Inhibition of thrombin, an unexplored function of retinoic acid.

Retinoic acid, a derivative of vitamin A, is known to possess in vivo anti-inflammatory, anti-platelet and fibrinolytic activities. We have investigated the in vitro thrombin and platelet aggregation inhibitory activities of vitamin A (retinol) and its derivatives, retinoic acid and retinaldehyde. The thrombin enzymatic assay was performed fluorimetrically to assess the inhibition of thrombin (Sigma and plasma). Retinoic acid, retinaldehyde and retinol exhibited potent inhibition of thrombin, with IC50 values of 67µg/ml, 74µg/ml and 152µg/ml, respectively for the inhibition of thrombin (Sigma); and 49µg/ml, 74µg/ml and 178µg/ml, respectively for the inhibition of thrombin (plasma). Amongst vitamin A and its derivatives, retinoic acid showed the highest inhibition of both the forms of thrombin. Vitamin A and its derivatives also displayed remarkable inhibition of platelet aggregation. This is the first report of vitamin A and its derivatives showing inhibition of thrombin and platelet aggregation in vitro.

Evaluation of Antithrombotic Activities of Solanum xanthocarpum and Tinospora cordifolia.

BACKGROUND: Solanum xanthocarpum and Tinospora cordifolia have been reported to exhibit anti-inflammatory, antiarthritic, antioxidant, antiallergic, and hepatoprotective activities. The origins of many of the currently available antithrombotic treatments are from natural products and natural sources. OBJECTIVE: To investigate the antithrombotic activities of methanolic leaf extracts of S. xanthocarpum(SXME) and T. cordifolia(TCME). MATERIALS AND METHODS: Antithrombotic activities were assessed by thrombin inhibition assay, thrombin generation assay, platelet adhesion assay on collagen-coated surface, and platelet PAC1-FITC binding by flow cytometry. RESULTS: SXME significantly inhibited thrombin activity at 5-20 mg/ml concentrations, whereas TCME inhibited thrombin activity at 500 µg/ml-5 mg/ml concentrations. Further, SXME inhibited thrombin generation at 2-20 mg/ml concentrations, whereas TCME exhibited significant inhibition at 200 µg/ml, suggesting that TCME has higher efficacy as compared to SXME. Moreover, SXME did not inhibit platelet adhesion on collagen-coated surface, whereas TCME inhibited platelet adhesion on collagen-coated surface at 5 mg/ml. Indomethacin showed significant inhibition in platelet adhesion at 300 µM. Further, SXME inhibited thrombin-induced platelet activation (PAC1-FITC binding) significantly at 1 mg/ml by about 80%, whereas TCME inhibited thrombin-induced platelet activation (PAC1-FITC binding) by about 40% at 1 mg/ml. CONCLUSION: These results strongly suggested that SXME and TCME possess antithrombotic activities. However, further studies are essential to find out the active constituent responsible for antithrombotic effect. SUMMARY: The methanolic extracts obtained from the leaves of Tinospora cordifolia and Solanum xanthocarpum were evaluated for antithrombotic activity by thrombin inhibition assay, thrombin generation assay, platelet adhesion assay and platelet activation assay by flow cytometry. These extracts inhibited thrombin activity and thrombin generation in rat plasma. Also, these extracts inhibited thrombin induced platelet activation in PAC1-FITC binding study in flow cytometry. Abbreviation Used: DVT: Deep vein thrombosis, TCME: Tinospora cordifolia methanolic extract, SXME: Solanum xanthocarpum methanolic extract, IL-1ß: interleukin-1ß, DMSO: dimethyl sulfoxide, PRP: Platelet rich plasma.

Assessment of raloxifene, estradiol-17ß, dl-ormeloxifene and levormeloxifene on thrombin activity.

Abstract Background: Cancer is one of the leading causes of morbidity and mortality globally. Cancer-associated thrombosis is well established in clinical settings, and thrombin has been found to induce angiogenesis at cancer sites. This establishes a link between cardiovascular diseases and cancer, where cancer and thrombin have been intricately associated. Various selective estrogen receptor modulators (SERMs) have been reported to exhibit anticancer activity. Therefore, we investigated estradiol-17ß and SERMs dl-ormeloxifene (centchroman), raloxifene and levormeloxifene (l-centchroman) for their anticancer effects and their effect on thrombin activity. Methods: Anticancer activity was assessed against PC-3 cell line by flow cytometry following treatment with estradiol-17ß and SERMs at 10 nM-1 mM concentrations. The cells were stained with propidium iodide and the percentage of cells in the sub-G0/G1 region was considered apoptotic. Thrombin inhibitory effect was evaluated by thrombin inhibition assay in vitro following incubation with 100 nM-3 mM concentrations of estradiol-17ß or various SERMs. Further, the effect of estradiol-17ß and SERMs on endogenous thrombin generation potential (ETP) was assessed by thrombin generation assay on rat plasma in vitro. Results: These compounds exhibited >90% cell death in PC-3 cell lines at 1 mM concentration except estradiol-17ß. Neither estradiol-17ß, dl-ormeloxifene and levormeloxifene showed any thrombin inhibitory or enhancing activity in thrombin inhibition assay, nor did they show any effect on ETP on rat plasma in vitro. However, raloxifene inhibited thrombin activity in a concentration-dependent manner. Raloxifene decreased ETP of the plasma at 3 and 1 mM,which is equivalent to that of 30-100 U/mL of heparin. Interestingly, raloxifene increased thrombin generation at lower concentrations and it inhibited thrombin generation at higher concentrations. Conclusions: These observations suggest that dl-ormeloxifene, estradiol-17ß and levormeloxifene do not possess thrombin inhibitory activity. Raloxifene possesses thrombin modulatory effect in addition to its anticancer activity, and this observation may help us in understanding the thromboembolic complications associated with raloxifene.

