Inhibitory effects of lysine analogues on t-PA induced whole blood clot lysis

The lysine analogues epsilon-aminocaproic acid (EACA) and trans-4-amino-methyl cyclohexane carboxylic acid (AMCA) are used to prevent excessive bleeding in patients with coagulopathies, such as hemophilia and thrombocytopenia, or in those who have received tissue plasminogen activator (t-PA). However, their relative efficacy in inhibiting lysis of clots that have been formed in the presence of exogenous t-PA or that have been formed and then exposed to exogenous t-PA has not been well characterized. The present study utilized blood from normal volunteers and ¹²⁵I-fibrinogen in a dilute whole blood clot assay to determine the relative concentrations of lysine analogues required for inhibition of clot lysis induced by exogenous t-PA. AMCA (0.06 mM) and EACA (0.6 mM) were effective in prolonging clot lysis if (1) whole blood clots were formed and then exposed to a lysine analogue and exogenous t-PA or if (2) whole blood clots were formed in the presence of exogenous t-PA and a lysine analogue. However, their inhibitory effect was markedly reduced if clots were formed in the presence of t-PA and then exposed to either of the lysine analogues. The analogues did not inhibit the initial binding of t-PA to fibrin. They did inhibit binding of plasminogen to fibrin as well as the activation of plasminogen by t-PA in the absence of fibrin. The data suggest that lysine analogues, even at low concentrations, reduce the rate of t-PA induced whole blood clot lysis by several mechanisms.     

Comparative study of the efficacy and specificity of tissue plasminogen activator and pro-urokinase: demonstration of synergism and of different thresholds of non-selectivity

Clot lysis and non-specific plasminogen activation in human plasma by tissue tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) were studied. The fibrinolytic activity of pro-UK was expressed as latent units, i.e. measured after activation with plasmin on a fibrin plate against the reference standard. The t-PA unitage was assigned on a weight basis of a similar equivalence of 100,000 IU/mg. To simplify comparison, both activators were expressed in IU (1 IU = −10 ng). At low concentration (1–50 IU/ml), t-PA induced more effective and more linear clot lysis, whereas pro-UK induced lysis was preceded by a lag phase. The two activators were equivalently effective at higher concentrations and saturated at the same lysis rate. Clots made from platelet rich plasma or whole blood were more responsive to lysis by pro-UK but not t-PA than corresponding platelet poor clots. At very low concentrations (2.5–5 IU/ml) of t-PA combined with moderate concentrations (25–50 IU/ml) of pro-UK, a synergistic effect on clot lysis, which was fibrin-specific, was observed. Plasminogen and fibrinogen and the appearance of plasmin-inhibitor complexes in plasma were measured after incubation with either activator with and without a clot present. Non-specific plasminogen activation occurred above a certain concentration of either activator but was found at lower concentrations of t-PA than pro-UK. In the absence of a clot, plasmin generation occurred with t-PA at about 30% of the concentration at which pro-UK induced a corresponding effect. It is concluded that there are important differences in the fibrinolytic and clot selective properties of t-PA and pro-UK, and that some of these properties may be complementary resulting in a fibrin specific, synergistic fibrinolytic effect.     

Influence of exogenous and endogenous tissue-type plasminogen activator on the lysability of clots in a plasma milieu in vitro

