EPCR deficiency ameliorates inflammatory arthritis in mice by suppressing the activation and migration of T cells and dendritic cells.

OBJECTIVES: Endothelial protein C receptor (EPCR) is highly expressed in synovial tissues of patients with RA, but the function of this receptor remains unknown in RA. This study investigated the effect of EPCR on the onset and development of inflammatory arthritis and its underlying mechanisms. METHODS: CIA was induced in EPCR gene knockout (KO) and matched wild-type (WT) mice. The onset and development of arthritis was monitored clinically and histologically. T cells, dendritic cells (DCs), EPCR and cytokines from EPCR KO and WT mice, RA patients and healthy controls (HCs) were detected by flow cytometry and ELISA. RESULTS: EPCR KO mice displayed >40% lower arthritis incidence and 50% less disease severity than WT mice. EPCR KO mice also had significantly fewer Th1/Th17 cells in synovial tissues with more DCs in circulation. Lymph nodes and synovial CD4 T cells from EPCR KO mice expressed fewer chemokine receptors CXCR3, CXCR5 and CCR6 than WT mice. In vitro, EPCR KO spleen cells contained fewer Th1 and more Th2 and Th17 cells than WT and, in concordance, blocking EPCR in WT cells stimulated Th2 and Th17 cells. DCs generated from EPCR KO bone marrow were less mature and produced less MMP-9. Circulating T cells from RA patients expressed higher levels of EPCR than HC cells; blocking EPCR stimulated Th2 and Treg cells in vitro. CONCLUSION: Deficiency of EPCR ameliorates arthritis in CIA via inhibition of the activation and migration of pathogenic Th cells and DCs. Targeting EPCR may constitute a novel strategy for future RA treatment.

Platelets from patients with myeloproliferative neoplasms have increased numbers of mitochondria that are hypersensitive to depolarization by thrombin.

Thrombosis is one of the cardinal manifestations of myeloproliferative neoplasms (MPN). The mechanisms leading to a prothrombotic state in MPN are complex and remain poorly understood. Platelet mitochondria play a role in platelet activation, but their number and function have not been extensively explored in MPN to date. We observed an increased number of mitochondria in platelets from MPN patients compared with healthy donors. MPN patients had an increased proportion of dysfunctional platelet mitochondria. The fraction of platelets with depolarized mitochondria at rest was increased in essential thrombocythemia (ET) patients and the mitochondria were hypersensitive to depolarization following thrombin agonist stimulation. Live microscopy showed a stochastic process in which a higher proportion of individual ET platelets underwent mitochondrial depolarization and after a shorter agonist exposure compared to healthy donors. Depolarization was immediately followed by ballooning of the platelet membrane, which is a feature of procoagulant platelets. We also noted that the mitochondria of MPN patients were on average located nearer the platelet surface and we observed extrusion of mitochondria from the platelet surface as microparticles. These data implicate platelet mitochondria in a number of prothrombotic phenomena. Further studies are warranted to assess whether these findings correlate with clinical thrombotic events.

A novel flow cytometry procoagulant assay for diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe prothrombotic complication of adenoviral vaccines, including the ChAdOx1 nCoV-19 (Vaxzevria) vaccine. The putative mechanism involves formation of pathological anti-platelet factor 4 (PF4) antibodies that activate platelets via the low-affinity immunoglobulin G receptor FcγRIIa to drive thrombosis and thrombocytopenia. Functional assays are important for VITT diagnosis, as not all detectable anti-PF4 antibodies are pathogenic, and immunoassays have varying sensitivity. Combination of ligand binding of G protein-coupled receptors (protease-activated receptor-1) and immunoreceptor tyrosine-based activation motif-linked receptors (FcγRIIa) synergistically induce procoagulant platelet formation, which supports thrombin generation. Here, we describe a flow cytometry-based procoagulant platelet assay using cell death marker GSAO and P-selectin to diagnose VITT by exposing donor whole blood to patient plasma in the presence of a protease-activated receptor-1 agonist. Consecutive patients triaged for confirmatory functional VITT testing after screening using PF4/heparin ELISA were evaluated. In a development cohort of 47 patients with suspected VITT, plasma from ELISA-positive patients (n = 23), but not healthy donors (n = 32) or individuals exposed to the ChAdOx1 nCov-19 vaccine without VITT (n = 24), significantly increased the procoagulant platelet response. In a validation cohort of 99 VITT patients identified according to clinicopathologic adjudication, procoagulant flow cytometry identified 93% of VITT cases, including ELISA-negative and serotonin release assay-negative patients. The in vitro effect of intravenous immunoglobulin (IVIg) and fondaparinux trended with the clinical response seen in patients. Induction of FcγRIIa-dependent procoagulant response by patient plasma, suppressible by heparin and IVIg, is highly indicative of VITT, resulting in a sensitive and specific assay that has been adopted as part of a national diagnostic algorithm to identify vaccinated patients with platelet-activating antibodies.

