Effect of Thrombin on Factor VIII/VON Willebrand Factor (FVIII/vWF)

Abstract

The sodium dodecyl sulfate (SDS) gels and immunological properties of FVIII/vWF and thrombin-activated (thr-act) FVIII/vWF are identical. Hence thrombin must either modify FVIII/vWF by very minor proteolysis or cleave only a few FVIII/vWF molecules. The procoagulant (PC) activity of FVIII/vWF and thr-act FVIII/vWF eluted sharply in the void volume (Vo) from 4% agarose in 0.15 M NaCl, with >65% loss of the PC activity of the thr-act FVIII/vWF by 3 hrs. The PC activity of FVIII/vWF and thr-act FVIII/vWF was stabilized by 0.25 M CaCl2. When FVIII/vWF and thr-act FVIII/vWF were filtered on 4% agarose in 0.25 M CaCl2, the protein eluted in the Vo, but most of the PC activity eluted later in a region of little detectable protein; however, the delayed PC peak from thr-act FVIII/vWF was greatly enhanced above control levels. Nonreduced, neither activity peak protein entered a 7.5% SDS gel. After reduction, the gel pattern for the PC activity peak protein from FVIII/vWF showed major bands of 195x, 79x, 61x, 51x and 18x (103) molecular weight (MW) and several minor bands >100,000 MW. The reduced PC protein from thr-act FVIII/vWF lacked all bands >100,000 MW, but the four lower MW bands were present and well-resolved. Thrombin activation did not affect the vWF activity which was proportional to protein concentration throughout any chromatogram; the 195,000-dalton subunit was not necessary for vWF activity. FVIII/vWF, which had the peak PC activity in the Vo and no other PC activity peak on 4% agarose-0.25 M CaCl2, was thr-act before filtration under the same conditions. Then, the greatly enhanced PC activity eluted well after the Vo, with <10% PC activity in the Vo. We conclude that modification of a few FVIII/vWF molecules by thrombin and their stabilization by 0.25 M CaCl2 causes the PC peak that elutes aberrantly from 4% agarose-0.25 M CaCl2.