The interaction between components of the fibrinolytic system and GPIb/V/IX of platelets thrombus formation in mice

The interaction of fibrinolytic components with GPIb/V/IX of platelets on thrombus formation, was investigated in mice deficient in tissue type (tPA-/-), urokinase type plasminogen activator (uPA-/-) or plasminogen activator inhibitor-1 (PAI-1-/-) and in their wild type control (tPA+/+, uPA+/+, PAI-1+/+). A thrombus was induced in the murine carotid artery using a photochemical reaction. The times to occlusion after the initiation of endothelial injury in all wild type mice was within 12 min, and no significant changes in occlusion delay were observed in uPA-/- and tPA-/- mice compared to wild type mice, whereas that of PAI-1 mice were significantly prolonged (16.9+/-2.9 min, P<0.05). When high molecular weight aurintricarboxylic acid (ATA), an inhibitor of platelet glycoprotein Ib/V/IX, was administered, the time to occlusion was prolonged in a dose-dependent manner in all types of mice. However, when this compound was injected in tPA-/- mice, the most significant changes were observed: i.e. the estimated ED(50) was 20.2 times higher than that in tPA+/+ mice, but the estimated ED(50) in uPA-/- mice was not changed as compared with that of wild type mice. On the other hand, when ATA was injected in PAI-1-/- mice, the estimated ED(50) was significantly decreased (P<0.05). Platelet aggregation induced by botrocetin was not significantly different among all types of mice. The bleeding time was prolonged in a dose dependent-manner when ATA was injected in all types of mice. In conclusion, the antithrombotic effect of inhibition of platelet GPIb/V/IX is severely affected by the absence or presence of tPA-production on thrombus formation and the inhibition of PAI-1 could enhance this antithrombotic effect.     

Inhibition of PAI-1: a new anti-thrombotic approach

Proteolytic degradation of fibrin (fibrinolysis) is mediated by plasminogen and its activators, tissue-type plasminogen activator (tPA(1)) and urokinase (uPA). Fibrinolysis is critical for preventing thrombus growth and restoring blood flow following thrombotic vascular occlusion. Plasminogen activator inhibitor-1 (PAI-1), a member of the serine protease inhibitor (serpin) superfamily, is the principal inhibitor of tPA and uPA in the fibrinolytic system. High levels of circulating PAI-1 are associated with a number of thrombotic diseases. In animal studies, transgenic mice overexpressing human PAI-1 develop spontaneous thrombosis, whereas PAI-1-deficient mice are more resistant to venous or arterial thrombosis. Furthermore, inhibition of PAI-1 activity prevents thrombus formation in animal models. The antithrombotic effects of PAI-1 inhibition are achieved by enhancing endogenous fibrinolytic activity without directly affecting blood coagulation and platelet function. Phenotypic analysis of PAI-1 deficiency in both human and mouse suggests that inhibition of PAI-1 will not lead to severe bleeding or other major adverse effects. Thus, PAI-1 inhibitors represent a new class of antithrombotic drugs with a possible wider therapeutic index than conventional antiplatelet and anticoagulant agents. This review summarizes the role of PAI-1 in thrombotic diseases and recent progress in the development of small molecule PAI-1 inhibitors.     

Comparative antiplatelet effects of aspirin, vapiprost and GR144053, a GPIIb/IIIa antagonist, with a special reference to the role of platelet microaggregates.

