Increased Circulating Cell-Free DNA in Eosinophilic Granulomatosis With Polyangiitis: Implications for Eosinophil Extracellular Traps and Immunothrombosis

BackgroundEndogenous DNA derived from nuclei or mitochondria is released into the blood circulation as cell-free DNA (cfDNA) following cell damage or death. cfDNA is associated with various pathological conditions; however, its clinical significance in antineutrophil cytoplasmic antibody-associated vasculitis (AAV) remains unclear. This study aimed to evaluate the clinical significance of cfDNA in AAV.MethodsWe enrolled 35 patients with AAV, including 10 with eosinophilic granulomatosis with polyangiitis (EGPA), 13 with microscopic polyangiitis, and 12 with granulomatosis with polyangiitis. Serum cf-nuclear DNA (cf-nDNA) and cf-mitochondrial DNA (cf-mtDNA) levels were measured by quantitative polymerase chain reaction before and after the initiation of immunosuppressive therapy. Tissue samples from EGPA patients were examined by immunofluorescence and transmission electron microscopy. The structure of eosinophil extracellular traps (EETs) and neutrophil extracellular traps (NETs) and stability against DNase were assessed in vitro. Platelet adhesion of EETs were also assessed.ResultsSerum cf-nDNA and cf-mtDNA levels were significantly higher in AAV than in healthy controls, with the highest levels in EGPA; however, serum DNase activities were comparable among all groups. cf-nDNA and cf-mtDNA decreased after treatment and were associated with disease activity only in EGPA. Blood eosinophil count and plasma D-dimer levels were significantly correlated with cf-nDNA in EGPA and cf-mtDNA. EGPA tissue samples showed lytic eosinophils and EETs in small-vessel thrombi. The structure of EETs showed bolder net-like chromatin threads in vitro and EETs showed greater stability against DNase than NETs. EETs provided a scaffold for platelet adhesion.ConclusioncfDNA was increased in EGPA, associated with disease activity. The presence of DNase-resistant EETs in small-vessel thrombi might contribute to higher concentration of cfDNA and the occurrence of immunothrombosis in EGPA.

Agitation-dependent biomechanical forces modulate GPVI receptor expression and platelet adhesion capacity during storage

Background Continuous agitation during storage slows down the platelet storage lesions. However, in special circumstances, manual-mixing can be alternatively used to store products for short time periods without compromising platelet quality. Based on this finding, and given the role of shear stress in modulating receptor expression, we were interested in comparing the levels of platelet adhesion receptor, GPVI and platelet adhesion capacity under each storage condition. Methods Platelet concentrates (PCs) were divided into three groups: continuously-agitated PCs (CAG-PCs) with or without PP2 (Src kinase inhibitor) and manually-mixed PCs (MM-PCs). Platelet count/MPV, swirling, GPVI and P-selectin expression, GPVI shedding, platelet adhesion/spreading to collagen were examined during 5 days of storage. Results While MM- and CAG-PCs showed similar levels of P-selectin expression, GPVI expression was significantly elevated in MM-PCs with lower GPVI shedding/expression ratios, enhanced platelet adhesion/spreading and swirling in manually-mixed PCs. Of note, CAG-PCs treated with PP2 also demonstrated lower P-selectin expression and GPVI shedding, higher GPVI expression and attenuated swirling and spreading capability. Conclusion Given the comparable platelet activation state in MM and CAG-PCs as indicated by P-selectin expression, enhanced platelet adhesion/spreading in MM-PCs, along with relatively higher GPVI expression here, supports previous studies demonstrating a role for biomechanical forces in modulating GPVI-dependent function. Thus, lower GPVI expression in CAG-PCs may be due to shear forces induced by agitation, which keeps this receptor down-regulated while also attenuating platelet adhesion/spreading capacities during storage. Low platelet function in PP2-CAG-PCs also highlights the importance of Src-kinases threshold activity in maintaining platelets quality.

The Proteoglycan Biglycan Modulates Platelet Adhesion and Thrombus Formation in a GPVI-Dependent Manner

Background: Vascular injury induces the exposure of subendothelial extracellular matrix (ECM) important to serve as substrate for platelets to adhere to the injured vessel wall to avoid massive blood loss. Different ECM proteins are known to initiate platelet adhesion and activation. In atherosclerotic mice, the small, leucine-rich proteoglycan biglycan is important for the regulation of thrombin activity via heparin cofactor II. However, nothing is known about the role of biglycan for hemostasis and thrombosis under nonatherosclerotic conditions. Methods: The role of biglycan for platelet adhesion and thrombus formation was investigated using a recombinant protein and biglycan knockout mice. Results: The present study identified biglycan as important ECM protein for the adhesion and activation of platelets, and the formation of three-dimensional thrombi under flow conditions. Platelet adhesion to immobilized biglycan induces the reorganization of the platelet cytoskeleton. Mechanistically, biglycan binds and activates the major collagen receptor glycoprotein (GP)VI, because reduced platelet adhesion to recombinant biglycan was observed when GPVI was blocked and enhanced tyrosine phosphorylation in a GPVI-dependent manner was observed when platelets were stimulated with biglycan. In vivo, the deficiency of biglycan resulted in reduced platelet adhesion to the injured carotid artery and prolonged bleeding times. Conclusions: Loss of biglycan in the vessel wall of mice but not in platelets led to reduced platelet adhesion at the injured carotid artery and prolonged bleeding times, suggesting a crucial role for biglycan as ECM protein that binds and activates platelets via GPVI upon vessel injury.

