Biochemical and immunocytochemical characterization of coronins in platelets

Riley, David R.J.; Khalil, Jawad S.; Naseem, Khalid M.; Rivero, Francisco

Abstract

Results Platelets express at least four coronins Proteomics and transcriptomics studies indicate that both human and mouse platelets express Coro1, 2, 3 and 7, while other coronins are practically undetectable (Supplemental Table 1 and 2). To demonstrate the presence of coronins in platelets we resolved human and mouse platelet lysates by SDS-PAGE, followed by western blot with a panel of antibodies specific for various coronins (see Supplemental Fig. 1 for antibody specificity). Coro1, 2 and 3 appeared as single bands with apparent molecular weights of or above 56 kDa whereas Coro7 appeared as a single band of 100 kDa (Fig. 1A). While Coro1, 2 and 3 appear relatively abundant, Coro 7 is expressed at much lower levels in both human and mouse platelets. In this study we will mainly focus on human Coro1 as a paradigm of class I coronins, but will also address Coro3 and Coro2 is some assays and will verify if our findings apply to mouse coronins. Subcellular distribution of Coro1 To investigate the distribution of Coro1 we carried out a simple subcellular fractionation in human platelets. Resting platelets were lysed in an isotonic sucrose solution and cytosol and membrane fractions separated by ultracentrifugation and analyzed by immunoblot. As shown in Fig. 1B, most of Coro1 (64%) was recovered in the cytosolic fraction and the rest associated with the membrane fraction. The blot was reprobed for β-actin and 77% of actin was cytosolic and the rest membrane-associated. Since Coro1 is an actin-binding protein, we further investigated whether this membrane association is mediated by actin. Resting platelets were treated with 20 μM latrunculin B (LatB) to depolymerise F-actin prior to subcellular fractionation. As expected, under these conditions almost all actin was recovered in the cytosolic fraction. There was no statistically significant difference in Coro1 association to the membrane fraction in the absence (35.7 ± 9.6%) or presence (27.1 ± 11.7%) of LatB, indicating that the association of Coro1 to platelet membranes is independent of its association with actin. In these experiments probing for the cytosolic marker in resting platelets spleen tyrosine kinase (Syk) and the membrane marker CD36 confirmed that each fractionation was free from cross-contamination.

Citation

Riley, D. R., Khalil, J. S., Naseem, K. M., & Rivero, F. (in press). Biochemical and immunocytochemical characterization of coronins in platelets. Platelets, 1-12. https://doi.org/10.1080/09537104.2019.1696457

• Journal Article Type: Article
• Acceptance Date: Nov 16, 2019
• Online Publication Date: Dec 4, 2019
• Deposit Date: Nov 17, 2019
• Publicly Available Date: Dec 5, 2020
• Journal: Platelets
• Print ISSN: 0953-7104
• Publisher: Taylor and Francis
• Peer Reviewed: Peer Reviewed
• Pages: 1-12
• DOI: https://doi.org/10.1080/09537104.2019.1696457
• Keywords: Hematology; General Medicine; Actin cytoskeleton; actin nodule; Arp2/3 complex; collagen; coronin; platelets; thrombin; Triton insoluble pellet
• Public URL: https://hull-repository.worktribe.com/output/3182109
• Additional Information: Peer Review Statement: The publishing and review policy for this title is described in its Aims & Scope.; Aim & Scope: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=iplt20; Received: 2019-09-22; Revised: 2019-11-05; Accepted: 2019-11-16; Published: 2019-12-04
• Contract Date: Dec 19, 2019