Microthrombi produced have a potential to form larger thrombi, leading to vascular occlusions. Recently, a new device to easily detect microaggregates using laser-light scattering (LS) has been developed. We adopted this device to comparatively evaluate the inhibitory effects of aspirin (1,3 or 10 mg kg(-1)), vapiprost (0.3, 1 or 3 mg kg(-1)) or GR144053 (0.1, 0.3 or 1 mg kg(-1)) on ex vivo aggregation of hamster platelets in relation to their in vivo antithrombotic effects. A transluminal thrombus was produced in the hamster femoral artery by the photochemical reaction. Each compound was injected i.v. as a bolus 10 min prior to the reaction, showing a dose-dependent antithrombotic effect, i.e. they prolonged the time before the artery occluded. At that time cyclic flow reductions occurred more marked when aspirin or vapiprost was given. At the end of experiments, blood was collected to evaluate the platelet aggregation using both the new LS device and the conventional optical density (OD) method. Many more small aggregates were still formed when the highest dose of aspirin or vapiprost was used as compared with that of GR144053, although suppression of the platelet aggregation using the OD method, prolongation of the occlusion time and the bleeding time were quite similar. In conclusion, a GPIIb/IIIa antagonist markedly suppressed the microthrombi and reduced the cyclic flow reduction. This further indicates the importance of small aggregates as triggers of thrombosis and shows that prevention of their formation may result in improved vascular patency after thrombotic insult.     

DRL performance in mice with deletion of tPA, uPA or PAI-1 genes.

Plasminogen activators are serine proteases induced in the brain by electrical activity leading to synaptic remodelling. They are classified into two distinct subtypes, tissue plasminogen activating factor and urokinase plasminogen activating factor (tPA and uPA, respectively), which are both expressed in brain areas thought to be important in learning and memory. Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of tPA and uPA activity, and is expressed in corresponding brain areas. Mice lacking tPA show a deficit in the acquisition of a 15 s differential reinforcement of low rate of responding (DRL15") task relative to their wild types (WTs) under certain conditions. The current set of experiments were designed to investigate further the role of tPA and to extend our knowledge to uPA and PAI-1, using mice with the respective genes deleted (uPA -/- and PAI-1 -/- mice) in the DRL15" task. uPA -/- mice showed no disruption of DRL acquisition, but PAI-1 -/- mice showed a deficit similar to that seen in tPA -/- mice. In an attempt to compensate for this deficit, experiments using a fixed number of reinforcers or a signalled-DRL15" schedule, similar to that used in rat lesion studies of DRL, were performed. tPA -/- mice were able to complete the signalled-DRL task as well as their WTs, and, similarly, PAI-1 -/- mice were able to learn the fixed-number-of-reinforcers-DRL15" schedule and the signalled-DRL schedule. These data indicate that uPA deletion does not affect performance of a standard DRL15" task, whereas deletion of PAI-1 has the same behavioural consequences in these tasks as deletion of tPA. Deficits of both genotypes can be attenuated by providing either external information on completion of the delay or by equalizing the number of reinforcers obtained.     

The antithrombotic efficacy of the GP IIb/IIIa antagonist SR121787 is potentiated by antithrombin-dependent factor Xa inhibition without an increase in the bleeding risk in the rabbit

Current antithrombotic therapy in acute coronary symptoms is only partially effective and exhibits bleeding complications. These experiments were designed to address the antithrombotic and hemorrhagic interactions of the novel glycoprotein (GP) IIb/IIIa antagonist SR121787 in combination with the indirect inhibitor of factor Xa, SR90107/ORG31540. Thrombogenesis was initiated by electrolytic injury of the intimal surface of the carotid artery, and thrombus formation was assessed by recording carotid blood flow and by measuring thrombus weight 45 min after electrical stimulation. SR121787 was an efficacious antithrombotic agent in this model (ED50 = 16.3+/-0.3 mg/kg, p.o.), whereas heparin (4.5 mg/kg, i.v.) and SR90107/ORG31540 [1 mg/kg (850 aXa anti-units/kg), i.v.] were only marginally effective (17 and 27% inhibition of carotid blood flow reduction, respectively). Coadministrations of heparin (4.5 mg/kg, i.v.) or SR90107/ORG31540 (0.5 mg/kg, i.v.) were found to potentiate the antithrombotic efficacy of threshold doses of SR121787 (5 or 10 mg/kg, p.o.). The enhancement of the antithrombotic efficacy of SR121787 by SR90107/ORG31540 was--contrary to the association of SR121787 with heparin--not accompanied by an increased blood loss from the incised rabbit ear. Coadministrations of heparin or SR90107/ORG31540 did not influence the ex vivo antiaggregatory activity of SR121787. SR121787 coadministration did not alter the systemic anticoagulant activities in heparin or SR90107/ORG31540-treated animals. These data suggest the potential for optimized antithrombotic treatment in acute coronary syndromes when a GP IIb/IIIa antagonist (SR121787) is combined with an antithrombin-dependent factor Xa inhibitor (SR90107/ORG31540).     

