Identification of AcAP5 as a novel factor Xa inhibitor with both direct and allosteric inhibition.

Ancylostoma caninum anticoagulant peptide 5 (AcAP5) is a potent inhibitor for coagulation factor Xa (FXa). Previous studies show that AcAP5 binds to FXa at the active site, and/or the exosite. The active site-binding contributes to direct blocking of FXa catalytic activity, but the effect of exosite-binding and the underlying mechanism remain unknown. To investigate whether and how the exosite-binding affects FXa function, we prepared several AcAP5 mutants with modifications to the active site-binding or exosite-binding region. Their FXa-inhibiting and anticoagulant activities were examined both in vitro and in rabbit plasma, and the interactions with FXa were analyzed using in silico molecular modeling, docking, and molecular dynamics simulation. Mutants abolishing either active site- or exosite-binding resulted in a dramatic decrease in their anti-FXa and anticoagulant activities. Elongation of AcAP5 exosite-binding region also impaired the FXa-inhibiting activity. Computational analysis demonstrated that the conformation of FXa becomes more rigid due to exosite-binding with AcAP5, which consequently affects its catalytic activity. Our results suggest that both active site- and exosite-binding contribute to the FXa inhibitory activity of AcAP5.

Increased autophagic degradation contributes to the neuroprotection of hydrogen sulfide against cerebral ischemia/reperfusion injury.

Hydrogen sulfide (H2S), an endogenous gaseous signal molecule, exhibits protective effect against ischemic injury. However, its underlying mechanism is not fully understood. We have recently reported that exogenous H2S decreases the accumulation of autophagic vacuoles in mouse brain with ischemia/reperfusion (I/R) injury. To further investigate whether this H2S-induced reduction of autophagic vacuoles is caused by the decreased autophagosome synthesis and/or the increased autophagic degradation inautophagic flux, we performed in vitro and in vivo studies using SH-SY5Y cells for the oxygen and glucose deprivation/reoxygenation (OGD/R) and mice for the cerebral I/R, respectively. NaHS (a donor of H2S) treatment significantly increased cell viability and reduced cerebral infarct volume. NaHS treatment reduced the OGD/R-induced elevation in LC3-II (an autophagic marker), which was completely reversed by co-treatment with an autophagic flux inhibitor bafilomycin A1 (BafA1). However, H2S did not affect the OGD/R-induced increase of the ULK1 self-association and decrease of the ATG13 phosphorylation, which are the critical steps for the initiation of autophagosome formation. Cerebral I/R injury caused an increase in LC3-II, a decrease in p62 and the accumulation of autophagosomes in the cortex and the hippocampus, which were inhibited by NaHS treatment. This H2S-induced decline of LC3-II in ischemic brain was reversed by BafA1. Moreover, BafA1 treatment abolished the protection of H2S on the cerebral infarction. Collectively, the neuroprotection of exogenous H2S against ischemia/hypoxia and reperfusion/reoxygenation injury is mediated by the enhancement of autophagic degradation.

Effect of magnolol on cerebral injury and blood brain barrier dysfunction induced by ischemia-reperfusion in vivo and in vitro.

Magnolol, a neolignan compound isolated from traditional Chinese medicine Magnolia officinalis, has a potentially therapeutic influence on ischemic stroke. Previous studies have demonstrated that cerebral ischemia-reperfusion (I-R) and blood-brain barrier (BBB) are involved in the pathogeneses of stroke. Therefore, in vivo and in vitro studies were designed to investigate the effects of magnolol on I-R-induced neural injury and BBB dysfunction. In cerebral I-R model of mice, cerebral infarct volumes, brain water content, and the exudation of Evans blue were significantly reduced by intravenous injection with magnolol at the doses of 1.4, 7.0, and 35.0 µg/kg. When primary cultured microglial cells were treated with 1 µg/ml lipopolysaccharide (LPS) plus increasing concentrations of magnolol, ranging from 0.01 to 10 µmol/L, magnolol could statistically inhibit LPS-induced NO release, TNF-α secretion, and expression of p65 subunit of NF-κB in the nucleus of microglial cells. In the media of brain microvascular endothelial cells (BMECs), oxygen and glucose deprivation-reperfusion (OGD-R) could remarkably lead to the elevation of TNF-α and IL-1ß levels, while magnolol evidently reversed these effects. In BBB model in vitro, magnolol dose- and time-dependently declined BBB hyperpermeability induced by oxygen and glucose deprivation (OGD), OGD-R, and ephrin-A1 treatment. More importantly, magnolol could obviously inhibit phosphorylation of EphA2 (p-EphA2) not only in ephrin-A1-treated BMECs but also in cerebral I-R model of mice. In contrast to p-EphA2, magnolol significantly increased ZO-1 and occludin levels in BMECs subjected to OGD. Taken together, magnolol can protect neural damage from cerebral ischemia- and OGD-reperfusion, which may be associated with suppressing cerebral inflammation and improving BBB function.

