Process for separation and isolation of ahf, von willebrand's ristocetin

Abstract

A process for the separation and isolation of AHF, fibronectin and von Willebrand's ristocetin cofactor from blood plasma by adding a sulfated mucopolysaccharide to blood plasma to a concentration of from about 0.05 to about 0.4 mg/ml of plasma, cooling the plasma to 0.degree. C.-15.degree. C., centrifuging the plasma to isolate a precipitate which forms and then removing the AHF-rich supernatant liquid from the precipitate. The precipitate is washed in a slightly basic solution of dilute salts and then dissolved in a buffered solution of chaotrophic salts of the Hofmeister series having an ionic strength from 0.01-1. Fibronectin is separated by chromotographic means from the dissolved precipitate which contains fibronectin and fibrinogen. The dissolved precipitate may also be applied to a glass or plastic column containing a 4% agarose gel (E. G. Sepharose 4B or Biogel A-15m) in a 0.25 molar solution sodium phosphate titrated to pH 7.0 with any base, preferably TRIS. The von Willebrand's ristocetin cofactor elutes in the void volume of the column, whereas the fibronectin-rich fraction elutes later. Both fractions may be collected and concentrated. in a preferred embodiment, 0.20 mg of sodium heparin per milliliter of human plasma is employed with the heparinized plasma solution cooled for 3 hours at a temperature of 2.degree. C.-4.degree. C.