PF4 activates the c-Mpl-Jak2 pathway in platelets.

ABSTRACT: Platelet factor 4 (PF4) is an abundant chemokine that is released from platelet α-granules on activation. PF4 is central to the pathophysiology of vaccine-induced immune thrombocytopenia and thrombosis (VITT) in which antibodies to PF4 form immune complexes with PF4, which activate platelets and neutrophils through Fc receptors. In this study, we show that PF4 binds and activates the thrombopoietin receptor, cellular myeloproliferative leukemia protein (c-Mpl), on platelets. This leads to the activation of Janus kinase 2 (JAK2) and phosphorylation of signal transducer and activator of transcription (STAT) 3 and STAT5, leading to platelet aggregation. Inhibition of the c-Mpl-JAK2 pathway inhibits platelet aggregation to PF4, VITT sera, and the combination of PF4 and IgG isolated from VITT patient plasma. The results support a model in which PF4-based immune complexes activate platelets through binding of the Fc domain to FcγRIIA and PF4 to c-Mpl.

Trivalent nanobody-based ligands mediate powerful activation of GPVI, CLEC-2, and PEAR1 in human platelets whereas FcγRIIA requires a tetravalent ligand.

BACKGROUND: Clustering of the receptors glycoprotein receptor VI (GPVI), C-type lectin-like receptor 2 (CLEC-2), low-affinity immunoglobulin γ Fc region receptor II-a (FcγRIIA), and platelet endothelial aggregation receptor 1 (PEAR1) leads to powerful activation of platelets through phosphorylation of tyrosine in their cytosolic tails and initiation of downstream signaling cascades. GPVI, CLEC-2, and FcγRIIA signal through YxxL motifs that activate Syk. PEAR1 signals through a YxxM motif that activates phosphoinositide 3-kinase. Current ligands for these receptors have an undefined valency and show significant batch variation and, for some, uncertain specificity. OBJECTIVES: We have raised nanobodies against each of these receptors and multimerized them to identify the minimum number of epitopes to achieve robust activation of human platelets. METHODS: Divalent and trivalent nanobodies were generated using a flexible glycine-serine linker. Tetravalent nanobodies utilize a mouse Fc domain (IgG2a, which does not bind to FcγRIIA) to dimerize the divalent nanobody. Ligand affinity measurements were determined by surface plasmon resonance. Platelet aggregation, adenosine triphosphate secretion, and protein phosphorylation were analyzed using standardized methods. RESULTS: Multimerization of the nanobodies led to a stepwise increase in affinity with divalent and higher-order nanobody oligomers having sub-nanomolar affinity. The trivalent nanobodies to GPVI, CLEC-2, and PEAR1 stimulated powerful and robust platelet aggregation, secretion, and protein phosphorylation at low nanomolar concentrations. A tetravalent nanobody was required to activate FcγRIIA with the concentration-response relationship showing a greater variability and reduced sensitivity compared with the other nanobody-based ligands, despite a sub-nanomolar binding affinity. CONCLUSION: The multivalent nanobodies represent a series of standardized, potent agonists for platelet glycoprotein receptors. They have applications as research tools and in clinical assays.

Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists.

CLEC-2 is a target for a new class of antiplatelet agent. Clustering of CLEC-2 leads to phosphorylation of a cytosolic YxxL and binding of the tandem SH2 domains in Syk, crosslinking two receptors. We have raised 48 nanobodies to CLEC-2 and crosslinked the most potent of these to generate divalent and tetravalent nanobody ligands. Fluorescence correlation spectroscopy (FCS) was used to show that the multivalent nanobodies cluster CLEC-2 in the membrane and that clustering is reduced by inhibition of Syk. Strikingly, the tetravalent nanobody stimulated aggregation of human platelets, whereas the divalent nanobody was an antagonist. In contrast, in human CLEC-2 knock-in mouse platelets, the divalent nanobody stimulated aggregation. Mouse platelets express a higher level of CLEC-2 than human platelets. In line with this, the divalent nanobody was an agonist in high-expressing transfected DT40 cells and an antagonist in low-expressing cells. FCS, stepwise photobleaching and non-detergent membrane extraction show that CLEC-2 is a mixture of monomers and dimers, with the degree of dimerisation increasing with expression thereby favouring crosslinking of CLEC-2 dimers. These results identify ligand valency, receptor expression/dimerisation and Syk as variables that govern activation of CLEC-2 and suggest that divalent ligands should be considered as partial agonists.

Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins.

The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects.

Inhibition of Src but not Syk causes weak reversal of GPVI-mediated platelet aggregation measured by light transmission aggregometry.

Src tyrosine kinases and spleen tyrosine kinase (Syk) have recently been shown to contribute to sustained platelet aggregation on collagen under arterial shear. In the present study, we have investigated whether Src and Syk are required for aggregation under minimal shear following activation of glycoprotein VI (GPVI) and have extended this to C-type lectin-like receptor-2 (CLEC-2) which signals through the same pathway. Aggregation was induced by the GPVI ligand collagen-related peptide (CRP) and the CLEC-2 ligand rhodocytin and monitored by light transmission aggregometry (LTA). Aggregation and tyrosine phosphorylation by both receptors were sustained for up to 50 min. The addition of inhibitors of Src, Syk or Bruton's tyrosine kinase (Btk) at 150 sec, by which time aggregation was maximal, induced rapid loss of tyrosine phosphorylation of their downstream proteins, but only Src kinase inhibition caused a weak (~10%) reversal in light transmission. A similar effect was observed when the inhibitors were combined with apyrase and indomethacin or glycoprotein IIb-IIIa (GPIIb-IIIa) antagonist, eptifibatide. On the other hand, activation of GPIIb-IIIa by GPVI in a diluted platelet suspension, as measured by binding of fluorescein isothiocyanate-labeled antibody specific for the activated GPIIb-IIIa (FITC-PAC1), was reversed on the addition of Src and Syk inhibitors showing that integrin activation is rapidly reversible in the absence of outside-in signals. The results demonstrate that Src but not Syk and Btk contribute to sustained aggregation as monitored by LTA, possibly as a result of inhibition of outside-in signaling from GPIIb-IIIa to the cytoskeleton through a Syk-independent pathway. This is in contrast to the role of Syk in supporting sustained aggregation on collagen under arterial shear.

Katacine Is a New Ligand of CLEC-2 that Acts as a Platelet Agonist.

BACKGROUND: CLEC-2 is a platelet receptor with an important role in thromboinflammation but a minor role in hemostasis. Two endogenous ligands of CLEC-2 have been identified, the transmembrane protein podoplanin and iron-containing porphyrin hemin, which is formed following hemolysis from red blood cells. Other exogenous ligands such as rhodocytin have contributed to our understanding of the role of CLEC-2. OBJECTIVES: To identify novel CLEC-2 small-molecule ligands to aid therapeutic targeting of CLEC-2. METHODS: ALPHA screen technology has been used for the development of a high-throughput screening (HTS) assay recapitulating the podoplanin-CLEC-2 interaction. Light transmission aggregometry was used to evaluate platelet aggregation. Immunoprecipitation and western blot were used to evaluate direct phosphorylation of CLEC-2 and downstream protein phosphorylation. Autodock vina software was used to predict the molecular binding site of katacine and mass spectrometry to determine the polymeric nature of the ligand. RESULTS AND CONCLUSION: We developed a CLEC-2-podoplanin interaction assay in a HTS format and screened 5,016 compounds from a European Union-open screen library. We identified katacine, a mixture of polymers of proanthocyanidins, as a novel ligand for CLEC-2 and showed that it induces platelet aggregation and CLEC-2 phosphorylation via Syk and Src kinases. Platelet aggregation induced by katacine is inhibited by the anti-CLEC-2 monoclonal antibody fragment AYP1 F(ab)'2. Katacine is a novel nonprotein ligand of CLEC-2 that could contribute to a better understanding of CLEC-2 activation in human platelets.

Galectin-9 activates platelet ITAM receptors glycoprotein VI and C-type lectin-like receptor-2.