Exogenous hydrogen sulfide attenuates cerebral ischemia-reperfusion injury by inhibiting autophagy in mice.

Hydrogen sulfide (H2S), the third gas transmitter, has been proven to be neuroprotective in cerebral ischemic injury, but whether its effect is mediated by regulating autophagy is not yet clear. The present study was undertaken to explore the underlying mechanisms of exogenous H2S on autophagy regulation in cerebral ischemia. The effects and its connection with autophagy of NaHS, a H2S donor, were observed through neurological deficits and cerebral infarct volume in middle cerebral artery occlusion (MCAO) mice; autophagy-related proteins and autophagy complex levels in the ischemic hemisphere were detected with Western blot assay. Compared with the model group, NaHS significantly decreased infarct volume and improved neurological deficits; rapamycin, an autophagy activator, abolished the effect of NaHS; NaHS decreased the expression of LC3-II and up-regulated p62 expression in the ischemic cortex 24 h after ischemia. However, NaHS did not significantly influence Beclin-1 expression. H2S has a neuroprotective effect on ischemic injury in MCAO mice; this effect is associated with its influence in down-regulating autophagosome accumulation.

Design, synthesis and biological evaluation of pyridazino[3,4,5-de]quinazolin-3(2H)-one as a new class of PARP-1 inhibitors.

A series of pyridazino[3,4,5-de]quinazolin-3(2H)-one derivatives were designed and synthesized as PARP-1 inhibitors. Most of the synthesized compounds showed good inhibitory activities of PARP-1 and four of them achieved at the IC50 values ranging from 0.0914 µM to 0.244 µM. Two compounds, 1a and 1b, were further tested for their neuroprotective effect in the PC12 cell model injured by H2O2 and both of them exhibited excellent activities.

Engineering a "three-in-one" hirudin prodrug to reduce bleeding risk: A proof-of-concept study.

An ideal anticoagulant should have at least three properties including targeted delivery to the thrombosis site, local activation or releasing to centralize the anti-thrombosis effects and thus reduce the bleeding risks, and long persistence in circulation to avoid repeated administration. In the present study, we sought to test a "three-in-one" strategy to design new protein anticoagulants. Based on these criteria, we constructed two hirudin prodrugs, R824-HV-ABD and ABD-HV-R824. The R824 peptide can bind phosphatidylserine on the surface of the procoagulant platelets and thus guide the prodrug to the thrombosis sites; albumin-binding domain (ABDs) can bind the prodrug to albumin, and thereby increase its persistence in circulation; the hirudin (HV) core in the prodrug is flanked by factor Xa recognition sites, thus factor Xa at the thrombosis site can cleave the fusion proteins and release the activated hirudin locally. Hirudin prodrugs were able to bind with procoagulant platelets and human serum albumin in vitro with high affinity, targeted concentrated and prevented the formation of occlusive thrombi in rat carotid artery injury model. Their effective time was significantly extended compared to native hirudin, and R824-HV-ABD showed a significantly improved half-life of about 24 h in rats. The bleeding time of prodrug-treated mice was much shorter than that of hirudin-treated mice. The results from the proof-of-concept studies, for the first time, demonstrate that "three-in-one" prodrug strategy may be a good solution for protein or peptide anticoagulants to reduce their bleeding risks.