Restoration of middle cerebral artery thrombosis by novel glycoprotein IIb/IIIa antagonist FK419 in guinea pig

We compared the antithrombotic efficacy of FK419 [(S)-2-acetylamino-3-[(R)-[1-[3-(piperidin-4-yl)propionyl]piperidin-3-ylcarbonyl]amino] propionic acid trihydrate], a novel nonpeptide glycoprotein IIb/IIIa antagonist, with recombinant tissue plasminogen activator (rt-PA) and other antithrombotic agents (aspirin, ozagrel, argatroban and heparin). FK419 not only inhibited ADP- and collagen-induced guinea pig platelet aggregation, but also induced disaggregation for ADP-induced aggregated platelets in vitro. In the photochemically induced middle cerebral artery thrombosis model in guinea pigs, FK419 dose-dependently shortened the time to first reperfusion and the total middle cerebral artery occlusion time and reduced ischemic brain damage and ameliorated neurological deficits measured 24 h after middle cerebral artery occlusion. Rt-PA similarly improved the middle cerebral artery patency, brain damage and neurological deficits. Neither aspirin, ozagrel, argatroban nor heparin restored the middle cerebral artery blood flow and improved the brain damage or neurological deficits. These results demonstrated that novel glycoprotein IIb/IIIa antagonist FK419 could disperse thrombus and ameliorated ischemic brain damage, suggesting that FK419 would be an attractive intervention for stroke patients.     

T-686, a novel inhibitor of plasminogen activator inhibitor-1, inhibits thrombosis without impairment of hemostasis in rats

The aim of this study was to evaluate the antithrombotic potential of T-686 ((3E,4E)-3-benzylidene-4-(3,4,5-trimethoxy-benzylidene)-pyrrolidine-2,5-dione), a novel inhibitor of plasminogen activator inhibitor-1 (PAI-1), in rat thrombosis models. T-686 (0.1–100 mg/kg per day, p.o.) dose dependently decreased the weight of venous thrombi induced by a combination of stasis and hypercoagulability. The antithrombotic effect was enhanced by repeated administration of T-686. Warfarin (0.1 mg/kg per day for 3 days) also prevented thrombus formation. The antithrombotic action by warfarin was accompanied by prolongation of coagulation time, while no effect on coagulation time was observed in T-686-treated rats. T-686 lowered the activity of PAI-1 in plasma. In the arterio-venous shunt model, pretreatment with T-686 (10 mg/kg per day) or ticlopidine (100 mg/kg per day) for 8 days inhibited thrombus formation by 33% and 44%, respectively. T-686 had no effect on collagen-induced platelet aggregation ex vivo, while ticlopidine inhibited platelet aggregation. T-686 did not affect bleeding time at 10–100 times the antithrombotic dose, while warfarin dose dependently prolonged bleeding time at and around the antithrombotic dose. These results suggest that T-686 prevents thrombus formation in rats without impairment of hemostasis.     