BACKGROUND: Platelets are multifunctional cellular mediators in many physiological and pathophysiological processes such as thrombosis, angiogenesis, and inflammation. Several members of galectins, a family of carbohydrate-binding proteins with a broad range of immunomodulatory actions, have been reported to activate platelets. OBJECTIVE: In this study, we investigated the role of galectin-9 (Gal-9) as a novel ligand for platelet glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2). METHODS: Platelet spreading, aggregation, and P-selectin expression in response to Gal-9 were measured in washed platelet suspensions via static adhesion assay, light transmission aggregometry, and flow cytometry, respectively. Solid-phase binding assay and protein phosphorylation studies were utilized to validate the interaction between Gal-9 and GPVI, and immunoprecipitation for detecting CLEC-2 phosphorylation. Wild-type (WT), GPVI-knockout (Gp6-/- ), and GPVI and CLEC-2-double knockout (Gp6-/- /Gp1ba-Cre-Clec1bfl/fl ) mice were used. RESULTS: We have shown that recombinant Gal-9 stimulates aggregation in human and mouse washed platelets dose-dependently. Platelets from both species adhere and spread on immobilized Gal-9 and express P-selectin. Gal-9 competitively inhibited the binding of human recombinant D1 and D2 domains of GPVI to collagen. Gal-9 stimulated tyrosine phosphorylation of CLEC-2 and proteins known to lie downstream of GPVI and CLEC-2 including spleen tyrosine kinase and linker of activated T cells in human platelets. GPVI-deficient murine platelets exhibited significantly impaired aggregation in response to Gal-9, which was further abrogated in GPVI and CLEC-2-double-deficient platelets. CONCLUSIONS: We have identified Gal-9 as a novel platelet agonist that induces activation through interaction with GPVI and CLEC-2.

The PI3Kδ Inhibitor Idelalisib Diminishes Platelet Function and Shows Antithrombotic Potential.

BACKGROUND: Clinical management of ischemic events and prevention of vascular disease is based on antiplatelet drugs. Given the relevance of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) as a candidate target in thrombosis, the main goal of the present study was to identify novel antiplatelet agents within the existing inhibitors blocking PI3K isoforms. METHODS: We performed a biological evaluation of the pharmacological activity of PI3K inhibitors in platelets. The effect of the inhibitors was evaluated in intracellular calcium release and platelet functional assays, the latter including aggregation, adhesion, and viability assays. The in vivo drug antithrombotic potential was assessed in mice undergoing chemically induced arterial occlusion, and the associated hemorrhagic risk evaluated by measuring the tail bleeding time. RESULTS: We show that PI3K Class IA inhibitors potently block calcium mobilization in human platelets. The PI3K p110δ inhibitor Idelalisib inhibits platelet aggregation mediated by ITAM receptors GPVI and CLEC-2, preferentially by the former. Moreover, Idelalisib also inhibits platelet adhesion and aggregation under shear and adhesion to collagen. Interestingly, an antithrombotic effect was observed in mice treated with Idelalisib, with mild bleeding effects at high doses of the drug. CONCLUSION: Idelalisib may have antiplatelet effects with minor bleeding effects, which provides a rationale to evaluate its antithrombotic efficacy in humans.

The structure of CLEC-2: mechanisms of dimerization and higher-order clustering.

The platelet C-type lectin-like receptor CLEC-2 drives inflammation-driven venous thrombosis in mouse models of thrombo-inflammatory disease with a minimal effect on hemostasis identifying it as a target for a new class of antiplatelet agent. Here, we discuss how the protein structure and dynamic arrangement of CLEC-2 on the platelet membrane helps the receptor, which has a single YxxL motif (known as a hemITAM), to trigger intracellular signaling. CLEC-2 exists as a monomer and homo-dimer within resting platelets and forms higher-order oligomers following ligand activation, a process that is mediated by the multivalent nature of its ligands and the binding of the tandem SH2 domains of Syk to the phosphorylated hemITAM and concomitantly to PIP2 or PIP3 to localize it to the membrane. We propose that a low level of active Syk is present at the membrane in resting platelets due to phosphorylation by Src family kinases and that clustering of receptors disturbs the equilibrium between kinases and phosphatases, triggering phosphorylation of the CLEC-2 hemITAM and recruitment of Syk. Knowledge of the structure of CLEC-2 and the mechanism of platelet activation has important implications for development of therapeutics.

Overcoming challenges in developing small molecule inhibitors for GPVI and CLEC-2.

GPVI and CLEC-2 have emerged as promising targets for long-term prevention of both arterial thrombosis and thrombo-inflammation with a decreased bleeding risk relative to current drugs. However, while there are potent blocking antibodies of both receptors, their protein nature comes with decreased bioavailability, making formulation for oral medication challenging. Small molecules are able to overcome these limitations, but there are many challenges in developing antagonists of nanomolar potency, which is necessary when considering the structural features that underlie the interaction of CLEC-2 and GPVI with their protein ligands. In this review, we describe current small-molecule inhibitors for both receptors and strategies to overcome such limitations, including considerations when it comes to in silico drug design and the importance of complex compound library selection.

