Antithrombotic effects and related mechanisms of Salvia deserta Schang root EtOAc extracts.

Salvia deserta Schang (SDS) belongs to the same family as Salvia miltiorrhiza bunge, one of the antithrombotic Chinese herbal medicines. In our study, EtOAc root extracts were analyzed for their effects on adenosine diphosphate (ADP)-induced platelet aggregation in rabbits and FeCl3-induced rat common carotid artery thrombosis as well as on rat blood plasma concentrations of thromboxane B2 (TXB2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1α), antithrombin-III (AT-III), protein C (PC), plasminogen (PLG), plasminogen activator inhibitor (PAI-1), von Willebrand factor (vWF) and tissue-type plasminogen activator (t-PA). EtOAc extracts from SDS roots had significant inhibitory effects on ADP-induced maximum platelet aggregation rate (10.2 ± 2.6 vs control 35.7 ± 5.2; P < 0.05), reduced the FeCl3-induced rat common carotid artery thrombus weight and thrombus area ratio (P < 0.05), significantly decreased plasma TXB2, vWF and PAI-1 levels and increased 6-keto-PGF1α and t-PA levels in a dose dependent manner (all P < 0.05). Thus, the ratio of TXB2/6-keto-PGF1α was significantly decreased (P < 0.05), while the ratio of t-PA/PAI-1 was significantly increased (P < 0.05). In addition, enhanced AT-III and PC activities indicated coagulation inactivation effects of EtOAc SDS root extracts. EtOAc extraction from SDS showed antithrombotic effects, which are likely due to platelet adhesion and aggregation inhibition as well as anticoagulant activities.

Antithrombotic Activity of DAB-15, a Novel Diazepinobenzimidazole Compound.

Antithrombotic activity of a new orally administered antiplatelet compound DAB-15 was compared to that of acetylsalicylic acid, ticlopidine, and clopidogrel in the experimental model of arterial thrombosis in rats caused by surface application of 50% ferric chloride (III) on the carotid artery. Compound DAB-15 exerted a dose-dependent antithrombotic effect and was superior to acetylsalicylic acid, ticlopidine and clopidogrel by 5, 7, and 4.9 times, respectively (by ED50). This necessitates studying of the action mechanism of this antiplatelet compound with consideration of its influence on different stages of the pathogenesis of platelet aggregation.

A rabbit model of cerebral microembolic signals for translational research: preclinical validation for aspirin and clopidogrel.

UNLABELLED: Essentials Microembolic signal (MES) is an independent predictor of stroke risk in patients. A rabbit model of cerebral microembolic signals was established. Therapeutic efficacy was demonstrated for aspirin and clopidogrel on microembolic signals. Potential translational value of this preclinical model of MES was demonstrated. SUMMARY: Objectives Cerebral microembolic signals (MESs) detected by transcranial Doppler (TCD) ultrasound constitute an independent predictor of stroke risk and prognosis. The aim of this study was to develop a novel preclinical model of MESs to facilitate translational research. Methods A clinical TCD ultrasound machine was used to detect MESs in the cerebral circulation of New Zealand White rabbits. Technical feasibility was assessed for the measurement of MESs in the middle cerebral artery (MCA) by TCD. FeCl3 -induced carotid arterial thrombosis was optimized for the generation of endogenous microemboli. Ascending doses of two antithrombotic agents (aspirin and clopidogrel) were evaluated individually and in combination for their effects on both arterial thrombosis and MESs in a 30% FeCl3 -induced carotid arterial thrombosis model, along with ex vivo functional assays. Results Dose-dependent FeCl3 -induced arterial thrombosis studies showed that 30% FeCl3 resulted in the most consistent and reproducible MESs in the MCA (3.3 ± 0.7 MESs h(-1) ). Ascending-dose studies showed that the effective doses for 50% inhibition (ED50 ) of thrombus formation, based on integrated blood flow and thrombus weight, respectively, were 3.1 mg kg(-1) and 4.2 mg kg(-1) orally for aspirin, and 0.3 mg kg(-1) and 0.28 mg kg(-1) orally for clopidogrel. The ED50 values for MES incidence were 12.7 mg kg(-1) orally for aspirin, and 0.25 mg kg(-1) orally for clopidogrel. Dual treatment with aspirin (5 mg kg(-1) ) and clopidogel (0.3 mg kg(-1) ) resulted in significant reductions in cerebral MESs (P < 0.05) as compared with monotherapy with either agent. Conclusions Our study demonstrated the successful establishment of the MES model in rabbits, and it may provide translational value for MESs and ischemic stroke research.

