Affimer reagents as tool molecules to modulate platelet GPVI-ligand interactions and specifically bind GPVI dimer.

Glycoprotein (GP)VI 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 (KD in the nM range). These Affimers inhibited GPVI-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 ADP. D22 but not M17/D18 displaced nanobody2 (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, while M17 targets a site on the D2-domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody Fab-fragment. D18 targets a new region on the D2-domain. We found that D18 is a stable non-covalent 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.

Ticagrelor inverse agonist activity at the P2Y12 receptor is non-reversible versus its endogenous agonist adenosine 5´-diphosphate.

BACKGROUND AND PURPOSE: Ticagrelor is labelled as a reversible, direct-acting platelet P2Y12 receptor (P2Y12 R) antagonist that is indicated clinically for the prevention of thrombotic events in patients with acute coronary syndrome (ACS). As with many antiplatelet drugs, ticagrelor therapy increases bleeding risk in patients, which may require platelet transfusion in emergency situations. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y12 R. EXPERIMENTAL APPROACH: Studies were performed in human platelets, with P2Y12 R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were undertaken to assess G protein-subunit activation downstream of P2Y12 R activation. KEY RESULTS: Initial studies revealed that a range of P2Y12 R ligands, including ticagrelor, displayed inverse agonist activity at P2Y12 R. Only ticagrelor was resistant to washout and, in human platelet and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y12 R function. The P2Y12 R agonist 2MeSADP, which was also resistant to washout, was able to effectively compete with ticagrelor. In silico docking revealed that ticagrelor and 2MeSADP penetrated more deeply into the orthosteric binding pocket of the P2Y12 R than other P2Y12 R ligands. CONCLUSION AND IMPLICATIONS: Ticagrelor binding to P2Y12 R is prolonged and more akin to that of an irreversible antagonist, especially versus the endogenous P2Y12 R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding, and for bleeding associated with urgent invasive procedures.

Preanalytical conditions for multiparameter platelet flow cytometry.

Background: 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. Objectives: We aimed to characterize the effects of several preanalytical variables on the analysis of blood platelets by multiparameter fluorescent flow cytometry. Methods: 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. Results: 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. Conclusion: 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.

Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability, and resistance to fibrinolysis.

Fibrinogen is essential for blood coagulation. The C-terminus of the fibrinogen α-chain (αC-region) is composed of an αC-domain and αC-connector. Two recombinant fibrinogen variants (α390 and α220) were produced to investigate the role of subregions in modulating clot stability and resistance to lysis. The α390 variant, truncated before the αC-domain, produced clots with a denser structure and thinner fibres. In contrast, the α220 variant, truncated at the start of the αC-connector, produced clots that were porous with short, stunted fibres and visible fibre ends. These clots were mechanically weak and susceptible to lysis. Our data demonstrate differential effects for the αC-subregions in fibrin polymerisation, clot mechanical strength, and fibrinolytic susceptibility. Furthermore, we demonstrate that the αC-subregions are key for promoting longitudinal fibre growth. Together, these findings highlight critical functions of the αC-subregions in relation to clot structure and stability, with future implications for development of novel therapeutics for thrombosis.

Multidimensional flow cytometry reveals novel platelet subpopulations in response to prostacyclin.

BACKGROUND: Robust platelet activation leads to the generation of subpopulations characterized by differential expression of phosphatidylserine (PS). Prostacyclin (PGI2 ) modulates many aspects of platelet function, but its influence on platelet subpopulations is unknown. OBJECTIVES AND METHODS: We used fluorescent flow cytometry coupled to multidimensional fast Fourier transform-accelerated interpolation-based t-stochastic neighborhood embedding analysis to examine the influence of PGI2 on platelet subpopulations. RESULTS: Platelet activation (SFLLRN/CRP-XL) in whole blood revealed three platelet subpopulations with unique combinations of fibrinogen (fb) binding and PS exposure. These subsets, PSlo /fbhi (68%), PShi /fblo (23%), and PShi /fbhi (8%), all expressed CD62P and partially shed CD42b. PGI2 significantly reduced fibrinogen binding and prevented the majority of PS exposure, but did not significantly reduce CD62P, CD154, or CD63 leading to the generation of four novel subpopulations, CD62Phi /PSlo /fblo (64%), CD62Phi /PSlo /fbhi (22%), CD62Phi /PShi /fblo (3%), and CD62Plo /PSlo /fblo (12%). Mechanistically this was linked to PGI2 -mediated inhibition of mitochondrial depolarization upstream of PS exposure. Combining phosphoflow with surface staining, we showed that PGI2 -treated platelets were characterized by both elevated vasodilator-stimulated phosphoprotein phosphorylation and CD62P. The resistance to cyclic AMP signaling was also observed for CD154 and CD63 expression. Consistent with the functional role of CD62P, exposure of blood to PGI2 failed to prevent SFLLRN/CRP-XL-induced platelet-monocyte aggregation despite reducing markers of hemostatic function. CONCLUSION: The combination of multicolor flow cytometry assays with unbiased computational tools has identified novel platelet subpopulations that suggest differential regulation of platelet functions by PGI2 . Development of this approach with increased surface and intracellular markers will allow the identification of rare platelet subtypes and novel biomarkers.