Phosphoproteomic Analysis of Platelets in Severe Obesity Uncovers Platelet Reactivity and Signaling Pathways Alterations.

OBJECTIVE: Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and Results: We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients. CONCLUSIONS: Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.

Deciphering the secretome of leukocyte-platelet rich fibrin: towards a better understanding of its wound healing properties.

Leukocyte-platelet rich fibrin (L-PRF) is extensively used in the dentistry field and other clinical scenarios due to its regeneration properties. The goal of the present study was to depict the L-PRF secretome and how it changes over time. We obtained L-PRF membranes and cultured them in DMEM. The secretome was collected at days 3, 7 and 21. The secretome at day 3 was analysed by LC-MS/MS and differences over time were analysed by Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH). Overall, 705 proteins were identified in the secretome of L-PRF membranes after 3 days of culture, including growth factors (EGF, PDGFA) and proteins related to platelet and neutrophil degranulation. A total of 202 differentially secreted proteins were quantified by SWATH when comparing secretomes at days 3, 7 and 21. Most of them were enriched at day 3 such as MMP9, TSP1 and CO3. On the contrary, fibrinogen and CATS were found down-regulated at day 3. Growth factor and western blotting analysis corroborated the proteomic results. This is the most detailed proteome analysis of the L-PRF secretome to date. Proteins and growth factors identified, and their kinetics, provide novel information to further understand the wound healing properties of L-PRF.

Analysis of platelets from a diet-induced obesity rat model: elucidating platelet dysfunction in obesity.

Obesity is one of the main health problems in industrialized countries. The contribution of multiple factors developed in obesity can hardly be modeled in vitro. In this context, the development of animal models mimicking human obesity could be essential. The aim of the present study was to compare platelets from a diet-induced obesity (DIO) rat model with their lean control group in order to elucidate platelet dysfunction mechanisms in obesity and correlate the results with previous data from morbid obese patients. In parallel, we also established a blood collection and platelet isolation methodology to study the DIO rat model at biochemical and functional level. Optimal blood collection was obtained from vena cava and platelet isolation was based on a serial of centrifugations avoiding platelet activation. Our results show that the DIO rat model simulate obesity pathologically since weight gain, fasting glucose and platelet counts are increased in obese rats. Interestingly, platelet levels of the active form of Src (pTyr419) showed a tendency to increase in DIO rats pointing towards a potential dysfunction in Src family kinases-related signalling pathways in obesity. Moreover, platelets from DIO rats adhere more to collagen compared with the control group, pointing towards Glycoprotein VI (GPVI) as one of the dysregulated receptors in obesity, in agreement with our recent studies in humans. These results confirm that obesity, in line with human studies, present a platelet dysregulation, and highlight the relevance of considering novel antithrombotic drug targets in these patients, such as GPVI.

Thrombo-Inflammation in Cardiovascular Disease: An Expert Consensus Document from the Third Maastricht Consensus Conference on Thrombosis.

Thrombo-inflammation describes the complex interplay between blood coagulation and inflammation that plays a critical role in cardiovascular diseases. The third Maastricht Consensus Conference on Thrombosis assembled basic, translational, and clinical scientists to discuss the origin and potential consequences of thrombo-inflammation in the etiology, diagnostics, and management of patients with cardiovascular disease, including myocardial infarction, stroke, and peripheral artery disease. This article presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following topics: (1) challenges of the endothelial cell barrier; (2) circulating cells and thrombo-inflammation, focused on platelets, neutrophils, and neutrophil extracellular traps; (3) procoagulant mechanisms; (4) arterial vascular changes in atherogenesis; attenuating atherosclerosis and ischemia/reperfusion injury; (5) management of patients with arterial vascular disease; and (6) pathogenesis of venous thrombosis and late consequences of venous thromboembolism.

A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade.

C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA2 at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr525+526) and Src(p-Tyr419), and for PLCγ2 activity (p-Tyr759). We demonstrated that Syk phosphorylation at Tyr525+526 also happens in the presence of ADP and TXA2 inhibitors, which is not the case for Src-pTyr419 and PLCγ2-pTyr759. Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca2+ mobilization assays confirmed the relevance of ADP and TXA2 for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosome.