Use of Aspirin in normalization of recombinant human erythropoietin-mediated hyper-reactivity of platelets in rats.

OBJECTIVES: The cytokine erythropoietin is the primary stimulator of erythropoiesis and recombinant human erythropoietin (rHuEPO), which is widely used in the treatment of anemia associated with advanced chronic kidney disease (CKD). Adverse cardiovascular outcomes have been observed during clinical trials of anemia correction with rHuEPO in CKD patients. We investigated the effects of short-term, high-dose treatment with rHuEPO on platelet reactivity and effects of aspirin on platelet reactivity in healthy rats.
MATERIALS AND METHODS: Animals received three daily dose of rHuEPO (25 μg/kg s.c.). Platelets were isolated after 48 h of last dose of rHuEPO to study the arachidonic acid-induced platelet aggregation. Aspirin (75 mg/kg p.o.) was given to animals just before 1 h of isolation of platelets.
RESULTS: In rats, treatment with rHuEPO increased platelet reactivity and platelet count. The increased platelet reactivity was paralleled by decreased time-to-occlusion (TTO) in arterial thrombosis model, and decreased bleeding time after tail transection in rats. Treatment with rHuEPO followed by single dose of aspirin showed significant reduction in TTO and bleeding time as compared with aspirin-treated group.
CONCLUSIONS: These findings suggest that rHuEPO increases platelet reactivity and aspirin normalizes the hyper-reactive platelet and may reduce the cardiovascular events associated with rHuEPO in CKD patients.

Introduction

Correction of renal anemia by recombinant human erythropoietin (rHuEPO) has been proven to be an effective treatment with several beneficial effects on exercise capacity due to reduction of hypoxia and improvement of cardiac function.[1] At present, rHuEPO is approved for the treatment of anemia associated with renal failure, chemotherapy, and HIV antiviral treatment or to reduce the need for transfusion in preoperative surgical patients. However, besides these beneficial effects, potential risk factors of a rHuEPO treatment have been reported. Treatment with rHuEPO has been linked to an increased risk of thrombotic events, and it seems to affect various cardiovascular and hemostatic parameters unrelated result of increase in hematocrit (HCT).[2] EPO may also be prothrombotic in humans by increasing platelet reactivity, systemic blood pressure through vasoconstriction and calcium uptake stored in platelets.[34] A study in dogs suggested that rHuEPO not only promotes the synthesis of increased numbers of reticulated platelets but these newly produced platelets are also hyper reactive compared with controls.[5] Treatment with rHuEPO has been reported to increase the mean platelet volume (MPV) correlated to a worsened outcome after cerebrovascular and cardiovascular events as well as increased risk of recurrent events of myocardial infarction (MI) and stroke.[67] Targeting a specific receptor having altered aggregatory responses towards agonist would be beneficial to prevent thrombovascular accidents due to rHuEPO therapy. Concerns have been raised regarding the application of these experimental findings in patients with acute coronary syndrome (ACS) because the administration of rHuEPO is associated with increased platelet reactivity in healthy subjects and increased risks of thrombotic events in anemic patients with end stage renal disease (ESRD).[8910]

There are few existing data on the effect of short-term administration of rHuEPO on platelet function and no prior studies to determine whether rHuEPO may mitigate the effects of aspirin used in ACS. This preclinical study was designed to find altered aggregatory response of platelets due to rHuEPO treatment and to normalize recombinant human erythropoietin-mediated hyper-reactivity of platelets using suitable antiplatelet agent.

Materials and Methods

Chemicals

Recombinant Human Erythropoietin injection (4000 IU) and aspirin were obtained from Cadila Healthcare Ltd., Ahmedabad, India. Arachidonic acid, Bovine Serum Albumin and some other reagents were obtained from Sigma Aldrich Co., USA.

Animals

Six to eight weeks old male Wistar rats (Animal Research Facility, Zydus Research Centre, Ahmedabad) weighing 230-280 grams, were housed into groups of 4 in polypropylene cages with stainless steel top grill containing autoclaved rice husk at an ambient temperature of 22 ± 3 ºC and relative humidity of 55 ± 10%. They were exposed to 12-h light dark cycle and had free access to food and water ad libitum. Animals were fed standard laboratory diet (Chakkan, Hyderabad). The methods and procedures described in the present report has been reviewed and approved by the Institutional Animal Ethics Committee (IAEC) and experiments were performed in accordance with the guidelines laid down by the committee for the Control and Supervision of Experimentation on Animals (CPCSEA).

