A B S T R A C T Human factor VIII froml nornmals anid hemophiliacs was partially purified by ethaniol and polyethylene glycol precipitations. Final purification was achieved by gel filtration on 2 or 4%Xc agarose or ion1 exchange chromatography on diethylaminoethyl cellulose. Comparable amounts of highly purified protein were obtained from normal and hemophilic plasma following the agarose chromatographv step. Highly purified factor VIII was not dissociated by 6 -MI guanidine hydrochloride or 1% sodium dodecyl sulfate. However, when reduced by P-mercaptoethanol and analyzed by sodium dodecyl sulfate polvacrylamide gel electrophoresis, a single subunit species with an estimiiated 19-5,000 molecular weight was found for both normcal an(d hemiiophilic factor VIII. By sedimiientation equilibrium analysis, the normal factor V-III subunit was homogeneous and had an estimated nmolecular wveight of 202,000. The subunit polypeptides from niormal or hemoplilic factor VIII contained carbohy\drate. Each was homogeneous by isoelectric focusing. Immilllunodiffusion of purified normal and hemoplhilic factor VIII against rabbit aintiserum to purified normiial liuman factor VIII slhowed a single line of precipitationi. V-erv low concenitr-ations of purified humiian thrombin initially increased the activitv of normal factor VIII about threefold and tlhen progressively destroyed activity by 3 h. Only-minimal activation occurred with hemiiophilic factor VIII. Both the activation and inactivation of normal alnd hemo-

The activated partial thromboplastin time (APTT) has been advocated for monitoring heparin effect. This study was designed to determine the in vitro sensitivity to heparin of commercially available APTT reagents. Heparin was added in increasing concentrations to pooled citrated plasma. Fibrometer APTT determinations were performed at each concentration using General Diagnostics, Ortho, Dade, Hyland, and BBL reagents. A tilt-tube kaolin-activated partial thromboplastin time was also tested using a Sigma partial thromboplastin prepared by the method of Bell and Alton. The General Diagnostics, Sigma, and Ortho reagents displayed linear heparin sensitivity; the General Diagnostics APTT was prolonged 1 1/2-2 1/2 times in a plasma heparin range that prolonged a modified in-vitro Lee-White clotting times 2-3 times. The other reagents were either insensitive, too sensitive, or nonlinear in heparin response. Thus, commercial reagents vary widely in their in-vitro sensitivity to heparin.