Antithrombotic efficacy of RPR208566, a novel factor Xa inhibitor, in a rat model of carotid artery thrombosis

Coagulation factor Xa is the sole enzyme responsible for activating the zymogen prothrombin to thrombin, resulting in fibrin generation, platelet activation, and subsequent thrombus formation. Our objective was to evaluate the antithrombotic efficacy of the novel factor Xa inhibitor, 2-(3-carbamimidoyl-benzyl)-3-[(3', 4'dimethoxy-biphenyl-4-carbonyl)-amino]-butyric acid methyl ester-trifluoroacetate (RPR208566), in a well-established rat model of arterial thrombosis, and to compare the results with those obtained with argatroban and heparin, direct and indirect inhibitors of thrombin, respectively. Thrombus formation was initiated by placing a filter paper saturated with FeCl(2) on the adventia of the carotid artery for 10 min. Time-to-occlusion was measured from initiation of injury until blood flow reached zero. Formed thrombi were removed and weighed 60 min after the placement of the filter paper. RPR208566, heparin, and argatroban dose-dependently increased time-to-occlusion and reduced thrombus mass. When administered at 500 microgram/kg+50 microgram/kg/min, RPR208566 prolonged time-to-occlusion to 56+/-4 min (vs. 18+/-2 min for vehicle) and reduced thrombus mass to 3.0+/-0.7 mg (vs. 7.3+/-0.6 mg for vehicle). The highest doses of argatroban (500 microgram/kg+50 microgram/kg/min) and heparin (300 U/kg+10 U/kg/min) increased time-to-occlusion to the maximum of 60 min and decreased thrombus mass to 5.5+/-0.8 and 2.6+/-0.3, respectively. The antithrombotic effects of heparin and argatroban at these doses were associated with increases in activated partial thromboplastin time of 5.6+/-0.9- and 2.9+/-0.3-fold over baseline, respectively. However, the highest dose of RPR208566 produced a modest 1.3+/-0.1-fold increase in activated partial thromboplastin time. These results indicate that factor Xa inhibition with compounds such as RPR208566 may be an attractive mechanism for novel antithrombotic drug therapy.     

Metabolism-directed optimisation of antithrombotics: the prodrug principle

Thromboembolic disorders are the major cause of mortality and morbidity in Western societies. Coagulation enzymes, such as thrombin, factor Xa and a tissue factor/factor VIIa complex, together with platelet GPIIb/IIIa receptors, are the focal point of attention in pharmaceutical research aimed at finding new antithrombotic agents. However, finding orally active drugs for these particular molecular targets has proved to be anything but straightforward. Thrombin, factor Xa, tissue factor/factor VIIa and platelet GPIIb/IIIa receptors display a preference for molecules containing highly basic arginine and/or acidic aspartate moieties, which are, however, associated with poor bioavailability after oral application. Different approaches have been taken to achieve favourable absorption, metabolism, distribution and clearance, without compromising the antithrombotic activity of the compounds. This review highlights the use of the prodrug principle in optimising antithrombotic agents.     

Effects of fibrinogen receptor antagonist GR144053F and aurintricarboxylic acid on platelet activation and degranulation.

Activated blood platelets play crucial role in restenosis due to their fundamental significance in thrombus formation. Therefore, platelets are attractive targets for the inhibition with a variety of antagonists. In this study, we present direct evidence that GR144053F [non-peptide antagonist of glycoprotein IIb-IIIa complex (GPIIb-IIIa)] inhibits activation and degranulation of human platelets, and opposes the action of aurintricarboxylic acid (ATA), the antagonist of von Willebrand factor, which augments platelet secretion. The effects of both drugs on platelet function were monitored by using various instrumental methods. Platelet-rich plasma and whole-blood aggregation was measured by using ADP and collagen as agonists. Platelet degranulation was assessed based on the expression of surface membrane activation markers: P-selectin, glycoprotein Ib, and activated GPIIb-IIIa complex. Measurements of closure time with platelet function analyzer PFA-100 enabled us to reason on primary hemostatic capacity and reflected both aggregability and adhesiveness. GR144053F markedly reduced primary hemostatic platelet response (IC(50) = 114.0 +/- 9.6 nM) under conditions that closely mimicked natural blood flow in circulation, and inhibited aggregation in platelet-rich plasma (IC(50) = 17.7 +/- 7.0 nM). It was equally potent inhibitor of platelet activation, degranulation, fibrinogen binding, platelet consumption, and aggregate formation. Also, ATA was efficient in inhibition of platelet aggregation and adhesion (by up to 50% at 100 microM), but the combined action of both drugs on primary haemostatic capacity was not additive. GR144053F suppressed the activating effects of ATA on platelet degranulation and secretion. Overall, our data indicate that GR144053F is not only the efficient blocker of fibrinogen binding to GPIIb-IIIa, but also hampers platelet degranulation and may attenuate the activating effects of ATA.     

