Aspirin Enhance ADP-induced Platelet Aggregation By Unfractionated Heparin And The Possible Mechanisms

Objective:To investigate the effects of unfractionated heparin on platelet aggregation in vivo and in vitro, as well as the possible enhancement effect of aspirin on ADP-induced platelet aggregation by UFH.Methods:1 In vivo animal experiments16 Rabbits whole blood samples were collected before and 20 minutes after intravenous injection of 200 IU/Kg UFH and used to measure baseline ADP- and AA- platelet aggregation. Then rabbits were divided into two groups: ASA group and clopidogrel group. Rabbits in ASA group were intragastrically administrated 12 mg/Kg.d ASA. Seven days later, rabbit whole blood samples were similarly collected and immediately used to determine ADP- and AA-induced platelet aggregation. Those rabbits were then intragastrically administrated 12 mg/Kg.d ASA and 4 mg/Kg.d clopidogrel. Seven days later, rabbit whole blood samples were similarly collected to measure ADP- and AA-induced platelet aggregation. So did the clopidogrel group. 3.2% sodium citrate was used as anticoagulation at 1:9 ratio.Similarly, 12 Rabbits whole blood samples were collected before and 20 minutes after intravenous injection of 200 IU/Kg dalteparin and used to measure baseline ADP- and AA- platelet aggregation. Then rabbits were divided into two groups: ASA group and clopidogrel group. Rabbits in ASA group were intragastrically administrated 12 mg/Kg.d ASA. Seven days later, rabbit whole blood samples were similarly collected and immediately used to determine ADP- and AA-induced platelet aggregation. Those rabbits were then intragastrically administrated 12 mg/Kg.d ASA and 4 mg/Kg.d clopidogrel. Seven days later, rabbit whole blood samples were similarly collected to measure ADP- and AA- induced platelet aggregation. So did the clopidogrel group.The platelet aggregation test was conducted by impedance aggregometry using the Chrono-log aggregometer model 590. Each sample was tested for the maximum aggregation in response to 10μmol/L ADP and 0.5 mmol/L AA. Each sample was tested twice within 2 hours after sampling. Data were presented as mean of the two tests.2 In vitro aggregation of human platelet-rich plasmaTwenty-four samples of 5 mL platelet-rich plasma were collected from healthy adults using an automatic platelet collector. One mL of each sample was incubated with UFH at final concentration of 0, 1, 2, 3 and 4 IU/mL at 37 oC for 10 minutes, respectively, and used to detect 10μmol/L ADP-induced platelet aggregation.Similarly, 36 samples of platelet-rich plasma collected from different healthy adults were used to examine the effects of UFH on ADP-induced platelet aggregation after incubation for 20 minutes.28 samples of 5 mL platelet-rich plasma were collected. One ml of each sample were pre-incubated with no-drug or 100umol/L ASA or 100umol/L ASA and 300umol/L A2P5P or 300umol/L A2P5P respectively at 37oC for 5 minutes and then incubated with UFH 4IU/mL 37oC for 10 minutes. The other one ml of each sample was incubated with saline equivalent for 15 minutes.Then all samples were used to measured ADP-induced platelet aggregation, Another 28 platelet-rich plasma samples were used to perform similar experiments under same conditions except incubation with UFH for 20 minutes and with saline for 25 minutes.The platelet aggregation test was conducted by impedance aggregometry using the Chrono-log aggregometer model 590. Each sample was tested for the maximum aggregation and 5'residual aggregation in response to 10μmol/L ADP. Each sample was tested twice within 2 hours after sampling. Data were presented as mean of the two tests.4 VASP phosphorylation Forty samples of 4 mL platelet-rich plasma were used in the following experiments. One mL of each sample was pre-incubated with no-drug or 100μmol/L ASA or 100μmol/L ASA and 300μmol/L A2P5P at 37 oC for 5 minutes, respectively, and then incubated with 4 IU/mL UFH for 20min. Other one ml of each sample was incubated with saline equivalent for 25min. Then all the above samples were used to measure VASP phosphorylation by flow cytometry. In brief, 10μL of each sample was added into three Falcon tubes numbered T1 containing 10μl PGE1, and T2 as well as T3 containing 10μl mixtures of PGE1 and ADP, respectively, and vertexed for 2 seconds at low speed. After incubation at room temperature for 10 minutes, samples were fixed with 10μl 3% paraformaldehyde for 5 minutes at room temperature. Tenμl of mouse monoclonal antibody directed against phospho-VASP was added into T1 and T2, and 10μl control mouse monoclonal antibody was added into T3. After addition of 10μl IgG-FITC and CD61-PE in each tube, samples were incubated for 5 minutes at room temperature in dark, immediately placed at 2~8 oC after addition of 2 ml diluent, and analyzed by flow cytometry within 2 hours.Platelet reactivity index (PRI) was calculated using the following equation: PRI = [(MFIPGE1-MFIPGE1+ADP)/MFIPGE1] x 100Where MFIPGE1 and MFIPGE1+ADP were the mean fluorescent intensity of samples under resting state and activated state, respectively.5 Alteration of platelet ultrastructureSix samples of 7 mL platelet-rich plasma were used to observe platelet ultrastructure by TEM after the following treatments: one mL of each sample was pre-incubated with no-drug at 37 oC for 5 minutes and then incubated with 1, 2, 3 and 4 IU/mL UFH at 37 oC for 20 minutes, respectively; one mL of each sample was pre-incubated with 100μmol/L ASA or 100μmol/L ASA and 300μmol/L A2P5P at 37 oC for 5 minutes, then incubated with 4 IU/mL UFH for 20 minutes; one mL of each sample was incubated at 37 oC for 25 minutes as control. All samples were fixed by incubating with equal volume of 0.3% glutaraldehyde at room temperature for 10 minutes. Platelets were