Inhibitory Effects of Dimeric Copper(II) bis(o-acetoxybenzoate) on Platelet-neutrophil adhesion and Thrombosis

Antithrombotic effect of the copper-aspirin complex (dimeric copper(II) bis(o-acetoxybenzoate) was evaluated in the model of venous thrombosis; its effects on platelet-neutrophil adhesion were investigated by use of rosette assay. The results showed that the intragastrically administered copper-aspirin complex (5, 7, and 10 mg kg-1) dose-dependently lowered the wet and dry thrombus weight; it significantly decreased the binding of arachidonic acid-activated platelets to neutrophils with an IC50 value of 41.5 μmol L-1. The results suggested that copper aspirinate inhibited platelet-neutrophil adhesion and resulted in a more potent antithrombotic activity.

Copper aspirinate (Cu 14.99%, C 51.21%, and H 3.32%; purity > 98%) was synthesized by Kunming Institute of Precious Metals. It was dissolved in 0.9 % saline (pH 6.5). Crystalline aspirin was dissolved in 1% NazCO3 before use. Arachidonic acid (AA) was from Sigma Chemical Co. It was dissolved in 100 mmol L "1 Na2CO3 before use. Z Shen, L. Wu, W. Liu, J. Liu and Z Chen Inhibitory Effects ofDimeric Copper(ll) Bis(o-Acetoxybenzoate) on Platelet-neutrophil Adhesion and Thrombosis Preparation of ligated venous thrombosis SD rats were divided into 5 groups with 10 rats each. A: 0.9 % saline as vehicle group; B: aspirin 20.0 mg / kg as reference; and C E: 5, 7, and 10 mg / kg copper-aspirin complex treated groups. All the above substances were administered intragastrically h before experiment. The method of Chert Changxun et al [4] was used to produce the model of venous thrombosis. Briefly, the rats were anesthetized by ip 30.0 mg / kg sodium pentobarbital. Then a midline incision of the abdomen was made and the inferior vena cava was isolated and ligated below the left renal vein level. The abdomen was then closed. One hour later, the abdomen was reopened. The thrombus in the inferior vena cava was collected into a glass dish for measurement of wet weight. It was then placed in a drying oven at 60C for 20 h before measuring the dry weight. Significance was analyzed by t test.

Preparation of platelets
Blood sample from rat carotid artery was collected into plastic tubes, anticoagulated with 2.7 % EDTA. This sample was spun for 10 min at 180 Ixg to obtain platelet-rich plasma (PRP). PRP was fluter spun to pellet platelets at 1000 tg for 10 rain. Platelet pellets were washed three times and resusroended in phosphate buffer solution (PBS, containing 1.0 % bovine serum albumin and 1.4 mmol L EDNA). CII viability by Typan blue exclusion was above 95 % and cell counter was adjusted to 10 cell L.

Preparation of neutrophils
Neutrophils were isolated from the resulting blood by dextran sedimentation and followed by Ficoll-Hypaque (special gravity 1.077) and hypotonic lysis of erythrocytes. The cell pellet was resuspended in an erythrocyte lysis buffer composed of 155 mmol L q NH4CI, 2.96 mmol L "l KHCO3, and 3.72 mmol L EDTA. The tube was gently inverted and after 5 rain the suasion was centrifuged at 350 tg for 10 rain, and the cell pellet was washed in PBS lacking calcium; then resuded in Hanks' solution (containing mmol L q CaCI2 or 5 mmol L q EGTA in vehicle, reflecting the situation with or without external calcium). Cells were adjusted to a count of 2x106 cell mL q. Cells prepared in this manner contained 98 % neutrophils and were 96 % viable.

Rosette assay
The method of Hamburger et al [5] was modified. Briefly, 50 tL aliquots of platelet suspension mmol L) for 15 min at room were placed in microtiter wells and exposed to arachidonic acid (0. temperature without stirring. Fifty gL of 0.9 % saline or drug solution was added and incubated for 15 mm at 37C. Then 100 xL of neutrophils was added to the platelet suspension and incubated for 30 min at 4C under rocking condition. One hundred neutrophils were scored for the presence (two or more platelets per neutrophil) or absence (zero or one platelet per neutrophil) of platelets. Neutrophils bearing two or more platelets were thus defined as rosettes. For each assay, done in triplicate, the rosetting score was assessed by two different observers.
Platelet-neutrophil adhesion may play key factors in thromboembolic processes and adhesion of these two kinds of blood cells is involved in the process of thrombomodulation [1]. Activation of platelets increases neutrophil adhesion to foreign surfaces, neutrophile aggregation, lysosomal enzyme release, etc. Platelet-derived products are able to promote neutrophil chemotaxsis, enzyme release, and phagocytosis and to inhibit oxidative burst [6]. On the other hand, neutrophil-derived products can enhance platelet aggregation, semtonin release and cytoplasmic Ca -+ movement [7]. It is necessary, therefore, to study an antithrombotic drug based on its influence on multiple cellular interactions.
In venous thrombosis, the copper-aspirin complex showed a dose-dependently inhibition, seven mg kg 1 of copper aspirinate obtained nearly equal antithrombotic effect to 20 mg kg 1 of its parent compoundmaspirin. Obviously, copper aspir;mate exhibited a more potent antithrombosis.
The percentage of rosettes in vehicle was 71.8 or 11.6 % in a condition of external mmol L "1 CaC12 or 5 mmol L EGTA. Copper aspirinate and aspirin significantly decreased the binding of platelets to neutrophils with mmol L " external Ca2+, giving IC50 values 41.5 and 51.4 tmol L , respectively (Table II). Thrombus formation is mediated by the platelet-neutrophil interactions including cell binding and platelet aggregation. In vehicle (0.9 % saline) containing external mmol L I CaCI2 or 5 mmol L EGTA, the percentage of rosettes induced by AA was 71.8 or 11.2 %, demonstrating a calcium dependent relationship between platelet-neutrophil adhesion. Both the copper-aspirin complex and aspirin showed significant inhibition on rosetting between AA-activated platelets and neutrophils, and was more active than aspirin based on the IC50 value. It is suggested that the copper-aspirin complex showed more potent antithrombotic activity than aspirin due to its higher activity on platelet-neutrophil adhesion.
In conclusion, the copper-aspirin complex may be more potential in treating thromboembolic diseases because of its lower gastrointestinal side effects.