Publications by authors named "Steve P Watson"

224 Publications

AVEXIS technology identifies novel platelet-leukocyte binding partners including CD148-CD300a.

Blood Adv 2021 Sep 23. Epub 2021 Sep 23.

University of Birmingham, Edgbaston, Birmingham, United Kingdom.

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http://dx.doi.org/10.1182/bloodadvances.2021004546DOI Listing
September 2021

Platelet GPVI (Glycoprotein VI) and Thrombotic Complications in the Venous System.

Arterioscler Thromb Vasc Biol 2021 Sep 9:ATVBAHA121316108. Epub 2021 Sep 9.

Department of Biochemistry, CARIM, Maastricht University, The Netherlands (G.P., M.N., J.W.M.H.).

The immunoglobulin receptor GPVI (glycoprotein VI) is selectively expressed on megakaryocytes and platelets and is currently recognized as a receptor for not only collagen but also a variety of plasma and vascular proteins, including fibrin, fibrinogen, laminin, fibronectin, and galectin-3. Deficiency of GPVI is protective in mouse models of experimental thrombosis, pulmonary thromboembolism as well as in thromboinflammation, suggesting a role of GPVI in arterial and venous thrombus formation. In humans, platelet GPVI deficiency is associated with a mild bleeding phenotype, whereas a common variant rs1613662 in the gene is considered a risk factor for venous thromboembolism. However, preclinical studies on the inhibition of GPVI-ligand interactions are focused on arterial thrombotic complications. In this review we discuss the emerging evidence for GPVI in venous thrombus formation and leukocyte-dependent thromboinflammation, extending to venous thromboembolism, pulmonary thromboembolism, and cancer metastasis. We also recapitulate indications for circulating soluble GPVI as a biomarker of thrombosis-related complications. Collectively, we conclude that the current evidence suggests that platelet GPVI is also a suitable cotarget in the prevention of venous thrombosis due to its role in thrombus consolidation and platelet-leukocyte complex formation.
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http://dx.doi.org/10.1161/ATVBAHA.121.316108DOI Listing
September 2021

Platelet activation by charged ligands and nanoparticles: platelet glycoprotein receptors as pattern recognition receptors.

Platelets 2021 Jul 16:1-13. Epub 2021 Jul 16.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Charge interactions play a critical role in the activation of the innate immune system by damage- and pathogen-associated molecular pattern receptors. The ability of these receptors to recognize a wide spectrum of ligands through a common mechanism is critical in host defense. In this article, we argue that platelet glycoprotein receptors that signal through conserved tyrosine-based motifs function as pattern recognition receptors (PRRs) for charged endogenous and exogenous ligands, including sulfated polysaccharides, charged proteins and nanoparticles. This is exemplified by GPVI, CLEC-2 and PEAR1 which are activated by a wide spectrum of endogenous and exogenous ligands, including diesel exhaust particles, sulfated polysaccharides and charged surfaces. We propose that this mechanism has evolved to drive rapid activation of platelets at sites of injury, but that under some conditions it can drive occlusive thrombosis, for example, when blood comes into contact with infectious agents or toxins. In this Opinion Article, we discuss mechanisms behind charge-mediated platelet activation and opportunities for designing nanoparticles and related agents such as dendrimers as novel antithrombotics.
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http://dx.doi.org/10.1080/09537104.2021.1945571DOI Listing
July 2021

Characterisation of inverse agonism of the orphan-G protein-coupled receptor GPR52 by cannabinoid ligands Cannabidiol and O-1918.

Heliyon 2021 Jun 1;7(6):e07201. Epub 2021 Jun 1.

Sosei Heptares, Steinmetz Building, Granta Park, Great Abington, Cambridge CB21 6DG, United Kingdom.

The identification of cannabinoid ligands Cannabidiol and O-1918 as inverse agonists of the orphan receptor GPR52 is reported. Detailed characterisation of GPR52 pharmacology and modelling of the proposed receptor interaction is described. The identification of a novel and further CNS pharmacology for the polypharmacological agent and marketed drug Cannabidiol is noteworthy.
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http://dx.doi.org/10.1016/j.heliyon.2021.e07201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8219759PMC
June 2021

CLEC-2 Prevents Accumulation and Retention of Inflammatory Macrophages During Murine Peritonitis.