collected by centrifugation at 2000 r/min for 8 minutes and further fixed with 4% glutaraldehyde for 30 minutes at room temperature. The yellow films of platelets on the top of the tubes were carefully collected and washed with 0.1 mol/L phosphate buffer for three times. These yellow films were cut into 1.5×2 mm pieces, fixed with 3% glutaraldehyde at 4 oC for 3 hours and 1% osmium tetroxide for 2 hours, dehydrated through a series of graded ethanol and acetone, embedded with epoxy Epon 812 and sliced into ultrathin sections. After stained with uranyl acetate and lead nitrate, they were examined by TEM to observe platelet ultrastructure and photographed.Results:1. UFH can enhance ADP-induced platelet aggregationWe first examined the effects of intravenous injection of UFH on rabbit platelet aggregation. It was shown that injection of UFH significantly increased baseline ADP-, but not AA-induced platelet aggregation from 6.82±2.28 ? to 8.88±2.88 ?. Then we further studied the effect of UFH on human platelet aggregation in vitro. It was seen that incubation with 4 IU/mL UFH for 10 minutes significantly enhanced platelet aggregation compared with control untreated platelet; Incubation with 1 to 4 IU/mL UFH for 20 minutes also significantly enhanced ADP-induced platelet aggregation in a dose dependent manner (P<0.05). Five-minute residual platelet aggregation showed the same results.We also observed the effects of UFH on platelet ultrastructure by TEM. As shown in Figure 2.1, normal platelets were in disk- or elliptical disk-like shapes with smooth and integrate membrane, small and short processes were occasionally seen on some platelet surfaces; in cytoplasm, clear open canalicular system (OCS) and dense tubular system (DTS) and few expanding bubbles can be found, and large amounts of glycogen granules were randomly distributed. After incubation with 1 IU/ml UFH, platelet membrane was intact and otherwise normal except for a small amount of protuberance on the surface and mild glycogen accumulation in cytoplasm. After incubation with 2 IU/ml UFH, pseudopodia on some platelet surfaces and mild glycogen accumulation in cytoplasm were found; After incubation with 3 IU/mL UFH, platelet morphology was significantly changed: they appeared in different shapes with broken membrane and pseudopodia. Bubbles appeared on the surface. fewer granules, Large vesicles and accumulated glycogen were in cytoplasm. Some platelets appeared to be aggregation tendency. After incubation with 4 IU/mL UFH, large number of debris and granule were seen outside platelets, platelet were in different shape and sizes, more pseudopodia formed, more platelet membrane was broken; amounts ofαgranules, OCS were reduced, large and irregular vesicles appeared, glycogen was accumulated in almost every platelet, and platelet aggregation was obvious.2. ASA might further enhances ADP-induced platelet aggregation by UFHIn rabbits intragastrically administrated ASA for a week, ADP-induced platelet aggregation was enhanced by intravenous injection of UFH from 6.99±2.10 ? to 9.93±3.03 ?, whereas AA-induced platelet aggregation was not significantly changed. So did the dalteparin group. Therefore, we further explored the effect of UFH and ASA plus UFH on human platelet aggregation in vitro. We found that pre-incubation with ASA significantly enhanced ADP-induced human platelet aggregation after incubation with UFH for 10 minutes from 14.05±5.04 ? to 18.18±4.33 ? and 20 minutes from 14.57±3.27? to 20.09±6.91 ?, which is more prominent than that of the former. Similar results were found in 5-minute residual platelet aggregation.We also examined ultrastructure of platelet after incubation with ASA and UFH by TEM. The results indicated that platelets were in different shapes and sizes ; many platelets lost integrate membrane while pseudopodia and membrane vesicles on their surface; fewer platelet granules and OCS in cytoplasm and more large irregular vesicles in cytoplasm. Platelets aggregation and degranulation were found.3. Effect of A2P5P, a P2Y1 receptor antagonist, on ADP-induced platelet aggregation after incubation with UFH and ASA plus UFH Pre-incubation with 300μmol/L A2P5P, a P2Y1 receptor antagonist, significantly decreased ADP-induced human platelet aggregation by ASA plus UFH whether incubation for 10 minutes or incubation for 20 minutes. Similar results were also obtained in 5-minute residual platelet aggregation. These results suggest that P2Y1 is involved in ASA enhancement of ADP-induced platelet aggregation by UFH. Compared with incubation for 10 minutes with UFH, that with A2P5P plus UFH significantly decreased ADP-induced platelet aggregation, which shows that P2Y1 is also involved in ADP-induced platelet aggregation by UFH.TEM showed that after pre-incubation with ASA plus A2P5P, platelet ultrastructure was not significantly altered, showing round or oblong shape, integrated membrane, more visible granules inside platelet except few membrane vesicles and mild glucogon accumulation. Aggregated platelet and platelet aggregation tendency were not observed.4. Role of P2Y12 receptor in the ADP-induced platelet aggregation after incubation with UFH or ASA plus UFHFlow cytometry showed that PRI of the platelets incubated with UFH was obviously higher than that of control group, while PRI was no significant difference between UFH group and ASA plus UFH group.Intragastrically administrated ASA plus CLO for a week, ADP-induced platelet aggregation was still enhanced by intravenous injection of UFH in rabbits.Conclusions: UFH can dose-dependently enhance ADP-induced platelet aggregation through ADP P2Y1 receptor and P2Y12 receptor passway; ASA might further enhanced ADP-induced platelet aggregation by UFH, which is possibly related to ADP P2Y1 receptor passway.