The fibrinolytic potential of tissue-type plasminogen activator (t-PA) either incorporated in a clot (endogenous) or added to the surrounding plasma (exogenous), was studied in an in vitro system consisting of 125I-labeled human plasma clots (200 microliters) immersed in human plasma (2 ml). Clot lysis was measured as a function of endogenous t-PA concentration (in the absence of added exogenous t-PA), as a function of exogenous t-PA concentration (without added endogenous t-PA) and as a function of the same concentration of both endogenous and exogenous t-PA. Equivalent clot lysis was obtained with a 2 to 4 times lower concentration of endogenous t-PA as compared to exogenous t-PA, corresponding to a 20 to 40 times smaller total amount of endogenous versus exogenous t-PA. Fifty percent lysis in 5 hrs was obtained with about 5 IU/ml of endogenous t-PA or with 10 IU/ml of exogenous t-PA. The presence of both exogenous (10 IU/ml) and endogenous (5 IU/ml) t-PA resulted in 50 percent lysis in 1.5 hrs, indicating that t-PA incorporated in a thrombus contributes significantly to its lysis by exogenous t-PA. Similar results were obtained with plasma obtained after 10 min of venous occlusion in seven healthy subjects. Spontaneous clot lysis within 5 hrs was only observed with post-occlusion clots in pre- or post- occlusion plasma in two subjects in whom the t-PA level rose to 10-15 IU t-PA/ml. In the five other subjects with post-occlusion t-PA levels below 2 IU/ml, no clot lysis was observed within 24 hrs. The influence of the fast-acting inhibitor of t-PA on clot lysability by endogenous or exogenous t-PA was investigated by immersing clots prepared from normal or inhibitor-rich plasma (endogenous inhibitor) in normal or inhibitor-rich plasma (exogenous inhibitor). Exogenous t-PA inhibitor efficiently neutralizes clot lysis by both exogenous and endogenous t-PA. Endogenous t-PA inhibitor, however, efficiently neutralizes endogenous t-PA but has little influence on clot lysis by exogenous t-PA. These findings indicate that t-PA inhibitor is not concentrated into a clot and that t-PA inhibitor in plasma efficiently neutralizes t-PA incorporated in a clot. alpha 2-Antiplasmin depleted plasma clots were more susceptible to lysis by both endogenous and exogenous t-PA than normal clots.(ABSTRACT TRUNCATED AT 400 WORDS)     

Clot structure modification by fondaparinux and consequence on fibrinolysis: a new mechanism of antithrombotic activity

Fondaparinux is a synthetic pentasaccharide consisting of the minimal sequence of heparin which interacts with antithrombin (AT). It represents a new class of selective factor Xa inhibitors without any antithrombin activity. It has been shown to exhibit potent antithrombotic properties in clinical studies. However, the mechanism of its antithrombotic action has not yet been fully established. In the present study it was shown that fondaparinux, used at pharmacological concentration (500 ng/ml), rendered the clot more susceptible to fibrinolysis induced by t-PA: plasma fibrin clots formed in the presence of fondaparinux and perfused with t-PA were degraded at a faster rate than those formed in the absence of fondaparinux. This fibrinolytic activity of fondaparinux is mainly due to a modification of clot structure characterized by a loose fibrin conformation with less branched fibers and the presence of large pores in comparison to control clots which present a tighter conformation. The difference in fibrin structure was responsible for an increase in clot porosity leading to a better availability of t-PA to the fibrin network. It is related to the decrease in thrombin generation, in an AT-dependent pathway. It was also demonstrated that in the presence of exogenous thrombomodulin, the inhibition of TAFI activation by fondaparinux could contribute, to a lesser extent, to the increased thrombus lysis. The increase in t-PA induced thrombus lysis could contribute to the antithrombotic activity of fondaparinux.     

Effects of fibrin on the enhanced activation of plasminogen by urokinase and tissue plasminogen activator: role of cross-link

When human plasma was activated by urokinase (UK) in the presence of thrombin, thrombin plus Ca++, Ca++ or in their absence and the plasmin activity was measured by the hydrolysis of S-2251, plasmin activity was higher in the presence of cross-linked or non cross-linked plasma clot. The results of similar experiments utilizing plasma after severe exercise indicated that the hydrolysis of S-2251 by plasma containing tissue plasminogen activator (t-PA) was also higher in cross-linked or non cross-linked plasma clot. Fibrinolysis was faster in thrombin-induced plasma clot, but was later shown significantly in plasma clot induced by thrombin and Ca++, whereas practically no fibrinogenolysis was shown in plasma. When Glu-plasminogen (Glu-plg) was activated by UK in the presence of cross-linked or non cross-linked fibrin and alpha 2 antiplasmin (alpha 2AP), fibrinolysis was faster in cross-linked fibrin than non cross-linked fibrin in the presence of alpha 2AP. No fibrinogenolysis was shown either. Plasmin activity measured by the hydrolysis of S-2251 was also higher in cross-linked or non cross-linked fibrin than in fibrinogen in the presence of alpha 2AP. These results indicate that enhanced activation of Glu-plg by UK or t-PA in the presence of fibrin was a more significant event than the inactivation of plasmin in the plasma clot or purified clot by alpha 2AP cross-linked to fibrin.     

