VWF-dependent platelet ‘priming’ potentiates novel leukocyte interactions and mediates NETosis under flow

Platelet-leukocyte interactions are important in diverse pathophysiological settings from infection to DVT. Previously characterised interactions require robust activation of platelets (e.g. via P-selectin) and/or leukocytes (e.g. via certain β2-integrins). However, recent studies reveal that platelets captured by von Willebrand factor (VWF) under flow can acquire the ability to bind leukocytes. I hypothesised that, under flow, VWF ‘primes’ platelets, in turn facilitating an uncharacterised platelet-leukocyte interaction. My aim was to characterise the interaction between VWF-‘primed’ platelets and leukocytes under flow. Using microfluidic assays, I demonstrated that, under flow, binding of platelets to the VWF A1 domain causes intracellular Ca2+-release and αIIbβ3 activation. VWF-‘primed’ platelets captured neutrophils and T-cells (but not monocytes and B-cells) under low shear. Leukocyte binding was independent of P-selectin and β2-integrins, but significantly reduced by αIIbβ3 blockade, and was enhanced in regions of turbulent flow. Bound neutrophils underwent Ca2+-release and formed neutrophil extracellular traps (NETs), in a Ca2+, NADPH-oxidase and shear-dependent manner. The neutrophil receptor was identified as SLC44A2 through differential gene expression analysis using RNA-sequencing data from the Blueprint consortium. Neutrophils and SLC44A2-transfected HEK293T cells bound activated αIIbβ3, in a manner that was inhibited by blocking the first extracellular loop of SLC44A2. A SNP in SLC44A2 (rs2288904-G/A, M.A.F.-0.22) encoding the R154Q substitution was recently shown to be protective against DVT. Neutrophils homozygous for SLC44A2 rs2288904-A and SLC44A2(R154Q)-transfected HEK293T cells exhibited a significant reduction in the ability to bind VWF-‘primed’ platelets. Platelets from a novel transgenic mouse (GpIbasig/sig) exhibit a decreased ability to become ‘primed’ by VWF and recruit neutrophils under flow. Taken together, these data reveal a previously unreported interaction between platelets and neutrophils, while providing novel mechanistic insights into platelet-mediated NET formation and into the protective effect of the SLC44A2 rs2288904-A polymorphism in venous thrombosis.Open Acces

The role of CD8+ T cell clones in immune thrombocytopenia

Immune thrombocytopenia (ITP) is traditionally considered an antibody-mediated disease. However, a number of features suggest alternative mechanisms of platelet destruction. In this study, we use a multi-dimensional approach to explore the role of cytotoxic CD8+ T cells in ITP. We characterised patients with ITP and compared them to age-matched controls using immunophenotyping, next-generation sequencing of T cell receptor (TCR) genes, single-cell RNA sequencing, and functional T cell and platelet assays. We found that adults with chronic ITP have increased polyfunctional, terminally differentiated effector memory CD8+ T cells (CD45RA+CD62L-) expressing intracellular interferon-g, tumour necrosis factor-a, and Granzyme B defining them as TEMRA cells. These TEMRA cells expand when the platelet count falls and show no evidence of physiological exhaustion. Deep sequencing of the T cell receptor showed expanded T cell clones in patients with ITP. T cell clones persisted over many years, were more prominent in patients with refractory disease, and expanded when the platelet count was low. Combined single-cell RNA and TCR sequencing of CD8+ T cells confirmed that the expanded clones are TEMRA cells. Using in vitro model systems, we show that CD8+ T cells from patients with ITP form aggregates with autologous platelets, release interferon-g and trigger platelet activation and apoptosis through TCR-mediated release of cytotoxic granules. These findings of clonally expanded CD8+ T cells causing platelet activation and apoptosis provide an antibody-independent mechanism of platelet destruction, indicating that targeting specific T-cell clones could be a novel therapeutic approach for patients with refractory ITP

Platelet–Neutrophil Crosstalk in Thrombosis

Platelets are essential for the formation of a haemostatic plug to prevent bleeding, while neutrophils are the guardians of our immune defences against invading pathogens. The interplay between platelets and innate immunity, and subsequent triggering of the activation of coagulation is part of the host system to prevent systemic spread of pathogen in the blood stream. Aberrant immunothrombosis and excessive inflammation can however, contribute to the thrombotic burden observed in many cardiovascular diseases. In this review, we highlight how platelets and neutrophils interact with each other and how their crosstalk is central to both arterial and venous thrombosis and in COVID-19. While targeting platelets and coagulation enables efficient antithrombotic treatments, they are often accompanied with a bleeding risk. We also discuss how novel approaches to reduce platelet-mediated recruitment of neutrophils could represent promising therapies to treat thrombosis without affecting haemostasis