic activity) was observed. Secondary plasmin cleavage was not observed when fibrin was present.     

Albumin concentration influences fibrinolytic activity in plasma and purified systems

Delayed clot lysis was observed in 9 patients with nephrotic syndrome. This delay, assessed by fibrin degradation product release, was corrected by supplementing patients' plasma with purified human albumin to reach the normal albumin concentration of 45g/1. The effect of albumin on clot lysis was further assessed in the presence of albumin-depleted plasma and in a purified system containing tissue-type plasminogen activator (t-PA), plasminogen, fibrin monomers and increasing albumin concentrations. An increase in plasminogen uptake was observed when clots were prepared in the presence of such concentrations. We conclude that low albumin levels may alter clot structure and reduce spontaneous thrombolysis, thereby increasing the risk of thrombosis.     

Fibrinolytic protease from Bacillus cereus S46: Purification,characterization, and evaluation of its in vitro thrombolytic potential

Intravascular thrombosis is a prime cause of cardiac complications worldwide. Microbial fibrinolytic proteases are of clinical significance in thrombosis treatment. The present study discusses the purification and characterization of a protease from Bacillus cereus S46, ascertaining its in vitro thrombolytic activity against a blood clot. By the three-step purification involving precipitation, dialysis, and diethylaminoethyl-cellulose ion-exchange chromatography, a 12.37-fold purification of the enzyme to homogeneity was achieved. The apparent molecular mass of the protease was 30 kDa, as found by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The optimum activity of the enzyme was observed at pH 8.0 and 40°C. The enzyme retained an 82.19% residual activity at pH 8.0 and 40°C for 1 h. The K_m and V_max values of the protease with casein were 0.0027 mM and 9.712 µmol/min, respectively. In an in vitro assay, the purified protease resulted in 97.02% lysis of the blood clot. The fibrinolytic potential of the enzyme, together with its characteristics of being active and stable under near-physiological conditions, may suggest its application as a therapeutic agent.     

Accelerating thrombolysis with chitosan-coated plasminogen activators encapsulated in poly-(lactide-co-glycolide) (PLGA) nanoparticles

Accelerating thrombolysis using plasminogen activators (PAs) encapsulated liposomes or PEG microparticles by pressure-driven permeation have been demonstrated in vitro and in vivo in animal models. However, designing and delivering PA-encapsulated nanoparticles (NPs) to enhance thrombolysis by applying electrostatic forces or ligand-receptor interactions between the NPs and blood clots has not been proposed. Therefore, without a pressure-driving factor, tissue-plasminogen activator (t-PA) encapsulated in PLGA NPs with chitosan (CS) and CS-GRGD coating and their thrombolysis capabilities in a blood clot-occluded tube model were evaluated by determining clot lysis times and the masses of the digested clots. The characteristics and release profiles of t-PA-encapsulated PLGA, PLGA/CS and PLGA/CS-GRGD NPs are determined by FT-IR, a laser particle/zeta potential analyzer and HPLC. Additionally, the permeation capacities of the NPs into flat blood clots were examined. For example, the mean particle sizes and encapsulation efficacies of t-PA for the NPs are in the ranges 260-320 nm and 65.5-70.5%, respectively. The results reveal that the NPs for the shortest clot lysis time and the highest weight percentages of digested clot are PLGA/CS (20.7 +/- 0.7 min) and PLGA/CS-GRGD (25.7 +/- 1.3 wt%), respectively. Compared with t-PA solution, the NPs can significantly shorten clot lysis times in the following order: PLGA/CS NPs (38.8 +/- 1.5%) > PLGA/CS-GRGD NPs (16.3 +/- 1.0%) > PLGA NPs (7.7 +/- 1.2%). Compared with t-PA solution, the NPs significantly increase the weight of digested clots in the order, PLGA/CS-GRGD (40.9 +/- 1.5%) > PLGA/CS (27.8 +/- 1.2%) > PLGA (8.6 +/- 0.6%). The highest release rate of t-PA in the fast release phase and the highest permeability into intra-clots of PLGA/CS and PLGA/CS-GRGD NPs, respectively, correspond to the shortest clot lysis time and the largest increase in weight of the digested clots among the NP system. In conclusion, the NPs designed based on new concepts significantly accelerate thrombolysis in vitro in this model, and may be useful in clinical study.     