Atherogenic lipid stress induces platelet hyperactivity through CD36-mediated hyposensitivity to prostacyclin: the role of phosphodiesterase 3A.

Prostacyclin (PGI2) controls platelet activation and thrombosis through a cyclic adenosine monophosphate (cAMP) signaling cascade. However, in patients with cardiovascular diseases this protective mechanism fails for reasons that are unclear. Using both pharmacological and genetic approaches we describe a mechanism by which oxidized low density lipoproteins (oxLDL) associated with dyslipidemia promote platelet activation through impaired PGI2 sensitivity and diminished cAMP signaling. In functional assays using human platelets, oxLDL modulated the inhibitory effects of PGI2, but not a phosphodiesterase (PDE)-insensitive cAMP analog, on platelet aggregation, granule secretion and in vitro thrombosis. Examination of the mechanism revealed that oxLDL promoted the hydrolysis of cAMP through the phosphorylation and activation of PDE3A, leading to diminished cAMP signaling. PDE3A activation by oxLDL required Src family kinases, Syk and protein kinase C. The effects of oxLDL on platelet function and cAMP signaling were blocked by pharmacological inhibition of CD36, mimicked by CD36-specific oxidized phospholipids and ablated in CD36-/- murine platelets. The injection of oxLDL into wild-type mice strongly promoted FeCl3-induced carotid thrombosis in vivo, which was prevented by pharmacological inhibition of PDE3A. Furthermore, blood from dyslipidemic mice was associated with increased oxidative lipid stress, reduced platelet sensitivity to PGI2 ex vivo and diminished PKA signaling. In contrast, platelet sensitivity to a PDE-resistant cAMP analog remained normal. Genetic deletion of CD36 protected dyslipidemic animals from PGI2 hyposensitivity and restored PKA signaling. These data suggest that CD36 can translate atherogenic lipid stress into platelet hyperactivity through modulation of inhibitory cAMP signaling.

Dyslipidemia-associated atherogenic oxidized lipids induce platelet hyperactivity through phospholipase Cγ2-dependent reactive oxygen species generation.

Oxidized low-density lipoprotein (oxLDL) and associated oxidized phosphocholine-headgroup phospholipids (oxPCs) activate blood platelets through ligation of the scavenger receptor CD36. Previously, we found that oxLDL stimulated phosphorylation of phospholipase Cγ2 (PLCγ2). However, the functional relevance of PLCγ2 phosphorylation in oxLDL-mediated platelet hyperactivity remained elusive. Here, we set out to explore the functional importance of PLCγ2 in oxLDL-mediated platelet activation using human and genetically modified murine platelets. The CD36-specific oxidized phospholipid (oxPCCD36) triggered the generation of reactive oxygen species (ROS) in platelets under static and arterial flow conditions. The ROS generation in response to oxPCCD36 was sustained for up to 3 h but ablated in CD36- and PLCγ2-deficient platelets. The functional importance of ROS generation in response to atherogenic lipid stress was examined through measurement of P-selectin expression. OxPCCD36 induced P-selectin expression, but required up to 60 min incubation, consistent with the timeline for ROS generation. P-selectin expression was not observed in CD36- and PLCγ2-deficient mice. The ability of oxPCCD36 and oxLDL to stimulate P-selectin expression was prevented by incubation of platelets with the ROS scavenger N-acetyl-cysteine (NAC) and the NOX-2 inhibitor gp91ds-tat, but not with the NOX-1 inhibitor ML171. In summary, we provide evidence that prolonged exposure to oxLDL-associated oxidized phospholipids induces platelet activation via NOX-2-mediated ROS production in a CD36- and PLCγ2-dependent manner.