Dr Matt Hindle

OBJECTIVE: Platelets are blood cells which are critical for effective haemostasis and thrombosis. Beyond these classic functions of platelets, a range of roles for them in infectious diseases have also emerged. However little is currently understood about platelet-virus interactions. This study was performed to assess if human platelets are permissive to, and support replication of Semliki Forest virus (SFV), a commonly used model alphavirus. RESULTS: Uptake of the virus by washed human platelets was demonstrated with fluorescent flow cytometry using eGFP fused SFV. To explore if uptake of virus leads to transcriptional replication, RT-qPCR for SFV nsP4 was performed on platelets infected with SFV. This showed a significant increase in nsP4 RNA which increased further over time. This is the first demonstration of SFV RNA increasing within platelets and suggests that platelets may be able to support viral replication, however further work is required to clarify if this leads to production of functional virions.

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.

Background Chronic inflammation is a cardiovascular risk factor and interleukin-1® (IL-1®) is central to the inflammatory host response. Platelets contain the NLRP3 inflammasome and are able to translate IL-1® mRNA and secrete mature IL-1® upon activation. However, the role of a chronic inflammatory environment on platelet IL-1® mRNA and protein content remains unclear. Methods Sixty-five patients with stable inflammation (i.e. hsCRP within predefined margins in 2 separate measurements) were stratified according to hsCRP levels in a low (0.0 – 0.9 mg/L), medium (1.0 – 2.9 mg/L) and high (3.0 – 9.9 mg/L) risk group. Platelet reactivity as well as platelet IL-1® protein synthesis was studied. Results The highest risk group was characterised by a distinct cardiovascular risk profile and approximately 20% higher platelet counts. While platelet reactivity was not different, a reduction of intracellular platelet IL-1® mRNA and IL-1® protein levels was observed in the highest risk group and linked to decreased platelet-size and granularity. This signature suggests a phenotype of chronic IL-1® secretion and could be experimentally phenocopied by stimulation of platelets from healthy volunteers with either TRAP-6 or collagen related peptide (CRP-XL). Conclusion Our data suggests a phenotype of chronic IL1β- secretion by platelets in patients with chronic sterile inflammation.

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.

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.

Background Ticagrelor is labelled as a reversible, direct-acting platelet P2Y12 receptor (P2Y12R) 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 in emergency situations requires platelet transfusion although there is ongoing debate on its effectiveness following ticagrelor therapy. The aim of this study was to further examine the reversibility of ticagrelor at the P2Y12R. Methods Studies were performed in human platelets with both P2Y12R-stimulated GTPase activity and platelet aggregation assessed. Cell-based bioluminescence resonance energy transfer (BRET) assays were also undertaken to assess G protein subunit activation downstream of P2Y12R activation. Results Initial studies revealed a range of P2Y12R ligands including ticagrelor displayed inverse agonist activity at the P2Y12R. Of these only ticagrelor was resistant to wash-out. In both human platelets and cell-based assays, washing failed to reverse ticagrelor-dependent inhibition of ADP-stimulated P2Y12R function in contrast to other P2Y12R antagonists. The P2Y12R agonist 2MeSADP, which was also resistant to wash-out, 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 P2Y12R than other P2Y12R ligands. Conclusion Ticagrelor binding to the P2Y12R is prolonged and more akin to that of an irreversible antagonist especially versus the endogenous P2Y12R agonist ADP. This study highlights the potential clinical need for novel ticagrelor reversal strategies in patients with spontaneous major bleeding and bleeding associated with urgent invasive procedures.

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.

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.

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 optimisation and careful consideration of pre-analytical 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 characterise the effects of several pre-analytical 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 four distinct and commonly used markers of platelet activation including fibrinogen binding, expression of CD62P and CD42b, and phosphatidylserine exposure. Results The use of sub-optimal conditions led to increases in basal platelet activity and reduced sensitivities to stimulation, however the use of optimal conditions protected the platelets from artefactual stimulation and preserved basal activity and sensitivity to activation. Summary The optimal pre-analytical conditions identified here for the measurement of platelet phenotype by flow cytometry suggests a framework for future development of multiparameter platelet assays for high quality datasets and advanced analysis.