Improvement and Application of Acute Blood Stasis Rat Model Aligned with the 3Rs (Reduction, Refinement and Replacement) of Humane Animal Experimentation.

OBJECTIVE: To establish a novel cardiocentesis method for withdrawing venous blood from the right atrium, and to improve an acute blood stasis rat model using an ice bath and epinephrine hydrochloride (Epi) while considering the 3Rs (reduction, refinement, and replacement) of humane animal experimentation. METHODS: An acute blood stasis model was established in male Sprague-Dawley rats by subcutaneous injection (s.c.) Epi (1.2 mg/kg) administration at 0 h, followed by a 5-min exposure to an ice-bath at 2 h and s.c. Epi administration at 4 h. Control rats received physiological saline. Rats were fasted overnight and treated with Angelicae Sinensis Lateralis Radix (ASLR) and Pheretima the following day. Venous blood was collected using our novel cardiocentesis method and used to test whole blood viscosity (WBV), prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen (FIB) content. RESULTS: The rats survived the novel cardiocentesis technique; WBV value returned to normal while hematological parameters such as hemoglobin level and red blood cell count were restored to >94% of the corresponding values in normal rats following a 14-day recovery. Epi (1.2 mg/kg, s.c.) combined with a 5-min exposure to the ice bath replicated the acute blood stasis rat model and was associated with the highest WBV value. In rats showing acute blood stasis, ASLR treatment [4 g/(kg·d) for 8 days] decreased WBV by 9.98%, 11.09%, 9.34%, 9.00%, 7.66%, and 7.03% (P<0.05), while Pheretima treatment [2.6 g/(kg·d), for 8 days] decreased WBV by 25.49%, 25.94%, 16.28%, 17.76%, 11.07%, and 7.89% (P<0.01) at shear rates of 1, 3, 10, 30, 100, and 180 s-1, respectively. Furthermore, Pheretima treatment increased APTT significantly (P<0.01). CONCLUSIONS: We presented a stable, reproducible, and improved acute blood stasis rat model, which could be applied to screen drugs for promoting blood circulation and eliminating blood stasis.

Improving long circulation and procoagulant platelet targeting by engineering of hirudin prodrug.

To reduce systemic bleeding risks during anticoagulant treatment, a new concept named "precise anticoagulation" was proposed to localize the effects of anticoagulants via the targeted delivery of prodrugs to the coagulation site. In this study, the fusion protein Annexin V-hirudin 3-ABD (hAvHA) was constructed to achieve the prolonged circulation and targeted delivery of hirudin to coagulation sites. hAvHA was inactive as a prodrug, and it could bind to albumin during circulation. The drug was quickly activated via factor Xa-mediated cleavage once coagulation occurred, and hirudin was efficiently released to exert antithrombin activity in vitro. The hAvHA protein could be activated in mouse blood and exert significant anticoagulation effects. The results of FITC labeling illustrated that hAvHA bound to procoagulant platelets, suggesting the Annexin V modification permits targeted delivery to sites of thrombosis. hAvHA bound to albumin in vitro with an equilibrium dissociation constant of 8 pM, suggesting the ABD modification permitted prolonged circulation in vivo. Moreover, the bleeding time was much shorter in hAvHA-treated mice than in hirudin-treated mice. Therefore, our results suggested that that hAvHA is a potential and promising anticoagulant in vivo.

[Constituents of Millettia nitida var. hirsutissima].

OBJECTIVE: To separate effective constituents from Millettia nitida var. hirsutissima. METHOD: Compounds were isolated by chromatography methods, structures were identified by spectroscopic means. RESULT: Eight flavonoids (1-8) and two triterpenes (9-10) were isolated from this plant. They were identified as calycosin (1), genistin (2), gliricidin (3), 8-O-methylretusin (4), afromosin-7-O-beta-D-glucopyranoside (5), lanceolarin (6), soliquiritigenin (7), symplocoside (8), lupeol (9), 3beta-friedelanol (10). CONCLUSION: The compounds (1-10) were obtained from M. nitida var. hirsutissima for the first time. The 13C-NMR dada of 1 were correct assignment on the basis of 2D-NMR spectral analysis.