Effects of Cydonia oblonga Miller extracts on blood hemostasis, coagulation and fibrinolysis in mice, and experimental thrombosis in rats.

INTRODUCTION: Cydonia oblonga Miller (COM) is traditionally used in Uyghur medicine for the prevention of cardiovascular disease. The present study is designed to explore the effects of COM extracts on models and markers of thrombosis and related biomarkers. MATERIALS AND METHODS: 20, 40, 80 mg/kg/day COM aqueous extracts and 5mg/kg/day aspirin, orally for 14 days were compared to untreated controls in mice on bleeding and clotting times, using the tail cutting and glass slide methods and for death rates in collagen-epinephrine pulmonary thrombosis, thrombolysis in vitro and euglobulin lysis time (ELT). In rats, common carotid artery FeCl3-induced thrombus and inferior vena cava thrombosis occlusion time, plasma concentrations of thromboxane B2 (TXB2) and 6-keto-prostaglandine F1α (6-keto-PGF1α) were measured. RESULTS AND CONCLUSION: Compared to controls, COM extracts dose-dependently prolonged bleeding by 2.17, 2.78 and 3.63 times, vs. aspirin 2.58, and the clotting time by 1.44, 2.47 and 2.48 times, vs. aspirin 1.91. COM reduced pulmonary embolus mortality by 27, 40 and 53%, vs. 47% for aspirin. COM dose-dependently increased thrombolysis by 45, 55 and 63%, vs. 56% for aspirin, and shortened ELT to 71, 61 and 43%, vs. 43% for aspirin. In rats, venous occlusion time was prolonged. Arterial and venous thrombus weights were dose-dependently reduced in COM groups. TXB2 decreased and 6-keto-PGF1α increased with COM and aspirin, with an association between 6-keto-PGF1α/TXB2 and arterial or venous thrombus weight for all products, and for occlusion time with COM but not for aspirin. CONCLUSION: We confirm the experimental effects of COM on hemostasis and thrombosis. Further exploration of putative clinical effects appear justified.

Iridoid glycosides extracted from zhizi (fructus gardeniae) decrease collagen-induced platelet aggregation and reduce carotid artery thrombosis in an in vivo rat model.

OBJECTIVE: To investigate the anti-platelet aggregation and antithrombotic effects in rats of iridoid glycosides extracted from Zhizi (Fructus Gardeniae). METHODS: The present study evaluated the antithrombotic activity of iridoid glycosides (IGs) in a rat model of carotid artery thrombosis. The effects on coagulation, such as thromboplastin time (APTT), thrombin time (TT) and prothrombin time (PT), and the effect on collagen-induced platelet aggregation in vivo were investigated. Rats were intragastrically administered IGs (50, 100 or 200 mg/ kg) twice daily for 3 days. RESULTS: IGs were shown for the first time to have an antithrombotic action through the inhibition of platelet aggregation, with little effect on the coagulation time of peripheral blood. Our results also showed that IGs may significantly and dose-dependently reduce arterial thrombus load in a model of carotid artery thrombosis and inhibit collagen-induced platelet aggregation in rats. IGs (100 or 200 mg/kg) had no significant effect on APTT and PT, but did lengthen TT at a higher dose. CONCLUSION: These data, together with the previously reported neuroprotective effects of IGs in rats with cerebral ischemia, suggest that the antithrombotic action of IGs may potentially contribute to the treatment of cerebral ischemic diseases, including cerebral apoplexy.

[Study on prevention effect of huoluotongnao tablet on stroke (I)].