Nonpeptide factor Xa inhibitors: DPC423, a highly potent and orally bioavailable pyrazole antithrombotic agent

DPC423, 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'-(methylsulfonyl)[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide, is a synthetic, orally bioavailable, competitive, and selective inhibitor of human coagulation factor Xa (K(i) [nM]: factor Xa, 0.15; trypsin, 60; thrombin, 6000; plasma kallikrein, 61; activated protein C, 1800; factor IXa, 2200; factor VIIa, >15,000; chymotrypsin, >17,000; urokinase, >19,000; plasmin, >35,000; tissue plasminogen activator, >45,000; complement factor I, 44,000 [IC(50)]). In vitro, DPC423 produced anticoagulant effects in human plasma in which it doubled prothrombin time, activated partial thromboplastin time, and Heptest clotting time at 3.1 +/- 0.4, 3.1 +/- 0.4, and 1.1 +/- 0.5 microM, respectively. In dogs, DPC423 had a good pharmacokinetic profile with an oral bioavailability of 57%, a plasma clearance of 0.24 L/kg/h, and a plasma half-life of 7.5 h. In rabbit and rat models of arteriovenous shunt thrombosis, DPC423 was an effective antithrombotic agent with an IC(50) of 150 and 470 nM, respectively. The antithrombotic effect of DPC423 is likely to be related to the inhibition of factor Xa but not to the inhibition of thrombin or due to direct inhibition of platelet aggregation. Therefore, based on potency, selectivity, efficacy, and oral bioavailability, DPC423 was selected for clinical development as an oral anticoagulant for the potential treatment of thrombotic disorders. Preliminary human data suggest that DPC423 is orally bioavailable in humans and has a long plasma half-life.     

Tissue plasminogen activator is not involved in methamphetamine-induced neurotoxicity

To investigate the role of tissue plasminogen activator (tPA) in methamphetamine (METH)-induced dopaminergic neurotoxicity, we compared the changes in tyrosine hydroxylase (TH) and dopamine transporter (DAT) levels in the striatum after repetitive treatment of METH at 4 mg/kg among wild-type, tPA-deficient (tPA-/-), and protease activated receptor-1-deficient (PAR-1-/-) mice. METH treatment caused a marked decrease in TH and DAT levels in the striatum of those mice with a similar magnitude. No difference in METH-induced abnormal behavior and hyperthermia was observed among the three types of mice. These results suggest that neither tPA nor PAR-1 is involved in METH-induced dopaminergic neurotoxicity in vivo.     

Understanding the mechanism of platelet thrombus formation under blood flow conditions and the effect of new antiplatelet agents