Aims/hypothesis Individuals with type 1 diabetes are at increased cardiovascular risk, particularly in the presence of insulin resistance. A prothrombotic environment is believed to contribute to this risk but thrombotic pathways in type 1 diabetes are only partially understood and the role of platelets is incompletely studied. We hypothesised that platelets from individuals with type 1 diabetes exhibit platelet hyperactivity due to both increased propensity for activation and diminished sensitivity to inhibition, with an amplified maladaptive phenotype in those with insulin resistance. Methods Blood samples were obtained from individuals with type 1 diabetes enrolled on the ‘Double diabEtes and adVErse cLinical Outcome: identification of mechanistic Pathways’ (DEVELOP) study with insulin resistance assessed as estimated glucose disposal rate (eGDR), whereby eGDR >8 or <6 mg kg−1 min−1 indicates normal insulin sensitivity or advanced insulin resistance, respectively. Platelet function was analysed using whole blood multiparameter flow cytometry to simultaneously measure three distinct markers of activation, including integrin αIIbβ3 (PAC-1 binding), P-selectin (CD62P) and phosphatidylserine (PS) (Annexin V). Both activation and inhibition responses of the platelets were investigated, which were subjected to the machine learning tool Full Annotation Shape-constrained Trees (FAUST) to characterise platelet subpopulations. Results A total of 32 individuals with type 1 diabetes were studied (median age [range] of 24 [18–34] years, 59% male, diabetes duration [mean ± SD] of 14.0 ± 6.3 years and HbA1c of 65.3 ± 14.0 mmol/mol [8.1%]). An increased basal expression, measured as mean fluorescence intensity, of all three platelet activation markers was detected in the type 1 diabetes group compared with healthy control participants (CD62P expression 521 ± 246 vs 335 ± 67; p<0.001, PAC-1 370 ± 165 vs 231 ± 88; p=0.011 and PS 869 ± 762 vs 294 ± 109; p=0.001). Following platelet stimulation, an enhanced activation of these markers was found in the type 1 diabetes group. Within the type 1 diabetes group, those with advanced insulin resistance (eGDR<6 mg kg−1 min−1) showed increased platelet activation compared with individuals with normal insulin sensitivity (eGDR>8 mg kg−1 min−1) with single agonist stimulation CD62P expression (29,167 ± 2177 vs 22,829 ± 2535, p<0.001 and PAC-1 19,339 ± 11,749 and 5187 ± 2872, p=0.02). Moreover, individuals with type 1 diabetes showed reduced sensitivity to platelet inhibition by prostacyclin (PGI2) compared with control participants. Stratification of individuals with type 1 diabetes by insulin resistance demonstrated that in the presence of PGI2, suppression of stimulated CD62P was 17 ± 11% and 33 ± 12% (p=0.02) for advanced insulin resistance and normal insulin sensitivity groups, respectively, with even larger differences demonstrated for PAC-1 (48 ± 17% and 75 ± 7%; p=0.006) and PS exposure (33 ± 12% and 84 ± 10%; p=0.001). Furthermore, FAUST analysis showed that, under basal conditions, there was a different distribution of the eight platelet subpopulations comparing advanced insulin resistance and normal insulin sensitivity groups, with differences also detected following PGI2 inhibition. Conclusions/interpretation Our novel characterisation of platelets in type 1 diabetes shows a maladaptive phenotype with increased basal activity together with hyperactivation following stimulation and diminished responses to inhibition. Insulin resistance appears to further drive this adverse thrombotic phenotype, suggesting an enhanced platelet-driven cardiovascular risk in those with type 1 diabetes and reduced insulin sensitivity.

Blood platelets are anucleate cells that play a vital role in haemostasis, innate immunity, angiogenesis, and wound healing. However, the inappropriate activation of platelets also contributes to vascular inflammation, atherogenesis, and thrombosis. Platelet activation is a highly complex receptor-mediated process that involves a multitude of signalling intermediates in which Reactive Oxygen Species (ROS) are proposed to play an important role. However, like for many cells, changes in the balance of ROS generation and/or scavenging in disease states may lead to the adoption of maladaptive platelet phenotypes. Here, we review the diverse roles of ROS in platelet function and how ROS are linked to specific platelet activation pathways. We also examine how changes in disease, particularly the plasma oxidised low-density lipoprotein (oxLDL), affect platelet ROS generation and platelet function.

Background Platelet activation is constrained by endothelial-derived prostacyclin (PGI2) through cyclic adenosine-5’-monophosphate (cAMP) signalling involving multiple isoforms of adenylyl cyclase (AC). The roles of specific AC isoforms in controlling haemostasis remain unclear and require clarification. Objectives To understand the specific contribution of AC6 in platelet haemostatic and thrombotic function. Methods A platelet-specific AC6 knockout (AC6-KO) mouse was generated. Biochemical approaches were used to determine intracellular signalling, with flow cytometry, tail bleeding time assays and in vivo thrombosis by ferric chloride were used to measure the haemostatic and thrombotic importance of platelet AC6. Results Loss of AC6 resulted in diminished accumulation of platelet cAMP in response to PGI2, while basal cAMP was unaffected. We found no differences in phosphodiesterase 3A (PDE3A) activity, suggesting the defect was in generation rather than hydrolysis of cAMP. Consistent with this, phosphorylation of PKA substrates, vasodilator-stimulated phosphoprotein and glycogen synthase kinase were diminished but not ablated. Functional studies demonstrated that the inhibition of thrombin-induced fibrinogen binding and P-selectin expression by PGI2 was severely compromised, while inhibition of GPVI-mediated platelet activation was largely unaffected. Under conditions of flow formed stable thrombi, but in the absence of AC6, thrombi were insensitive to PGI2. In vivo diminished sensitivity to PGI2 manifested as significantly reduced tail bleeding and accelerated occlusive arterial thrombus formation in response to vascular injury that were highly unstable and prone to embolisation in AC6-KO mice. Conclusions These data demonstrate that AC6 is linked directly to PGI2-mediated platelet inhibition and regulation of haemostasis and thrombosis in vivo.

