Platelet glycoprotein VI in the regulation of thrombus growth

Platelet glycoprotein VI (GPVI) is the principal signalling receptor for collagen, which is exposed upon damage to the extracellular matrix and at site of rupture of an atherosclerotic plaque. GPVI has recently been shown to be also a receptor for fibrinogen and fibrin, which mediate platelet aggregation and clot formation. In mice, GPVI deficiency protects form arterial thrombosis without causing excessive bleeding. Individuals with an inherited deficiency in GPVI have a mild bleeding diathesis. Together, these observations suggest a more important role of GPVI in thrombosis over haemostasis. The research described in this thesis investigates the relative contribution of GPVI in adhesion and platelet aggregation under flow on collagen and fibrin(ogen). As overarching hypothesis, I propose that GVI is a relevant signalling receptor for fibrin and fibrinogen in supporting thrombus propagation and stability. The first part is a critical assessment of the evidence that fibrin and fibrinogen bind to monomeric or dimeric GPVI in view of the earlier existing controversies. This is followed by the exploration of the role of GPVI in platelets adhesion to collagen using blood from Chilean patients with a homozygous insertion mutation in the GP6 gene which causes lack of GPVI expression on the platelet surface. The results show a critical role for GPVI in supporting platelet aggregation and phosphtidylserine exposure on collagen and non-collagen surfaces, but not adhesion which, on collagen, is mediated by the integrin α2β1. Further, it is estimated that in Chile there are over 4,000 individuals GPVI-deficient, of whom only a handful are known to have a bleeding diathesis. The next study investigates the contribution of GPVI to platelet adhesion and thrombus growth on fibrin, fibrinogen, using the Syk inhibitor, PRT-060318, and an anti-GPVI Fab, 9O12. The results show that GPVI contributes to platelet activation in response to fibrin and fibrinogen, that induces platelet secretion but low level of Ca2+ rises and that it is not required for platelet adhesion. Further, they show that in response to fibrin, GPVI acts in concert with the integrin αIIbβ3 in a non-redundant way. Successively, the action of two small molecule inhibitors of GPVI, losartan and honokiol, on platelet activation is investigated. The results show that both compounds are not selective GPVI antagonists. The following study explores the contribution of GPVI and integrin αIIbβ3 to the stability of a preformed thrombus under flow conditions. A comparison is made between inhibitors of Syk, Src, Btk and those of secondary mediators, ADP and TxA2. The results demonstrate a critical role for Syk in supporting aggregate stability, likely through fibrinogen-induced activation of both GPVI and integrin αIIbβ3. In addition, Syk appears to act in synergy with ADP and TxA2 and to be independent of collagen-induced GVPI activation, as a blocking nanobody has minor effect. Overall, the research in this thesis provides evidence that the role of GPVI extends beyond the onset of thrombus formation. GPVI activation in response to fibrin(ogen) contributes to platelet aggregation and it is key to thrombus stability. This conclusion further supports the argument that blocking GPVI may effectively prevent arterial thrombosis. Furthermore, this research highlights the importance of the integrin αIIbβ3 in supporting GPVI activation, suggesting that blocking signalling downstream of both receptors, can provide an alternative therapeutic strategy for the treatment of arterial thrombosis

Comparison of the GPVI inhibitors losartan and honokiol

<p>Losartan and honokiol are small molecules which have been described to inhibit aggregation of platelets by collagen. Losartan has been proposed to block clustering of GPVI but not to affect binding of collagen. Honokiol has been reported to bind directly to GPVI but only at a concentration that is three orders of magnitude higher than that needed for inhibition of aggregation. The mechanism of action of both inhibitors is so far unclear. In the present study, we confirm the inhibitory effects of both agents on platelet aggregation by collagen and show that both also block the aggregation induced by the activation of CLEC-2 or the low affinity immune receptor FcγRIIa at similar concentrations. For GPVI and CLEC-2, this inhibition is associated with a reduction in protein tyrosine phosphorylation of multiple proteins including Syk. In contrast, on a collagen surface, spreading of platelets and clustering of GPVI (measured by single molecule localisation microscopy) was not altered by losartan or honokiol. Furthermore, in flow whole-blood, both inhibitors suppressed the formation of multi-layered platelet thrombi at arteriolar shear rates at concentrations that hardly affect collagen-induced platelet aggregation in platelet rich plasma. Together, these results demonstrate that losartan and honokiol have multiple effects on platelets which should be considered in the use of these compounds as anti-platelet agents.</p

Human Bone Marrow Organoids for Disease Modeling, Discovery, and Validation of Therapeutic Targets in Hematologic Malignancies

A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow— stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain ex vivo. Fibrosis of the organoid occurred following TGFβ stimulation and engraftment with myelofibrosis but not healthy donor–derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow–like milieu. This enabling technology is likely to accelerate the discovery and prioritization of novel targets for bone marrow disorders and blood cancers. SIGNIFICANCE: We present a human bone marrow organoid that supports the growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment and provides a much-needed ex vivo tool for the prioritization of new therapeutics.</p

SARS-CoV-2 susceptibility and COVID-19 disease severity are associated with genetic variants affecting gene expression in a variety of tissues

