Project Summary Von Willebrand factor (VWF) is a multimeric blood glycoprotein that plays an important role in hemostasis and thrombosis. Platelet glycoprotein (GP)Ibα binds to the A1 domain of VWF, but only under high shear stress. This event triggers platelet activation and clot formation. We do not know how A1 can respond to only high shear. Recent papers suggest that the flanking regions that surround the A1 domain are responsible. These regions work together to shield the GPIbα-binding site on A1, acting as an autoinhibitory module (AIM) during normal blood flow. We hypothesize that force will dissociate the AIM, and that the activation of A1 is actually dissolution of the AIM-A1 interface. As the AIM is a discontinuous sequence, a tensile force applied would stretch the entire sequence, and disrupt the AIM. This leads to the possibility that stabilizing the AIM would allow for increased resistance to activation but would not directly interfere with the GPIbα-VWF interaction. Therapeutics that target GPIbα or A1 exhibit a bleeding phenotype, as this crucial interaction is prevented. Targeting the AIM would exhibit indirect modulation of the GPIbα-VWF interaction, whereby the AIM could still be dissociated by immense shear stress during injury. We hypothesize that under high shear, a critical tensile force is applied to VWF, that would abolish the AIM-A1 interface, allowing for exposure of A1. Specific Aim 1 is to define the dynamic response of the A1-AIM interface to tensile force. By investigating the structure and positioning of the AIM at the molecular level, we will determine the forces necessary to disrupt the AIM-A1 interaction and observe A1 binding of GPIbα. Specific Aim 2 is to determine if stabilization of the AIM will modulate VWF response to shear flow. The AIM may be utilized by endogenous regulatory proteins, or exogenous therapeutics targeted to the AIM, to impede VWF activation, but not hinder the binding event of VWF to platelets. We hypothesize that stabilization of the AIM is dependent on the shear force applied to VWF, and under conditions of high shear force, these effects may be reversible. The proposed studies will allow for wide-reaching investigation from the single molecule level to ex vivo blood simulation and allow for training opportunities to in multiple disciplines. Overall, completion of the proposed studies would provide support for the AIM as a therapeutic target for thrombotic diseases with minimal bleeding side effects.