Polyaspartoyl.l-arginine inhibits platelet aggregation through stimulation of NO release from endothelial cells.

Polyaspartoyl.l-arginine (PDR) is an inhibitor of platelet aggregation ex vivo but in vitro. This study attempts to elucidate the target cell of PDR action and its action mechanism. PDR (1.7-170 microg/ml) significantly inhibited platelet aggregation in vitro in the presence of rat aortic endothelial cells (RAEC), NO synthase inhibitor N-nitro-l-arginine methyl ester (l-NAME) inhibited this effect, but it was ineffective in the RAEC absence. Correspondingly, PDR increased NO level in the supernatants of the platelet reactants in RAEC presence, but failed to influence NO level in RAEC absence, and these effects of PDR were more potent than those of l-arginine. Furthermore, PDR markedly elevated the intracellular level of l-arginine, and it (17-170 microg/ml) also augmented l-citrulline level in RAEC, argininosuccinate lyase (ASL) inhibitor succinate enhanced its effect on l-citrulline but l-NAME weakened it. 170 microg/ml of PDR slightly increased the l-aspartate level in RAEC, and succinate enhanced this effect. However l-arginine, l-aspartate or the combination of l-arginine and l-aspartate failed to change levels of these amino acids. In addition, PDR (170 microg/ml) stimulated the expression of argininosuccinate synthetase (ASS) protein. In conclusion, the endothelial cell is direct target cell of PDR's action; PDR facilitates the entry of l-arginine by serving as a carrier of l-arginine into RAEC; it also supplies aspartic acid and stimulates ASS expression, and then enhances the intracellular citrulline-NO cycle, thus increases the availability of l-arginine and NO synthesis. Therefore the effect of PDR on platelet aggregation is primarily attributed to its stimulation of NO synthesis in endothelial cells; PDR may be a better cardiovascular protective agent than l-arginine.

Polyaspartoyl l-arginine protects endothelial cells against injury.

Polyaspartoyl l-arginine (PDR) is an anti-thrombotic agent and its anti-thrombotic effect is related with endothelial cells. This study is to investigate the effect of PDR on the endothelial cells. In cell injury assay 1.7-170 microg/ml of PDR significantly increased the viability of rat aorta endothelial cells (RAECs) injured by H(2)O(2), this effect was comparable with that of 95 microg/ml of alpha-tocopherol, and was more powerful than that of l-arginine. Nitric oxide synthase(NOS) inhibitor, L-NAME, almost abolished the effect of PDR, but not influence the effect of alpha-tocopherol or l-arginine. PDR enhanced the viability of RAECs injured by oxidized- low density lipoprotein (ox-LDL) either, which was comparable to that of alpha-tocopherol, whereas l-arginine, l-aspartic acid alone or their combined use failed to showed effects. PDR (17-170 microg/ml) raised nitrite level in RAEC medium, which is the major end-product of NO, but l-arginine (170 microg/ml) produced insignificant nitrite level rise. In addition, in the absence of RAEC PDR and l-arginine but alpha-tocopherol failed to lower the concentration of oxidative product (Fe(3+)) in a cell free system, whereas in the presence of RAEC PDR, l-arginine or alpha-tocopherol all significantly reduced the concentration of Fe(3+). In cell apoptosis assay PDR (17-170 microg/ml) lowered the percentage of early apoptotic and late apoptotic RAECs, consequently increased the percentage of normal cells. Furthermore PDR significantly inhibited caspase-3 activity in RAECs; this effect is comparable with alpha-tocopherol and more potent than that of l-arginine. In conclusion, PDR is a cell protector, it protects endothelial cell against oxidative injury and apoptosis; its cell protective effect against H(2)O(2) injuries is NOS dependent and is related with NO production; PDR is anti-oxidant, its anti-oxidant effect needs endothelial cell's participation. The findings suggest PDR may play a much better beneficial role than l-arginine in the prevention and treatment for those diseases with endothelial dysfunction.