Front Immunol 2021 7;12:693974. Epub 2021 Jun 7.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

Platelets play a key role in the development, progression and resolution of the inflammatory response during sterile inflammation and infection, although the mechanism is not well understood. Here we show that platelet CLEC-2 reduces tissue inflammation by regulating inflammatory macrophage activation and trafficking from the inflamed tissues. The immune regulatory function of CLEC-2 depends on the expression of its ligand, podoplanin, upregulated on inflammatory macrophages and is independent of platelet activation and secretion. Mechanistically, platelet CLEC-2 and also recombinant CLEC-2-Fc accelerates actin rearrangement and macrophage migration by increasing the expression of podoplanin and CD44, and their interaction with the ERM proteins. During ongoing inflammation, induced by lipopolysaccharide, treatment with rCLEC-2-Fc induces the rapid emigration of peritoneal inflammatory macrophages to mesenteric lymph nodes, thus reducing the accumulation of inflammatory macrophages in the inflamed peritoneum. This is associated with a significant decrease in pro-inflammatory cytokine, TNF-α and an increase in levels of immunosuppressive, IL-10 in the peritoneum. Increased podoplanin expression and actin remodelling favour macrophage migration towards CCL21, a soluble ligand for podoplanin and chemoattractant secreted by lymph node lymphatic endothelial cells. Macrophage efflux to draining lymph nodes induces T cell priming. In conclusion, we show that platelet CLEC-2 reduces the inflammatory phenotype of macrophages and their accumulation, leading to diminished tissue inflammation. These immunomodulatory functions of CLEC-2 are a novel strategy to reduce tissue inflammation and could be therapeutically exploited through rCLEC-2-Fc, to limit the progression to chronic inflammation.
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http://dx.doi.org/10.3389/fimmu.2021.693974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215360PMC
June 2021

Editorial: structure-function relationships of tyrosine kinase- and tyrosine phosphatase-linked receptors in platelets and megakaryocytes.

Authors:
Steve P Watson

Platelets 2021 Aug 30;32(6):722-723. Epub 2021 May 30.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

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http://dx.doi.org/10.1080/09537104.2021.1920309DOI Listing
August 2021

Assessment of thrombotic risk during long-term treatment of immune thrombocytopenia with fostamatinib.

Ther Adv Hematol 2021 30;12:20406207211010875. Epub 2021 Apr 30.

Department of Pediatrics, Division of Hematology, Weill Medical College of Cornell University, 115 East 67th Street, New York, NY 10065, USA.

Background: Patients with immune thrombocytopenia (ITP) are at risk of bleeding and, paradoxically, thromboembolic events (TEEs), irrespective of thrombocytopenia. The risk of thrombosis is increased by advanced age, obesity, and prothrombotic comorbidities: cancer, hyperlipidemia, diabetes, hypertension, coronary artery disease, and chronic kidney disease, among others. Certain ITP treatments further increase the risk of TEE, especially splenectomy and thrombopoietin receptor agonists. Spleen tyrosine kinase (SYK) is a key signaling molecule common to thromboembolic and hemostatic (in addition to inflammatory) pathways. Fostamatinib is an orally administered SYK inhibitor approved in the USA and Europe for treatment of chronic ITP in adults.

Methods: The phase III and extension studies included heavily pretreated patients with long-standing ITP, many of whom had risk factors for thrombosis prior to initiating fostamatinib. This report describes long-term safety and efficacy of fostamatinib in 146 patients with up to 5 years of treatment, a total of 229 patient-years, and assesses the incidence of thromboembolic events (by standardized MedDRA query).

Results: Platelet counts ⩾50,000/µL were achieved in 54% of patients and the safety profile was as described in the phase III clinical studies with no new toxicities observed over the 5 years of follow-up. The only TEE occurred in one patient (0.7%, or 0.44/100 patient-years), who experienced a mild transient ischemic attack. This is a much lower rate than might be expected in ITP patients.

Conclusion: This report demonstrates durable efficacy and a very low incidence of TEE in patients receiving long-term treatment of ITP with the SYK inhibitor fostamatinib.

Clinicaltrialsgov Identifiers: NCT02076399, NCT02076412, and NCT02077192.
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http://dx.doi.org/10.1177/20406207211010875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8111531PMC
April 2021

Platelet count and disease - editorial policy.

Platelets 2020 11 7;31(8):969-970. Epub 2020 Aug 7.

Institute for Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

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http://dx.doi.org/10.1080/09537104.2020.1802002DOI Listing
November 2020

Editorial policy during the lockdown.