Kinetics of activation of variant plasminogens in a noncrosslinked fibrin clot: Abnormal plasmin generation with the plasminogen.Streptokinase complex,urokinase and tissue plasminogen activator

A purified clot lysis system (noncrosslinked) was developed to evaluate and study variant plasminogens from patients with a history of thrombophilia. Plasmin generation was studied in the soluble phase and in the clot lysis system with different activators, streptokinase and its derivatives, urokinase and tissue plasminogen activator. Five of the variant plasminogens (Dl, D2, K, S, and F) generated low levels of plasmin in the soluble phase, whereas two (M and Y) generated near normal levels of plasmin. Plasmin generation in the clot lysis system divided the variants into three groups: one variant (Dl) had a very prolonged lysis time or a negligible plasmin generation rate with three direct activators, three variants (D2, K and S) had moderately prolonged lysis times or low plasmin generation rates with these activators, and three variants (F, M, and Y) had normal lysis times and normal plasmin generation rates with these activators. Kinetics of activation parameters with three direct plasminogen activators were determined from a non-linear regression analysis of plots of velocity versus plasminogen concentration and were verified by Lineweaver-Buck plots where possible. With plasminogen.streptokinase, the four variants with prolonged lysis times, had normal apparent Michaelis constants and low catalytic rate constants. Three variants with normal lysis times had high apparent Michaelis constants showing decreased affinity for the plasminogen.streptokinase activator complex, and high catalytic rate constants. With urokinase, the variant with the longest clot lysis time had a high apparent Michaelis constant and a low catalytic rate constant, three variants with prolonged lysis times gave low catalytic rate constants; two of them gave normal apparent Michaelis constants and one gave a high apparent Michaelis constant. Of the three variants with normal lysis times, one gave normal kinetic constants and two had high apparent Michaelis constants with high catalytic rate constants. With tissue plasminogen activator, the variant with the longest clot lysis time gave a high apparent Michaelis constant and a high catalytic rate constant. The three variants with prolonged lysis times gave variable results, one had a slightly higher apparent Michaelis constant with a low catalytic rate constant, the second one had a very low apparent Michaelis constant with a low catalytic rate constant and the third one had a high apparent Michaelis constant with a normal catalytic rate constant. Three variants with normal lysis times had slightly higher Michaelis constants and near normal catalytic rate constants. With the noncrosslinked fibrin substrate, all of the variant plasminogens have lowered catalytic efficiencies with two or three plasminogen activators. The theoretical aspects of these plasmin generation data are discussed.     

Increased fibrinolytic potential after diet intervention in healthy coronary high-risk individuals

Twenty healthy individuals (15 men and 5 women) with initial fasting serum triglycerides greater than or equal to 1.80 mmol/l and euglobulin clot lysis time after venous occlusion greater than or equal to 60 min (upper normal limit 45 min) were tested for fibrinolytic response to venous occlusion and intravenous injection of desmopressin (DDAVP), serum lipids, serum glucose and relative body weight before and after a minimum of 3 to a maximum of 12 months' diet intervention. In order to be defined as a good diet responder, at least 20% reduction of the initial serum triglyceride concentration was required. At the end of the study, half of the participants (7 men and 3 women) met the criteria of good diet responders. All of these showed an improved fibrinolytic response to DDAVP injection, and 7 out of 10 had a normalized fibrinolytic response to venous occlusion. We conclude that, through dietary measures with substantial reduction of hypertriglyceridaemia, it is possible to improve and even normalize the fibrinolytic potential.     