Building platelet phenotypes: Diaphanous-related formin 1 (DIAPH1)-related disorder.

Variants of the Diaphanous-Related Formin 1 (DIAPH-1) gene have recently been reported causing inherited macrothrombocytopenia. The essential/"diagnostic" characteristics associated with the disorder are emerging; however, robust and complete criteria are not established. Here, we report the first cases of DIAPH1-related disorder in Australia caused by the autosomal dominant gain-of-function DIAPH1 R1213X variant formed by truncation of the protein within the diaphanous auto-regulatory domain (DAD) with loss of regulatory motifs responsible for autoinhibitory interactions within the DIAPH1 protein. We affirm phenotypic changes induced by the DIAPH1 R1213X variant to include macrothrombocytopenia, early-onset progressive sensorineural hearing loss, and mild asymptomatic neutropenia. High-resolution microscopy confirms perturbations of cytoskeletal dynamics caused by the DIAPH1 variant and we extend the repertoire of changes generated by this variant to include alteration of procoagulant platelet formation and possible dental anomalies.

Deficiency of protease-activated receptor (PAR) 1 and PAR2 exacerbates collagen-induced arthritis in mice via differing mechanisms.

OBJECTIVES: Protease-activated receptor (PAR) 1 and PAR2 have been implicated in RA, however their exact role is unclear. Here, we detailed the mechanistic impact of these receptors on the onset and development of inflammatory arthritis in murine CIA and antigen-induced arthritis (AIA) models. METHODS: CIA or AIA was induced in PAR1 or PAR2 gene knockout (KO) and matched wild type mice. The onset and development of arthritis was monitored clinically and histologically. Immune cells, cytokines and MMPs were detected by ELISA, zymography, flow cytometry, western blot or immunohistochemistry. RESULTS: In CIA, PAR1KO and PAR2KO exacerbated arthritis, in opposition to their effects in AIA. These deficient mice had high plasma levels of IL-17, IFN-γ, TGF-ß1 and MMP-13, and lower levels of TNF-α; T cells and B cells were higher in both KO spleen and thymus, and myeloid-derived suppressor cells were lower only in PAR1KO spleen, when compared with wild type cells. Th1, Th2 and Th17 cells were lower in PAR1KO spleens cells, whereas Th1 and Th2 cells were lower and Th17 cells higher in both KO thymus cells, when compared with wild type cells. PAR1KO synovial fibroblasts proliferated faster and produced the most abundant MMP-9 amongst three type cells in the control, lipopolysaccharides or TNF stimulated conditions. CONCLUSION: This is the first study demonstrated that deficiency of PAR1 or PAR2 aggravates inflammatory arthritis in CIA. Furthermore, the protective functions of PAR1 and PAR2 in CIA likely occur via differing mechanisms involving immune cell differentiation and cytokines/MMPs.

Role of thrombomodulin expression on hematopoietic stem cells.