Recombinant variants of tissue-type plasminogen activator containing amino acid substitutions in the finger domain.

Tissue-type plasminogen activator (t-PA) is a fibrin-specific agent which has been used to treat acute myocardial infarction. In an attempt to clarify the determinants for its rapid clearance in vivo and high affinity for fibrin clots, we produced five variants containing amino acid substitutions in the finger domain, at amino acid residues 7-9, 10-14, 15-19, 28-33, and 37-42. All the variants had a prolonged half-life and a decreased affinity for fibrin of various degrees. The 37-42 variant demonstrated about a 6-fold longer half-life with a lower affinity for fibrin. Human plasma clot lysis assay estimated the fibrinolytic activity of the 37-42 variant to be 1.4-fold less effective than that of the wild-type rt-PA. In a rabbit jugular vein clot lysis model, doses of 1.0 and 0.15 mg/kg were required for about 70% lysis in the wild-type and 37-42 variant, respectively. Fibrinogen was degraded only when the wild-type rt-PA was administered at a dose of 1.0 mg/kg. These findings suggest that the 37-42 variant can be employed at a lower dosage and that it is a more fibrin-specific thrombolytic agent than the wild-type rt-PA.     

Thrombin activatable fibrinolysis inhibitor (TAFI) does not inhibit in vitro thrombolysis by pharmacological concentrations of t-PA

TAFI (thrombin activatable fibrinolysis inhibitor) is a plasma procarboxypeptidase that upon activation inhibits the fibrinolytic process by removing the C-terminal lysines from partially degraded fibrin. The generation of activated TAFI (TAFIa) has been suggested to represent a mechanism of thrombus resistance to thrombolytic therapy. However, the ability of TAFI to inhibit fibrinolysis by pharmacological concentrations of t-PA has not been properly investigated. We used an in vitro model consisting of 125I-fibrin blood clots submerged in autologous defibrinated plasma. Upon addition of t-PA (125-5,000 ng/ml) and CaCl2 (25 mM), samples were incubated at 37 degrees C, and clot lysis was measured at intervals from the radioactivity released into solution. The role of TAFI was assessed either by neutralizing the generated TAFIa with the specific inhibitor PTI (50 microg/ml) or by enhancing TAFI activation through the addition of recombinant soluble thrombomodulin (solulin, 1 microg/ml). In our clot lysis model, activation of TAFI amounted to about 20% of inducible carboxypeptidase activity. Addition of PTI, however, produced a significant increase in the extent of lysis only at concentrations of t-PA equal to or lower than 250 ng/ml. When solulin was added to the plasma surrounding the clot, about 70% of TAFI was activated within 15 min. Under these conditions, inhibition of clot lysis was very marked in samples containing 125 or 250 ng/ml of t-PA, but negligible in those containing pharmacological concentrations of the activator (1,000 and 5,000 ng/ml). Additional experiments suggest that loss of fibrin-dependence by elevated concentrations of t-PA may be one of the mechanisms explaining the lack of effect of TAFIa. Our data indicate that, under our experimental conditions, clot lysis by pharmacological concentrations of t-PA is not influenced by TAFIa even after maximal activation of this procarboxy-peptidase.     

Availability of the B beta(15-21) epitope on cross-linked human fibrin and its plasmic degradation products.

The binding of radiolabeled monoclonal antifibrin antibody 59D8 (specific for fibrin but not fibrinogen) to a series of degraded fibrin clots showed that the availability of the B beta(15-21) epitope (against which 59D8 had been raised) was inversely proportional to the extent of clot lysis. Examination of digest supernatants revealed that the B beta(15-21) epitope was released from clots as a high molecular weight degradation product in the presence of calcium ions but that the generation of low molecular weight peptides occurred in the absence of calcium ions. To address the question of epitope accessibility, we compared levels of fibrin clot binding among four radioactively labeled antibodies: antifibrin monoclonal antibody 59D8, two antifibrinogen monoclonal antibodies that cross-reacted with fibrin, and an affinity-purified polyclonal antifibrinogen antibody. We expected that the antifibrinogen antibodies would show enhanced binding to clots in comparison with the antifibrin antibody. However, the epitope accessibility experiments showed that all four antibody preparations bound fibrin clots at comparable levels. Taken together, these studies demonstrated that one fibrin-specific epitope, B beta(15-21), remains available on clots as they undergo degradation by plasmin and, importantly, that the epitope is not solubilized at a rate faster than the rate at which the clot is itself solubilized. The availability of the B beta(15-21) epitope during the course of plasminolysis assures the potential utility of antifibrin antibodies such as 59D8 for detecting thrombi and targeting plasminogen activators.     

