Cell-autonomous regulation of complement C3 by factor H limits macrophage efferocytosis and exacerbates atherosclerosis.

Complement factor H (CFH) negatively regulates consumption of complement component 3 (C3), thereby restricting complement activation. Genetic variants in CFH predispose to chronic inflammatory disease. Here, we examined the impact of CFH on atherosclerosis development. In a mouse model of atherosclerosis, CFH deficiency limited plaque necrosis in a C3-dependent manner. Deletion of CFH in monocyte-derived inflammatory macrophages propagated uncontrolled cell-autonomous C3 consumption without downstream C5 activation and heightened efferocytotic capacity. Among leukocytes, Cfh expression was restricted to monocytes and macrophages, increased during inflammation, and coincided with the accumulation of intracellular C3. Macrophage-derived CFH was sufficient to dampen resolution of inflammation, and hematopoietic deletion of CFH in atherosclerosis-prone mice promoted lesional efferocytosis and reduced plaque size. Furthermore, we identified monocyte-derived inflammatory macrophages expressing C3 and CFH in human atherosclerotic plaques. Our findings reveal a regulatory axis wherein CFH controls intracellular C3 levels of macrophages in a cell-autonomous manner, evidencing the importance of on-site complement regulation in the pathogenesis of inflammatory diseases.

In situ FRET-based localization of the N terminus of myosin binding protein-C in heart muscle cells.

Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated regulatory protein frequently found mutated in patients suffering from hypertrophic cardiomyopathy (HCM). Recent in vitro experiments have highlighted the functional significance of its N-terminal region (NcMyBP-C) for heart muscle contraction, reporting regulatory interactions with both thick and thin filaments. To better understand the interactions of cMyBP-C in its native sarcomere environment, in situ Foerster resonance energy transfer-fluorescence lifetime imaging (FRET-FLIM) assays were developed to determine the spatial relationship between the NcMyBP-C and the thick and thin filaments in isolated neonatal rat cardiomyocytes (NRCs). In vitro studies showed that ligation of genetically encoded fluorophores to NcMyBP-C had no or little effect on its binding to thick and thin filament proteins. Using this assay, FRET between mTFP conjugated to NcMyBP-C and Phalloidin-iFluor 514 labeling the actin filaments in NRCs was detected by time-domain FLIM. The measured FRET efficiencies were intermediate between those observed when the donor was attached to the cardiac myosin regulatory light chain in the thick filaments and troponin T in the thin filaments. These results are consistent with the coexistence of multiple conformations of cMyBP-C, some with their N-terminal domains binding to the thin filament and others binding to the thick filament, supporting the hypothesis that the dynamic interchange between these conformations mediates interfilament signaling in the regulation of contractility. Moreover, stimulation of NRCs with ß-adrenergic agonists reduces FRET between NcMyBP-C and actin-bound Phalloidin, suggesting that cMyBP-C phosphorylation reduces its interaction with the thin filament.

Filamin B restricts vaccinia virus spread and is targeted by vaccinia virus protein C4.

Vaccinia virus (VACV) is a large DNA virus that encodes scores of proteins that modulate the host immune response. VACV protein C4 is one such immunomodulator known to inhibit the activation of both the NF-κB signaling cascade and the DNA-PK-mediated DNA sensing pathway. Here, we show that the N-terminal region of C4, which neither inhibits NF-κB nor mediates interaction with DNA-PK, still contributes to virus virulence. Furthermore, this domain interacts directly and with high affinity to the C-terminal domain of filamin B (FLNB). FLNB is a large actin-binding protein that stabilizes the F-actin network and is implicated in other cellular processes. Deletion of FLNB from cells results in larger VACV plaques and increased infectious viral yield, indicating that FLNB restricts VACV spread. These data demonstrate that C4 has a new function that contributes to virulence and engages the cytoskeleton. Furthermore, we show that the cytoskeleton performs further previously uncharacterized functions during VACV infection. IMPORTANCE: Vaccinia virus (VACV), the vaccine against smallpox and monkeypox, encodes many proteins to counteract the host immune response. Investigating these proteins provides insights into viral immune evasion mechanisms and thereby indicates how to engineer safer and more immunogenic VACV-based vaccines. Here, we report that the N-terminal domain of VACV protein C4 interacts directly with the cytoskeletal protein filamin B (FLNB), and this domain of C4 contributes to virus virulence. Furthermore, VACV replicates and spreads better in cells lacking FLNB, thus demonstrating that FLNB has antiviral activity. VACV utilizes the cytoskeleton for movement within and between cells; however, previous studies show no involvement of C4 in VACV replication or spread. Thus, C4 associates with FLNB for a different reason, suggesting that the cytoskeleton has further uncharacterized roles during virus infection.