Experimental Design

Pilot experiment was designed to determine the dose and frequency of rHuEPO administration to obtain statistically significant hyper reactivity of platelets. Each rat received three consecutive doses of rHuEPO i. e., 25 μg/kg, s. c. and 50 μg/kg, s. c. at an interval of 24 h and then arachidonic acid-induced platelet aggregation assay was performed 48 h after the last injection of rHuEPO. The increased platelet reactivity at the dose of 50 μg/kg, s. c. has been reported.[11] But, we found increase in platelet reactivity even at lower dose i. e., 25 μg/kg. Therefore, we selected the lower dose for our study.

Male Wistar rats were divided in 4 groups (N = 6); control group received vehicle alone (0.9% w/v NaCl); second group received three consecutive doses of rHuEPO (25 μg/kg, s. c.) at an interval of 24 hour followed by vehicle treatment before one hour of blood collection; third group received three consecutive dose of vehicle at an interval of 24 hour followed by single dose of aspirin (75 mg/kg, p. o.) before 1 hour of blood collection and fourth group received both, rHuEPO (25 μg/kg, s. c.) three consecutive dose at an interval of 24 hour and aspirin (75 μg/kg, p. o.) before one hour of blood collection. Blood collection for platelet isolation was performed 48 hours after the last injection of rHuEPO/vehicleor after 1 hour of aspirin/vehicle administration. Determination of ex-vivo platelet aggregation, time to thrombus formation and bleeding time were obtained by (1) arachidonic acid-induced ex-vivo platelet aggregation (2) FeCl3- induced arterial thrombosis and (3) Bleeding time using tail transection test, respectively. All methods used in the study were also described in published literature.[1213] All animals were euthanized using CO2 chamber after completion of study.

Arachidonic Acid-Induced Ex-vivo Platelet Aggregation Study

Treated rats were anesthetized with ether and the blood was collected from retro orbital plexus in centrifuge tubes pre-filled with tri sodium citrate (3.8% w/v, 1:9). Rat blood was centrifuged at 200× g for 15 minutes at 4ºC. Platelet rich plasma (PRP) was collected carefully to avoid contamination with red cells or leukocytes. PRP was then centrifuged at 1000× g for 15 min at 4ºC. Supernatant was thrown and pellets were re-suspended by adding 3 ml of modified tyrode solution (134 mM NaCl, 3 mM KCl, 2 mM MgCl2, 0.3 mM NaH2PO4, 12 mM NaHCO3, 1 mM EDTA, 3.5 mg/ml bovine serum albumin and 12 mM glucose, pH 7.4). Washing of platelet pellet was repeated twice. Washed platelets were re-suspended in modified tyrode buffer (without EDTA) to a concentration 3 × 108 cells/ml and allowed to rest for 15 minutes prior to use. To replenish the calcium concentration, CaCl2 (1 mM) has been added to platelet suspension just before starting the experiment. Washed platelets (180 μl each) were added into 96 well, flat-bottomed micro titer plates. Platelet aggregation assay was performed at 37ºC for 5 minutes in kinetic mode with orbital shaking using 20 μl arachidonic acid (175 μM final concentration) as aggregating agent. The concentration of arachidonic acid was previously standardized by performing pilot experiments and the minimum concentration at which it shows significant platelet aggregation was selected for the study (data not shown). Measurement of optical density was performed at 405 nm using a Spectramax 190 plate reader (Molecular Devices Corporation, Sunnyvale, California, USA). Percentage platelet aggregation was calculated by subtracting the final reading from the initial reading of the corresponding well.

