(618b) Shear-Induced Structural Changes in Blood Proteins

The physiological and pathological functions of blood proteins are imparted by changes in their structure. Von Willebrand factor (VWF) is a large, multimeric, multidomain human blood glycoprotein. The protein plays an important role in arterial thrombosis by aiding platelet deposition at sites of vascular injury. Motivated by this major disease, we examine the stability and organization of VWF in solution by a combination of biomedical techniques, polymer characterization and mathematical modeling. Of particular interest to our investigation are the identification of structural domains that are most susceptible to perturbation/change (fluid shear and denaturation), and the extent to which interactions between the individual domains within VWF contribute to its overall structure.

We examine the biophysical and biological features regulating the structure and the size of VWF in solution, both at static (no fluid flow) and under fluid shear conditions by employing small angle neutron scattering (SANS) and fluorescence methods. Here, we focus on shear-dependent conformational changes of VWF ascertained by the fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid dipotassium salt (bis-ANS) that partitions into the protein hydrophobic domains. The fluorescence intensity due to probe-VWF interaction increased, suggesting that new hydrophobic domains were exposed upon fluid shear application at shear rates greater than 2300-6000/s and at shear times greater than 1min. SANS studies at higher resolution revealed structural changes in VWF commencing at shear rates below 3000/s and at length scales less than 10nm. Such fluid shear-induced conformational changes are specific to VWF on the basis of control experiments on bovine serum albumin. Relaxation of the VWF structure was observed over the course of minutes following cessation of shear, giving lower signal due to bis-ANS binding to VWF. Taken together, our data suggest that changes in VWF conformation, at small-length scales and physiological shear stresses, likely precede protein unfolding and enhanced bis-ANS binding at higher shear rates. Changes in structure at high shear rates are partially reversible. The structural changes in VWF conformation reported here likely regulate protein function in response to fluid shear application. The findings emerging from this work may be applicable to other blood proteins also. Further, these shear-mediated features may regulate the cascade of events that contribute to arterial thrombosis and other blood coagulation processes.