Protraction of whole blood and plasma clot lysis in patients with high levels of an inhibitor of tissue-type plasminogen activator

The influence of the newly discovered, fast-acting inhibitor of tissue-type plasminogen activator (t-PA) on the lysis time of plasma clots was studied by visual observation of lysis of clotted citrated plasma after addition of purified t-PA. To a series of plasma samples with various concentrations of naturally occurring PA-inhibitor purified t-PA was added to a final concentration, which in pooled normal plasma is sufficient to induce clot lysis within a few hours. In those plasma samples with a high free inhibitor level, determined by measuring the recovery of the activity of added purified t-PA, clot lysis was retarded. Whole blood clots were made by clotting freshly collected non-anticoagulated blood with thrombin after admixture of a trace amount of radiolabeled fibrinogen and a fixed amount of t-PA. Lysis rate, read from the appearance of radioactivity in the serum after centrifugation, was significantly lower in clots obtained from subjects with a high free inhibitor level than in those with a low inhibitor level. It is concluded that the PA-inhibitor protracts clot lysis and may be relevant for physiological fibrinolysis.     

In vitro studies on the fibrinolytic, thrombolytic and fibrinogenolytic properties of a tissue plasminogen activator from guinea pig keratocytes

The fibrinolytic and thrombolytic properties of a tissue plasminogen activator (tPA) purified from the conditioned medium of an established guinea pig keratocyte (GPK) cell line were investigated in in vitro systems and compared with urokinase. Using the fibrin clot lysis assay, GPK activator appears to be similar to human melanoma tPA and not to human urokinase. GPK activator also caused negligible fibrinogen breakdown, when incubated with human plasma at 37 degrees C over 23 hr. Urokinase on the other hand caused significant fibrinogenolysis, under similar conditions. Comparison of the lysis of plasma clots by GPK activator and human urokinase have shown that GPK activator was a much more effective fibrinolytic agent than urokinase, especially at lower concentrations (less than 50 IU/ml). Studies on the thrombolytic effect of GPK activator on the lysis of aged and cross-linked whole human blood clots and plasma clots hanging in artificially circulating human plasma suggest that GPK activator can lyse both these types of clots equally well. The lysis is dose dependent, attaining complete lysis within 3-6 hr with the concentration of GPK activator in the range of 1-5 micrograms/ml plasma. It is concluded that GPK activator has a higher fibrinolytic and thrombolytic activity and lower fibrinogenolytic activity than urokinase.     

Effect of physiological concentrations of vasopressin on components of the fibrinolytic system

High physiological concentrations of plasma vasopressin (aVP) when achieved by infusion cause an increase in plasma factor VIII coagulant activity and shortening of the euglobulin clot lysis time (ECLT). To investigate the effects of aVP on components of the fibrinolytic pathway and on thrombin generation, 9 healthy volunteers were infused with saline for 30 min followed by aVP for 1 hour and blood samples taken every 30 min for measurement of aVP, ECLT, tissue-type plasminogen activator (t-PA), t-PA inhibition (tPA-I), plasminogen activator inhibitor 1 (PAI-1 Ag), activated partial thromboplastin time (APTT), fibrinopeptide A (FPA), fibrinopeptide B 15-42 (FPB beta 15-42) and cross-linked fibrin breakdown products (XL-FDP). Plasma aVP rose to a median of 75 pg/ml after 90 min and fell to 13.8 pg/ml 30 min later. The APTT fell from 43.5 to 35 sec (p less than 0.01) but there was no change in plasma FPA or in XL-FDP. Plasminogen activator activity (10(6)/ECLT2) increased from 25 to 736 units (p less than 0.01) and t-PA from 200 to 1012 mIU/ml (p less than 0.01). tPA-I fell from 8.0 to 2.7 IU/ml at 90 min (p less than 0.05) but PAI-1 Ag remained unchanged. Plasma FPB beta 15-42 was 2.4 and 1.2 pmol/ml before infusion with aVP and showed a small rise to 3.5 pmol/ml after 60 min (p less than 0.05). The results show the effects of aVP on fibrinolysis are mediated by an increase in t-PA. In the absence of thrombin generation the rise in t-PA was not accompanied by changes in XL-FDP.     