OBJECTIVE: To study the prevention effect of Huoluotongnao tablet on stroke. METHODS: Thrombosis on arteriovenous shunt rats model, platelet aggregation and hypertension combined high cholesterol rats model were used. RESULTS: Huoluotongnao tablet high and low dosage could inhibit the formation of arteriovenous thrombosis and platelet aggregation significantly ,the inhibition rate was 17.71%, 22.69%, 20.34% and 24.43%, respectively. Pretreatment of Huoluotongnao tablet could inhibit the formation of arteriovenous thrombosis significantly; The levels of CHOz in all treatment groups of hypertension combined high cholesterol rats model were decreased significantly,the levels of TGz and LDL-C were decreased in the high dosage group,the blood pressure was decreased in the middle dosage group. eta bL, eta P and eta r (B/P) were decreased in the middle and high dosage groups. eta bM, AI and CY were decreased in the middle and high dosage groups. Huoluotongnao tablet had effect on blood lipid,blood pressure and hemorheology and in a dose-dependence manner. Its minimal effecting dose was the middle dose. g/kg (crude drug) and has certain prevention effect on stroke. CONCLUSION: Huoluotongnao tablet's minimal effecting dose is 1.28

Troubleshooting the rabbit ferric chloride-induced arterial model of thrombosis to assess in vivo efficacy of antithrombotic drugs.

INTRODUCTION: The FeCl3-induced arterial model of thrombosis is one of the most widely used animal models to assess arterial efficacy of new antithrombotic drug candidates. This model is well-established in rodents but in a less extent in the rabbit. In this work, we present a methodology for a rabbit FeCl3-induced arterial model of thrombosis derived from our troubleshooting which allows the generation of reliable efficacy data for new antithrombotic drug candidates. METHODS: Rabbits were administered with heparin 4.5U/kg/min, argatroban 10µg/kg/min or saline by intravenous infusion. The blood flow was monitored using a Doppler flow probe. The time from the application of FeCl3 to the recorded zero blood flow was defined as the time to occlusion, with a maximum recording time of 60min post-FeCl3 application. After 30min of infusion, thrombosis was induced by wrapping a FeCl3-saturated filter paper around the carotid artery caudal to the flow probe. Animals were subject to exclusion criteria based on the visual aspect of the artery FeCl3-induced injury and based on changes in blood flow upon FeCl3 application. RESULTS: Following the application of FeCl3, a mean time to occlusion for saline, heparin and argatroban of 24.3±1.8, 52.5±4.8 and 53.5±4.5min was obtained, respectively. Mean time to occlusion for heparin and argatroban administered groups was significantly different when compared to the saline-treated group (p<0.05). These results for the test compounds represent approximately 80% of the maximum possible prolongation. DISCUSSION: The rabbit FeCl3-induced arterial model of thrombosis presented in this paper derived from our troubleshooting is sensitive and reproducible for the generation of accurate and reliable efficacy data in the assessment of new antithrombotic agents in preclinical drug development.

Endogenous and natural complement inhibitor attenuates myocardial injury and arterial thrombogenesis.

BACKGROUND: Coagulation disorders and reperfusion of ischemic myocardium are major causes of morbidity and mortality. Lectin pathway initiation complexes are composed of multimolecular carbohydrate recognition subcomponents and 3 lectin pathway-specific serine proteases. We have recently shown that the lectin pathway-specific carbohydrate recognition subcomponent mannose-binding lectin plays an essential role in the pathophysiology of thrombosis and ischemia/reperfusion injury. Thus, we hypothesized that the endogenous mannose-binding lectin (MBL)/ficolin-associated protein-1 (MAP-1) that inhibits complement activation in vitro also could be an in vivo regulator by attenuating myocardial schema/reperfusion injury and thrombogenesis when used at pharmacological doses in wild-type mice. METHODS AND RESULTS: In 2 mouse models, MAP-1 preserves cardiac function, decreases infarct size, decreases C3 deposition, inhibits MBL deposition, and prevents thrombogenesis. Furthermore, we demonstrate that MAP-1 displaces MBL/ficolin-associated serine protease (MASP)-1, MASP-2, and MASP-3 from the MBL complex. CONCLUSIONS: Our results suggest that the natural, endogenous inhibitor MAP-1 effectively inhibits lectin pathway activation in vivo. MAP-1 at pharmacological doses represents a novel therapeutic approach for human diseases involving the lectin pathway and its associated MASPs.