Platelets 2020 05 25;31(4):411. Epub 2020 Apr 25.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

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http://dx.doi.org/10.1080/09537104.2020.1758532DOI Listing
May 2020

Targeted Phosphoinositides Analysis Using High-Performance Ion Chromatography-Coupled Selected Reaction Monitoring Mass Spectrometry.

J Proteome Res 2021 06 3;20(6):3114-3123. Epub 2021 May 3.

Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V., 44227 Dortmund, Germany.

Phosphoinositides are minor components of cell membranes, but play crucial roles in numerous signal transduction pathways. To obtain quantitative measures of phosphoinositides, sensitive, accurate, and comprehensive methods are needed. Here, we present a quantitative targeted ion chromatography-mass spectrometry-based workflow that separates phosphoinositide isomers and increases the quantitative accuracy of measured phosphoinositides. Besides testing different analytical characteristics such as extraction and separation efficiency, the reproducibility of the developed workflow was also investigated. The workflow was verified in resting and stimulated human platelets, fat cells, and rat hippocampal brain tissue, where the LOD and LOQ for phosphoinositides were at 312.5 and 625 fmol, respectively. The robustness of the workflow is shown with different applications that confirms its suitability to analyze multiple less-abundant phosphoinositides.
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http://dx.doi.org/10.1021/acs.jproteome.1c00017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8280744PMC
June 2021

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

Platelets 2021 Aug 5;32(6):733-743. Epub 2021 Apr 5.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham.

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 PIP or PIP 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.
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http://dx.doi.org/10.1080/09537104.2021.1906407DOI Listing
August 2021

Evidence that GPVI is Expressed as a Mixture of Monomers and Dimers, and that the D2 Domain is not Essential for GPVI Activation.

Thromb Haemost 2021 Feb 26. Epub 2021 Feb 26.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

Collagen has been proposed to bind to a unique epitope in dimeric glycoprotein VI (GPVI) and the number of GPVI dimers has been reported to increase upon platelet activation. However, in contrast, the crystal structure of GPVI in complex with collagen-related peptide (CRP) showed binding distinct from the site of dimerization. Further fibrinogen has been reported to bind to monomeric but not dimeric GPVI. In the present study, we have used the advanced fluorescence microscopy techniques of single-molecule microscopy, fluorescence correlation spectroscopy (FCS) and bioluminescence resonance energy transfer (BRET), and mutagenesis studies in a transfected cell line model to show that GPVI is expressed as a mixture of monomers and dimers and that dimerization through the D2 domain is not critical for activation. As many of these techniques cannot be applied to platelets to resolve this issue, due to the high density of GPVI and its anucleate nature, we used Förster resonance energy transfer (FRET) to show that endogenous GPVI is at least partially expressed as a dimer on resting and activated platelet membranes. We propose that GPVI may be expressed as a monomer on the cell surface and it forms dimers in the membrane through diffusion, giving rise to a mixture of monomers and dimers. We speculate that the formation of dimers facilitates ligand binding through avidity.
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http://dx.doi.org/10.1055/a-1401-5014DOI Listing
February 2021

Structure-function relationship of the platelet glycoprotein VI (GPVI) receptor: does it matter if it is a dimer or monomer?

Platelets 2021 Aug 26;32(6):724-732. Epub 2021 Feb 26.

Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

GPVI is a critical signaling receptor responsible for collagen-induced platelet activation and a promising anti-thrombotic target in conditions such as coronary artery thrombosis, ischemic stroke, and atherothrombosis. This is due to the ability to block GPVI while having minimal effects on hemostasis, making it a more attractive target over current dual-antiplatelet therapy (DAPT) with acetyl salicylic acid and P2Y inhibitors where bleeding can be a problem. Our current understanding of how the structure of GPVI relates to function is inadequate and recent studies contradict each other. In this article, we summarize the structure-function relationships underlying the activation of GPVI by its major ligands, including collagen, fibrin(ogen), snake venom toxins and charged exogenous ligands such as diesel exhaust particles. We argue that contrary to popular belief dimerization of GPVI is not required for binding to collagen but serves to facilitate binding through increased avidity, and that GPVI is expressed as a mixture of monomers and dimers on resting platelets, with binding of multivalent ligands inducing higher order clustering.
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http://dx.doi.org/10.1080/09537104.2021.1887469DOI Listing
August 2021

Structural characterization of a novel GPVI-nanobody complex reveals a biologically active domain-swapped GPVI dimer.