The level of extrinsic plasminogen activator (t-PA) during clotting as a determinant of the rate of fibrinolysis; inefficiency of activators added afterwards

Dissolution of washed fibrin clots in vitro was found to depend on the concentration of extrinsic (tissue-type) plasminogen activator (t-PA) during clotting. Washed fibrin clots prepared from t-PA-rich plasma lyse spontaneously within a few hours, but similar clots, prepared from t-PA-poor plasma, do not lyse within 24 hours, neither spontaneously nor on addition of t-PA, streptokinase (SK) or urokinase (UK). When washed fibrin clots were suspended in buffered saline to which t-PA, SK or UK was added, lysis was enhanced by addition of lys-plasminogen to the buffer. This observation may have important consequences for the interpretation of fibrinolytic experiments in vitro and in vivo, for the management of a variety of diseases complicated by fibrin depositions, and eventually for thrombolytic therapy with t-PA.     

Factor XIII cross-linking and the rate of fibrinolysis induced by streptokinase and urokinase

The rate of lysis induced by streptokinase or urokinase of fibrin clots with varying degrees of Factor XIII cross-linking was investigated. Various techniques were used in which the plasminogen activator was uniformly distributed through the clot, or was present only at the surface. The clots were produced from purified fibrinogen solutions or from plasma. None of these ivestigations gave any evidence to indicate that lytic rates were decreased by the cross-linking of either the α or γ chains of fibrin.     

Comparison of affinities of urokinase and tissue plasminogen activator for fibrin clots

Affinities of low molecular weight two-chain urokinase (UK) and tissue plasminogen activator (t-PA) for fibrin clots were investigated by using clot lysis rates to estimate an affinity (Kd) between activator and fibrin clots. Lysis rates were obtained using a simple spectrophotometric based clot lysis assay which is described here. Fibrin clots, containing residual plasminogen, were suspended in a 1 ml cuvette and the increase in absorbance at 280 nm due to release of soluble fibrin peptides measured over a 150 to 250 minute time period. Lysis rates were obtained from plots of time squared vs absorbance change. Plots of activator concentration vs reciprocal rates yielded regression coefficients of 0.999 and Kd values of nM for the affinity of both activators for fibrin clots. Although both activators are known to differ in affinity for fibrin, they nonetheless had similar affinities and lysis rates for the insoluble fibrin clots. This assay also suggested possible synergism; rates over twice that expected by an additive effect were observed when the two activators were mixed at 0.3 to 7.6 nM each.     

Effect of a selective thrombin inhibitor MCI-9038 on fibrinolysis in vitro and in vivo

The effect of a selective thrombin inhibitor, (2R, 4R)-4-methyl-1-[N2-[(3-methyl-1,2,3,4-tetrahydro-8-quinolinyl)sulfonyl]- L-arginyl]-2-piperidinecarboxylic acid (MCI-9038), on the fibrinolysis induced by t-PA and u-PA was studied in vitro and in vivo. MCI-9038 remarkably reduced the lysis time of the plasma clot generated by the addition of calcium chloride to the plasma at the concentration ranging from 0.01 to 0.3 microM. Heparin also reduced the plasma clot lysis time with a lower effect than MCI-9038. The fibrin crosslinkage in the plasma clot was inhibited by MCI-9038 or heparin. MCI-9038 potently inhibited the factor XIIIa generation from factor XIII by thrombin. The effect on the in vivo thrombolysis was studied on the arterial thrombosis generated by the endothelial cell injury of the rabbit carotid artery by acetic acid. t-PA dissolved the thrombi with the infusion at 0.96 mg/kg over 2 h without a significant activation of a systemic fibrinolysis. u-PA dissolved the thrombi with the infusion at 180,000 and 360,000 IU/kg over 2 h. At a dose of 0.48 mg/kg t-PA or 90,000 IU/kg u-PA, the thrombi were not dissolved, but the combined use of MCI-9038 at 1.2 mg/kg over 2 h effectively dissolved the thrombi. Thus, combination of MCI-9038 with plasminogen activators accelerated thrombolysis of an experimental thrombosis in rabbits.     