[Study of dahuangzhechong pills on anti-arterial thrombosis with the orthogonal design].

OBJECTIVE: To screen the main component of Dahuangzhechong pill's anti-arterial thrombosis with the orthogonal design and refine Dahuangzhechong pills. METHODS: In accordance with the orthogonal design table (L(16)2(15)), divided herbs into 16 groups and made the appropriate liquid. The liquid was gave to SD rats by intragastric administration,the model group, normal control group received the same volume of physiological saline. Isolated rats' carotid artery after intragastric administration a week,modeled according to ferric chloride inducement the carotid artery thrombosis method, then collected blood, detected content of platelet, thromboxane B2 (TXB2) and 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), sheared and measured dry weight of the modeling artery, then placed arteries in 10% formalin fixation, observed morphological changes in vascular tissue by HE staining. RESULTS: Pathological examination revealed: each experimental group had thrombosis, softening, dissolution, absorption, and intimal injury, but the severity of thrombosis were diferent. Orthogonal analysis showed: 1, influence on dry weight of thrombus: rhubarb, ground beetle, leeches, peach seed, dry paint, except dry paint P<0.05, the others P<0.01.2, influence on plasma 6- keto-PGF1alpha level: peach seed, dry paint, ground beetle, gadfly, grubs, leeches, rhubarb, except rhubarb P<0.05, the others P<0.01.3, influence on plasma TXB2: ground beetle, peach seed, dried paint, rhubarb, leeches, except leech P<0.05, the others P<0.01.4, influence on platelet count: peach seed, dry paint, rhubarb, ground beetle, gadfly, leeches, except gadfly, leeches P<0.05, the others P<0.01. CONCLUSION: Anti-artery thrombosis of Dahuangzhechong Pill is most closely related with rhubarb, ground beetle, leeches, peach seed, dry paint and gadfly.

Effect of Chinese herbal medicine for activating blood circulation and detoxifying on expression of inflammatory reaction and tissue damage related factors in experimental carotid artery thrombosis rats.

OBJECTIVE: To observe the pharmaceutical effect of Chinese drugs for activating blood circulation (Xiongshao Capsule, XSC, ) and for activating blood circulation and detoxification (Xiongshao Capsule and Huanglian Capsule, XSHLC, ) in terms of the indices of thrombosis, inflammatory reaction and tissue damage related factors in experimental carotid artery thrombosis rats. METHODS: Fifty Wistar rats were randomly divided into the sham operation group, the model group, the Simvastatin group (SG), the activating blood circulation (ABC) group, and the activating blood circulation and detoxifying (ABCD) group, with 10 rats in each group. Simvastatin (1.8 mg/kg), XSC (0.135 g/kg) and XSHLC (0.135 g/kg) were administered to Simvastatin, ABC and ABCD group by gastrogavage, and an equal volume of normal saline was given to the sham operation group and the model group. After 2 weeks of successive medication, the rats in the model and all drug therapy groups were made into experimental carotid artery thrombosis model. The serum levels of matrix metalloproteinases (MMP-9), tissue inhibitors to metalloproteinase (TIMP-1), granule membrane protein-140 (GMP-140), tissue-type plasminogen activator (t-PA), high-sensitivity C-reactive protein (hs-CRP) and interleukin-6 (IL-6) were detected with enzyme-linked immunoassay 24 h later. RESULTS: Compared with the model group, the levels of serum GMP-140, hs-CRP, IL-6 and MMP-9 were significantly decreased, and the level of t-PA was significantly increased in the ABC and ABCD group ( P<0.05), while the level of serum hs-CRP in ABCD group decreased significantly compared with that in the ABC group (P<0.05). CONCLUSIONS: Chinese drugs both for activating blood circulation and for activating blood circulation and detoxifying have good effects on regulating indices of thrombosis, inflammatory reaction and tissue damage in experimental carotid artery thrombosis rats. The effect of activating blood circulation and detoxifying drugs on regulating the level of serum hs-CRP is superior to that of activating blood circulation drug alone.

