Effect of fluid shear on VWF size.
Purified VWF (125 μL) at approximately 0.1 mg/mL was maintained under static conditions or was sheared in a viscometer at specified shear rates for fixed times. (A) Samples were diluted 8-fold in HEPES buffer and immediately subjected to SLS. MWt and Rgwere determined from this plot. MWt equals 1/(y-axis intercept). An increase in VWF homotypic aggregate size and MWt was observed with shear and time. Data are mean ± SEM for 2 shear runs with a single batch of VWF. (B) Western blot analyses of unsheared and sheared samples demonstrate qualitative increases in VWF size. Lane 1 indicates unsheared VWF; lane 2, VWF sheared at 2155/s for 30 seconds; lane 3, VWF sheared at 6000/s for 120 seconds. An equal amount of protein was loaded in each lane. (C) Densitometry tracing of the Western blot in panel B. Dashed line corresponds to highest MWt of the unsheared sample. Application of shear causes larger aggregates to appear to the left of this line. Results are representative of 3 independent experiments.
