Sulfenylome analysis of pathogen‐inactivated platelets reveals the presence of cysteine oxidation in integrin signaling pathway and cytoskeleton regulation

Abstract

Cysteine oxidation to sulfenic acid plays a key role in redox regulation and signal transduction. Platelet sulfenylome was studied by quantitative proteomics in pathogen inactivated platelets. One hundred and seventy‐four sulfenylated proteins were identified in resting platelets. Pathogen inactivation oxidized integrin βIII, which could activate the mitogen‐activated protein kinases pathway. Abstract BackgroundCysteine‐containing protein modifications are involved in numerous biological processes such redox regulation or signal transduction. During the preparation and storage of platelet concentrates, cell functions and protein regulations are impacted. In spite of several proteomic investigations, the platelet sulfenylome, ie, the proteins containing cysteine residues (R‐SH) oxidized to sulfenic acid (R‐SOH), has not been characterized. MethodsA dimedone‐based sulfenic acid tagging and enrichment coupled to a mass spectrometry identification workflow was developed to identify and quantify the sulfenic acid‐containing proteins in platelet concentrates treated or not with an amotosalen/ultraviolet A (UVA) pathogen inactivation technique. ResultsOne hundred and seventy‐four sulfenylated proteins were identified belonging mainly to the integrin signal pathway and cytoskeletal regulation by Rho GTPase. The impact on pathogen inactivated platelet concentrates was weak compared to untreated ones where three sulfenylated proteins (myosin heavy chain 9, integrin βIII, and transgelin 2) were significantly affected by amotosalen/UVA treatment. Of particular interest, the reported oxidation of cysteine residues in integrin βIII is known to activate the receptor αIIbβIII. Following the pathogen inactivation, it might trigger the phosphorylation of p38MAPK and explain the lesions reported in the literature. Moreover, procaspase activating compound‐1 (PAC‐1) binding assays on platelet activation showed an increased response to adenosine diphosphate exacerbated by the tagging of proteins with dimedone. This result corroborates the hypothesis of an oxidation‐triggered activation of αIIbβIII by the pathogen inactivation treatment. ConclusionsThe present work completes missing information on the platelet proteome and provides new insights on the effect of pathogen inactivation linked to integrin signaling and cytoskeleton regulation.

Increase of Phosphoprotein Expressions in Amotosalen/UVA-Treated Platelet Concentrates

“…The present work focused on the impact of amotosalen/UVA treatment from a basal platelet stage perspective. 3,906 proteins and 7,334 phosphosites were detected and quantified, which is similar to the results of Burkhart and colleagues [18] with their 4,000 unique proteins and in agreement with the literature [19,26]. As it was decided to be restrictive and to consider only proteins and phosphosites present in at least 5 over 6 replicates, the curation ended up with 2,473 proteins and 2,214 phosphosites.…”

“…These two types of PTMs play a key role since they are linked together, and cross-talks have been demonstrated in several platelet mechanisms [25]. By targeting the oxidation of cysteine moieties (sulfenic acid), we identified an increase of sulfenylated sites, of which Cys412 is in integrin β3, following the amotosalen/UVA treatment [26]. This oxidation is in agreement with the activation of integrin αIIbβ3 and the phosphorylation of p38MAPK [27,28].…”

“…This cascade participates in integrin activation and aggregation, dense granule secretion, and microvesicle release in platelets [5,31,34]. We hypothesized that the activation of p38 within the integrin αIIbβ3 pathway could be a consequence of integrin β3 oxidation on Cys due to amotosalen/UVA treatment [26], a process known to induce oxidations [24,35,36]. This cross-talk between oxidation and phosphorylation could activate the MAPK cascade through PAK2 phosphorylation.…”

Two novel platelet biotinylation methods and their impact on stored platelet concentrates in a blood bank environment

“…Introducing this PI step in PCs production, enables to increase the storage duration from 5 to 7 days at 22°C. However, the PI process, 1–8 storage conditions, and duration 9–13 modify platelet functions 14 …”

“…Introducing this PI step in PCs production, enables to increase the storage duration from 5 to 7 days at 22°C. However, the PI process, [1][2][3][4][5][6][7][8] storage conditions, and duration [9][10][11][12][13] modify platelet functions. 14 Understanding the in vivo impact of transfused platelets on patients is then primordial.…”