Granule secretion is an essential platelet function that contributes not only to haemostasis but also to wound healing, inflammation, and atherosclerosis. Granule secretion from platelets is facilitated, at least in part, by Soluble N-ethylmaleimide-Sensitive Factor (NSF) Attachment Protein Receptor (SNARE) complex-mediated granule fusion. Although α-synuclein is a protein known to modulate the assembly of the SNARE complex in other cells, its role in platelet function remains poorly understood. In this study, we provide evidence that α-synuclein is critical for haemostasis using α-synuclein-deficient (−/−) mice. The genetic deletion of α-synuclein resulted in impaired platelet aggregation, secretion, and adhesion in vitro. In vivo haemostasis models showed that α-synuclein−/− mice had prolonged bleeding times and activated partial thromboplastin times (aPTTs). Mechanistically, platelet activation induced α-synuclein serine (ser) 129 phosphorylation and re-localisation to the platelet membrane, accompanied by an increased association with VAMP 8, syntaxin 4, and syntaxin 11. This phosphorylation was calcium (Ca2+)- and RhoA/ROCK-dependent and was inhibited by prostacyclin (PGI2). Our data suggest that α-synuclein regulates platelet secretion by facilitating SNARE complex formation.

Platelet activation is critical for haemostasis, but if unregulated can lead to pathological thrombosis. Endogenous platelet inhibitory mechanisms are mediated by prostacyclin (PGI2)-stimulated cAMP signalling, which is regulated by phosphodiesterase 3A (PDE3A). However, spatiotemporal regulation of PDE3A activity in platelets is unknown. Here, we report that platelets possess multiple PDE3A isoforms with seemingly identical molecular weights (100 kDa). One isoform contained a unique N-terminal sequence that corresponded to PDE3A1 in nucleated cells but with negligible contribution to overall PDE3A activity. The predominant cytosolic PDE3A isoform did not possess the unique N-terminal sequence and accounted for >99% of basal PDE3A activity. PGI2 treatment induced a dose and time-dependent increase in PDE3A phosphorylation which was PKA-dependent and associated with an increase in phosphodiesterase enzymatic activity. The effects of PGI2 on PDE3A were modulated by A-kinase anchoring protein (AKAP) disruptor peptides, suggesting an AKAP-mediated PDE3A signalosome. We identified AKAP7, AKAP9, AKAP12, AKAP13, and moesin expressed in platelets but focussed on AKAP7 as a potential PDE3A binding partner. Using a combination of immunoprecipitation, proximity ligation techniques, and activity assays, we identified a novel PDE3A/PKA RII/AKAP7 signalosome in platelets that integrates propagation and termination of cAMP signalling through coupling of PKA and PDE3A.

Severe blood loss due to trauma, anaemia, or chemotherapy necessitates immediate red blood cell (RBC) transfusions. The short shelf-life of RBCs at 4 °C complicates emergency supply management. Glycerol is the state-of-the-art cryopreservative for RBCs but the time from thawing to transfusion is more than 1 hour, due to the slow, extensive washing process. This presents a major barrier to blood on demand in emergency or military situations. Here we demonstrate that polyampholytes combined with DMSO and trehalose can cryopreserve human RBCs and allow rapid washout in under 30 minutes. Post wash-out, preserved RBCs exhibit comparable viability, morphological integrity, and function to glycerol-preserved RBCs. This method enhances processing and handling, facilitating the use of frozen RBCs in healthcare, military, and other fields reliant on constant donation. Rapid-washout solutions could unlock blood-on-demand from cryopreserved stocks, improving cold-chain management and reducing reliance on walking donors.

Platelets are anucleate cells that primarily facilitate thrombosis and hemostasis but can also act as mediators of vascular inflammation in disease. Platelets are typically understood to do this through the release of pre-formed chemokines coupled with direct heterotypic interactions with a variety of immune cells. However, an alternative mode of action has been described where platelets are able to undertake de novo synthesis of the cytokines interleukin-1β (IL-1β) and interleukin-18 (IL-18). The primary mechanism to produce these inflammatory mediators is the activation of the NACHT leucine-rich repeat pyrin domain-containing protein 3 (NLRP3) inflammasome, a multi-protein complex that processes IL-1β and IL-18 through caspase activation. The presence and characteristics of the NLRP3 inflammasome have been widely described in a variety of nucleated cells, although its role in anucleate platelets is less clear. In the last decade, the presence of the inflammasome has been reported in platelets and linked to several diseased states including sickle cell disease, acute coronary syndrome, sepsis, and viral hemorrhagic fever. This emerging new biology of platelets, its role in platelet function, vascular inflammation, and other related areas of exploration are critically reviewed here.