Biological and thrombolytic properties of proenzyme and active forms of human urokinase--IV. Variability in fibrinolytic response of plasma of several mammalian species

The fibrinolytic and fibrinogenolytic properties of recombinant pro-urokinase (Rec-pro-UK) and recombinant urokinase (Rec-UK) were compared with those of natural urokinase (Nat-UK) and of tissue-type plasminogen activator (t-PA) in an in vitro system consisting of 125I-labeled autologous plasma clots immersed in plasma of humans, five primate species, dogs, rabbits and pigs. With each of the four plasminogen activators, a dose-dependent clot lysis was observed, the degree of which differed, however, very markedly from one species to the other. At a concentration of 100 IU/ml of urokinase extensive plasma clot lysis was obtained in plasma of man, Macaca mulatta, Macaca fascicularis and Macaca radiata, while the plasma clots of Papio cynocephalus, Papio anubis and rabbit, dog and pig were much more resistant to lysis. No significant differences in the extent of lysis were observed between Rec-pro-UK and Rec-UK nor between Rec-UK and Nat-UK. Comparable degrees of lysis were obtained with t-PA at 3- to 5-fold lower concentrations. Lysis with Rec-UK or Nat-UK was always associated with extensive activation of the fibrinolytic system in plasma, evidenced by fibrinogen breakdown and plasminogen activation and alpha 2-antiplasmin consumption. With t-PA, extensive clot lysis was obtained in the absence of fibrinolytic activation in the plasma. With Rec-pro-UK the response was intermediate; at high concentrations (200 IU/ml) extensive lysis in the reactive species was associated with fibrinogen consumption, while at intermediate concentrations (50-100 IU/ml) significant clot lysis was obtained in the reactive species in the absence of marked activation of the fibrinolytic system in the plasma.     

Profibrinolytic activity of the direct thrombin inhibitor melagatran and unfractionated heparin in platelet-poor and platelet-rich clots

Anticoagulants have been shown to stimulate fibrinolysis principally via inhibition of thrombin-mediated activation of TAFI (thrombin activatable fibrinolysis inhibitor). Their profibrinolytic effect, however, may vary according to their mechanism of action and to the clot composition. We compared the fibrinolytic activity of the direct thrombin inhibitor melagatran with that of unfractionated heparin in platelet-poor (PPP) and platelet-rich (PRP) models consisting of tissue-factor-induced clots exposed to exogenous t-PA (25 ng/ml). In the PPP clot model, both heparin (0.1-0.6 U/ml) and melagatran (20-320 ng/ml) caused a concentration-dependent shortening of lysis time. However, when drug profibrinolytic activity (lysis ratio) was expressed in function of the aPTT prolongation (aPTT ratio), melagatran was more efficient than heparin. In the PRP clot model, melagatran displayed a fibrinolytic activity fairly comparable to that observed in PPP whilst heparin caused a modest reduction of lysis time only at the highest concentrations. Assay of thrombin and TAFIa generation in defibrinated plasma showed that the presence of platelets markedly reduced the ability of heparin, but not that of melagatran, to inhibit the formation of these enzymes. Altogether these data indicate that melagatran is more efficient than heparin in promoting fibrinolysis, particularly in plateletrich clots, and may thus grant a greater antithrombotic activity by enhancing thrombus dissolution.     