Platelets at the intersection of inflammation and coagulation in the APC-mediated response to myocardial ischemia/reperfusion injury.

Thromboinflammation is a complex pathology associated with inflammation and coagulation. In cases of cardiovascular disease, in particular ischemia-reperfusion injury, thromboinflammation is a common complication. Increased understanding of thromboinflammation depends on an improved concept of the mechanisms of cells and proteins at the axis of coagulation and inflammation. Among these elements are activated protein C and platelets. This review summarizes the complex interactions of activated protein C and platelets regulating thromboinflammation in cardiovascular disease. By unraveling the pathways of platelets and APC in the inflammatory and coagulation cascades, this review summarizes the role of these vital mediators in the development and perpetuation of heart disease and the thromboinflammation-driven complications of cardiovascular disease. Furthermore, this review emphasizes the significance of the counteracting effects of platelets and APC and their combined role in disease states.

Slower Calcium Handling Balances Faster Cross-Bridge Cycling in Human MYBPC3 HCM.

BACKGROUND: The pathogenesis of MYBPC3-associated hypertrophic cardiomyopathy (HCM) is still unresolved. In our HCM patient cohort, a large and well-characterized population carrying the MYBPC3:c772G>A variant (p.Glu258Lys, E258K) provides the unique opportunity to study the basic mechanisms of MYBPC3-HCM with a comprehensive translational approach. METHODS: We collected clinical and genetic data from 93 HCM patients carrying the MYBPC3:c772G>A variant. Functional perturbations were investigated using different biophysical techniques in left ventricular samples from 4 patients who underwent myectomy for refractory outflow obstruction, compared with samples from non-failing non-hypertrophic surgical patients and healthy donors. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and engineered heart tissues (EHTs) were also investigated. RESULTS: Haplotype analysis revealed MYBPC3:c772G>A as a founder mutation in Tuscany. In ventricular myocardium, the mutation leads to reduced cMyBP-C (cardiac myosin binding protein-C) expression, supporting haploinsufficiency as the main primary disease mechanism. Mechanical studies in single myofibrils and permeabilized muscle strips highlighted faster cross-bridge cycling, and higher energy cost of tension generation. A novel approach based on tissue clearing and advanced optical microscopy supported the idea that the sarcomere energetics dysfunction is intrinsically related with the reduction in cMyBP-C. Studies in single cardiomyocytes (native and hiPSC-derived), intact trabeculae and hiPSC-EHTs revealed prolonged action potentials, slower Ca2+ transients and preserved twitch duration, suggesting that the slower excitation-contraction coupling counterbalanced the faster sarcomere kinetics. This conclusion was strengthened by in silico simulations. CONCLUSIONS: HCM-related MYBPC3:c772G>A mutation invariably impairs sarcomere energetics and cross-bridge cycling. Compensatory electrophysiological changes (eg, reduced potassium channel expression) appear to preserve twitch contraction parameters, but may expose patients to greater arrhythmic propensity and disease progression. Therapeutic approaches correcting the primary sarcomeric defects may prevent secondary cardiomyocyte remodeling.

Disruption of proteostasis causes IRE1 mediated reprogramming of alveolar epithelial cells.

Disruption of alveolar type 2 cell (AEC2) protein quality control has been implicated in chronic lung diseases, including pulmonary fibrosis (PF). We previously reported the in vivo modeling of a clinical surfactant protein C (SP-C) mutation that led to AEC2 endoplasmic reticulum (ER) stress and spontaneous lung fibrosis, providing proof of concept for disruption to proteostasis as a proximal driver of PF. Using two clinical SP-C mutation models, we have now discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop a reprogrammed cell state that heretofore has been seen only as a response to lung injury. Using single-cell RNA sequencing in vivo and organoid-based modeling, we show that this state arises de novo from intrinsic AEC2 dysfunction. The cell-autonomous AEC2 reprogramming can be attenuated through inhibition of inositol-requiring enzyme 1 (IRE1α) signaling as the use of an IRE1α inhibitor reduced the development of the reprogrammed cell state and also diminished AEC2-driven recruitment of granulocytes, alveolitis, and lung injury. These findings identify AEC2 proteostasis, and specifically IRE1α signaling through its major product XBP-1, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.