Effects of polysaccharides enriched in 2,4-disulfated fucose units on coagulation, thrombosis and bleeding. Practical and conceptual implications.

Sulfated polysaccharides from marine invertebrates have well-defined structures and constitute a reliable class of molecules for structure-activity relationship studies. We tested the effects of two of these polysaccharides, namely a sulfated fucan and a fucosylated chondroitin sulfate, on coagulation, thrombosis and bleeding. The compounds share similar 2,4-disulfated fucose units, which are required for high anticoagulant activity in this class of polymer. These residues occur either as branches in fucosylated chondroitin sulfate or as components of the linear chain in the sulfated fucan. These polysaccharides possess anticoagulant activity but differ significantly in their mechanisms of action. The fucosylated chondroitin sulfate inhibits thrombin by heparin cofactor II, whereas sulfated fucan inhibits thrombin by both antithrombin and heparin cofactor II. In addition, these polysaccharides also have serpin-independent anticoagulant activities. Fucosylated chondroitin sulfate, but not sulfated fucan, activates factor XII. As a result of the complex anticoagulant mechanism, the invertebrate polysaccharides differ in their effects on experimental thrombosis. For instance, the sulfated fucan inhibits venous thrombosis at lower doses than fucosylated chondroitin sulfate. In contrast, fucosylated chondroitin sulfate is significantly more potent than sulfated fucan in arterial thrombosis. Finally, fucosylated chondroitin sulfate increases bleeding, while sulfated fucan has only a discrete effect. In conclusion, the location of 2,4-disulfated fucose units in the polysaccharide chains dictates the effects on coagulation, thrombosis and bleeding.

Effects of propyl gallate on carotid artery thrombosis and coagulation/fibrinolysis system in rats.

OBJECTIVE: To investigate the effects of propyl gallate (PrG) on the thrombus formation time and the coagulation/fibrinolysis system in an experimental carotid artery thrombosis model in rats. METHODS: Fifty SD rats were randomly divided into 5 groups (10 animals/group): the normal group (normal saline 2 mL/kg), the model group (normal saline, 2 mL/kg), the heparin control group (1,250 IU/kg), the low dose PrG group (30 mg/kg), and the high dose PrG group (60 mg/kg). Thirty minutes after intravenous injection of saline or the corresponding drugs, a carotid artery thrombus was induced by continuous electric stimulation in all rats except for those in the normal group. The duration from the initiation of the electric stimulation to the sudden drop in carotid temperature was recorded as the thrombus formation time. Levels of plasma tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were determined by ELISA. RESULTS: PrG (30 and 60 mg/kg) can prolong the thrombus formation time, but the effect was obviously weaker than that of heparin (P<0.05, P<0.01). Compared with the model group, PrG (30 and 60 mg/kg) elevated the plasma activity of t-PA (both P<0.05) and showed an increasing tendency in elevating the ratio of t-PA/PAI-1 (P>0.05), while it had no significant effect on the level of PAI-1. CONCLUSION: PrG has a certain antithrombotic effect and can slightly regulate the imbalance of the t-PA /PAI-1 ratio.

[Effect of Xuesaitong drop pills on experimerntal thrombosis and thrombolysis in rats].

