Lyophilized BV was prepared at the Venomous Animals Department, Razi Vaccine and Serum Institute, and was kept at — 20˚C. Sephadex G-50 was purchased from Pharmacia (Sweden). Calcium chloride (CaCl2) and protrombin time (PT) and partial thromboplastin time (PTT) Kit products were purchased from Fisher Diagnostics (Germany). The other reagents and chemicals of analytical grade were purchased from Fluka and Merck.
Blood samples were extracted from 10 healthy young rabbits of both sexes with no history of blood disease. Blood samples from these rabbits were centrifuged for 15 minutes at 3,000 rpm. The obtained plasma was separated and was used for the PT and PTT testing.
Tissue thromboplastin was reconstituted according to instructions and was labeled with the time, date and initials of the technician. The thromboplastin reagent was stabile for seven days after reconstitution. The sample was allowed to sit 10 — 15 minutes and was than inverted gently several times. The reagents were mixed well prior to pipetting any of them reagent at any step in this procedure.
One to two mls of the tissue thromboplastin- CaCl2 reagent (PT reagent) was pipetted into a test tube, and the test tube placed into an incubator at 37˚C. The level of the thromboplastin was not allowed to exceed the height of the heat block. Normal rabbit plasm, 100 μL was poured into the test tubes and at least one minute was allowed for the plasma to reach 37˚C. Then, 200 μL of the PT reagent was poured into the tube containing the rabbit plasma, and simultaneously stop watch was started.
The solution in the tube was mixed and left in the heat block for a minimum of 7 — 8 seconds. It was then removed, and its exterior was wiped. The tube was then tilted back and forth gently until a visible clot formed. As the clot formed, the mixture began to gelatinize and turn cloudy. The stop watch was immediately stopped when the clot began to form, and the time in seconds was recorded .
A sufficient quantity of CaCl2 reagent was heated to 37˚C for the tests to be performed. Normal rabbit plasma, 100 μL, was poured into a labeled test tube, and 100 μL of partial thromboplastin reagent was added. The plasma/partial thromboplastin mixture was incubated at 37˚C for a minimum of three minutes, and CaCl2 100 μL, was forcefully added into the plasma/partial thromboplastin mixture; a stop watch was started immediately.
The mixture in the tube was stirred once after the calcium reagent had been added, and the tube was allowed to remain in the heat block for approximately 20 seconds while being stirred occasionally. After 20 seconds, the tube was removed from the water bath/heat block, and its outside was dried. The tube was then gently tilted back and forth until a visible clot formed, at which time the stop watch was immediately stopped and the time in seconds was recorded .
The concentrations of the proteins in the BV were measured by using the method of Lowry with bovine serum albumin (BSA) as the standard . Standard curves were constructed by measuring the concentration of the BSA solution; then, the amounts of the proteins in the crude BV were obtained. In addition, 10 mg of BV were used to prepare samples with concentrations of 1 to 4 mg/mL. The samples with these concentrations were the used to assess the effect of BV on clotting with PT.
The lethal dose 50 (LD50) of the Iranian crude BV was determined in mice (18 to 20 g National Institutes of Health (NIH) strain; NIH mouse are albino outbred mice which were derived from a nucleus colony obtained from the NIH). The sample at each dose was prepared in physiological sterile serum and was injected into the mice. For the selection of an appropriate dose, several different doses were injected into two mice per dose up to lethality. The doses were then chosen so that for the first dose, no mice died, and for last dose, all animals died. When different doses were prepared for each sample, 2 mL of each dose (0.5 mL for each mouse) were intravenously into four mice. Then, the mortality of the mice was recorded for 24 hours. The LD50 values of each sample were calculated by using the Spearman-Karber Finney statistics .
The anti-coagulation factors from BV were purified by using chromatographic techniques. To separate the coagulation factor, we dissolved 300 mg of the crude BV in 4 mL of ammonium acetate buffer, which was then centrifuged for 15 minutes at 14,000 rpm. The impurities were removed by using a micro filter 0.45. The gel chromatography column was equilibrated by using 20 mM ammonium acetate buffer (pH 4.75). After the column had been equilibrated, the protein solution was applied on sephadex G-50 (3 × 200 cm); then, the column was eluted with the same buffer. The absorption of each fraction was read at 280 nm. All the fractions were dialyzed for 24 hours against distilled water and were concentrated at 4˚C. After concentration, the intensities of peaks were accurately measured. The PT test was done for peaks (fractions) with the same concentration and fractions that showed anti-coagulation activity were detected [16, 17].
To confirm the presence of coagulation and anti-coagulation activities, we performed PT tests with the same concentrations, 20 μg/mL, for the fractions obtained by using gel chromatography. For determining the molecular weight and the purity of the crude BV and the fractions obtained by using the gel chromatography technique, we performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the method of Laemmli .
Analyses to determine the means, standard deviations (SDs) and P-values of the test data were performed using the software mini tab. P-value was calculated, and a P-value less than 0.05 was meaningful.