Background: Type 2B VWD mutations share a common feature of enhanced association of VWF to platelet GPIbα, but lead to disparate extent of thrombocytopenia in patients. Concurrent homogeneous and heterogeneous effects dictate that a type 2B mutation affect at least two separable properties of VWF. Previous studies have posited that the flanking residues of the VWF A1 domain function as an autoinhibitory regulator of its accessibility, but whether they adopt inherent structural elements or function by steric hindrance has been contested.

Aims: We aim to characterize how type 2B mutations alter conformational dynamics of A1 and flanking residues, with the broader goal of defining additional alterable properties of VWF.

Methods: Stability and dynamics of mammalian A1 fragments (residues 1238-1493) bearing type 2B mutations were analyzed using thermal shift assays, hydrogen deuterium exchange mass spectrometry, and analytical ultracentrifugation. Binding and functional activity of these variants were assessed via biolayer interferometry and platelet aggregometry.

Results: Of the seven mutant A1 fragments that are being studied, all are primarily monomers and have unfolding temperatures significantly lower than WT. Interestingly, most mutants including H1268D and R1341Q, but not R1306W, undergo two thermal transitions. All mutants exhibit slower dissociation rates from immobilized recombinant GPIbα (Figure 1) and spontaneously aggregate washed platelets at 60 nM concentration albeit to various degrees. Lastly, compared to WT, some mutants such as H1268D has increased deuterium uptake in flanking residues beyond Asp1261 and Asp1472 and in residues of α1β2, β3α2, and α3β4 loops during 10-30s exchange time frame.

Conclusions: Type 2B mutations reduce global stability of the A1 domain (including flanking residues) and, in some cases, enhance exposure of the secondary GPIbα binding site. The increased deuterium uptake of flanking residues beyond Asp1261 and Asp1472 in some type 2B mutants suggest that these flanking residues adopt certain structural elements that may contribution to VWF autoinhibition.

[Figure 1. Binding Kinetics of A1 Variants]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/type-2b-mutations-differentially-alter-conformational-dynamics-of-vwf-a1-domain/