OBJECTIVE: To study the effect and the mechanism of Xuesaitong drop pills (total saponins in Radix Notoginseng; XDP) on experimental thrombosis, thrombolysis and blood theology. METHOD: First, the rats were randomly divided into five groups: control, XDP (90, 30, 10 mg x kg(-1)), Xuesaitong tablet (XP) 30 mg x kg(-1). Then the effect of the drugs on thrombus and thrombosis was studied after the ratsthrombosis was induced by the arteriovenous shunt. Second, the rats were randomly divided into seven groups: model, XDP (90, 30, 10 mg x kg(-1)), XT (90, 30 mg x kg(-1)), lumbrukinase capsule. Then the effect of the drugs on thrombus and thrombosis was studied after the rats'thrombosis was induced by the electrical stimulation of common carotid artery. Third, the rats were randomly divided into six groups: control, model, XDP (80, 40 mg x kg(-1)), XT (40, 20 mg x kg(-1)). Then the effect of the drugs on blood circulation promoting was observed after the rats'acute blood stasis induced by adrenalin and icy water. RESULT: XDP 90, 30 mg x kg(-1) could notably lighten the wet-weight and dry-weight of thrombus in the arteriovenous shunt model in rats in a dose-dependent manner (P < 0.01). XDP 90 mg x kg(-1) with intragastric administration for 3 days had the satisfactory effect on thrombolysis after the rat's thrombosis was induced by the electrical stimulation of common carotid artery (P < 0.01). XDP 80, 40 , 20 mg x kg(-1) reduced significantly erythrocyte aggregation (P < 0.01) and decreased the whole blood viscosity at low shear rate (P < 0.05). XDP 80, 40 mg x kg(-1) reduced the whole blood viscosity at high shear rate and plasma viscosity (P < 0.05). XDP 80 mg x kg(-1) decreased the whole blood viscosity at high shear rate (P < 0.05). CONCLUSION: XDP can significantly inhibit the thrombosis and has the satisfactory effect on thrombolysis. One kind of the mechanism is related to the effect on blood rheology.

Preclinical evaluation of S18886 in an experimental model of coronary arterial thrombosis.

The specific thromboxane receptor antagonist, S18886, was evaluated for prevention of coronary arterial thrombosis and myocardial ischemia-reperfusion in anesthetized canines. For the primary thrombosis study in left circumflex (LCX) coronary artery, 26 dogs were randomized to receive either vehicle (n = 7) or intravenous S18886 (0.3 mg/kg, n = 6; 1.0 mg/kg, n = 6; and 3.0 mg/kg, n = 7). The respective times to occlusion after S18886 were as follows: 56.8 +/- 9.3, 83.5 +/- 14.9, and 92.4 +/- 15.7 minutes compared to 43.3 +/- 8.2 minutes after vehicle. S18886 caused a minimal increase in tongue bleeding time and a significant decrease in ex vivo platelet aggregation to arachidonic acid or U46619. Another 37 dogs were randomized to receive placebo (n = 12), clopidogrel 1.0 mg/kg p.o. QDX3 (n = 9), clopidogrel + S18886 0.3 (n = 9) or 1.0 (n = 7) mg/kg intravenous. Clopidogrel produced a 50% reduction in adenosine diphosphate-induced platelet aggregation and a slight increase in the time to occlusion. However, clopidogrel + S18886 1.0 mg/kg prevented occlusive thrombus formation in most of the coronary vessels over 6 hours. S18886 did not alter myocardial infarct size in the ischemia-reperfusion model. In conclusion, S18886 alone caused a dose-dependent prolongation in the time to primary occlusive coronary artery thrombosis, whereas S18886 + clopidogrel displayed effective in preventing occlusive thrombus formation with only a moderate increase of tongue-bleeding time.

Murine model of ferric chloride-induced vena cava thrombosis: evidence for effect of potato carboxypeptidase inhibitor.

BACKGROUND/OBJECTIVE: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. In the present study, we describe the development of a murine model of vena cava thrombosis and its use to characterize the antithrombotic activity of potato carboxypeptidase inhibitor (PCI) of TAFIa (activated TAFI) in mice. METHODS/RESULTS: Vena cava thrombosis was induced by various concentrations of FeCl(3) in C57BL/6 mice. A relatively mild stimulus (3.5% FeCl(3)) induced thrombosis that was consistent and sensitive to reference antithrombotic agents such as clopidogrel and heparin. Dose-response studies identified a PCI dose (5 mg kg(-1) bolus plus 5 mg kg(-1) h(-1), i.v.) that produced a maximum 45% decrease in vena cava thrombus mass as assessed by protein content (n = 8, P < 0.01 compared to vehicle) in the 3.5% FeCl(3)-induced model without exogenous tissue plasminogen activator administration. In contrast, PCI had no effect on 3.5% FeCl(3)-induced carotid artery thrombosis in mice. In a tail transection bleeding model, the 5 mg kg(-1) bolus plus 5 mg kg(-1) h(-1) dose of PCI increased tail-bleeding time up to 3.5 times control (n = 8, P < 0.05). The ex vivo activity of antithrombotic doses of PCI was also demonstrated by the enhanced lysis of whole blood clots formed in a thrombelastograph with the addition of a sub-threshold concentration of tPA. CONCLUSION: These studies provide evidence for a role of TAFIa in venous thrombosis in mice, and describe an optimized vena cava injury model appropriate for the evaluation of antithrombotic drugs and the characterization of novel therapeutic targets.