Platelet aggregation induced by stirring of a platelet suspension after activation by the exogenous addition of stimulants, such as ADP, epinephrine and thrombin, has long been used as a platelet function test, and for the screening of antiplatelet agents. Since platelet aggregation has been demonstrated to be mediated exclusively by the binding of plasma fibrinogen to the activated GP IIb/IIIa, anti-GP IIb/IIIa agents, which can completely inhibit platelet aggregation, were expected to become among the most potent of antithrombotic agents. The strong preventive effects of anti-GP IIb/IIIa agents against thrombotic complications after coronary intervention, and the weaker preventive effects of the same agents against the onset of coronary thrombosis in unstable angina pectoris patients point to both the efficacy and the limitations of anti-GP IIb/IIIa antithrombotic agents. Recently, many investigators have reported that platelets play a major role in thrombus formation at sites exposed to blood flow. Several platelet-function assay systems have been developed to elucidate the mechanism of thrombus formation under blood flow conditions. Numerous studies have demonstrated that von Willebrand factor (VWF) and its interaction with its receptors on platelets, including GP Ibalpha and GP IIb/IIIa, play essential roles not only in platelet activation, but also in platelet adhesion and possibly platelet cohesion. These findings prompted us to explore whether the VWF-mediated process of thrombus formation could be exploited as a target for potential antiplatelet agents. Moreover, recent studies have demonstrated that multiple synergistic signals, including those mediated by catecholamine receptors, purine nucleotide receptors, as well as some types of collagen receptors are involved in the process of VWF-mediated platelet thrombus formation. We now have new antiplatelet agents on the horizon, targeted against thrombus formation under blood flow conditions.     

苯那普利对肾病幼鼠纤溶蛋白肾组织定位表达的影响

目的　探讨肾病幼年大鼠肾小球硬化及肾间质纤维化病变进展过程中 ,肾组织尿激酶型纤溶酶原激活物 (u PA )、组织型纤溶酶原激活物 (t PA)及其特异性抑制物 (PAI- 1)蛋白定位表达的特点 ,及予血管紧张素转换酶抑制剂 (ACEI)——苯那普利 (benazepril;lotensin)治疗的影响。方法　采用阿霉素诱导的肾病大鼠为动物模型 ,予 ACEI治疗 12周后测大鼠体重、血压、尿蛋白、及血生化各项指标的变化 ,同时用免疫组织化学染色等方法检测各组肾组织 u PA、t PA和 PAI- 1的蛋白表达的变化特点。结果　肾病大鼠肾组织 PAI- 1表达均高于正常对照组 ,而 u PA、t PA均低于正常组 ;经治疗后肾组织 PA I- 1趋于下降 ,而 u PA、t PA表达趋于增高 (P<0 .0 1)。结论　纤溶系统的平衡紊乱是肾病大鼠肾小球硬化和肾间质纤维化进展中的重要病生理变化之一 ,ACEI治疗可改善 PAs/ PAI- 1的异常表达 ,防止细胞外基质的异常沉积 ,阻止肾小球硬化和间质纤维化病变进展。     

Prevention of reocclusion following tissue type plasminogen activator-induced thrombolysis by the RGD-containing peptide, echistatin, in a canine model of coronary thrombosis

We evaluated the effect of the RGD-containing peptide, echistatin, on thrombolysis time and acute reocclusion in a canine model of coronary thrombosis/thrombolysis. Occlusive thrombus formation was induced by electrical injury, via a stimulating electrode, to the endothelial surface of the circumflex coronary artery in the open-chest, anesthetized dog in the presence of a critical stenosis. Fifteen minutes after occlusive thrombus formation, dogs received either an intravenous infusion of vehicle (saline at 0.1 ml/min) or echistatin (15 micrograms/kg/min i.v.). Heparin was given as an initial bolus (100 U/kg i.v.) 15 min after thrombus formation and repeated at hourly intervals (50 U/kg). This dose of heparin increased activated partial thromboplastin time to 1.5- to 2.5- fold over control. Thrombolysis was induced with recombinant tissue-type plasminogen activator (tPA) at a total dose of 1 mg/kg, intravenously administered over 90 min with 10% given as an initial bolus. The vehicle-treated animals reperfused at 48 +/- 9 min with a reperfusion incidence of 60% (3/5). The echistatin-treated animals reperfused at 46 +/- 5 min with a reperfusion incidence of 100% (5/5). After stopping the tPA infusion, acute reocclusion occurred in 100% (3/3) of the vehicle-treated dogs and in only 20% (1/5) of the echistatin-treated dogs. Echistatin caused a greater than 5-fold increase in buccal mucosa bleeding time and almost completely inhibited ex vivo platelet aggregation to ADP, collagen, and U-46619. Residual thrombus wet weight, determined at the end of the experiment, was significantly lower for the echistatin group (2.1 +/- 0.2 mg) compared to the vehicle group (5.8 +/- 0.7 mg).(ABSTRACT TRUNCATED AT 250 WORDS)     

