(64g) Effects of Fluid Shear on the Conformation of Human Von Willebrand Factor Studied by Neutron and Light Scattering

Von Willebrand factor (vWF) is the largest protein found in human blood circulation. It plays an important role during blood coagulation and thrombosis in arteries. This protein is unique in that like polymers, it has well-organized repeat structures. Each of these repeat structures or monomers has molecular weight of 250KDa. The final multimeric or polymeric vWF has molecular weight that exceeds 10,000kDa in normal humans. The function of vWF is known to be regulated by hydrodynamic stresses that are both physiological and pathological. Application of such shear forces has been shown to augment the binding affinity of multimeric vWF for its platelet glycoprotein receptor GpIb, increase vWF proteolysis via metalloprotease ADAMTS-13 and augment blood platelet adhesion in vascular injury models that mimic atherosclerotic plaque rupture. Thus there is considerable interest in understanding the effects of fluid forces on vWF structure and function.

In the current paper, we coupled static and dynamic light scattering with small-angle neutron scattering (SANS) to measure the solution structure of vWF that was isolated from human blood plasma. The response of this protein to defined hydrodynamic stresses was also measured. While light scattering allowed measurement of overall protein size, SANS allowed investigations at smaller length scales. Results suggest that vWF in solution, under static conditions, exists as an extended rod-like molecule with an ellipsoid minor axis radius of ~28nm and a major axis radius of ~175nm. These studies also reveal the presence of 5nm globular regions that correspond to micro-domains within the molecule. When laminar shear was applied to vWF in the shear rate range from 300/s-3000/s and protein structure was measured in real time using SANS, we observed for the first time that blood proteins may undergo conformation changes in response to hydrodynamic shear. These structural changes took place in a time-dependent manner at length scales<10nm. This suggests that protein domain level interactions may be altered by hydrodynamic shear. Seven different vWF pools from humans with different blood-groups displayed similar structural changes suggesting that our observations are blood-group independent. Further, changes in neutron scattering patterns were specific to vWF since a smaller protein bovine serum albumin (BSA) did not undergo similar changes upon application of fluid shear. In addition to these structural changes at small length scales, light scattering analysis of vWF subjected to shear in a cone-plate viscometer demonstrate that pathological shear stresses may cause the aggregation or self-association of vWF. Overall, our studies provide the first evidence of protein conformation changes in response to fluid shear. The findings support a model where domain level conformational changes in vWF caused by hydrodynamic shear may augment the exposure of protein hydrophobic domains that are involved in vWF self-association.