BACKGROUND: Activation of protease-activated receptor 1 (PAR1) by either thrombin or activated protein C (aPC) differentially regulate the quiescence and bone marrow (BM) retention of hematopoietic stem cells (HSC). Murine HSC co-express THBD, PAR1, and endothelial protein C receptor (EPCR), suggesting that HSC sustain quiescence in a quasi-cell autonomous manner due to the binding of thrombin present in the microenvironment to THBD, activation of EPCR-bound protein C by the thrombin-THBD-complex, and subsequent activation of PAR1 by the aPC-EPCR complex. OBJECTIVE: To determine the role of THBD expression on HSC for sustaining stem cell quiescence and BM retention under homeostatic conditions. METHODS: Hematopoietic stem cell function was analyzed in mice with constitutive or temporally controlled complete THBD-deficiency by flow cytometry, functional assays, and single cell RNA profiling. RESULTS: THBD was expressed in mouse, but not human, HSC, progenitors, and immature B cells. Expression in vascular endothelium was conserved in humans' BM. Mice with constitutive THBD deficiency had a normal peripheral blood profile, altered BM morphology, reduced numbers of progenitors and immature B cells, pronounced extramedullary hematopoiesis, increased HSC frequency, and marginally altered transcriptionally defined HSC stemness. Transplantation experiments indicated near normal engraftment and repopulating ability of THBD-deficient HSC. Transgenic aPC supplementation normalized BM histopathology and HSC abundance, and partially restored transcriptional stemness, but had no effect on B cell progenitors and extramedullary hematopoiesis. Temporally controlled THBD gene ablation in adult mice did not cause the above abnormalities. CONCLUSION: THBD expression on HSPC has minor effects on homeostatic hematopoiesis in mice, and is not conserved in humans.

The differential expression of protease activated receptors contributes to functional differences between dark and fair keratinocytes.

BACKGROUND: Dark skin has different properties in comparison to fair skin. Melanocytes have been shown to partly contribute to these differences, however, the involvement of keratinocytes from dark or fair skin is not well demonstrated. OBJECTIVES: This study investigated the proliferation and barrier function of dark keratinocytes (DK) and fair keratinocytes (FK), and the role of protease activated receptor (PAR)1 and PAR2. METHODS: DK and FK were isolated from human neonatal foreskins. Cells were treated with PAR1/PAR2 agonists or antagonists, proliferation was measured by BrdU assay; permeability by the flux of FITC-dextran; protein expression by immunostaining or western blot. RESULTS: When compared to FK, DK proliferated significantly slower; had higher cell permeability; expressed less phosphorylated (P)-ERK/ERK, caspase-14, E-cadherin, tissue growth factor (TGF)-ß3 and PAR1; and expressed more PAR2, and matrix metalloproteinase (MMP)-9. Activation of PAR1 or inhibition of PAR2 stimulated cell proliferation and ERK activation, and in concordance inhibition of PAR1 or activation of PAR2 suppressed cell proliferation and ERK activation in both DK and FK. Inhibition of PAR2 decreased and inhibition of PAR1 increased cell permeability. In foreskin sections, the epidermis of dark foreskin expressed less caspase-14 and the same level but different distribution of E-cadherin, when compared to fair foreskin. CONCLUSIONS: These data highlight functional differences in proliferation and barrier integrity between HK and FK that are partly associated with their differential expression of PAR1 and PAR2.

Redox properties of the tissue factor Cys186-Cys209 disulfide bond.

TF (tissue factor) is a transmembrane cofactor that initiates blood coagulation in mammals by binding Factor VIIa to activate Factors X and IX. The cofactor can reside in a cryptic configuration on primary cells and de-encryption may involve a redox change in the C-terminal domain Cys(186)-Cys(209) disulfide bond. The redox potential of the bond, the spacing of the reduced cysteine thiols and their oxidation by TF activators was investigated to test the involvement of the dithiol/disulfide in TF activation. A standard redox potential of -278 mV was determined for the Cys(186)-Cys(209) disulfide of recombinant soluble TF. Notably, ablating the N-terminal domain Cys(49)-Cys(57) disulfide markedly increased the redox potential of the Cys(186)-Cys(209) bond, suggesting that the N-terminal bond may be involved in the regulation of redox activity at the C-terminal bond. Using As(III) and dibromobimane as molecular rulers for closely spaced sulfur atoms, the reduced Cys(186) and Cys(209) sulfurs were found to be within 3-6 Å (1 Å=0.1 nm) of each other, which is close enough to reform the disulfide bond. HgCl2 is a very efficient activator of cellular TF and activating concentrations of HgCl2-mediated oxidation of the reduced Cys(186) and Cys(209) thiols of soluble TF. Moreover, PAO (phenylarsonous acid), which cross-links two cysteine thiols that are in close proximity, and MMTS (methyl methanethiolsulfonate), at concentrations where it oxidizes closely spaced cysteine residues to a cystine residue, were efficient activators of cellular TF. These findings further support a role for Cys(186) and Cys(209) in TF activation.

