Enhanced Local Disorder in a Clinically Elusive von Willebrand Factor Provokes High-Affinity Platelet Clumping

Abstract

Mutation of the cysteines forming the disulfide loop of the platelet GPIbα adhesive A1 domain of von Willebrand factor causes quantitative VWF deficiencies in the blood and von Willebrand disease. We report two cases of transient severe thrombocytopenia induced by DDAVP-treatment. Cys1272Trp and Cys1458Tyr mutations identified by genetic sequencing implicate an abnormal gain-of-function phenotype, evidenced by thrombocytopenia, that quickly relapses back to normal platelet counts and deficient plasma VWF. Using surface plasmon resonance, analytical rheology, and hydrogen-deuterium exchange mass spectrometry (HXMS), we decipher mechanisms of A1-GPIbα mediated platelet adhesion and resolve dynamic secondary structure elements that regulate the binding pathway. Constrained by the disulfide, conformational selection between weak and tight binding states of A1 takes precedence and drives normal platelet adhesion to VWF. Less restrained through mutation, loss of the disulfide preferentially diverts binding through an induced-fit disease pathway enabling high-affinity GPIbα binding and firm platelet adhesion to a partially disordered A1 domain. HXMS reveals a dynamic asymmetry of flexible and ordered regions common to both variants indicating that the partially disordered A1 lacking the disulfide retains native-like structural dynamics. Both binding mechanisms share common structural and thermodynamic properties, but the enhanced local disorder in the disease state perpetuates high-affinity platelet agglutination, characteristic of type 2B VWD, upon DDAVP-stimulated secretion of VWF leading to transient thrombocytopenia and a subsequent deficiency of plasma VWF, characteristic of type 2A VWD.

Platelet‐type von Willebrand disease: Local disorder of the platelet GPIbα β‐switch drives high‐affinity binding to von Willebrand factor

“…Viewed from a dynamics perspective, the β‐switch acts like an opposable thumb capable of regulating the binding of A1. The hydrogen‐deuterium exchange data support a mechanism by which the β‐switch exists as a coil which fluctuates between closed and open conformations (Figure ).…”

“…The A1 domain of VWF (Q 1238 ‐P 1471 ) was expressed in Escherichia coli M15 cells as fusion proteins containing an N‐terminal hexahistidine‐tag using BamHI and HindIII restriction sites in the Qiagen pQE‐9 plasmid . All proteins were purified and their quality was confirmed by RP‐HPLC and analytical gel filtration as described previously …”

“…Hydxogen‐deuterium exchange mass spectrometry involves steady‐state time‐dependent HX followed by acid quenching, pepsinolysis, reverse phase separation of proteolytic fragments, and electrospray ionization orbitrap mass spectrometry . HXMS was performed on WT GPIbα and on the mutants as recently published using a setup as described by Mayne et al HX samples were prepared by 1‐5 dilution of GPIbα into TBS in D 2 O (pD 7.4). After defined times (1 minute to oven incubation at 25°C) the exchange was stopped by a drop in pD to 2.7.…”

“…Prior to the HX experiments, peptide maps containing a pool of searchable peptides for all GPIbα variants were generated using Bioworks 3.3.1 (Thermo Fisher Scientific) and the Matlab‐based software EXMS 2 as previously described . Not all of the peptides in the pool are guaranteed to be identified in any given experimental measurement.…”

“…All structures of A1 are highly similar in the conformation of the backbone chain, but detailed structural analyses of the complex crystal structure suggest that the rearrangement of intermolecular electrostatics between residue side chains alter the dynamics of the α1β2 loop causing a pivot of the α2 helix . Such a conformational propensity is supported by recent hydrogen‐deuterium exchange experiments and is thought to be “on—pathway” toward high—affinity …”