MYBPC3-c.772G>A mutation results in haploinsufficiency and altered myosin cycling kinetics in a patient induced stem cell derived cardiomyocyte model of hypertrophic cardiomyopathy.

Approximately 40% of hypertrophic cardiomyopathy (HCM) mutations are linked to the sarcomere protein cardiac myosin binding protein-C (cMyBP-C). These mutations are either classified as missense mutations or truncation mutations. One mutation whose nature has been inconsistently reported in the literature is the MYBPC3-c.772G > A mutation. Using patient-derived human induced pluripotent stem cells differentiated to cardiomyocytes (hiPSC-CMs), we have performed a mechanistic study of the structure-function relationship for this MYBPC3-c.772G > A mutation versus a mutation corrected, isogenic cell line. Our results confirm that this mutation leads to exon skipping and mRNA truncation that ultimately suggests ∼20% less cMyBP-C protein (i.e., haploinsufficiency). This, in turn, results in increased myosin recruitment and accelerated myofibril cycling kinetics. Our mechanistic studies suggest that faster ADP release from myosin is a primary cause of accelerated myofibril cross-bridge cycling due to this mutation. Additionally, the reduction in force generating heads expected from faster ADP release during isometric contractions is outweighed by a cMyBP-C phosphorylation mediated increase in myosin recruitment that leads to a net increase of myofibril force, primarily at submaximal calcium activations. These results match well with our previous report on contractile properties from myectomy samples of the patients from whom the hiPSC-CMs were generated, demonstrating that these cell lines are a good model to study this pathological mutation and extends our understanding of the mechanisms of altered contractile properties of this HCM MYBPC3-c.772G > A mutation.

Cardiac myosin-binding protein C N-terminal interactions with myosin and actin filaments: Opposite effects of phosphorylation and M-domain mutations.

N-terminal cardiac myosin-binding protein C (cMyBP-C) domains (C0-C2) bind to thick (myosin) and thin (actin) filaments to coordinate contraction and relaxation of the heart. These interactions are regulated by phosphorylation of the M-domain situated between domains C1 and C2. In cardiomyopathies and heart failure, phosphorylation of cMyBP-C is significantly altered. We aimed to investigate how cMyBP-C interacts with myosin and actin. We developed complementary, high-throughput, C0-C2 FRET-based binding assays for myosin and actin to characterize the effects due to 5 HCM-linked variants or functional mutations in unphosphorylated and phosphorylated C0-C2. The assays indicated that phosphorylation decreases binding to both myosin and actin, whereas the HCM mutations in M-domain generally increase binding. The effects of mutations were greatest in phosphorylated C0-C2, and some mutations had a larger effect on actin than myosin binding. Phosphorylation also altered the spatial relationship of the probes on C0-C2 and actin. The magnitude of these structural changes was dependent on C0-C2 probe location (C0, C1, or M-domain). We conclude that binding can differ between myosin and actin due to phosphorylation or mutations. Additionally, these variables can change the mode of binding, affecting which of the interactions in cMyBP-C N-terminal domains with myosin or actin take place. The opposite effects of phosphorylation and M-domain mutations is consistent with the idea that cMyBP-C phosphorylation is critical for normal cardiac function. The precision of these assays is indicative of their usefulness in high-throughput screening of drug libraries for targeting cMyBP-C as therapy.

The W792R HCM missense mutation in the C6 domain of cardiac myosin binding protein-C increases contractility in neonatal mouse myocardium.