Honokiol up-regulates prostacyclin synthease protein expression and inhibits endothelial cell apoptosis.

Honokiol is a bioactive compound extracted from the Chinese medicinal herb Magnolia officinalis. We recently demonstrated that honokiol inhibited arterial thrombosis through stimulation of prostacyclin (PGI2) generation and endothelial cell protection. The current study is designed to investigate its mechanism of stimulation of PGI2 generation and cell protection. 6-keto-PGF1alpha, the stable metabolite of PGI2, in the media of rat aortic endothelial cells was measured with radioimmunoassay kits. Indomethacin, an inhibitor of cyclooxygenase (COX) and tranylcypromine, a prostacyclin synthease inhibitor were used to ascertain the target enzyme affected by honokiol. Prostacyclin synthease protein levels in endothelial cells were determined by Western blot analysis using an anti-PGI2 synthease rabbit polyclonal antibody. Flow cytometry was used to quantify the apoptotic cells and spectrophotometry was used to test the caspase-3 activity. Honokiol (0.376-37.6 microM) increased the level of 6-keto-PGF1alpha in the media of normal endothelial cells. It counteracted the inhibitory effect of tranylcypromine on the PGI2 generation, but did not influence the effect of indomethacin; evidently, honokiol up-regulated the expression of prostacyclin synthease in the endothelial cells. These effects showed perfect concentration-dependent behavior. In addition, at lower concentration (0.376-3.76 microM), honokiol significantly decreased the percentage of apoptotic endothelial cells induced by oxidized low-density lipoprotein (ox-LDL) and significantly lowered the activity of caspase-3 stimulated by ox-LDL. A high dose of honokiol (37.6 microM), however, failed to influence either of them. In conclusion, honokiol augments PGI2 generation in normal endothelial cells; its effect on PGI2 generation attributes to up-regulation of prostacyclin synthease expression; its cell protection may be correlated with its inhibition on apoptosis of endothelial cells. These findings have partly revealed the molecular mechanism of honokiol on inhibiting arterial thrombosis.

Cyclovirobuxine D ameliorates acute myocardial ischemia by K(ATP) channel opening, nitric oxide release and anti-thrombosis.

Cyclovirobuxine D is an active compound extracted from Buxus microphylla, which has been used for treating acute myocardial ischemia in China. The present study was to investigate its mechanism on myocardial ischemia. Cyclovirobuxine D significantly increased cardiomyocytes viability injured by oxidation or hypoxia. It significantly reduced the infarct size induced by ligating the coronary artery in rats, and the effect was almost abolished by glibenclamide, a blocker of ATP sensitive potassium channel, but it was not influenced by cyclooxygenase-2 inhibitor celecoxib or estrogen receptor antagonist tamoxifen. In addition, cyclovirobuxine D significantly protected rat aorta endothelial cells against hypoxia and enhanced nitric oxide (NO) release from endothelial cells, which was inhibited by nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME). Furthermore, cyclovirobuxine D significantly decreased the weight of venous thrombus in rats. In conclusion, the action mechanism of cyclovirobuxine D on myocardial ischemia may be related with its cytoprotection, K(ATP) channel opening, NO generation stimulating and venous thrombosis inhibiting.

Design and evaluation of EphrinA1 mutants with cerebral protective effect.

The activation of EphA2 receptor by its natural ligand EphrinA1 causes blood brain barrier dysfunction, and inactivation of EphA2 reduces BBB damage in ischemic stroke. Thus, EphA2 targeted antagonists may serve as neuroprotective agents. We engineered four mutants of EphrinA1, EM1, EM2, EM3 and EM4, respectively. The computational analysis showed that these four mutants were capable of interacting with EphA2. Their potential neuroprotective effects were examined in mouse focal ischemia/reperfusion (I/R) model. EM2 exhibited strong neuroprotective effects, including reduced brain infarct volume, neuronal apoptosis, cerebral edema, and improved neurological scores. The EM2-mediated protection was associated with a comparative decrease in BBB leakage, inflammatory infiltration, and higher expression levels of tight junction proteins, such as zonula occludens-1 and Occludin. I/R-induced high expression of Rho-associated protein kinase 2 (ROCK2) was down-regulated after EM2 treatment. Moreover, EM2 reduced agonist doxazosin-induced EphA2 phosphorylation and cells rounding in PC3 cells, indicating EphA2-antagonizing activity of EM2. These finding provided evidences of the neuroprotection of EphA2 antagonist and a novel approach for ischemic stroke treatment. These results also suggested that a receptor agonist can be switched to an antagonist by substituting one or more relevant residues.