Antithrombotic activity of a newly synthesized coumarin derivative 3-(5-hydroxy-2,2-dimethyl-chroman-6-yl)-N-{2-[3-(5-hydroxy-2,2-dimethyl-chroman-6-yl)-propionylamino]-ethyl}-propionamide.

Anti-platelet therapy is a useful strategy to prevent acute thromboembolic artery occlusions. This study was designed to assess the efficacy of seselin derivatives against murine pulmonary thromboembolism, bleeding time, platelet activation and thrombosis. Administration of C3 (16 mg/kg) offered 70% protection against collagen- and epinephrine-induced pulmonary thromboembolism and 30% protection against arachidonic acid-induced death in mice, without adversely affecting bleeding time. No significant difference was observed by C3 in ferric chloride-induced arterial thrombosis in rats. Significant reduction in thrombus weight was observed in arteriovenous shunt model. In rat PRP, C3 reduced ADP and collagen-induced platelet aggregation. In chronic hamster model of dyslipidemia, administration of C3 (16 mg/kg p.o. for 90 days) had no effect on plasma lipids, vasoreactivity and platelet adhesion. C3 fed hamsters showed reduced whole-blood aggregation response to ADP and collagen compared to HC-fed hamsters. In addition, C3 augmented thrombin time; however, time to occlusion was not increased. These results convincingly demonstrated that C3 is a novel molecule that reduces the risk of thrombosis and alleviates prothrombotic state associated with hyperlipidemia without any adverse effect on bleeding time. The high benefit/risk ratio of this compound makes it a suitable candidate for future valid studies.

Effect of ormeloxifene, a nonsteroidal once-a-week oral contraceptive, on systemic hemodynamics in adult female rats.

OBJECTIVE: To investigate the short-term effects of ormeloxifene on systemic hemodynamics, coagulation profile, and serum antioxidant activity in vivo in comparison with raloxifene. MATERIALS AND METHODS: Colony-bred adult female Sprague-Dawley rats were randomized into 19 groups of 10 each and received either ormeloxifene or raloxifene (0.25, 1.25, or 3 mg/kg/day) for 7, 15, or 30 days by the oral route. Animals of control group received vehicle (gum-acacia in distilled water) alone in a similar manner. Systemic hemodynamics and serum total antioxidant activity were assessed 24 h after the last treatment. RESULTS: There was no significant effect of ormeloxifene administered at these doses and schedules on hemodynamic parameters or antioxidant activity, except for increase in amplitude of R wave in rats treated with 3 mg/kg/day dose for 30 days. This effect with raloxifene was evident only 7 days after treatment at this dose. Overall response was, however, almost similar with both the agents. CONCLUSION: The findings demonstrate comparable pharmacological profile of ormeloxifene and raloxifene on short-term administration to rats. Based on changes observed in the ECG (R wave), long-term studies may lead to justifiable comparison of beneficial and harmful effects of ormeloxifene and raloxifene in relation to cardiovascular effects.

Anti-platelet effects of Curcuma oil in experimental models of myocardial ischemia-reperfusion and thrombosis.

Extensive research on the mechanism of action and medicinal importance of curcumin obtained from turmeric (Curcuma longa) has unfolded its potential therapeutic value against many chronic ailments. Curcuma oil (C.oil), the highly lipophilic component from Curcuma longa has been documented for its neuroprotective efficacy against rat cerebral ischemia-reperfusion injury; however its effect on myocardial reperfusion injury remains unexplored. In the present study, effect of C.oil (500 mg/kg, po) was evaluated against myocardial ischemia-reperfusion induced injury in the rat model. C.oil failed to confer protection against cardiac injury, however significant reversal of ADP induced platelet aggregation (p<0.05) was evident in the same animals. Moreover, collagen and thrombin induced platelet aggregation (p<0.001) as well as tyrosine phosphorylation of various proteins in activated platelets was also suppressed. C.oil also offered significant protection against collagen-epinephrine induced thromboembolism in mice as well as augmented total time to occlusion against FeCl(3) induced arterial thrombosis in rats. C.oil however had no effect on coagulation parameters (TT, PT and aPTT) and exerted a mild effect on the bleeding time. Bioavailability of C.oil, as assessed by monitoring ar-turmerone, α,ß-turmerone and curlone, was 13%, 11% and 7% respectively, indicating high systemic exposure. Moreover, longer mean residence time (MRT) of ar-turmerone (13.2h), α,ß-turmerone (11.6h) and Curlone (14.0 h) and plasma elimination half lives in the range of 5.5 to 7.2h correlated with single 500 mg/kg dose regimen of C.oil. In the present study, C.oil thus seems to be an efficacious and safe anti-platelet agent which was protective against intravascular thrombosis.