Anticoagulant properties of Apis mellifera (honey bee) venom

A vAlt>srY of animal venoms, mostly of snake origin, modify blood coagulation (HOUSSAY, 1930; L~ et al., 1955 ; OUYANG, 1957 ; BoQusr, 1964 ; MF,AiJME, 1966 ; ROSENFELD et al., 1968 ; JI z-PO1tRAS, 1970; LEE, 1975 ; Tu, 1977) . JOSHUA and I3HAY (1975) demonstrated that an extract from the venom sac of the oriental hornet (Vespa orientalis) had an anticoagulant effect on blood coagulation. HABERMANN (1954) reported that honey bee (Apia mellifera) venom inhibited blood coagulation through inactivation of tissue thromboplastin probably by its phospholipase A component . Since the effect of this venom on other clotting factors is not well known, it was of interest to investigate the anticoagulant action of this venom and elucidate its mode of action . Apis mellifera venom, crude cephalin (chloroform extract of acetone-dried rabbit brain) and Russell's viper venom in crude cephalin were obtained from Sigma Chem. Co . Thrombin (bovine) was purchased from Parke-Davis Co. and dissolved in 50~ glycerolsaline to give a stock solution of 1000 N.LH . p/ml (S~GGI~ts and SMrrH, 1942) . Fibrinogen (bovine) was purchased from Pentex Co. and dissolved in imidazole-saline (pH 7"4j and the stock solution of 20 mg/ml was stored at -70°C before use . Rabbit brain thromboplastin was prepared according to the method of BIGGS and MACi?A1tLAN13 (1962) . Plateletpoor plasma was obtained from citrated blood (1 :9, v/v) after centrifugation for 20 min at 3000 rpm and 4°C . Blood collected from the rabbit marginal ear vein through a 19-gauge needle was mixed with 0'1 M Na9EDTA (1 :14, v/v) as anticoagulant and centrifuged for 10 min at 800 g and at room temperature . The platelet pellet was washed with Tris-buffered saline (pH 7"4) containing NaEEDTA (3 mM), apyrase (ADPase activity, 0~5 U/ml) and bovine serum albumin (1 mg/ml) and recentrifuged (modification of the methods of MUSTARD et al., 1972 and ARDIJE et al., 1971) . This wash procedure was repeated two times and the washed platelets were suspended in Tris-buffered saline, ruptured with a stirrer and then frozen and thawed three times before use . Whole blood clotting time was determined by the method of LEe and WFnre (1913) . Calcium clotting time of plasma was carried out as described by BIGGS and MACFARLANE (1962). The one-stage method of Qulcx (1938) was used to measure the plasma prothrombin time . Partial thromboplastin time was measured by the method of NYS et al. (1962) .