Blood 2021 Jun;137(24):3443-3453

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.

Glycoprotein VI (GPVI) is the major signaling receptor for collagen on platelets. We have raised 54 nanobodies (Nb), grouped into 33 structural classes based on their complementary determining region 3 loops, against recombinant GPVI-Fc (dimeric GPVI) and have characterized their ability to bind recombinant GPVI, resting and activated platelets, and to inhibit platelet activation by collagen. Nbs from 6 different binding classes showed the strongest binding to recombinant GPVI-Fc, suggesting that there was not a single dominant class. The most potent 3, Nb2, 21, and 35, inhibited collagen-induced platelet aggregation with nanomolar half maximal inhibitory concentration (IC50) values and inhibited platelet aggregation under flow. The binding KD of the most potent Nb, Nb2, against recombinant monomeric and dimeric GPVI was 0.6 and 0.7 nM, respectively. The crystal structure of monomeric GPVI in complex with Nb2 revealed a binding epitope adjacent to the collagen-related peptide (CRP) binding groove within the D1 domain. In addition, a novel conformation of GPVI involving a domain swap between the D2 domains was observed. The domain swap is facilitated by the outward extension of the C-C' loop, which forms the domain swap hinge. The functional significance of this conformation was tested by truncating the hinge region so that the domain swap cannot occur. Nb2 was still able to displace collagen and CRP binding to the mutant, but signaling was abolished in a cell-based NFAT reporter assay. This demonstrates that the C-C' loop region is important for GPVI signaling but not ligand binding and suggests the domain-swapped structure may represent an active GPVI conformation.
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http://dx.doi.org/10.1182/blood.2020009440DOI Listing
June 2021

Heterozygous mutation SLFN14 K208N in mice mediates species-specific differences in platelet and erythroid lineage commitment.

Blood Adv 2021 01;5(2):377-390

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences.

Schlafen 14 (SLFN14) has recently been identified as an endoribonuclease responsible for cleaving RNA to regulate and inhibit protein synthesis. Early studies revealed that members of the SLFN family are capable of altering lineage commitment during T-cell differentiation by using cell-cycle arrest as a means of translational control by RNase activity. SLFN14 has been reported as a novel gene causing an inherited macrothrombocytopenia and bleeding in human patients; however, the role of this endoribonuclease in megakaryopoiesis and thrombopoiesis remains unknown. To investigate this, we report a CRISPR knock-in mouse model of SLFN14 K208N homologous to the K219N mutation observed in our previous patient studies. We used hematological analysis, in vitro and in vivo studies of platelet and erythrocyte function, and analysis of spleen and bone marrow progenitors. Mice homozygous for this mutation do not survive to weaning age, whereas heterozygotes exhibit microcytic erythrocytosis, hemolytic anemia, splenomegaly, and abnormal thrombus formation, as revealed by intravital microscopy, although platelet function and morphology remain unchanged. We also show that there are differences in erythroid progenitors in the spleens and bone marrow of these mice, indicative of an upregulation of erythropoiesis. This SLFN14 mutation presents distinct species-specific phenotypes, with a platelet defect reported in humans and a severe microcytic erythrocytosis in mice. Thus, we conclude that SLFN14 is a key regulator in mammalian hematopoiesis and a species-specific mediator of platelet and erythroid lineage commitment.
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http://dx.doi.org/10.1182/bloodadvances.2020002404DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839357PMC
January 2021

GPVI (Glycoprotein VI) Interaction With Fibrinogen Is Mediated by Avidity and the Fibrinogen αC-Region.

Arterioscler Thromb Vasc Biol 2021 03 21;41(3):1092-1104. Epub 2021 Jan 21.

Discovery and Translational Science Department, Institute of Cardiovascular and Metabolic Medicine (R.-G.X., J.S.G., S.R.B., H.R.M., C.D., R.A.S.A.).

Objective: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization.

Conclusions: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.
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http://dx.doi.org/10.1161/ATVBAHA.120.315030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7901536PMC
March 2021

Immobilized collagen prevents shedding and induces sustained GPVI clustering and signaling in platelets.

Platelets 2021 Jan;32(1):59-73

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham , Birmingham, UK.