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting.

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbß3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbß3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

Magnolol derivative 002C-3 protects brain against ischemia-reperfusion injury via inhibiting apoptosis and autophagy.

Neuroprotective agents can rescue ischemic penumbra in cerebral ischemia. However, the clinically effective neuroprotective agents for cerebral ischemic injury remain deficient in clinic so far. This study was undertaken to investigate the brain protective effect of 002C-3 and its potential mechanisms in rats, and its preliminary toxicity in mice. A transient middle cerebral artery occlusion (tMCAO) model in rats was used to evaluate its effect and mechanism, a dose limited experiment was used to evaluate its preliminary toxicity. 10-50µg/kg of 002C-3 (single iv bolus after reperfusion) significantly reduced neurological scores, infarct volumes and brain water contents, and the effect was more potent than that of magnolol under the same mole dose; 50µg/kg of 002C-3 significantly decreased the number of TUNEL-positive cells, reduced the activity of caspase-3, and lowered the autophagy-related proteins LC3-II and Beclin-1 level in I-R cerebral tissue. At 1000 times' dose of high effective dose (ip) 002C-3 failed to show evident toxicity in mice, and the mean body weight of mice treated with 002C-3 was almost the same as that of the vehicle control, but magnolol caused evident toxicity and death. In conclusion, 002C-3 has significant protective effect against cerebral ischemia-reperfusion injury; the effect is more potent than magnolol; this effect is maybe associated with its inhibition of both apoptosis and autophagy; its toxicity is greatly reduced compared to magnolol. These results provided data for its further research and development.

Structure-guided creation of AcAP5-derived and platelet targeted factor Xa inhibitors.

Anticoagulants and anti-platelet agents are simultaneously administrated in clinical practice (i.e. percutaneous coronary intervention), which cause significant risk of systemic bleeding. Targeted delivery of anticoagulants to the activated platelets at sites of vascular injuries may condense the site-specific anticoagulant effect and reduce the hemorrhage side effects in uninjured vessels. To this end, we prepared three ancylostoma caninum anticoagulant peptide 5 (AcAP5) variants NR1, NR2 and NR3 engineered with a platelet-binding Arg-Gly-Asp (RGD) motif and evaluated their anti-Factor Xa (FXa) and platelet-binding effects. These RGD-containing AcAP5 variants were capable of interacting with platelet receptor αIIbß3 as shown in computational analysis. All variants, especially NR2 and NR3, retained entirely the anti-FXa function of parent AcAP5. Moreover, they prevented the formation of occlusive thrombi in rat carotid artery injury model, suggesting that they inhibit platelet aggregation in vivo. Further functional investigation of NR3 demonstrated that NR3 inhibited platelet aggregation in vitro and FXa activity in vivo, and prolonged the coagulation time, all in a dose-dependent manner. Through flow cytometry assay, we confirmed the binding of NR3 to αIIbß3 receptor. In mouse model of carotid artery endothelium injury, NR3-treated mice showed less tail bleeding time than AcAP5-treated mice, and aspirin plus NR3 treatment exhibited moderate reduction of blood loss compared with aspirin plus AcAP5 treatment. These results indicate the feasibility to engineer a novel FXa inhibitor specifically targeting the activated platelets, which centralizes its anticoagulation efficacy in the injured vascular endothelium and reduces the risk of systemic bleeding.

Anti-thrombotic activity of PDR, a newly synthesized L-Arg derivative, on three thrombosis models in rats.