Potentiation by Lys-plasminogen of clot lysis by single or two chain urokinase-type plasminogen activator or tissue-type plasminogen activator

Study has been made of the influence of addition of human NH2 terminal glutamic acid plasminogen (Glu-Plg) or human NH2 terminal lysine plasminogen (Lys-Plg) to normal citrated plasma upon the rate of lysis of fully crosslinked plasma clots in the presence of single or two chain urokinase type plasminogen activator (scu-PA/tcu-PA) or tissue plasminogen activator (t-PA). The specificity of any thrombolytic property was evaluated by measurement of plasma fibrinogen levels. Lys-Plg added to a concentration of 20% of normal plasma plasminogen caused 5 to 6 fold increase in the extent of lysis observed at 6 hours by 100 units/ml of scu-PA and with a small increase in fibrinogenolysis. Glu-Plg added at 20% of normal level had no influence on thrombolysis but at 50% of normal caused increased thrombolysis with rapid depletion of plasma fibrinogen. An apparently synergistic effect of addition of tcu-PA on scu-PA activity was increased by addition of plasminogen (e.g. addition of 20% Lys-Plg increased the lysis rate 4 to 5 fold over the first hour equivalent to an increase of potency of approximately three to four fold). Addition of plasminogen up to double the normal plasma concentration was observed to have no influence on clot lysis in the presence of t-PA. Plasminogen potentiated the rate of lysis by scu-PA/t-PA synergic mixtures with an approximately 1.5 to 1.9 fold increase in potency. Potentiation occurred without increase in the depletion of plasma fibrinogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of thrombolytic agents on human platelet function

In this study, we have evaluated the effects of four different thrombolytic agents, including Streptokinase from Hoechst and from Kabivitrum, Urokinase from Abbott and tissue plasminogen activator (t-PA) from Genetech, on platelet-rich plasma clots and platelet aggregation. At concentrations lower than 50 ugs/ml, t-PA had no inhibitory effect on clot retraction or platelet aggregation induced by weak or potent agonist. At a higher concentration (greater than 100 ugs/ml), t-PA specifically antagonized the action of thrombin on clot formation and platelet aggregation. Streptokinase (Kabivitrum) potentiated the action of weak agonists on platelet aggregation, but the same agent from Hoechst had no negative or positive influence. None of the drugs tested had an adverse effect on platelet function at suggested therapeutic levels. None of the thrombolytic agents were capable of dissociating preformed clots made from platelet-rich plasma. However, all of them caused lysis of whole blood clots. Also, prior incubation of plasma alone or platelet-rich plasma with any of the agents prevented subsequent clot formation. The studies demonstrate that thrombolytic drugs at therapeutic concentrations do not affect platelet function adversely. They have a potent effect on whole blood clots, but not on clots from platelet-rich plasma. Therefore, platelets may play a critical role in determining the degree of reperfusion and the frequency of reocclusion following treatment with thrombolytic agents in vivo.     

Clot penetration and fibrin binding of amediplase,a chimeric plasminogen activator (K2 tu-PA)

Amediplase (K(2) tu-PA) is a hybrid plasminogen activator, consisting of the kringle 2 domain of alteplase and the protease domain of urokinase. The objective of this study was to determine the in vitro clot penetration of amediplase in relation to its fibrin binding and to compare the properties with those of alteplase. The clot lysis activity of amediplase in internal clot lysis models (both purified system and plasma system) was about 10 times less than that of alteplase. The clot lysis activity of amediplase in an external clot lysis model (plasma system) was similar to that of alteplase at therapeutic concentrations around 1 micro g/ml. The fibrin-clot binding properties of amediplase and alteplase were studied in a purified system as well as in a plasma system. In both systems amediplase bound to fibrin although to a significantly lower extent than alteplase. The binding of amediplase or alteplase did not increase during plasmin-mediated degradation of fibrin. The binding of amediplase was fully inhibited by epsilon-aminocaproic acid, indicating that the observed binding was specific and occurred via the lysine binding site in the kringle of amediplase. Clot penetration was studied during pressure-driven fluid permeation using syringes containing plasma clots. Amediplase was able to enter the clot without significant hindrance, while alteplase was concentrated on the top of the plasma clot and hardly entered into the inner parts of the clot. Diffusion-driven clot penetration was studied during clot lysis using confocal microscopy. Alteplase was detected on or close to the clot surface, while two-chain urokinase, which has no affinity to fibrin, was also detected deep inside the clot. Amediplase showed a penetration behaviour, which was distinct from that of alteplase and similar to that of two-chain urokinase. We concluded that the fibrin binding of amediplase is moderate and does not hinder clot penetration under permeation-driven or diffusion-driven transport conditions. Enhanced clot penetration, especially in large clots, could allow a more efficient lysis during thrombolytic therapy.