Variability in SARS-CoV-2 susceptibility and COVID-19 disease severity between individuals is partly due to genetic factors. Here, we identify 4 genomic loci with suggestive associations for SARS-CoV-2 susceptibility and 19 for COVID-19 disease severity. Four of these 23 loci likely have an ethnicity-specific component. Genome-wide association study (GWAS) signals in 11 loci colocalize with expression quantitative trait loci (eQTLs) associated with the expression of 20 genes in 62 tissues/cell types (range: 1:43 tissues/gene), including lung, brain, heart, muscle, and skin as well as the digestive system and immune system. We perform genetic fine mapping to compute 99% credible SNP sets, which identify 10 GWAS loci that have eight or fewer SNPs in the credible set, including three loci with one single likely causal SNP. Our study suggests that the diverse symptoms and disease severity of COVID-19 observed between individuals is associated with variants across the genome, affecting gene expression levels in a wide variety of tissue types

A first update on mapping the human genetic architecture of COVID-19

peer reviewe

Role of Tyrosine Kinase Syk in Thrombus Stabilisation at High Shear

Understanding the pathways involved in the formation and stability of the core and shell regions of a platelet-rich arterial thrombus may result in new ways to treat arterial thrombosis. The distinguishing feature between these two regions is the absence of fibrin in the shell which indicates that in vitro flow-based assays over thrombogenic surfaces, in the absence of coagulation, can be used to resemble this region. In this study, we have investigated the contribution of Syk tyrosine kinase in the stability of platelet aggregates (or thrombi) formed on collagen or atherosclerotic plaque homogenate at arterial shear (1000 s&minus;1). We show that post-perfusion of the Syk inhibitor PRT-060318 over preformed thrombi on both surfaces enhances thrombus breakdown and platelet detachment. The resulting loss of thrombus stability led to a reduction in thrombus contractile score which could be detected as early as 3 min after perfusion of the Syk inhibitor. A similar loss of thrombus stability was observed with ticagrelor and indomethacin, inhibitors of platelet adenosine diphosphate (ADP) receptor and thromboxane A2 (TxA2), respectively, and in the presence of the Src inhibitor, dasatinib. In contrast, the Btk inhibitor, ibrutinib, causes only a minor decrease in thrombus contractile score. Weak thrombus breakdown is also seen with the blocking GPVI nanobody, Nb21, which indicates, at best, a minor contribution of collagen to the stability of the platelet aggregate. These results show that Syk regulates thrombus stability in the absence of fibrin in human platelets under flow and provide evidence that this involves pathways additional to activation of GPVI by collagen

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

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

Hydroxychloroquine inhibits hemolysis-induced arterial thrombosis ex vivo and improves lung perfusion in hemin-treated mice

Background: Free labile hemin acts as a damage-associated molecular pattern during acute and chronic hemolysis and muscle injury supporting platelet activation and thrombosis.Aim: We investigated the anti-thrombotic potential of hydroxychloroquine on hemolysis-induced arterial thrombosis ex vivo, hemin-induced platelet activation, ferric-chloride (FeCl3)-induced arterial thrombosis and lung perfusion following hemin injection in mice.Results: Erythrocyte lysis and endothelial cell activation cooperatively supported platelet aggregation and thrombosis at arterial shear stress. This thrombotic effect was reversed by hydroxychloroquine. In a purified system, hydroxychloroquine inhibited platelet build-up on immobilized von Willebrand factor in hemolyzed blood without altering initial platelet recruitment. Hydroxychloroquine inhibited hemin-induced platelet activation and phosphatidylserine exposure independently of reactive oxygen species generation. In the presence of hemin, hydroxychloroquine did not alter glycoprotein VI shedding but reduced C-type-lectin-like-2 expression on platelets. In vivo, hydroxychloroquine reversed pulmonary perfusion decline induced by exogenous administration of hemin. In arterial thrombosis models, hydroxychloroquine inhibited FeCl3-induced thrombosis in the carotid artery and reduced von Willebrand factor accumulation in the thrombi.Conclusion: Hydroxychloroquine inhibited hemolysis-induced arterial thrombosis ex-vivo and improved pulmonary perfusion in hemin-treated mice, supporting a potential benefit of its use as an adjuvant therapy in hemolytic diseases to limit arterial thrombosis and to improve organ perfusion

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

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 g-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.M.N. and J.W.M.H. thank the Interreg V Euregio Meuse-Rhine Program (Poly-Valve) for financial support. S.P.W. holds a British Heart Foundation Chair (CH03/003). E.E.G. receives funding from the National Health and Medical Research Council of Australia and the Australian Research Council. This work was supported by a Birmingham-Maastricht Studentship (G.P.) and by the BHF Accelerator Award (AA/18/2/34218)

Nonredundant roles of platelet glycoprotein VI and integrin αIIbβ3 in fibrin-mediated microthrombus formation

Objective: Fibrin is considered to strengthen thrombus formation via integrin alpha IIb beta 3, 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(-1)) 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 alpha IIb beta 3 in Glanzmann thrombasthenia annulled platelet adhesion. Blocking of glycoprotein VI with Fab 9O12 substantially, but incompletely reduced platelet secretion, Ca2+ 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 alpha IIb beta 3 through signaling via Syk and low-level Ca2+ rises