Initial optical density = Optical Density at 0 second

Final optical density = Optical Density at 300 seconds

FeCl3-induced Arterial Thrombosis in Wistar Rats

FeCl3-induced chemical injury was used as a model of arterial thrombosis as described previously.[13] Treated rats were anesthetized with urethane (1 gm/kg, i. p.) and secured in supine position. A midline cervical incision was made on ventral side of the neck and left carotid artery was isolated by blunt dissection. A 2 × 3 mm strip of whatmann filter paper # 1 saturated with 35% (w/v) FeCl3 was placed on the carotid artery for 5 minutes. A temperature probe (Thermalert-TH8, Physitemp Instruments Inc, New Jersey, USA) was placed distal to filter paper to monitor the temperature of carotid artery. A sudden fall in temperature (about 2ºC) was taken as an indication of cessation of blood flow as a consequence to thrombus formation. The time from FeCl3 application till the time of thrombus formation was noted as time to occlusion. A cutoff time was fixed at 45 minutes if no thrombus formation was seen in drug-treated animals. All animals were euthanized after completion of study.

Bleeding Time Using Tail Transection Test in Wistar Rats

Male Wistar rats were treated as described previously and anesthetized with urethane (1 gm/kg, i. p.). Rat tail has been cleaned with saline and transected 5 mm from the tip with a sterile surgical scalpel blade. The resultant wound was gently blotted with whatmann filter paper # 1 at time 0 and at 30-second time intervals thereafter until bleeding stopped. The time when no blood could be blotted on the filter paper was considered as the bleeding time. In case, the bleeding did not stop, a maximum cutoff bleeding time was fixed at 20 minutes. All animals were euthanized after completion of experiment.

Statistical Analysis

Data were presented as means ± SEM and analyzed by one-way ANOVA followed by Tukey's multiple comparison tests using Graph Pad Prism 5.0 software. P <0.05 was considered to be statistically significant.

Results

Initially we designed pilot experiments and found that rHuEPO at three consecutive doses of 25 μg/kg s. c. and 50 μg/kg, s. c. significantly altered arachidonic acid induced platelet aggregatory response using the arachidonic acid (175 μM) as an aggregating agent [Figure 1a]. Therefore, 25 μg/kg dose of rHuEPO was considered for further study. We used aspirin (75 mg/kg, p. o.) as an antiplatelet agent and dose of aspirin (75 mg/kg, p. o.) has been selected after performing pilot experiments. We found that single dose of aspirin (75 mg/kg, p. o.) showed significant antiplatelet response in rat when platelets were isolated one hour after drug administration (data not shown).

Figure 1

(a) Effects of Recombinant Human Erythropoietin on arachidonic-induced % change in platelet aggregation using rat-washed platelets (N = 6). All values are expressed as mean ± SEM. a indicates P < 0.05 Vs vehicle control. (b) Effects of Recombinant Human Erythropoietin (rHuEPO) and aspirin on arachidonic-induced % change in platelet aggregation using rat washed platelets (N = 6). All values are expressed as mean ± SEM. a indicates P < 0.05 Vs vehicle control, b indicates P < 0.05 Vs HuEPO-treated group

Effects of Recombinant Human Erythropoietin and Aspirin on Arachidonic-induced Platelet Aggregation

Animals were randomized on body weight basis and grouped as described previously. We found that rHuEPO treatment (25 μg/kg, s. c.) for 3 consecutive days produced significant increase in platelet aggregatory response, which is an indicative of platelet hyper-reactivity [Figure 1b]. Treatment with rHuEPO followed by single dose of aspirin showed significant decrease in platelet aggregatory response as compared with rHuEPO-alone group and bring down the platelets to the normal, which has been reflected by non-significant reduction in platelet aggregatory response as compared with vehicle control group [Figure 1b].

Effects of Recombinant Human Erythropoietin and Aspirin on Time to Carotid Artery Occlusion

Subsequently, we also explored the above observations of platelet aggregation studies in in vivo model of arterial thrombosis in rats. Treatment with rHuEPO for 3 consecutive days produced significant decrease in time-to-occlusion (TTO). Treatment with aspirin alone produced significant rise in TTO and all animals showed no clot formation till cutoff time. Interestingly, treatment with rHuEPO followed by aspirin showed significant reduction in TTO as compared to aspirin-treated group [Figure 2].