Tiplaxtinin, a novel, orally efficacious inhibitor of plasminogen activator inhibitor-1: design, synthesis, and preclinical characterization.

Indole oxoacetic acid derivatives were prepared and evaluated for in vitro binding to and inactivation of human plasminogen activator inhibitor-1 (PAI-1). SAR based on biochemical, physiological, and pharmacokinetic attributes led to identification of tiplaxtinin as the optimal selective PAI-1 inhibitor. Tiplaxtinin exhibited in vivo oral efficacy in two different models of acute arterial thrombosis. The remarkable preclinical safety and metabolic stability profiles of tiplaxtinin led to advancing the compound to clinical trials.

Orally administered heparins prevent arterial thrombosis in a rat model.

Our previous studies demonstrated that orally administered heparins prevent thrombosis in a rat jugular vein thrombosis model, where bovine unfractionated heparin (UFH) and the low molecular weight heparin tinzaparin reduced thrombotic incidence by 50% at 7.5 and 0.1 mg/kg, respectively. Our objectives were to determine if similar antithrombotic effects of oral heparin could be observed in an arterial thrombosis model. In this model, filter paper soaked in 30% ferric chloride was applied to the exposed rat carotid artery. A flowmeter recorded blood flow over a 60 min period determining time when the thrombus began forming (TTB) and time till occlusion (TTO). Immediately following, the thrombus was removed, dried and weighed 24 h later. Bovine UFH (7.5 mg/kg), tinzaparin (0.1 mg/kg) or saline was administered by stomach tube at 2, 5 and 25 h prior to thrombus initiation. TTB was significantly increased when UFH was given at 5 and 25 h but not 2 h prior, and when tinzaparin was given at 5 but not 2 or 25 h prior compared to rats given oral saline. TTO was significantly increased for both UFH and tinzaparin when given 5 and 25 h but not 2 h prior (one-way ANOVA). There was no difference in TTO and TTB between UFH and tinzaparin treated groups. A trend in reduction in thrombus weight was observed for UFH at 5 and 25 h prior and tinzaparin at 5 h prior to thrombus initiation (one-way ANOVA). Although no significant changes were observed in activated partial thromboplastin times, Heptest or anti-Xa activity from plasma of heparin treated rats, endothelial heparin concentrations were significantly greater than controls for UFH at 5 h and for tinzaparin at 2, 5, and 24 h. Thus, heparins administered by the oral route are effective antithrombotic agents in arterial as well as venous models.

Accelerated thrombolysis in a rabbit model of carotid artery thrombosis with liposome-encapsulated and microencapsulated streptokinase.

The present study compares the efficacy of two formulations of encapsulated streptokinase to streptokinase in a rabbit model of carotid artery thrombosis. Arterial thrombosis followed the injection of thrombin mixed with autologous whole blood into a carotid artery of New Zealand white rabbits. Thirty minutes after the confirmation of an occlusive thrombus, one of four streptokinase formulations was infused at a dosage of 6000 IU/kg into the jugular vein. Free streptokinase (FREE SK) was compared to identical dosages of streptokinase encapsulated in a liposome (LESK), streptokinase entrapped in a water-soluble polymer (MESK), and free streptokinase admixed with blank microparticles (FREE SK + BLANK). Carotid arterial blood flow was determined by pulsed Doppler flowmetry to confirm clot formation and reperfusion. Two hours after drug infusion, the rabbits were killed and the residual thrombus mass was determined. Compared to FREE SK (74.5 +/- 16.9 min; mean +/- SEM), LESK demonstrated significantly reduced reperfusion times (19.3 +/- 4.6 min) while MESK exhibited even greater improvement (7.3 +/- 1.6 min). FREE SK + BLANK showed no statistical improvement versus FREE SK. LESK and MESK also resulted in reduced residual clot mass and greater return of arterial blood flow. These studies suggest that encapsulation of streptokinase offers a potential method of improved fibrinolytic treatment for patients with clot-based disorders. MESK performed slightly better than LESK with improved production and storage characteristics.

