Desialylation of O-glycans activates von Willebrand factor by destabilizing its autoinhibitory module.

BACKGROUND: The binding of the A1 domain of von Willebrand factor (VWF) to platelet receptor glycoprotein (GP)Ibα defines the VWF activity in hemostasis. Recent studies suggest that sequences flanking A1 form cooperatively an autoinhibitory module (AIM) that reduces the accessibility of the GPIbα binding site on A1. Application of a tensile force induces unfolding of the AIM. Desialylation induces spontaneous binding of plasma VWF to platelets. Most O-glycans in VWF are located around the A1 domain. Removing certain O-glycans in the flanking sequences by site-directed mutagenesis enhances A1 binding to GPIbα and produces an effect similar to type 2B von Willebrand disease in animals. OBJECTIVES: To understand if and how desialylation of O-glycans in the flanking sequences increases A1 activity. METHODS: A recombinant AIM-A1 fragment encompassing VWF residues 1238-1493 and only O-glycans was treated with neuraminidase to produce desialylated protein. The glycan structure, dynamics, stability, and function of the desialylated protein was characterized by biochemical and biophysical methods and compared to the sialylated fragment. RESULTS: Asialo-AIM-A1 exhibited increased binding activity and induced more apparent platelet aggregation than its sialylated counterpart. It exhibited a lower melting temperature, and increased hydrogen-deuterium exchange rates at residues near the secondary GPIbα binding site and the N-terminal flanking sequence. Asialo-AIM-A1 is less mechanically stable than sialo-AIM-A1, with its unstressed unfolding rate approximately 3-fold greater than the latter. CONCLUSIONS: Desialylation of O-glycans around A1 increases its activity by destabilizing the AIM.

Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A.

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

Short-Acting Anti-VWF (von Willebrand Factor) Aptamer Improves the Recovery, Survival, and Hemostatic Functions of Refrigerated Platelets.

OBJECTIVE: Refrigeration-induced binding of VWF (von Willebrand factor) to platelets contributes to the rapid clearance of refrigerated platelets. In this study, we investigate whether inhibiting VWF binding by a DNA-based aptamer ameliorates the clearance of refrigerated platelets without significantly impeding hemostatic functions. Approach and Results: Platelets were refrigerated with or without aptamer ARC1779 for 48 hours. VWF binding, the effective lifetime of ARC1779, platelet post-transfusion recovery and survival, and the hemostatic function were measured. ARC1779 treatment during refrigeration inhibited the platelet-VWF interaction. ARC1779-treated refrigerated murine platelets exhibited increased post-transfusion recovery and survival than untreated ones (recovery of ARC1779-treated platelets: 76.7±5.5%; untreated: 63.7±0.8%; P<0.01. Half-life: 31.4±2.36 hours versus 28.1±0.86 hours; P<0.05). A similar increase was observed for refrigerated human platelets (recovery: 49.4±4.4% versus 36.8±2.1%, P<0.01; half-life: 9.2±1.5 hours versus 8.7±0.9 hours, ns). The effective lifetime of ARC1779 in mice was 2 hours. Additionally, ARC1779 improved the long-term (2 hours after transfusion) hemostatic function of refrigerated platelets (tail bleeding time of mice transfused with ARC1779-treated refrigerated platelets: 160±65 seconds; untreated: 373±96 seconds; P<0.01). The addition of an ARC1779 antidote before transfusion improved the immediate (15 minutes after transfusion) hemostatic function (bleeding time of treated platelets: 149±21 seconds; untreated: 320±36 seconds; P<0.01). CONCLUSIONS: ARC1779 improves the post-transfusion recovery of refrigerated platelets and preserves the long-term hemostatic function of refrigerated platelets. These results suggest that a short-acting inhibitor of the platelet-VWF interaction may be a potential therapeutic option to improve refrigeration of platelets for transfusion treatment.

Plasma kallikrein structure reveals apple domain disc rotated conformation compared to factor XI.

Essentials Zymogen PK is activated to PKa and cleaves substrates kininogen and FXII contributing to bradykinin generation. Monomeric PKa and dimeric homologue FXI utilize the N-terminal apple domains to recruit substrates. A high-resolution 1.3 Å structure of full-length PKa reveals an active conformation of the protease and apple domains. The PKa protease and four-apple domain disc organization is 180° rotated compared to FXI. SUMMARY: Background Plasma prekallikrein (PK) and factor XI (FXI) are apple domain-containing serine proteases that when activated to PKa and FXIa cleave substrates kininogen, factor XII, and factor IX, respectively, directing plasma coagulation, bradykinin release, inflammation, and thrombosis pathways. Objective To investigate the three-dimensional structure of full-length PKa and perform a comparison with FXI. Methods A series of recombinant full-length PKa and FXI constructs and variants were developed and the crystal structures determined. Results and conclusions A 1.3 Å structure of full-length PKa reveals the protease domain positioned above a disc-shaped assemblage of four apple domains in an active conformation. A comparison with the homologous FXI structure reveals the intramolecular disulfide and structural differences in the apple 4 domain that prevents dimer formation in PK as opposed to FXI. Two latchlike loops (LL1 and LL2) extend from the PKa protease domain to form interactions with the apple 1 and apple 3 domains, respectively. A major unexpected difference in the PKa structure compared to FXI is the 180° disc rotation of the apple domains relative to the protease domain. This results in a switched configuration of the latch loops such that LL2 interacts and buries portions of the apple 3 domain in the FXI zymogen whereas in PKa LL2 interacts with the apple 1 domain. Hydrogen-deuterium exchange mass spectrometry on plasma purified human PK and PKa determined that regions of the apple 3 domain have increased surface exposure in PKa compared to the zymogen PK, suggesting conformational change upon activation.