The possibility of a newly synthesized L-arginine derivative, polyaspartoyl-L-arginine (PDR), as a novel anti-thrombotic agent and its mode of action were investigated. The anti-platelet effects of PDR in rats ex vivo, anti-thrombotic effects in three thrombosis models in rats and its effect on some autacoids (nitric oxide [NO], thromboxane [TXA2] and prostacyclin [PGI2]) were studied. PDR (i.g.) significantly inhibited ADP-, collagen- or thrombin-induced rat platelet aggregation. In arteriovenous shunt model and ferric chloride-induced arterial thrombosis model in rats, PDR (i.g.) significantly reduced the thrombus weight. In electrical stimulation-induced arterial thrombosis in rats, PDR (i.v.) dose-dependently prolonged the thrombus occlusion time (OT). PDR increased the concentration of NO in plasma. In contrast with aspirin (ASA), PDR did not influence on the TXA2 and PGI2 levels in plasma. In conclusion, PDR is provided with significant inhibitory effect on platelet aggregation and prevention effect on platelet related thrombosis, which is probably attributed to its inhibition on platelet function by L-arginine-NO pathway. The results demonstrate that PDR is a novel, oral and venous effective platelet aggregation inhibitor and has a possibility used as an anti-thrombotic agent.

Hypolipidemic and anti-atherogenic effects of long-term Cholestin (Monascus purpureus-fermented rice, red yeast rice) in cholesterol fed rabbits.

Long-term effects of Cholestin (Monascus purpureus rice; red yeast rice) on serum lipids and severity of atherosclerosis were examined in rabbits fed for 200 days on a semi-purified diet containing 0.25% cholesterol. Serum total cholesterol was 25 and 40% lower, respectively, in rabbits fed 0.4 or 1.35 g/kg/day of Cholestin (Monascus purpureus rice; red yeast rice) compared to controls. This treatment also lowered serum LDL cholesterol. This 200-day treatment significantly reduced serum triglycerides and atherosclerotic index (ratio of non-HDL-cholesterol to HDL-cholesterol). Although similar reductions of total, LDL-cholesterol and triglycerides were observed, a parallel group of rabbits fed lovastatin (0.0024 g/kg/day) failed to reduce the index significantly. Apolipoprotein A(1) was increased and apolipoprotein B was reduced in all treatment groups. Severity of atherosclerosis was reduced significantly in all treatment groups. The sudanophilic area of involvement was 80.6% in controls, and reduced significantly; to 30.1% on the low dose of Cholestin (Monascus purpureus rice; red yeast rice), and 17.2% on the high dose. Lovastatin reduced severity of lesions by 89% (sudanophilia) and 84% (visual). Visual grading of lesion severity showed reduction by 38% and 68%.

[Studies on anti-inflammatory and immunologic enhancement of kangning granules].

OBJECTIVE: To investigate the effect of Kangning granules on inflammatory and immunology. METHOD: Pedal swelling induced by 1% agar in mice, dermal capillary hyper-permeability induced by histamine in rats were used to evaluate the anti-inflammatory of Kangning granules, and the clearance rate of intravenous charcoal particles was used to study its effect on non-specific immunologic functions. Serum haemolysin level and the number of antibody secretory cells in spleen were determined to assess its effects of specific immunologic functions. MTT colored method was used to determine the effect of Kangning granules on T- or B-Lymphocytes proliferation. RESULT: 5, 10, 20 g x kg(-1) of Kangning Granules significantly inhibited pedal swelling induced by agar in mice (P < 0.05, P < 0.01). 2.4, 4.8, 9.6 g x kg(-1) of Kangning Granules evidently decreased dermal capillary hyper-permeability induced by histamine in rats (P < 0.05, P < 0.01). 10, 20 g x kg(-1) of Kangning Granules enhanced the phagocytic function and phagocytosing velocity in mice (P < 0.05). 5, 10, 20 g x kg(-1) of Kangning Granules obviously increased serum haemolysin level and the number of antibody secretory cells in spleen (P < 0.05, P < 0.01). 20 g x kg(-1) of Kangning Granules promoted B-Lymphocytes proliferation (P < 0.05). CONCLUSION: Kangning granules significantly inhibited inflammatory reaction and increase immune functions in animals.