Ethanol plus caffeine (caffeinol) for treatment of ischemic stroke: preclinical experience.

BACKGROUND AND PURPOSE: Ethanol and caffeine are 2 common psychoactive dietary components. We have recently shown that low-dose ethanol plus caffeine results in a 70% to 80% reduction of infarct volume after reversible common carotid/middle cerebral artery (CCA/MCA) occlusion in rats. The combination (caffeinol) was effective after either oral pretreatment or intravenous administration starting up to 2 hours after stroke onset. Ethanol alone aggravated ischemic damage, while caffeine alone was without effect. Daily caffeinol for 2 weeks before ischemia eliminated the neuroprotection seen with acute treatment (tolerance). The purpose of our present study was to further characterize the properties of caffeinol as a possible treatment for ischemic stroke. METHODS: The transient CCA/MCA occlusion model was used in all experiments. Five sets of experiments were conducted (1) to test the effectiveness of various doses of ethanol (0.2 to 0.65 g/kg) and caffeine (3 to 10 mg/kg) in the caffeinol mixture; (2) to test whether the neuroprotective dose of caffeinol can improve behavioral dysfunction; (3) to test whether chronic ethanol or caffeine before ischemia will affect efficacy of caffeinol treatment; (4) to test whether the protective effect of caffeinol can be improved by pairing it with 35 degrees C hypothermia; and (5) to test whether caffeinol affects frequency of hemorrhage after administration of recombinant tissue plasminogen activator (rtPA) in ischemic animals. RESULTS: Doses as low as 0.2 g/kg of ethanol and 6 mg/kg of caffeine in the caffeinol were effective in reducing cortical infarct volume and behavioral dysfunction after transient CCA/MCA occlusion. Daily exposure to ethanol but not caffeine before CCA/MCA occlusion eliminated the therapeutic efficacy of acute caffeinol treatment, similar to the tolerance observed after chronic exposure to caffeinol. The therapeutic effect of caffeinol could be further improved by pairing it with mild intraischemic hypothermia, and caffeinol did not increase hemorrhagic infarction when given in combination with rtPA. CONCLUSIONS: Low doses of caffeinol, equivalent to no more than 2 to 3 cups of strong coffee and 1 cocktail, are consistently and highly neuroprotective, are well tolerated, can be added to other therapies to increase the effect of each, and do not interfere with or complicate rtPA therapy. Caffeinol is an appropriate candidate for clinical trial in stroke patients, although it may be less effective in patients with regular alcohol intake.

Potent dibasic GPIIb/IIIa antagonists with reduced prolongation of bleeding time: synthesis and pharmacological evaluation of 2-oxopiperazine derivatives.

A series of 2-oxopiperazine derivatives, possessing basic moieties at the 3- and the 4-positions, were synthesized and evaluated for their abilities to inhibit platelet aggregation and for their effects on bleeding time. Among the compounds, 2-[(3S)-4-[2-[(4-guanidinobenzoyl)amino]acetyl]-3-[3-[(4-guanidinobenzoyl)amino]propyl]-2-oxopiperazinyl]acetic acid (12c) showed a potent inhibitory effect on platelet aggregation and good dissociation between the efficacy and the bleeding side effect. Intravenous infusion of compound 12c at 1.6 microg/mL/min completely prevented arterial thrombus formation induced by endothelial injury in guinea pigs. The dose of 12c that prolonged the bleeding time to three times the control value was 5.8 microg/mL/min. These results suggest that compound 12c might be useful in the clinical treatment of thrombotic diseases, and we selected 12c (TAK-024) as a candidate for the clinical trials.