Wdr1-Mediated Actin Reorganization Is Essential for Integrin αIIbβ3 Activation in Platelets
Authors: Dasgupta Swapan Kumar , Da Qi , Le Anhquyen , Cruz Miguel A. , Thiagarajan Perumal 
Published: October 2019

Abstract
In resting platelets, the heterodimeric integrin αIIbβ3 is present in a low-affinity state. During platelet activation, the intracytoplasmic signals induce conformational changes that results in a swung-out conformation of the extracellular domain competent to bind ligands such as fibrinogen with high affinity to mediate platelet aggregation. Actin turnover is essential for this process and dynamic assembly and disassembly of actin filaments regulate it. We have identified Wdr1, a cofilin and actin binding protein containing WD40 repeats, as an essential component of the machinery that orchestrates actin fiber reorganization that leads to integrin αIIbβ3 activation.
Methods: Wdr1-deficient mouse strain, Wdr1rd/rd. was obtained through an N-ethyl-N-nitrosourea mutagenesis screen in Baylor College of Medicine. The mutant mouse has a T>A transversion in the second dinucleotide of the intron 9 splice donor site and it produces a mutant transcript containing a 6-bp in-frame deletion that results in a incorrectly folded, nonfunctional protein. Normal splicing produces a small amount of Wdr1 protein (~2%) resulting in a hypomorphic allele. Wdr1-deficient mice are moderately thrombocytopenic (85± 11 x 106 ml Wdr1 deficient versus 427± 52 x 106/ml for wild-type). Platelets were isolated from Wdr1-deficient and control mice. Platelet aggregation was carried out by standard turbidometric methods. Calcium mobilization was measured by incubating Wdr1-deficient and WT (wild-type) platelets with Fura 2 AM and measuring the Fura 2 fluorescence after collagen treatment. Conformational change in αIIbβ3 was determined by flow cytometry with a conformation-specific anti-αIIbβ3 antibody JON/A. In vivo hemostasis was assessed by tail bleeding time and FeCl3-induced endothelial carotid injury/thrombosis model was used to assess the occlusion in carotid artery of mice.
Results: Aggregation response of Wdr1-deficient platelets to different doses of collagen was significantly impaired compared to WT platelets. Under similar conditions, the calcium response was similar to the WT. In a parallel-plate flow chamber assay, WT platelets stably adhered to collagen surface and formed stable thrombus. On the other hand, significantly less number of Wdr1 deficient platelets were stably attach to the collagen surface and it did not form stable thrombus. As expected the tail bleeding time of Wdr1 deficient mice is significantly prolonged (> 10 minutes) compared to WT mice (<2 min). In vivo, in FeCl3 induced carotid artery thrombosis model, vessel occlusion in Wdr1 deficient was prolonged significantly compared wild type mice (15.8 ± 12.6 minutes versus 9.0 ± 10.5 minutes (p=0.041, Mann-Whitney non parametric comparison).
To examine directly the activation of αIIbβ3, we used JON/A antibody, which selectively binds to activated αIIbβ3 integrins on mouse platelets. Binding of collagen treated Wdr1-deficient platelets to JON/A as determined by flow cytometry, is significantly less compared to WT platelets (6.1±0.3 fluorescence units (FU) versus 17.4±0.6 FU, p≤0.05) indicating impaired inside-out activation of αIIbβ3. Since, Wdr1 promotes actin disassembly, which is essential for the rearrangement of the actin fibers that occurs during platelet activation, we measured actin turn over by measuring F-actin and G-actin ratios of collagen treated platelets at various time points. Actin turnover is highly impaired in Wdr1 deficient platelets compared to WT platelets. Furthermore, integrin αIIbβ3 association with actin cytoskeleton was markedly impaired in Wdr1 deficient mice compared to their WT controls.
These studies show that Wdr1 mediated actin cytoskeleton reorganization is essential for integrin αIIbβ3 activation.

Disclosures
No relevant conflicts of interest to declare.