Missense mutations in cardiac myosin binding protein C (cMyBP-C) are known to cause hypertrophic cardiomyopathy (HCM). The W792R mutation in the C6 domain of cMyBP-C causes severe, early onset HCM in humans, yet its impact on the function of cMyBP-C and the mechanism through which it causes disease remain unknown. To fully characterize the effect of the W792R mutation on cardiac morphology and function in vivo, we generated a murine knock-in model. We crossed heterozygous W792RWR mice to produce homozygous mutant W792RRR, heterozygous W792RWR, and control W792RWW mice. W792RRR mice present with cardiac hypertrophy, myofibrillar disarray and fibrosis by postnatal day 10 (PND10), and do not survive past PND21. Full-length cMyBP-C is present at similar levels in W792RWW, W792RWR and W792RRR mice and is properly incorporated into the sarcomere. Heterozygous W792RWR mice displayed normal heart morphology and contractility. Permeabilized myocardium from PND10 W792RRR mice showed increased Ca2+ sensitivity, accelerated cross-bridge cycling kinetics, decreased cooperativity in the activation of force, and increased expression of hypertrophy-related genes. In silico modeling suggests that the W792R mutation destabilizes the fold of the C6 domain and increases torsion in the C5-C7 region, possibly impacting regulatory interactions of cMyBP-C with myosin and actin. Based on the data presented here, we propose a model in which mutant W792R cMyBP-C preferentially forms Ca2+ sensitizing interactions with actin, rather than inhibitory interactions with myosin. The W792R-cMyBP-C mouse model provides mechanistic insights into the pathology of this mutation and may provide a mechanism by which other central domain missense mutations in cMyBP-C may alter contractility, leading to HCM.

Phosphorylation-dependent interactions of myosin-binding protein C and troponin coordinate the myofilament response to protein kinase A.

PKA-mediated phosphorylation of sarcomeric proteins enhances heart muscle performance in response to ß-adrenergic stimulation and is associated with accelerated relaxation and increased cardiac output for a given preload. At the cellular level, the latter translates to a greater dependence of Ca2+ sensitivity and maximum force on sarcomere length (SL), that is, enhanced length-dependent activation. However, the mechanisms by which PKA phosphorylation of the most notable sarcomeric PKA targets, troponin I (cTnI) and myosin-binding protein C (cMyBP-C), lead to these effects remain elusive. Here, we specifically altered the phosphorylation level of cTnI in heart muscle cells and characterized the structural and functional effects at different levels of background phosphorylation of cMyBP-C and with two different SLs. We found Ser22/23 bisphosphorylation of cTnI was indispensable for the enhancement of length-dependent activation by PKA, as was cMyBP-C phosphorylation. This high level of coordination between cTnI and cMyBP-C may suggest coupling between their regulatory mechanisms. Further evidence for this was provided by our finding that cardiac troponin (cTn) can directly interact with cMyBP-C in vitro, in a phosphorylation- and Ca2+-dependent manner. In addition, bisphosphorylation at Ser22/Ser23 increased Ca2+ sensitivity at long SL in the presence of endogenously phosphorylated cMyBP-C. When cMyBP-C was dephosphorylated, bisphosphorylation of cTnI increased Ca2+ sensitivity and decreased cooperativity at both SLs, which may translate to deleterious effects in physiological settings. Our results could have clinical relevance for disease pathways, where PKA phosphorylation of cTnI may be functionally uncoupled from cMyBP-C phosphorylation due to mutations or haploinsufficiency.

Drug discovery for heart failure targeting myosin-binding protein C.

Cardiac MyBP-C (cMyBP-C) interacts with actin and myosin to fine-tune cardiac muscle contractility. Phosphorylation of cMyBP-C, which reduces the binding of cMyBP-C to actin and myosin, is often decreased in patients with heart failure (HF) and is cardioprotective in model systems of HF. Therefore, cMyBP-C is a potential target for HF drugs that mimic its phosphorylation and/or perturb its interactions with actin or myosin. We labeled actin with fluorescein-5-maleimide (FMAL) and the C0-C2 fragment of cMyBP-C (cC0-C2) with tetramethylrhodamine (TMR). We performed two complementary high-throughput screens (HTS) on an FDA-approved drug library, to discover small molecules that specifically bind to cMyBP-C and affect its interactions with actin or myosin, using fluorescence lifetime (FLT) detection. We first excited FMAL and detected its FLT, to measure changes in fluorescence resonance energy transfer (FRET) from FMAL (donor) to TMR (acceptor), indicating binding. Using the same samples, we then excited TMR directly, using a longer wavelength laser, to detect the effects of compounds on the environmentally sensitive FLT of TMR, to identify compounds that bind directly to cC0-C2. Secondary assays, performed on selected modulators with the most promising effects in the primary HTS assays, characterized the specificity of these compounds for phosphorylated versus unphosphorylated cC0-C2 and for cC0-C2 versus C1-C2 of fast skeletal muscle (fC1-C2). A subset of identified compounds modulated ATPase activity in cardiac and/or skeletal myofibrils. These assays establish the feasibility of the discovery of small-molecule modulators of the cMyBP-C-actin/myosin interaction, with the ultimate goal of developing therapies for HF.