Figure 2

Effects of Recombinant Human Erythropoietin and aspirin on time to carotid artery occlusion using FeCl3-induced arterial thrombosis in male wistar rats (N = 6). All values are expressed as mean ± SEM. a indicates P < 0.05 Vs vehicle control, b indicates P < 0.05 Vs rHuEPOtreated group, c indicates P < 0.05 Vs aspirin-treated group

Effects of Recombinant Human Erythropoietin and Aspirin on Bleeding Time Profile

Bleeding profile plays a major role in safety assessment of antiplatelet or anticoagulant agents. Therefore, we have investigated bleeding profile of treated animals. The rHuEPO treatment showed significant reduction in bleeding time as compared to vehicle-treated animals. Treatment with aspirin alone produced significant increase in bleeding time. Further, bleeding profile of rHuEPO and aspirin-treated group was significantly lower as compared with aspirin-treated group [Figure 3].

Figure 3

Effects of Recombinant Human Erythropoietin (rHuEPO) and aspirin on bleeding time profile using tail transection in male wistar rats (N = 6). All values are expressed as mean ± SEM. a indicates P < 0.05 Vs vehicle control, b indicates P < 0.05 Vs rHuEPO-treated group, c indicates P < 0.05 Vs aspirin-treated group

Treatment once daily with rHuEPO for 3 consecutive days increased red blood cells (RBC) count, HCT and hemoglobin when measured 48 hours after the rHuEPO treatment [Table 1]. A rise in RBC, HCT and Hb confirms the desired activity of erythropoietin. Platelet count has also increased significantly in rHuEPO and rHuEPO + aspirin-treated groups.

Table 1

Effects of Recombinant Human Erythropoietin and aspirin on hematological profile using male wistar rats (N=6)

Discussion

Chronic kidney disease (CKD) is associated with anemia primarily due to reduced production of erythropoietin (EPO).[14] The deficiency of EPO and iron is routinely corrected with the use of rHuEPO and iron preparations. However, oxidative stress and inflammation in CKD patients hampers erythropoietic activity of rHuEPO and iron. Severe anemia despite higher dose of rHuEPO and iron supplements is commonly due to oxidative stress and inflammation.[15] Several studies have revealed a strong association between severity of renal anemia and risk of mortality from cardiovascular disease in CKD patients.[1617] Meta-analysis of randomized clinical trials have been shown significantly higher cardiovascular and thrombosis events among patients assigned to the higher than those randomized to the lower Hb targets.[18] Based on these observations, Kidney Disease Outcomes Quality Initiative (K/DOQI) guidelines were revised by reducing the optimum target Hb to 11-12 g/dl.[19]

It has been reported that rHuEPO administration significantly increases platelet count in ESRD patients.[20] Higher platelet count in rHuEPO-treated patients is due to complication of the thrombopoietic action by EPO. Further, rHuEPO enhances platelet reactivity by increasing intracellular calcium stores following platelet activation that can cause a pro-thrombotic state.[4] In addition, rHuEPO can stimulate blood coagulation pathway by increasing the production of P selectin, E selectin, plasminogen activator inhibitor-1 and von Willebrand factor.[212223] EPO is potentially beneficial for its anti-apoptotic, mitogenic and angiogenic activities.[24] On the other hand, some reports suggest that rHuEPO may have pro-thrombotic or platelet-activating effects. It has been hypothesized that short-term high dose treatment with rHuEPO may alter platelet function and hyper reactivity of platelet could be normalized using suitable antiplatelet agent.

