Blood platelets are anucleate cells that play a vital role in haemostasis, innate immunity, angiogenesis, and wound healing. However, the inappropriate activation of platelets also contributes to vascular inflammation, atherogenesis, and thrombosis. Platelet activation is a highly complex receptor-mediated process that involves a multitude of signalling intermediates in which Reactive Oxygen Species (ROS) are proposed to play an important role. However, like for many cells, changes in the balance of ROS generation and/or scavenging in disease states may lead to the adoption of maladaptive platelet phenotypes. Here, we review the diverse roles of ROS in platelet function and how ROS are linked to specific platelet activation pathways. We also examine how changes in disease, particularly the plasma oxidised low-density lipoprotein (oxLDL), affect platelet ROS generation and platelet function.

Flow cytometry is an important technique for understanding multiple aspects of blood platelet biology. Despite the widespread use of the platform for assessing platelet function, the optimization and careful consideration of preanalytical conditions, sample processing techniques, and data analysis strategies should be regularly assessed. When set up and designed with optimal conditions, it can ensure the acquisition of robust and reproducible flow cytometry data. However, these parameters are rarely described despite their importance. We aimed to characterize the effects of several preanalytical variables on the analysis of blood platelets by multiparameter fluorescent flow cytometry. We assessed anticoagulant choice, sample material, sample processing, and storage times on 4 distinct and commonly used markers of platelet activation, including fibrinogen binding, expression of CD62P and CD42b, and phosphatidylserine exposure. The use of suboptimal conditions led to increases in basal platelet activity and reduced sensitivities to stimulation; however, the use of optimal conditions protected the platelets from artifactual stimulation and preserved basal activity and sensitivity to activation. The optimal preanalytical conditions identified here for the measurement of platelet phenotype by flow cytometry suggest a framework for future development of multiparameter platelet assays for high-quality data sets and advanced analysis.

Glycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5′-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets.

Platelet activation is critical for haemostasis, but if unregulated can lead to pathological thrombosis. Endogenous platelet inhibitory mechanisms are mediated by prostacyclin (PGI₂)-stimulated cAMP signalling, which is regulated by phosphodiesterase 3A (PDE3A). However, spatiotemporal regulation of PDE3A activity in platelets is unknown. Here, we report that platelets possess multiple PDE3A isoforms with seemingly identical molecular weights (100 kDa). One isoform contained a unique N-terminal sequence that corresponded to PDE3A1 in nucleated cells but with negligible contribution to overall PDE3A activity. The predominant cytosolic PDE3A isoform did not possess the unique N-terminal sequence and accounted for >99% of basal PDE3A activity. PGI₂ treatment induced a dose and time-dependent increase in PDE3A phosphorylation which was PKA-dependent and associated with an increase in phosphodiesterase enzymatic activity. The effects of PGI₂ on PDE3A were modulated by A-kinase anchoring protein (AKAP) disruptor peptides, suggesting an AKAP-mediated PDE3A signalosome. We identified AKAP7, AKAP9, AKAP12, AKAP13, and moesin expressed in platelets but focussed on AKAP7 as a potential PDE3A binding partner. Using a combination of immunoprecipitation, proximity ligation techniques, and activity assays, we identified a novel PDE3A/PKA RII/AKAP7 signalosome in platelets that integrates propagation and termination of cAMP signalling through coupling of PKA and PDE3A.

Platelets are anucleate cells that primarily facilitate thrombosis and hemostasis but can also act as mediators of vascular inflammation in disease. Platelets are typically understood to do this through the release of pre-formed chemokines coupled with direct heterotypic interactions with a variety of immune cells. However, an alternative mode of action has been described where platelets are able to undertake de novo synthesis of the cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18). The primary mechanism to produce these inflammatory mediators is the activation of the NACHT leucine-rich repeat pyrin domain-containing protein 3 (NLRP3) inflammasome, a multi-protein complex that processes IL-1β and IL-18 through caspase activation. The presence and characteristics of the NLRP3 inflammasome have been widely described in a variety of nucleated cells, although its role in anucleate platelets is less clear. In the last decade, the presence of the inflammasome has been reported in platelets and linked to several diseased states including sickle cell disease, acute coronary syndrome, sepsis, and viral hemorrhagic fever. This emerging new biology of platelets, its role in platelet function, vascular inflammation, and other related areas of exploration are critically reviewed here.

The scavenger receptor CD36 links atherogenic dyslipidemia to platelet hyperactivity and accelerated thrombosis through the binding of oxidized low-density lipoproteins (oxLDL). The signaling mechanism(s) that facilitates CD36 translation of oxidative lipid stress into platelet activation is unclear. We examined the role of immunoreceptor tyrosine-based activation motif (ITAM) adapter proteins in CD36-mediated platelet activity. oxLDL induced the phosphorylation of the ITAM-containing adapter Fc receptor γ-chain (FcRγ) in human and murine platelets. Phosphorylation of FcRγ was blocked by Src family kinase (SFK) inhibitors, mimicked by CD36-specific oxidized phospholipids and ablated in CD36−/− murine platelets. Under basal conditions, a pool of CD36 formed a multiprotein complex that included FcRγ and the SFKs Lyn and Fyn. CD36 ligation by oxLDL resulted in the recruitment, phosphorylation, and activation of the tyrosine kinase Syk. To explore the functional cooperativity of this CD36-FcRγ complex, we used murine platelets deficient in FcRγ. The genetic ablation of FcRγ prevented oxLDL-induced tyrosine phosphorylation of Syk and downstream adapter SLP-76, but not SFKs. Moreover, platelet aggregation, in vitro thrombosis, and in vivo carotid thrombosis stimulated by oxLDL were lost in the absence of FcRγ. This study establishes FcRγ as a first functional coreceptor for CD36 in platelets, which enables lipid platelet hyperactivity and arterial thrombosis.