Heterozygous loss of platelet glycoprotein (GP) Ib-V-IX variably affects platelet function in velocardiofacial syndrome (VCFS) patients.

Velocardiofacial syndrome (VCFS) is a common, phenotypically heterogeneous developmental disorder caused by an interstitial microdeletion within human chromosome 22q11. The deleted chromosomal region in >90% of VCFS patients includes the GPIb beta gene, encoding for one subunit of the platelet GPIb-V-IX receptor, which is critical for platelet adhesion under shear, and important in aggregation and thrombin-mediated activation. Complete loss of GPIb-V-IX due to autosomal recessive inheritance of two GPIb alpha, Ib beta or GP9 gene mutations, results in a severe bleeding disorder, Bernard-Soulier syndrome (BSS). In this study, twenty-one confirmedVCFS patients were analyzed for platelet morphological and functional alterations, resulting from the heterozygous loss of one GPIb beta gene allele. Compared to unaffected family members, VCFS patients showed a significant decrease in platelet count; VCFS platelet size and mean platelet volume were increased, but not as markedly as in BSS. As expected from obligatory heterozygotes for GPIb beta deficiency, VCFS patients showed reduced platelet GPIb-V-IX surface expression and total GPIb content, but with considerable variation between cases. Platelet function tested using the PFA-100 trade mark analyzer was impaired in 70% of patients. Platelet aggregation was reduced in response to a GPIb-dependent agonist, ristocetin, in 50% of VCFS patients, with 35% showing a reduced response to thrombin receptor activating peptide. Genomic screening was performed to exclude mutations of the subunit genes, indicating that these platelet abnormalities were due to GPIb beta heterozygosity and not spontaneous BSS. In conclusion, many VCFS patients have in-vitro defects in platelet function that may increase their risk of bleeding during surgery.

A common ancestral glycoprotein (GP) 9 1828A>G (Asn45Ser) gene mutation occurring in European families from Australia and Northern Europe with Bernard-Soulier Syndrome (BSS).

Bernard-Soulier syndrome (BSS) is an extremely rare hereditary bleeding disorder, caused by mutations occurring in the Glycoprotein (GP) Ibalpha, GPIbbeta and GP9 genes that encode for the corresponding subunits of platelet GPIb-V-IX adhesion receptor complex. BSS has been reported in many populations, mostly behaving in an autosomal-recessive manner.While the great majority of BSS mutations are unique to a single individual or family, the GP9 1828A>G Asn45Ser mutation, which we have identified in an undocumented Australian Caucasian, has already been reported in multiple unrelated Caucasian families from various Northern and Central European countries. Haplotype analysis of 19 BSS patients from 15 unrelated Northern European families (including 2 compound heterozygote siblings from a British family previously published, and 17 1828A>G Asn45Ser homozygotes), showed that 14 of these BSS patients from 11 of the 1828A>G Asn45Ser homozygote families share a common haplotype at the chromosomal region 3' to the GP9 gene. Hence, the results suggest that the GP9 1828A>GAsn45Ser mutation in these families is ancient, and its frequent emergence in the European population is the result of a founder effect rather than recurrent mutational events. Association of the 1828A>G Asn45Ser mutation with variant haplotypes in 4 other Northern European BSS families raised the possibility of a second founder event, or rare recombinations in these families. Additional members from these 'atypical' lineages would need to be screened to resolve this question.