The data presented here show that short term high dose treatment with rHuEPO significantly affected platelet reactivity and blood parameters. To investigate whether the rHuEPO-induced effect on platelet was related to the high doses of rHuEPO, rats were also treated with 5-fold lower dose (5 μg/kg) of rHuEPO using the same treatment schedule. Three doses of 5 μg/kg EPO was sufficient to raise the HCT, but rHuEPO-mediated platelet aggregation was dose-dependent and more pronounced only at higher doses i. e., 25 μg/kg and 50 μg/kg. We used 25 μg/kg, s. c. dose three times in a week. EPO dosing interval we used was similar to the human rHuEPO regimen. One mg of EPO is equivalent to 120000 IU as per in-house potency ratio data of EPO. This corresponds to 3000 IU/kg of rat/dose in our study. As per formula reported in literature for dose translation based on body surface area, human equivalent dose (HED) (IU/kg) = animal dose (IU/kg) * Rat factor/Human Factor. As per the reference, rat factor is 6 and human factor is 37.[25] Therefore, human equivalent dose (IU/kg) =3000 * 6/37. Therefore, HED = 486.5 IU/kg or 4.05 μg/kg. In clinic, response range of dose of EPO in patients is from 75 IU/kg, s. c. to 300 IU/kg, s. c. per week. Dose varies to get target hemoglobin level that means our dose of rHuEPO in rat is higher than human dose. However, the duration of treatment of EPO in CKD patients is life-long, once the low hemoglobin is detected and the primary goal is to achieve target hemoglobin, which is 12 g/dl. Limitation of our study is that we studied short duration along with high dose of EPO presuming that low dose of EPO in human for long duration causes higher platelet reactivity. Another limitation of our study is that we used healthy rats and not the rat model of CKD. However, some of the published clinical studies reported 100, 200 and 400 IU/kg in healthy subjects to study the role of aspirin. They also reported that even aspirin or clopidogrel did not reduce the effects of both drugs on platelet function at the dose of 200 IU/kg of EPO but the dose of 400 IU/kg of EPO in healthy subjects attenuated the effects of aspirin on bleeding time only.[26] In our study, we used 486.5 IU/kg human equivalent dose of EPO in rat and found that there was increase in platelet reactivity and decrease in bleeding time in EPO-treated rats, which can be normalized using aspirin.

It has been reported that EPO has cytoprotective activity at higher dose. However, there is higher risk of cardiovascular events at high dose of EPO. Cardiovascular risk associated with EPO treatment may be reduced by using other EPO derivatives.[2728] We observed that in vitro stimulation of rat whole blood with high concentration of rHuEPO did not alter any agonist-induced aggregation. Therefore it seems feasible that rHuEPOdoes not exert direct effects on circulating platelets, but rather modulates platelet during synthesis and maturation in the bone marrow. EPO interacted with EPO-R on the cell surface, triggering activation of the janus-associated kinase-signal transducers and activators of transcription, phosphatidyl-inositol-3 kinase and mitogen activated protein kinase pathways.[29] It was observed that platelets from animals pre-treated with rHuEPO significantly altered arachidonic acid-induced ex-vivo platelet aggregation. The mechanisms of such early change of platelet function with rHuEPO are unknown but identifying receptors on platelet surface more prone for aggregation in presence of agonist and targeting with specific antagonist would be beneficial. In FeCl3-induced arterial thrombosis model, the time required for thrombus formation in carotid artery was found to be significantly increased in aspirin-treated rats till the cutoff period where as rHuEPO-treated rats showed a significant decrease in time to occlusion as compared to control group. This phenomenon may be due to increased HCT and altered platelet reactivity. Bleeding time was assessed as the time from the transection of tail tip to the cessation of blood flow. We used bleeding time as an in vivo measure of integrated platelet function and tissue hemostasis. Lower doses of rHuEPO (5 μg/kg, s. c.) did not modify bleeding time compared with control rats, while higher dose (25 μg/kg, s. c.) attenuated aspirin-induced increase in bleeding time compared with control group. It correlates with increased HCT and increased platelet aggregatory responses. In aspirin (75 mg/kg, p. o.)-treated group alone, there was significantly higher bleeding time. However, when aspirin was given to rHuEPO (25 μg/kg, s. c.)-treated rats, there was significantly less bleeding as compared with aspirin-alone group. The bleeding time takes into account the intrinsic platelet receptor function, granule release, platelet interaction with von Willebrand factor and fibrinogen, and the platelet concentration itself. Our findings suggest that the change in the bleeding time values may reflect changes in platelet dependent hemostasis.

Conclusion

We have shown that rHuEPO significantly increased platelet reactivity because it alters arachidonic acid-induced platelet aggregation in Wistar rats. We have also shown that rHuEPO attenuated aspirin induced further increase in bleeding time. We conclude that aspirin can control rHuEPO-induced increase in platelet hyper-reactivity and leads to normalization of platelet function. In CKD patients, who need higher dose of rHuEPO to achieve target Hb, rHuEPO-related cardiovascular mortality can be reduced if we use suitable antiplatelet agent such as aspirin, to normalize the platelet reactivity-induced by rHuEPO.