Collagen, the most thrombogenic constituent of blood vessel walls, activates platelets through glycoprotein VI (GPVI). In suspension, following platelet activation by collagen, GPVI is cleaved by A Disintegrin And Metalloproteinase (ADAM)10 and ADAM17. In this study, we use single-molecule localization microscopy and a 2-level DBSCAN-based clustering tool to show that GPVI remains clustered along immobilized collagen fibers for at least 3 hours in the absence of significant shedding. Tyrosine phosphorylation of spleen tyrosine kinase (Syk) and Linker of Activated T cells (LAT), and elevation of intracellular Ca, are sustained over this period. Syk, but not Src kinase-dependent signaling is required to maintain clustering of the collagen integrin α2β1, whilst neither is required for GPVI. We propose that clustering of GPVI on immobilized collagen protects GPVI from shedding in order to maintain sustained Src and Syk-kinases dependent signaling, activation of integrin α2β1, and continued adhesion.
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http://dx.doi.org/10.1080/09537104.2020.1849607DOI Listing
January 2021

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

Platelets 2021 Aug 7;32(6):744-752. Epub 2021 Jan 7.

Institute of Cardiovascular Sciences, Level 1 IBR, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

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 drug design and the importance of complex compound library selection.
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http://dx.doi.org/10.1080/09537104.2020.1863939DOI Listing
August 2021

Novel antiplatelet strategies targeting GPVI, CLEC-2 and tyrosine kinases.

Platelets 2021 Jan 13;32(1):29-41. Epub 2020 Dec 13.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham , Birmingham, UK.

Antiplatelet medications comprise the cornerstone of treatment for diseases that involve arterial thrombosis, including acute coronary syndromes (ACS), stroke and peripheral arterial disease. However, antiplatelet medications may cause bleeding and, furthermore, thrombotic events may still recur despite treatment. The interaction of collagen with GPVI receptors on the surface of platelets has been identified as one of the major players in the pathophysiology of arterial thrombosis that occurs following atherosclerotic plaque rupture. Promisingly, GPVI deficiency in humans appears to have a minimal impact on bleeding. These findings together suggest that targeting platelet GPVI may provide a novel treatment strategy that provides additional antithrombotic efficacy with minimal disruption of normal hemostasis compared to conventional antiplatelet medications. CLEC-2 is gaining interest as a therapeutic target for a variety of thrombo-inflammatory disorders including deep vein thrombosis (DVT) with treatment also predicted to cause minimal disruption to hemostasis. GPVI and CLEC-2 signal through Src, Syk and Tec family tyrosine kinases, providing additional strategies for inhibiting both receptors. In this review, we summarize the evidence regarding GPVI and CLEC-2 and strategies for inhibiting these receptors to inhibit platelet recruitment and activation in thrombotic diseases.
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http://dx.doi.org/10.1080/09537104.2020.1849600DOI Listing
January 2021

Nonredundant Roles of Platelet Glycoprotein VI and Integrin αIIbβ3 in Fibrin-Mediated Microthrombus Formation.

Arterioscler Thromb Vasc Biol 2021 02 3;41(2):e97-e111. Epub 2020 Dec 3.

Department of Biochemistry, CARIM, Maastricht University, the Netherlands (G.P., J.H., I.P., F.S., P.E.J.v.d.M., R.A.S.A., S.P.W., J.W.M.H.).

Objective: Fibrin is considered to strengthen thrombus formation via integrin αIIbβ3, but recent findings indicate that fibrin can also act as ligand for platelet glycoprotein VI. Approach and Results: To investigate the thrombus-forming potential of fibrin and the roles of platelet receptors herein, we generated a range of immobilized fibrin surfaces, some of which were cross-linked with factor XIIIa and contained VWF-BP (von Willebrand factor-binding peptide). Multicolor microfluidics assays with whole-blood flowed at high shear rate (1000 s) indicated that the fibrin surfaces, regardless of the presence of factor XIIIa or VWF-BP, supported platelet adhesion and activation (P-selectin expression), but only microthrombi were formed consisting of bilayers of platelets. Fibrinogen surfaces produced similar microthrombi. Markedly, tiggering of coagulation with tissue factor or blocking of thrombin no more than moderately affected the fibrin-induced microthrombus formation. Absence of αIIbβ3 in Glanzmann thrombasthenia annulled platelet adhesion. Blocking of glycoprotein VI with Fab 9O12 substantially, but incompletely reduced platelet secretion, Ca signaling and aggregation, while inhibition of Syk further reduced these responses. In platelet suspension, glycoprotein VI blockage or Syk inhibition prevented fibrin-induced platelet aggregation. Microthrombi on fibrin surfaces triggered only minimal thrombin generation, in spite of thrombin binding to the fibrin fibers.