Lipid-binding antiphospholipid antibodies: significance for pathophysiology and diagnosis of the antiphospholipid syndrome.

The antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence of pathogenic antiphospholipid antibodies (aPL). Since approximately 30 years ago, lipid-binding aPL, which do not require a protein cofactor, have been regarded as irrelevant for APS pathogenesis even though anticardiolipin are a diagnostic criterion of APS. In this review, we will summarize the available evidence from in vitro studies, animal models, and epidemiologic studies, which suggest that this concept is no longer tenable. Accordingly, we will only briefly touch on the role of other aPL in APS. This topic has been amply reviewed in detail elsewhere. We will discuss the consequences for laboratory diagnostics and future research required to resolve open questions related to the pathogenic role of different aPL specificities.

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.

Analysis of PROC mutations and clinical features in 22 unrelated families with inherited protein C deficiency.

Currently, limited information is available in the literature regarding the relationships between PROC mutations and clinical features in Chinese individuals. We aimed to characterize severe congenital Protein C deficiency in 22 unrelated Chinese families in a tertiary hospital by analyzing its clinical manifestation, associated risk factors, and gene mutations. We measured protein C activity and antigen levels for all participants, screened them for mutations in the PROC gene, and analyzed the clinical features of each family to identify commonalities and differences. The analysis revealed a total of 75 individuals with PCD and 16 different PROC mutations, including 12 missense mutations and 4 deletion mutations. Among them, 11 who were compound heterozygotes or homozygotes for mutations tended to develop symptoms at a younger age without any clear triggers. In contrast, the remaining 64 individuals who were heterozygotes for mutations often had clear triggers for their symptoms and experienced a milder course of the disease. It is worth noting that the mutation c.565C > T occurred most frequently, being identified in 8 out of 22 families (36%). Our team also reported five novel mutations, including c.742-744delAAG, c.383G > A, c.997G > A, c.1318C > T, and c.833T > C mutations. The identification of five novel mutations adds to the richness of the Human Genome Database. Asymptomatic heterozygotes are not uncommon, and they are prone to develop symptoms with obvious triggers. The evidence presented strongly suggest that asymptomatic individuals with family history of protein C deficiency can benefit from mutational analysis of PROC gene.

Venous thromboembolism risk in adults with hereditary thrombophilia: a systematic review and meta-analysis.

This systematic review and meta-analysis assesses venous thromboembolism (VTE) risk in adults with hereditary thrombophilia, including Factor V Leiden (FVL) mutation, prothrombin G20210A (FII) mutation, compound heterozygosity, protein C (PC), protein S (PS), and antithrombin (AT) deficiency. Eligibility criteria included studies suitable for quantitative synthesis with extractable information on VTE risk in adults (> 15 years). There were no restrictions on VTE type, location, or occurrence. Two authors reviewed all studies and extracted data from 107 publications, encompassing 107,130 individuals (21,560 experiencing VTE). We used a random effects model and calculated odds ratios (ORs) with 95% confidence intervals (CIs). The highest risk was associated with homozygous FVL (OR 5.58, 95% CI 4.61-6.74), homozygous FII (OR 5.16, 95% CI 3.12-8.52), and compound heterozygosity (OR 4.64, 95% CI 2.25-9.58). In contrast, VTE risk was lowest for FVL heterozygosity (OR 2.97, 95% CI 2.41-3.67) and FII heterozygosity (OR 2.21, 95% CI 1.70-2.87), whereas PC (OR 3.23, 95% CI 2.05-5.08), PS (OR 3.01, 95% CI 2.26-4.02), and AT deficiency (OR 4.01, 95% CI 2.50-6.44) demonstrated an intermediate VTE risk. These results highlight an increased risk of venous thromboembolism in adults with hereditary thrombophilia. However, the risk for patients with PC, PS, and AT deficiency appears to be lower than previously stated, likely due to varying thrombogeneity of the underlying genetic mutations. Further research addressing this aspect of VTE risk in hereditary thrombophilia is imperative to improve patient management. TRIAL REGISTRATION: PROSPERO registration number CRD42022376757.

Analysis of four hereditary protein C deficiencies associated with vascular thromboembolism.

OBJECTIVE: To analyze the clinical features and gene mutations in four families with hereditary protein C (PC) deficiency and explore their association with vascular thromboembolism. METHODS: The clinical data of four patients with PC deficiency were retrospectively analyzed. Venous blood samples were collected from the four affected patients and their family members, and relevant coagulation indexes and thrombin production and inhibition tests were performed. PCR was used to amplify and directly sequence the PROC gene of the probands. Software analysis was conducted to assess the conservativeness and pathogenicity of the mutated loci. Protein models were constructed to analyze the spatial structure before and after the mutation. RESULTS: Thrombin generation and inhibition assays demonstrated impaired anticoagulation in all four probands. Proband 1 and 4 presented clinically with pulmonary embolism and lower extremity deep vein thrombosis (DVT), Proband 2 with cerebral infarction, and Proband 3 with DVT. Genetic analysis revealed the presence of the following mutations: c.541T > G heterozygous missense mutation, c.577-579delAAG heterozygous deletion mutation, c.247-248insCT heterozygous insertion mutation, c.659G > A heterozygous missense mutation, and a new variant locus c.1146_1146delT heterozygous deletion mutation in the four probands, respectively. In particular, c.1146_1146delT heterozygous deletion mutations not reported previously. Conservativeness and pathogenicity analyses confirmed that most of these amino acid residues were conserved, and all the mutations were found to be pathogenic. Analysis of protein modeling revealed that these mutations induced structural alterations in the protein or led to the formation of truncated proteins. According to the American College of Medical Genetics and Genomics (ACMG) classification criteria and guidelines for genetic variants, c.1146_1146delT was rated as pathogenic (PVS1 + M2 + PM4 + PP1 + PP3 + PP4). CONCLUSION: The identified mutations are likely associated with decreased PC levels in each of the four families. The clinical manifestations of hereditary PC deficiency exhibit considerable diversity.

Coagulation and Inflammation in COVID-19: Reciprocal Relationship between Inflammatory and Coagulation Markers.

The coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), formerly known as 2019-nCoV. Numerous cellular and biochemical issues arise after COVID-19 infection. The severe inflammation that is caused by a number of cytokines appears to be one of the key hallmarks of COVID-19. Additionally, people with severe COVID-19 have coagulopathy and fulminant thrombotic events. We briefly reviewed the COVID-19 disease at the beginning of this paper. The inflammation and coagulation markers and their alterations in COVID-19 illness are briefly discussed in the parts that follow. Next, we talked about NETosis, which is a crucial relationship between coagulation and inflammation. In the end, we mentioned the two-way relationship between inflammation and coagulation, as well as the factors involved in it. We suggest that inflammation and coagulation are integrated systems in COVID-19 that act on each other in such a way that not only inflammation can activate coagulation but also coagulation can activate inflammation.

Activated Protein C Strengthens Cardiac Tolerance to Ischemic Insults in Aging.