Thiol Isomerase Activity of β 3 Integrin Psi Domain and L33P Polymorphism: Implications in Blood Coagulation and Anti-Thrombotic Therapy

Background: Integrins are α and β subunit heterodimeric receptors expressed ubiquitously on metazoan cells that play key roles in cell adhesion, movement, and signaling. The αIIbβ3 integrin on platelets is essential for thrombosis and hemostasis through its binding of fibrinogen and other ligands to mediate platelet adhesion and aggregation. The plexin-semaphorin-integrin (PSI) domain is an approximately 54 amino acid sequence at the N-terminus of the β3 subunit located in close proximity to the "knee" region. Our lab recently discovered that two "CXXC" motifs within PSI domains in the integrin family exert endogenous thiol isomerase activity, and blockade of this activity in β3 integrin with our novel anti-PSI domain monoclonal antibodies (mAbs) significantly attenuated platelet adhesion and aggregation without impairing hemostasis (Blood, 2017). Interestingly, intravital microscopy studies demonstrate that our anti-PSI antibodies inhibited thrombosis in vivo 10-30 fold more potent than their effects in vitro under anti-coagulant conditions, suggesting a possible inhibitory effect on blood coagulation. Notably, A L33P polymorphism (HPA-1b) within the β3 integrin PSI domain is linked to an approximately two-fold increased risk for cardiovascular disease (CVD), however, the underlying mechanism for this remains unclear. Methods/Results: To investigate the role of PSI domain in blood coagulation, we first employed thromboelastography (TEG) to compare our anti-PSI domain mAbs with other anti-β3 mAbs that do not directly bind to β3 PSI domain. Results show that anti-PSI domain mAbs inhibited blood coagulation in human and murine whole blood or platelet-rich plasma (PRP) significantly more than anti-αIIbβ3 antibodies JAN-D1, M1, and Abciximab precursor 7E3. To address whether the L33P polymorphism affects PSI domains thiol isomerase activity, we generated L33P PSI domain via site-directed mutagenesis in E. coli. Using a scrambled RNase assay, we found that L33P polymorphism enhanced thiol-isomerase activity relative to WT PSI domain. We further corroborated these findings through an insulin β chain reduction assay, and a MPB (N-Maleimidopropionyl-biocytin) western blot assay, which quantifies thiol isomerase activity through MPB binding to free thiols that have not been oxidized into RNase. Interestingly, TEG results show that recombinant human PSI domain enhanced blood coagulation in platelet-microparticle (PMP) free plasma, which was generated through high-speed centrifugation (17,000 x g) of platelet poor plasma (PPP)for 15 minutes that removed residual platelets and microparticles . Conclusion: We have discovered a novel role of integrin β3 PSI domain in blood coagulation, which is enhanced by the L33P polymorphism (HPA-1b). These data highlight the β3 PSI domain as a suitable therapeutic target for its roles in both platelet adhesion/aggregation, and blood coagulation. Furthermore, these data may explain the increased risk of CVD such as myocardial infarction and deep vein thrombosis for individuals with the L33P polymorphism. Disclosures No relevant conflicts of interest to declare.

Glycidyl methacrylate-crosslinked fish swim bladder as a novel cardiovascular biomaterial with improved antithrombotic and anticalcification properties

At present, commercial artificial biological valves are mostly prepared by crosslinking bovine or porcine pericardia with glutaraldehyde. Swim bladder has similar components and lower immunogenicity compared to bovine or porcine pericardium. In this study, we used a glycidyl methacrylate (GMA)–based radical polymerization method to crosslink decellularized swim bladders. Amino and carboxyl groups in the swim bladder were reacted with epoxy groups on GMA to introduce carbon–carbon double bonds to the swim bladder. The results showed that the platelet adhesion of GMA-crosslinked swim bladders (GMA-SBs) decreased by 35%, as compared to that of glutaraldehyde-crosslinked swim bladders (GLUT-SBs). Moreover, the superior anticoagulant property was further verified by the ex vivo arteriovenous shunt assay. Meanwhile, the subcutaneous implantation in rats showed that GMA-SBs were able to effectively inhibit the calcification compared with GLUT-SBs. In conclusion, GMA-SBs showed improved antithrombotic and anticalcification properties compared to GLUT-SBs.