Development and validation of models for the investigation of blood clotting in idealized stenoses and cerebral aneurysms

An in vitro model of blood clotting is presented using hypercoaguable milk as an analog for blood. Milk clot formation was studied for periods of 2, 5, 10, 20, and 30 min within an idealized stenosis geometry. Clot formation was recorded using photography, clot casting, and clot mass calculation. The distribution of clot within the fluid was seen to be in good agreement with a previous study that used a residence time model to predict areas of clot formation in thrombin solution. A numerical model was formulated within computational fluid dynamics package CFX that allowed local activation of blood clotting to be simulated. This model was applied to the analysis of an idealized cerebral aneurysm geometry. An idealized coil geometry was included within the aneurysm and clotting fluid concentration and fluid residence time were modeled using transport equations within CFX. The viscosity of the fluid was defined as a function of both residence time and clotting fluid concentration. The model was seen to produce features consistent with observations of thrombosis within cerebral aneurysms, while avoiding the unrealistic build up of clot in near-wall regions that is associated with a pure residence time model.This work was presented at the International Congress on Computational Bioengineering, September 24–26, 2003, Zaragoza, Spain, and the 41st Annual Meeting of the Japanese Society for Artificial Organs, October 30 to November 1, 2003, Sendai, Japan     

Highly stable plasminogen activator inhibitor type one (VLHL PAI-1) protects fibrin clots from tissue plasminogen activator-mediated fibrinolysis

Plasminogen activator inhibitor-1 (PAI-1) is the major specific inhibitor of tissue-type plasminogen activator (tPA) which mediates fibrin clot lysis through activation of plasminogen. Wild-type-PAI-1 (wPAI-1) is rapidly converted to the latent form (half-life of approximately 2 h) and loses its ability to inhibit tPA. We developed a very long half-life PAI-1 (VLHL PAI-1), a recombinant protein with a half-life >700 h compared with wPAI-1. In this study, VLHL PAI-1 was assessed for its ability to inhibit clot lysis in vitro. Clot formation was initiated in normal plasma supplemented with tPA by the addition of either tissue factor or human recombinant FVIIa. Clot lysis time, monitored turbidimetrically in a microtiter plate reader, was determined at various concentrations of wPAI-1 and VLHL PAI-1. Both wPAI-1 and VLHL PAI-1 caused a significant increase in clot lysis time, although the latter was somewhat less effective at lower concentrations. The VLHL PAI-1, but not wPAI-1, maintained its anti-fibrinolytic activity after preincubation overnight at 37 degrees. These studies demonstrate that VLHL PAI-1 is an effective inhibitor of fibrin clot degradation. Due to the high stability of VLHL PAI-1 compared with wPAI-1, this novel inhibitor of tPA-mediated fibrinolysis may have therapeutic applications for treating surgical and trauma patients when used directly or in conjunction with the procoagulant recombinant FVIIa.     

The euglobulin lysis time test: An ineffectual monitor of the therapeutic inhibition of fibrinolysis

In two clinical situations associated with hyperfibrinolysis the administration of antifibrinolytic drugs resulted in clinical haemostasis. The dilute clot lysis time and fibrin plate activity test but not the euglobulin lysis time reflected this control of excessive fibrinolysis by the antifibrinolytic drugs. The inhibition by epsilon-amino-caproic acid (EACA) of hyperfibrinolysis induced in vitro was reflected by the dilute clot lysis time but not by the euglobulin lysis time. Paper chromatography of the supernatant and euglobulin fractions as prepared for the euglobulin lysis time test from plasma with added EACA demonstrated that some 85% of the EACA was present in the supernatant, normally discarded during the test. Similarly, cellulose-acetate electrophoresis of the supernatant and euglobulin fractions from plasma containing Trasylol demonstrated the drug in the supernatant only. These findings indicate that when acetic acid is added to plasma containing EACA or Trasylol only a small proportion of the drug is precipitated with the euglobulin fraction. The euglobulin lysis time is thus an inaccurate index of the neutralization of hyperfibrinolysis by antifibrinolytic drugs.