Ellagic Acid-induced Hypercoagulable State in Animals:a Potentially Useful Animal Hypercoagulable Model for Evaluation of Anticoagulants

Na Liu,Jun-tian Liu＊,and Qiang-zong Zhang

Department of Pharmacology,Xi’an Jiaotong University School of Medicine,Xi’an 710061,China

HYPERCOAGULABLE state or prothrombotic state is a manifestation of the comprehensive abnormity of vessels,blood flow,and the components in blood,and may induce thrombosis.1As a pivotal pathological foundation and potential risk factor,hypercoagulability is now considered critical to initiation and development of cardiovascular and cerebrovascular diseases.Anticoagulants are able to prevent formation and progress of thrombosisviainhibiting the coagulant process,and therefore used for prevention of ischemic cardiovascular and cerebrovascular diseases in clinical practice.For assessment of their efficiency,it is necessary to establish a stable and effective animal model of hypercoagulable state mimicking clinical condition.Previous studies showed that some coagulation factors and endotoxin may induce hypercoagulable state in animals.2

Ellagic acid (EA),a plant phenol found in various fruits and nuts,has been documented for its ability to activate coagulation factor XII to trigger intrinsic coagulation system.3,4As an effective coagulant,EA has been applied to induce hypercoaguable state.5-9However,systematic and overall evaluation has not been conducted for hypercoaguable state models.The present study presents a comprehensive evaluation of EA-induced hypercoagulable state in different animals through observing coagulant and platelet functions.

MATERIALS AND METHODS

Animal models

Each type of animal was randomly divided into one control group and three EA groups (10 animals in each group in mice,8 in rats,and 6 in rabbits).The animals in EA groups received intravenous injection of different doses of EA (4.5 mg/kg,15 mg/kg,and 45 mg/kg in mice;3.15 mg/kg,10.5 mg/kg,and 31.5 mg/kg in rats;1.67 mg/kg,5.56 mg/kg,and 16.68 mg/kg in rabbits10);and those in control groups received saline instead.

Reagents

EA (Sigma-Aldrich,St.Louis,MO,USA) was dissolved in 1 mol/L sodium hydroxide solution,and the pH was adjusted to 7.4 with 1 mol/L hydrochloric acid.Prothrombin time (PT)and the activated partial thromboplastin time (APTT) assay kits were provided by Shanghai Sun Bio-Tech Development Incorporation (Shanghai,China).Thrombin assay kit was manufactured by Pacific Hemostasis (Huntersville,NC,USA).

Measurement of bleeding time (BT) and clotting time(CT) in mice

BT was assessed with an adapted tail transaction method previously described.11Briefly,each mouse was positioned in a special immobilization cage with the tail kept steady and immersed in saline at 37°C.The 3-mm end section of the tail was transected with a razor blade 5 minutes after the injection and immediately reimmersed in the warm saline.BT wasrecorded as the time from the transaction to the cessation of bleeding.

CT was determined with the template method.12Briefly,one eyeball of each mouse was quickly removed with a pair of curved tweezers 5 minutes after the injection.Two drops of blood were dripped in diameter of 5 mm at both ends on a glass slide for recording CT.At the same time,the drops were poked up from the edges with a sterile needle at intervals until blood streak appeared.

Determination of PT,APTT,and thrombin activity in rats

PT,APTT,and thrombin activity were measured with the standard methods.13,14In brief,rats were anesthetized with intraperitoneal injection of sodium pentobarbital (30 mg/kg),and then injected with EA or saline.Blood was collected from the abdominal aorta into the siliconized tube containing 3.8% sodium citrate [sodium citrate∶blood=1∶9 (v∶v)]5 minutes after injection of EA or saline.Plasma was immediately obtained by centrifuging the collected blood at 560 ×gfor 10 minutes.Finally,PT was measured by means of Quick’s one-stage prothrombin test,and APTT by means of modified APTT assay using an EA-containing APTT reagent.Thrombin activity was determined by PT assay with a PK-B hemagglutination analyzer (Zhongshan Peikang Limited Company for Medical Electronic Instruments,Zhongshan,China).

Examination of the circulating platelet aggregates in rats

The circulating platelet aggregates were detected with a previously described method.15In brief,blood was drawn from the abdominal aorta 5 minutes after injection of EA or saline,and separately anticoagulated with EDTA or EDTA containing formalin.Platelet-rich plasma(PRP) was prepared by centrifuging the blood at 150 ×gfor 8 minutes.The platelets in PRP were counted with a bright-line hemacytometer (American Optical,Buffalo,NY,USA) and a phase contrast microscope (Olympus,Tokyo,Japan),and the circulating platelet aggregate ratio (CPAR) was calculated according to the following formula∶16

Time-effect relationships of EA in rabbits

Blood samples were drawn from the ear artery of the rabbits at six different time points∶before injecting EA,and 5,15,30,60,and 120 minutes after injecting EA.The blood was anticoagulated with 3.8% sodium citrate in the ratio of 1∶9 (v∶v),and immediately centrifuged at 560 ×gfor 10 minutes to obtain the plasma.PT and APTT were measured with the same methods as in rats.

