Abstract

Von Willebrand Factor (VWF) initiates platelet adhesion at sites of vessel injury via the binding of platelet glycoprotein (GP) Ib-IX-V surface receptor to the VWF A1 domain. Congenital mutations occurring in the A1 domain result in the clinical bleeding diathesis of several subtypes of Type 2 Von Willebrand Disease. These mutations can either enhance VWF-platelet interactions (Type 2B) or cause a defect in this interaction (Type 2M). We hypothesize that these functional types of mutations could differentially alter VWF-platelet interactions in part by affecting the intrinsic stability of the A1 domain structure and consequently, a conformational transition induced by these interactions. In this study, we have characterized the thermodynamics of unfolding the A1 domain containing the Type 2B mutations (R1306Q, R1308L, and I1309V), the Type 2M mutation (G1324S) and a blocked disulfide variant of A1, (reduced and carboxyamidated, RCAM A1), that is representative of the Type 2M disulfide bond mutations, C1458Y and C1272(G,R,S). Using a combination of chemical and thermal denaturation monitored by circular dichroism spectroscopy and differential scanning calorimetry, we obtained a complete description of the thermodynamic stability of A1. Urea and guanidine-HCl denaturation show that the native conformation of A1 reversibly unfolds through a structurally stable intermediate, N·I·D, with two distinct conformational transitions that are widely separated in denaturant concentration. Thermal denaturation in the absence of denaturant shows that A1 unfolds via the two-step Lumry-Eyring mechanism, N·I→F, in which the reversible native to intermediate conformational transition is followed by the irreversible conversion of the intermediate to a final state that is unable to fold back to the native conformation. The clinically relevant mutations investigated affect only the thermodynamics of the first transition, N·I, suggesting that this conformational transition is important for the binding of A1 to the platelet GP1bα component of the GPIb-IX-V surface receptor. While the effects of Type 2B mutations on stability are moderate, the effects of the Type 2M mutations are extreme. G1324S increases the stability two-fold over wild type resulting in an increased concentration of urea required for unfolding and an increased melting temperature. Conversely, RCAM A1 completely destabilizes the native conformation resulting in a structure that resembles the intermediate conformation in the absence of denaturant. In conclusion, these thermodynamic observations parallel the clinical phenotypes associated with the mutations studied. The moderate effects of Type 2B mutations on A1 stability suggest that these mutations may impair quaternary interactions between A1 and neighboring VWF domains and enhance VWF-platelet interaction by exposing the GP1bα binding site. In contrast, Type 2M mutations cause extreme effects on stability. G1324S increases A1 stability and specifically reduces its conformational entropy, resulting in a more rigid structure with deficient capacity to interact with platelet GP1bα. RCAM-induced loss of the A1 disulfide bond dramatically destabilizes the native conformation, resulting in improper domain folding and defective interaction with platelet GP1bα.

Disclosures: No relevant conflicts of interest to declare.

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