BACKGROUND: APC (activated protein C) is a plasma serine protease with anticoagulant and anti-inflammatory activities. EPCR (Endothelial protein C receptor) is associated with APC's activity and mediates its downstream signaling events. APC exerts cardioprotective effects during ischemia and reperfusion (I/R). This study aims to characterize the role of the APC-EPCR axis in ischemic insults in aging. METHODS: Young (3-4 months) and aged (24-26 months) wild-type C57BL/6J mice, as well as EPCR point mutation (EPCRR84A/R84A) knockin C57BL/6J mice incapable of interaction with APC and its wild type of littermate C57BL/6J mice, were subjected to I/R. Wild-type APC, signaling-selective APC-2Cys, or anticoagulant-selective APC-E170A were administrated before reperfusion. RESULTS: The results demonstrated that cardiac I/R reduces APC activity, and the APC activity was impaired in the aged versus young hearts possibly attributable to the declined EPCR level with aging. Serum EPCR measurement showed that I/R triggered the shedding of membrane EPCR into circulation, while administration of APC attenuated the I/R-induced EPCR shedding in both young and aged hearts. Subsequent echocardiography showed that APC and APC-2Cys but not APC-E170A ameliorated cardiac dysfunction during I/R in both young and aged mice. Importantly, APC elevated the resistance of the aged heart to ischemic insults through stabilizing EPCR. However, all these cardioprotective effects of APC were blunted in the EPCRR84A/R84A mice versus its wild-type littermates. The ex vivo working heart and metabolomics results demonstrated that AMPK (AMP-activated protein kinase) mediates acute adaptive response while AKT (protein kinase B) is involved in chronic metabolic programming in the hearts with APC treatment. CONCLUSIONS: I/R stress causes shedding of the membrane EPCR in the heart, and administration of APC prevents I/R-induced cardiac EPCR shedding that is critical for limiting cardiac damage in aging.

New plasma protein C and protein S concentrate: A synergy for therapeutic purposes.

BACKGROUND AND OBJECTIVES: Deficiencies of protein C (PC) or protein S (PS) are rare diseases, characterized by mutations in the PC or PS genes, which encode plasma serine proteases with anti-coagulant activity. Severe PC or PS deficiencies manifest in early life as neonatal purpura fulminans, a life-threatening heamorrhagic condition requiring immediate treatment. First-line treatment involves replacement therapy, followed by maintenance with anti-coagulants. Replacement therapy with specific protein concentrates is currently only limited to PC, and therefore, a PC + PS concentrate represents a useful addition to therapeutic options, particularly for severe PS deficiency. Further, the production of a PC + PS concentrate from unused plasma fractionation intermediates would impact favourably on manufacturing costs, and consequently therapy prices for patients and health systems. MATERIALS AND METHODS: Several chromatographic runs were performed on the same unused plasma fractionation intermediates using different supports to obtain a PC/PS concentrate. The best chromatographic mediums were chosen, in terms of specific activity and recovery. A full process of purification including virus inactivation/removal and lyophilization steps was set up. RESULTS: The final freeze-dried product had a mean PC concentration of 47.75 IU/mL with 11% of PS, and a mean specific activity of 202.5 IU/mg protein, corresponding to over 12,000-fold purification from plasma. CONCLUSION: The development of a novel concentrated PC/PS mixture obtained from a waste fraction of other commercial products could be used for its potential therapeutic role in the management of neonatal purpura fulminans pathology.

A recombinant signalling-selective activated protein C that lacks anticoagulant activity is efficacious and safe in cutaneous wound preclinical models.

Various preclinical and clinical studies have demonstrated the robust wound healing capacity of the natural anticoagulant activated protein C (APC). A bioengineered APC variant designated 3K3A-APC retains APC's cytoprotective cell signalling actions with <10% anticoagulant activity. This study was aimed to provide preclinical evidence that 3K3A-APC is efficacious and safe as a wound healing agent. 3K3A-APC, like wild-type APC, demonstrated positive effects on proliferation of human skin cells (keratinocytes, endothelial cells and fibroblasts). Similarly it also increased matrix metollaproteinase-2 activation in keratinocytes and fibroblasts. Topical 3K3A-APC treatment at 10 or 30 µg both accelerated mouse wound healing when culled on Day 11. And at 10 µg, it was superior to APC and had half the dermal wound gape compared to control. Further testing was conducted in excisional porcine wounds due to their congruence to human skin. Here, 3K3A-APC advanced macroscopic healing in a dose-dependent manner (100, 250 and 500 µg) when culled on Day 21. This was histologically corroborated by greater collagen maturity, suggesting more advanced remodelling. A non-interference arm of this study found no evidence that topical 3K3A-APC caused either any significant systemic side-effects or any significant leakage into the circulation. However the female pigs exhibited transient and mild local reactions after treatments in week three, which did not impact healing. Overall these preclinical studies support the hypothesis that 3K3A-APC merits future human wound studies.