Statistical analysis

Data were expressed as means±SD.One-way analysis of variance (ANOVA) followed by Dunnett’s test was performed with SPSS 13.0 for multiple comparisons.APvalue lower than 0.05 was considered statistically significant.

RESULTS

Effects of EA on BT and CT in mice

EA led to decrease of BT and CT in mice in a dose-dependent manner (Fig.1,allP＜0.01).As the EA dose increased,BT was shortened by 32.0%,51.5%,and 84.5%,and CT shortened by 23.6%,36.3%,and 48.9% compared with the control.These results demonstrate that EA could significantly reduce BT and CT in normal mouse.

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Effects of EA on PT,APTT,thrombin activity,and the circulating platelet aggregates in rats

EA at the doses used in this study also diminished PT and APTT in rats in a dose-dependent manner (Figs.2A,B,allP＜0.05).PT was decreased by 26.7%,42.2%,and 65.2%,and APTT by 15.9%,25.9%,and 35.0% compared with the control along with the increasing dose.EA elevated thrombin activity in a dose-dependent way as well (P＜0.01),as the maximal increase of 63.2% was achieved by the highest dose (Fig.2C).CPAR was enlarged by EA at the three different doses in comparison with the control (Fig.2D,P＜0.05 in middle and high dose groups).The data suggest that EA could remarkably shorten PT and APTT,and enhances thrombin activity and the circulating platelet aggregation in normal rat.

Time-effect relationship of the procoagulant activity of EA in rabbits

A time-effect relationship was observed in the effects of EA on PT and APTT in rabbits (Fig.3).PT and APTT did not show significant changes in the observed time in control group,implying the stability of coagulation system in normal rabbits.In contrast,EA treatment caused a dose-dependent shortening of PT and APTT.Those effects appeared immediately after intravenous administration of EA,reached the peak after 15 minutes,and lasted for 30-60 minutes.The results indicate that EA may induce decline of PT and APTT in normal rabbit quickly and transiently.

DISCUSSION

As a risk factor for ischemic cardiovascular and cerebrovascular diseases,hypercoagulable state has received much attention due to its potential in inducing thrombosis.Hypercoagulable state is substantially the result of the interaction of different mechanisms involving the activation of various hemostatic components,including coagulation and fibrinolytic systems,vascular endothelium,and platelets.17It is known that BT is related to the conditions of vascular contraction,platelets,and coagulation functions,while CT is mainly associated with coagulation functions.PT and APTT reflect functions of intrinsic and extrinsic coagulation pathways,respectively.The results of the present study showed that EA shortened BT and CT in mice,PT and APTT in rats,which may be explained by the procoagulant activity of EA,consistent with the findings of Botti and Ratnoff.18However,part of our observations appear to be different from the data reported by Girolami et al,19as they found no EA-induced change in PT or partial thromboplastin time in rats,cats,rabbits,and dogs.This discrepancy may be due to the difference in doses of EA used.In our study,EA was administered to rats at 3.15 mg/kg at least,while Girolami et al used only 0.302 mg/kg(10-4mol/L,10 mL/kg).

Figure 1.Effects of ellagic acid (EA) at different concentrations on bleeding time (BT) and clotting time (CT) in mice.The control group was injected with saline instead of EA.(n=10)

Figure 2.Effects of EA on prothrombin time (PT),the activated partial thromboplastin time (APTT),thrombin activity,and the circulating platelet aggregates in rats.(n=8)

Figure 3.Time-effect relationship of the effect of EA on PT and APTT in rabbits.(n=6)

In the process of blood clotting,both intrinsic and extrinsic coagulation systems lead to thrombin production and blood clotting.It has been known that EA is able to activate coagulation factor XII to trigger intrinsic coagulation system.3,4The present study found that EA also enhanced thrombin activity,which may be attributed to its direct activation of coagulation factor XII.

Platelet hyperfunction is another factor responsible for hypercoagulable state as platelets participate in hemostatic process through adhesion and aggregation.Our investigation on the effect of EA on platelet aggregationin vivomanifested that EA increased circulating platelet aggregates,implying the ability of the procoagulant to activate platelets,possibly indirectly through promoting thrombin production.

The results about the time-effect relationship of EA indicate that EA could also reduce PT and APTT in rabbits,promptly but briefly (60 minutes in the present study).Further studies are crucial to confirm whether that relationship exists in other animal species.

For the successful establishment of a model,EA dose and time of blood collection are important factors.The findings of the present study suggest that middle dose of EA may produce a profound enough influence on the observed parameters.Therefore,the middle dose (15 mg/kg in mice,10.5 mg/kg in rats,and 5.56 mg/kg in rabbits) is recommended for the model of hypercoagulable state.The optimal time for blood collection in mice,rats,and rabbits is 5,5,and 15 minutes after injection of EA,respectively.10

We have previously established a model to evaluate the effect of leech,a traditional Chinese drug,on coagulation.20The results verify that non-heating extract of leech produces significant anticoagulant and antiplatelet actions in the hypercoagulable model.

In summary,the present research demonstrates that EA is able to induce hypercoagulable state through activating coagulation system and platelets in mice,rats,and rabbits.Since the model is simple and stable,and the parameters are specific and objective,the application of the model could be extensive in pharmacological and pathological investigations,especially in screening and evaluation of anticoagulants.

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