Diphenyl phosphonate inhibitors for the urokinase-type plasminogen activator: optimization of the P4 position

This paper describes the structure-activity relationship in a series of tripeptidyl diphenyl phosphonate irreversible urokinase plasminogen activator (uPA) inhibitors, originally derived from an arginyltripeptide. uPA is considered an interesting target in anticancer drug design. The selectivity of these inhibitors for uPA is enhanced by the optimization of the P4 position. The most interesting compound shows an IC(50) of 5 nM, with a selectivity index of more than 3000 toward other Arg/Lys-specific proteases such as tissue-type plasminogen activator, plasmin, factor Xa, and thrombin. A synthetic strategy for the preparation of small libraries of diphenyl phosphonate analogues of capped tripeptides is described. It is shown that uPA is irreversibly inhibited, and interactions with the active site were modeled. Finally, a diparacetamol phosphonate analogue was developed to circumvent the release of cytotoxic phenol.     

Adipocytes derived fibrinolytic components in peritoneum — a pilot study

The proteins of the fibrinolytic system — urokinase plasminogen activator(uPA), tissue plasminogen activator (tPA)and plasminogen activator inhibitor type IPAI-I) — play important roles in fibrotization in various organs and including peritoneum. To study the cellular localization of PAI-1, tPA and uPA within the adipose tissue of the peritoneal membrane in patients at the onset of peritoneal dialysis(PD) we determined the initial expression of these proteins in relationship to multiple clinical variables. Methods: routinely performed parietal peritoneal biopsies in 12 patients undergoing peritoneal catheter implantation were examined. We used formalinfixed, paraffin-embedded specimens for immunohistochemical localization of these proteins along with the stereological pointcounting method for quantification of their expression within the peritoneal adipose tissue. Results: strong positive mutual correlation between the expression of PAI-1 and both uPA (SpearmanR=0.66) and tPA (R=0.59) as well as between the expression of uPA and tPA (R=0.77) was found without any relatioship to BMI, age, peritoneal transport characteristic or diabetes status. Conclusion: Adipose tissue within the peritoneum is capable of producing fibrinolysis regulators (independently on clinical parameters) thus possibly affecting the fibrotization and function of peritoneum as dialysis membrane. The effect of dialysis solution dosing, composition and other dialysis related factors should be clarified in future studies.     

Role of tissue plasminogen activator in the rewarding effect of morphine]

Tissue plasminogen activator (tPA) is a serine protease that catalyzes the conversion of plasminogen (plg) to plasmin. The tPA-plasmin system plays a role in synaptic plasticity and remodeling. In this review, we focused on the role of tPA-plasmin system in the rewarding effect of morphine. A single morphine treatment induced tPA mRNA and protein expression in a naloxone-sensitive manner, which was associated with an increase in the enzyme activity in the nucleus accumbens (NAc). The acute effect of morphine in inducing tPA expression was diminished after repeated administration. No differences were observed in the morphine-induced antinociceptive effect between wild-type and tPA knockout (tPA-/-) mice. Morphine-induced conditioned place preference and hyperlocomotion were significantly reduced in tPA-/- and pLg-/- mice, being accompanied by a loss of morphine-induced dopamine release in the NAc. Microinjection of either exogenous tPA or plasmin into the NAc significantly potentiated morphine-induced dopamine release in the NAc of ICR mice. In contrast, plasminogen activator inhibitor-1 (PAI-1) dose-dependently reduced morphine-induced dopamine release. Furthermore, the defect of morphine-induced dopamine release and hyperlocomotion in tPA-/- mice was reversed by microinjections of either exogenous tPA or plasmin into the NAc. Our findings demonstrate a novel function of the tPA-plasmin system in regulating dopamine release in the NAc, which is involved in the morphine reward.     

