A STAINING METHOD FOR ELECTROPHORETICALLY SEPARATED CLOTTINGFACTORS

THIS PROJECT WILL DEVELOP AN ELECTROPHORETIC STAINING METHOD FOR CLOTTING FACTORS. THE METHOD MEASURES COAGULATION USING A COLORIMETRIC SYSTEM KNOWN AS ENZYME-LINKED COAGULATION ASSAY (ELCA). ELCA IS BASED ON THE SIMULTANEOUS CONVERSION OF SOLID-PHASE-ASSOCIATED FIBRINOGEN AND ENZYME-LABELED FIBRINOGEN IN SOLUTION BY THROMBIN, WHICH IS GENERATED IN OR ADDED TO THE SOLID-PHASE-ASSOCIATED MIXTURE. IN THIS STUDY, NITROCELLULOSE MEMBRANES WILL EITHER BIND THE CLOTTING FACTOR BY BLOTTING TRANSFER OR BE USED AS A SUBSTRATE VEHICLE FOR IDENTIFICATION OF THE LOCATION OF BANDS FROM ELECTROPHEROGRAMS; THE MEMBRANES WILL BE FIBRINOGEN COATED AND BLOCKED WITH CASEIN. ELCA ASSAYS WILLBE PERFORMED BY ADDING SUBSTRATE CONTAINING ALL FACTORS NEEDED FOR ASSAY OF A PARTICULAR FACTOR INCLUDING LABELED FIBRINOGEN. TWO PROTOCOLS FOR IDENTIFICATION OF THE SEPARATED COMPONENTSWILL BE USED. IN THE FIRST, THE SOLID PHASE WILL HAVE BOUND TO IT CLOTTING FACTORS TRANSFERRED FROM NONDENATURING ELECTROPHEROGRAMS; IN THE SECOND, THE SOLID PHASE WILL BE OVERLAID DIRECTLY ONTO POLYACRYLAMIDE GELS WITH SEPARATED CLOTTING FACTORS. THIS WILL PERMIT THE FUNCTIONAL IDENTIFICATION OF ACTIVE CLOTTING FACTORS ON ELECTROPHEROGRAMS. THIS WILL BE OF USE IN THE IDENTIFICATION OF GENETIC VARIANTS, ACTIVE AND ACTIVATED CLOTTING FACTORS IN PLASMA SAMPLES, AND PARTIALLY CARBOXYLATED CLOTTING FACTORS FROM COUMADIN-TREATED PATIENTS.