Regulation of protein structure and function under fluid shear: Role of Von Willebrand factor in thrombosis and hemostasis.

摘要

Cardiovascular diseases are one of the major causes of morbidity and mortality in the developed nations. Continuous efforts by numerous laboratories all around the world have been devoted to find the perfect cure for these diseases, but still perfect drug targets for diseases such as heart attack and stroke have still not been identified. As of today this field stands in its early development stages only. There are numerous questions in this field of science which still require answers and solution to this growing problem.;Related to this vast area of science, we tried to study the role of blood proteins and their interaction in the context of thrombosis and hemostasis. My Ph.D. dissertation examines the effect of fluid or hydrodynamic forces on protein structure and function. During the course of these investigations, we have developed novel spectroscopy tools to determine the role of fluid flow in regulating protein structure and self-association/aggregation properties. Many of the studies are performed with a large multimeric protein isolated from human blood called Von Willebrand Factor (VWF). The study is important since it is established that the level and activity of VWF is associated with many vascular diseases including acute coronary syndromes. VWF also plays a key role during thrombosis that is associated with myocardial infarction and stroke. Further, strategies to control the interaction of VWF with its receptor on blood platelets (integrin GpIba) are of interest in the biotech community since this is a druggable target.;Using Small Angle Neutron Scattering(SANS) for the first time we have provided the solution structure of Von Willebrand Factor and we have also shown that this multi domain protein structure is stabilized by non covalent inter domain interaction. We further applied the combined usage of SANS and fluorescence spectroscopy to elucidate for the first time that blood protein can undergo conformational changes in solution under the effect of fluid shear forces (shear rate 2300/s). Depending on the amount of shear forces applied blood proteins can undergo changes from smaller length scales to protein unfolding and hydrophobic domain exposures which may have physiological significance. Using various cell adhesion assays and the application of multi color flow cytometry we have also shown that blood protein interaction with platelets are specific in nature and platelet activation primarily follows the platelet receptor GpIb and VWF-A1 domain interaction which is further supported by VWF-platelet GpIIbIIIa interaction. We have also shown for the first time that under high fluid shear (shear rate >6000/s) conditions VWF binding to platelets follows a pathway where at first VWF self associates either on platelet surface or in solution and this large protein aggregate binding on platelet surface results in firm VWF binding to platelet receptor GpIb and augments platelet activation.;These findings support the idea that shear forces play a critical role in thrombus formation by inducing conformational changes in blood proteins. Opposed to the traditional belief of only surface mediated protein conformational changes our findings also indicate towards the development of a new hypothesis where protein conformational changes in solution are also of physiological relevance and these changes may have a substantial role in thrombosis.