Disintegrin Assays

Sonoclot Analyzer

  • A glass bead activated test (gbACT + Kit obtained from Sienco, Inc.) is used to monitor clot detection, fibrin formation and platelet function (clot retraction) on a Sonoclot® Coagulation and Platelet Function Analyzer (Sienco, Inc.).

  • The 10% citrated human blood is incubated at 37˚C for least 5 min prior to use.

  • While blood is incubating, the cuvette is placed into the cuvette holder and 13 µl of 0.25M CaCl2 is added to one side of the cuvette.

  • Ten microliters of venom fraction is added to the other side of the cuvette.

  • Three hundred microliters of warm citrated human blood is added to the cuvette.

  • The head assembly of the analyzer is closed 10 s after the start button is depressed.

  • Data acquisition is analyzed with Signature Viewer™ software provided by Sienco, Inc. on an iMac computer.

Inhibition of Platelet Aggregation Assay

  • A Chronolog™ is used to monitor platelet aggregation, by impedance, of whole human blood when venom samples are added.

  • Four hundred and fifty microliters of 10% citrated human blood is incubated to 37˚C at least 5 min prior to use with equal amounts of 0.15M Saline solution.

  • Ten microliters of venom sample are incubated with the blood sample for 2 min.

  • An electrode is inserted in the blood sample, and a minute and a half later, 20 µl of a 1mM ADP solution is added to the blood sample to promote platelet aggregation.

Cellular Adhesion Inhibition Using T24 Cells

  • Triplicate wells of a 96-well plate are coated with matrix ligands (fibronectin, fibrinogen, collegan IV, laminin or vitronectin) at 1, 10, and 100 µg in 0.01M Phosphate buffer saline (PBS), pH 7.4, and incubated overnight at 4˚C.

  • The plate is blocked with 0.2 mL of PBS in 5% Bovine serum albumin (BSA) and incubated at 37˚C for 1 h.

  • Cells are harvested, counted and resuspended in medium containing 1% BSA at 5 x 105 cells/mL.

  • Disintegrins are added to the cell suspension at various concentrations and allowed to incubated at 37˚C for 1 h.

  • The blocking solution is aspirated and the cell/disintegrin suspensions (0.2 mL) are added to the wells coated with matrix and incubated at 37˚C for 1 h.

  • The solution is aspirated with the exception of the positive controls and washed three times with PBS-1%BSA by filling and aspirating.

  • The plate is spun for 5 min at 1000 rpm on a Beckman Coulter AllegraTM 6R centrifuge and the supernatant is aspirated from the positive control group.

  • A total of 0.2 mL of medium in 1% BSA containing 3-[4,5-Dimethylthiazol-2-yl]2,5-diphenltetrazolium bromide (MTT) (5:1 vol/vol) is added to the wells containing cells and incubated at 37˚C for 2 h.

  • The plate is spun at 1000 rpm for 5 min to pellet cells and the medium is aspirated.

  • A total of 0.1 mL of Dimethyl sulfoxide (DMSO) is added to the wells to lyse the cells.

  • The plate is shaken gently and the absorbance is read at 570 nm using a Beckman Coulter AD340 reader.