Neutralization of plasminogen activator inhibitor I (PAI-1) by the synthetic antagonist PAI-749 via a dual mechanism of action

PAI-749 is a potent and selective synthetic antagonist of plasminogen activator inhibitor 1 (PAI-1) that preserved tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) activities in the presence of PAI-1 (IC(50) values, 157 and 87 nM, respectively). The fluorescence (Fl) of fluorophore-tagged PAI-1 (PAI-NBD119) was quenched by PAI-749; the apparent K(d) (254 nM) was similar to the IC(50) (140 nM) for PAI-NBD119 inactivation. PAI-749 analogs displayed the same potency rank order for neutralizing PAI-1 activity and perturbing PAI-NBD119 Fl; hence, binding of PAI-749 to PAI-1 and inactivation of PAI-1 activity are tightly linked. Exposure of PAI-1 to PAI-749 for 5 min (sufficient for full inactivation) followed by PAI-749 sequestration with Tween 80 micelles yielded active PAI-1; thus, PAI-749 did not irreversibly inactivate PAI-1, a known metastable protein. Treatment of PAI-1 with a PAI-749 homolog (producing less assay interference) blocked the ability of PAI-1 to displace p-aminobenzamidine from the uPA active site. Consistent with this observation, PAI-749 abolished formation of the SDS-stable tPA/PAI-1 complex. PAI-749-mediated neutralization of PAI-1 was associated with induction of PAI-1 polymerization as assessed by native gel electrophoresis. PAI-749 did not turn PAI-1 into a substrate for tPA; however, PAI-749 promoted plasmin-mediated degradation of PAI-1. In conclusion, PAI-1 inactivation by PAI-749 using purified components can result from a dual mechanism of action. First, PAI-749 binds directly to PAI-1, blocks PAI-1 from accessing the active site of tPA, and abrogates formation of the SDS-stable tPA/PAI-1 complex. Second, binding of PAI-749 to PAI-1 renders PAI-1 vulnerable to plasmin-mediated proteolytic degradation.     

Modulation of PAI-1 and tPA activity and thrombolytic effects of corilagin

In this study, Charlton's and Tomihisa's methods were modified to investigate the thrombolytic effect of corilagin from the Chinese herbal plant Phyllanthus urinaria L., as well as its effect on carotid artery patency status. The activity of type 1 plasminogen activator inhibitor (PAI-1) in rat plasma or platelet-released substances and tissue-type plasminogen activator (tPA) in rat plasma was assayed by use of a chromogenic substrate. The results showed that corilagin had a dose-dependent thrombolytic effect in rats. 5 mg/kg of corilagin produced a nearly similar reperfusion rate to that of 20000 U/kg of urokinase, whereas it produced a lower reocclusion rate than urokinase. Corilagin significantly inhibited PAI-1 activity in rat plasma or platelet-released substances while it elevated plasma tPA activity, in a concentration-dependent manner. Corilagin, however, had no influence on rabbit platelet aggregation. It is indicated that corilagin inhibited PAI-1 activity and increased tPA activity, and this property of corilagin is assumed to be responsible for the thrombolytic effect. Abbreviations. PO:persistent occlusion CR:cyclic reflow PP:persistent patency PAI-1:type 1 plasminogen activator inhibitor tPA:tissue-type plasminogen activator PBS:phosphate buffer solution IC (50):50 % of inhibitory concentration PRP:platelet-rich plasma ADP:adenosine diphosphate AA:arachidonic acid PAF:platelet-activating factor