Conclusions: Together, these results indicate that fibrin fibers, regardless of their way of formation, act as a consolidating surface in microthrombus formation via nonredundant roles of platelet glycoprotein VI and integrin αIIbβ3 through signaling via Syk and low-level Ca rises.
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http://dx.doi.org/10.1161/ATVBAHA.120.314641DOI Listing
February 2021

Editorial Platelets 2021: toward a brighter year.

Platelets 2021 01 1;32(1):1-2. Epub 2020 Dec 1.

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http://dx.doi.org/10.1080/09537104.2020.1854544DOI Listing
January 2021

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

Arterioscler Thromb Vasc Biol 2021 01 5;41(1):478-490. Epub 2020 Nov 5.

Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade Santiago de Compostela (M.N.B., L.H.-N., L.A.M., Á.G.).

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.
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http://dx.doi.org/10.1161/ATVBAHA.120.314485DOI Listing
January 2021

The collagen receptor glycoprotein VI promotes platelet-mediated aggregation of β-amyloid.

Sci Signal 2020 08 4;13(643). Epub 2020 Aug 4.

Department of Vascular and Endovascular Surgery, Heinrich-Heine University Medical Center, Moorenstrasse 5, 40225 Düsseldorf, Germany.

Cerebral amyloid angiopathy (CAA) and β-amyloid (Aβ) deposition in the brain parenchyma are hallmarks of Alzheimer's disease (AD). We previously reported that platelets contribute to Aβ aggregation in cerebral vessels by secreting the factor clusterin upon binding of Aβ40 to the fibrinogen receptor integrin αβ Here, we investigated the contribution of the collagen receptor GPVI (glycoprotein VI) in platelet-induced amyloid aggregation. Using platelets isolated from GPVI-wild type and GPVI-deficient human donors and mice, we found that Aβ40 bound to GPVI, which induced the release of ATP and fibrinogen, resulting in platelet aggregation. Binding of Aβ40 to integrin αβ, fibrinogen, and GPVI collectively contributed to the formation of amyloid clusters at the platelet surface. Consequently, blockade of αβ or genetic loss of GPVI reduced amyloid fibril formation in cultured platelets and decreased the adhesion of Aβ-activated platelets to injured carotid arteries in mice. Application of losartan to inhibit collagen binding to GPVI resulted in decreased Aβ40-stimulated platelet activation, factor secretion, and platelet aggregation. Furthermore, the application of GPVI- or integrin-blocking antibodies reduced the formation of platelet-associated amyloid aggregates. Our findings indicate that Aβ40 promotes platelet-mediated amyloid aggregation by binding to both GPVI and integrin αβ Blocking these pathways may therapeutically reduce amyloid plaque formation in cerebral vessels and the brain parenchyma of patients.
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http://dx.doi.org/10.1126/scisignal.aba9872DOI Listing
August 2020

Flow studies on human GPVI-deficient blood under coagulating and noncoagulating conditions.

Blood Adv 2020 07;4(13):2953-2961

Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands.

The role of glycoprotein VI (GPVI) in platelets was investigated in 3 families bearing an insertion within the GP6 gene that introduces a premature stop codon prior to the transmembrane domain, leading to expression of a truncated protein in the cytoplasm devoid of the transmembrane region. Western blotting and flow cytometry of GP6hom (homozygous) platelets confirmed loss of the full protein. The level of the Fc receptor γ-chain, which associates with GPVI in the membrane, was partially reduced, but expression of other receptors and signaling proteins was not altered. Spreading of platelets on collagen and von Willebrand factor (which supports partial spreading) was abolished in GP6hom platelets, and spreading on uncoated glass was reduced. Anticoagulated whole blood flowed over immobilized collagen or a mixture of von Willebrand factor, laminin, and rhodocytin (noncollagen surface) generated stable platelet aggregates that express phosphatidylserine (PS). Both responses were blocked on the 2 surfaces in GP6hom individuals, but adhesion was not altered. Thrombin generation was partially reduced in GP6hom blood. The frequency of the GP6het (heterozygous) variant in a representative sample of the Chilean population (1212 donors) is 2.9%, indicating that there are ∼4000 GP6hom individuals in Chile. These results demonstrate that GPVI supports aggregation and PS exposure under flow on collagen and noncollagen surfaces, but not adhesion. The retention of adhesion may contribute to the mild bleeding diathesis of GP6hom patients and account for why so few of the estimated 4000 GP6hom individuals in Chile have been identified.
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http://dx.doi.org/10.1182/bloodadvances.2020001761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7362345PMC
July 2020

Mice Deficient in T-bet Form Inducible NO Synthase-Positive Granulomas That Fail to Constrain .

J Immunol 2020 08 26;205(3):708-719. Epub 2020 Jun 26.

Institute of Immunology and Immunotherapy, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom;

Clearance of intracellular infections caused by Typhimurium (STm) requires IFN-γ and the Th1-associated transcription factor T-bet. Nevertheless, whereas IFN-γ mice succumb rapidly to STm infections, T-bet mice do not. In this study, we assess the anatomy of immune responses and the relationship with bacterial localization in the spleens and livers of STm-infected IFN-γ and T-bet mice. In IFN-γ mice, there is deficient granuloma formation and inducible NO synthase (iNOS) induction, increased dissemination of bacteria throughout the organs, and rapid death. The provision of a source of IFN-γ reverses this, coincident with subsequent granuloma formation and substantially extends survival when compared with mice deficient in all sources of IFN-γ. T-bet mice induce significant levels of IFN-γ after challenge. Moreover, T-bet mice have augmented IL-17 and neutrophil numbers, and neutralizing IL-17 reduces the neutrophilia but does not affect numbers of bacteria detected. Surprisingly, T-bet mice exhibit surprisingly wild-type-like immune cell organization postinfection, including extensive iNOS granuloma formation. In wild-type mice, most bacteria are within iNOS granulomas, but in T-bet mice, most bacteria are outside these sites. Therefore, Th1 cells act to restrict bacteria within IFN-γ-dependent iNOS granulomas and prevent dissemination.
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http://dx.doi.org/10.4049/jimmunol.2000089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372318PMC
August 2020

A rationale for blocking thromboinflammation in COVID-19 with Btk inhibitors.

Platelets 2020 07 19;31(5):685-690. Epub 2020 Jun 19.

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham , Birmingham, UK.

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http://dx.doi.org/10.1080/09537104.2020.1775189DOI Listing
July 2020

The platelet receptor CLEC-2 blocks neutrophil mediated hepatic recovery in acetaminophen induced acute liver failure.

Nat Commun 2020 04 22;11(1):1939. Epub 2020 Apr 22.

Centre for Liver and Gastrointestinal Research, Institute of Immunology and Inflammation, and National Institute for Health Research (NIHR) Birmingham Biomedical Research Centre, The Medical School, University of Birmingham, Birmingham, B15 2TT, UK.

Acetaminophen (APAP) is the main cause of acute liver failure in the West. Specific efficacious therapies for acute liver failure (ALF) are limited and time-dependent. The mechanisms that drive irreversible acute liver failure remain poorly characterized. Here we report that the recently discovered platelet receptor CLEC-2 (C-type lectin-like receptor) perpetuates and worsens liver damage after toxic liver injury. Our data demonstrate that blocking platelet CLEC-2 signalling enhances liver recovery from acute toxic liver injuries (APAP and carbon tetrachloride) by increasing tumour necrosis factor-α (TNF-α) production which then enhances reparative hepatic neutrophil recruitment. We provide data from humans and mice demonstrating that platelet CLEC-2 influences the hepatic sterile inflammatory response and that this can be manipulated for therapeutic benefit in acute liver injury. Since CLEC-2 mediated platelet activation is independent of major haemostatic pathways, blocking this pathway represents a coagulopathy-sparing, specific and novel therapy in acute liver failure.
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http://dx.doi.org/10.1038/s41467-020-15584-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7176690PMC
April 2020

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

Thromb Haemost 2020 Apr 14;120(4):538-564. Epub 2020 Apr 14.

Department of Hematology and Central Hematology Laboratory, Inselspital, Bern University Hospital, University of Bern, Bern, Switzerland; Center for Thrombosis and Hemostasis, University Medical Center of the Johannes Gutenberg University, Mainz, Germany; Haemostasis Research Unit, University College London, London, United Kingdom.

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.
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http://dx.doi.org/10.1055/s-0040-1708035DOI Listing
April 2020
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