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Severe protein C deficiency (SPCD) is a rare inherited thrombotic disease associated with high morbidity and mortality. In the current study, we established a viable murine model of SPCD, enabling preclinical gene therapy studies. By creating SPCD mice with severe hemophilia A (PROC -/- /F8 - ), the multi-month survival of SPCD mice enabled the exploration of recombinant adeno-associated viral vector-PC (rAAV8-PC) gene therapy (GT). rAAV8- PC (10 12 vg/kg of AAV8-PC) was injected via the tail vein into 6-8-week-old PROC -/- /F8 - mice. Their plasma PC antigen levels (median of 714 ng/mL, range 166-2488 ng/mL) and activity (303.5 ± 59%) significantly increased to the normal range after GT compared to untreated control animals. PC's presence in the liver after GT was also confirmed by immunofluorescence staining. Our translational research results provide the first proof of concept that an infusion of rAAV8-PC increases PC antigen and activity in mice and may contribute to future GT in SPCD. Further basic research of SPCD mice with prolonged survival due to the rebalancing of this disorder using severe hemophilia A may provide essential data regarding PC's contribution to specific tissues' development, local PC generation, and its regulation in inflammatory conditions.

Background: Protein C (PC) pathway serves as a major defense mechanism against thrombosis by the activation of PC through the thrombin-thrombomodulin complex and subsequent inactivation of the activated factor (F)V (FVa) and FVIII (FVIIIa) with the assistance of protein S, thereby contributing to hemostatic balance. We identified 2 unrelated patients who suffered from recurrent thrombosis and carried the same heterozygous mutation c.1153A>G, p.Met343Val (M343V), in PROC gene. This mutation had not been previously reported., Objectives: To explore the molecular basis underlying the anticoagulant defect in patients carrying the M343V mutation in PROC., Methods: We expressed PC-M343V variant in mammalian cells and characterized its properties through coagulation assays., Results: Our findings demonstrated that while activation of mutant zymogen by thrombin-thrombomodulin complex was slightly affected, cleavage of chromogenic substrate by APC-M343V was significantly impaired. However, Ca 2+ increased the cleavage efficiency by approximately 50%. Additionally, there was a severe reduction in affinity between APC-M343V and Na + . Furthermore, the inhibitory ability of APC-M343V toward FVa was markedly impaired. Structural and simulation analyses suggested that Val343 might disrupt the potential hydrogen bonds with Trp380 and cause Trp380 to orient closer to His211, potentially interfering with substrate binding and destabilizing the catalytic triad of APC., Conclusion: The M343V mutation in patients adversely affects the reactivity and/or folding of the active site as well as the binding of the physiological substrate to the protease, resulting in impaired protein C anticoagulant activity and ultimately leading to thrombosis., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2024 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Protein C (PC), a vitamin K-dependent serine protease zymogen in plasma, can be activated by thrombin-thrombomodulin(TM) complex, resulting in the formation of activated protein C (APC). APC functions to downregulate thrombin generation by inactivating active coagulation factors V(FVa) and VIII(FVIIIa). Deficiency in PC increases the risk of venous thromboembolism (VTE). We have identified two unrelated VTE patients with the same heterozygous mutation (c.1384 T > C, p.Ter462GlnextTer17) in PROC. To comprehend the role of this mutation in VTE development, we expressed recombinant PC-Ter462GlnextTer17 in mammalian cells and evaluated its characteristics using established coagulation assay systems. Functional studies revealed a significant impairment in the activation of the mutant by thrombin or thrombin-TM complex. Furthermore, APC-Ter462GlnextTer17 demonstrated diminished hydrolytic activity towards the chromogenic substrate S2366. APTT and FVa degradation assays showed that both the anticoagulant activity of the mutant protein was markedly impaired, regardless of whether protein S was present or absent. These results were further supported by a thrombin generation assay conducted using purified and plasma-based systems. In conclusion, the Ter462GlnextTer17 mutation introduces a novel tail at the C-terminus of PC, leading to impaired activity in both PC zymogen activation and APC's anticoagulant function. This impairment contributes to thrombosis in individuals carrying this heterozygous mutation and represents a genetic risk factor for VTE., Competing Interests: Declaration of competing interest The authors state that there are no conflicts of interest to disclose. This study was supported by Shanghai Pujiang Program (21PJ1409800), Shanghai Natural Science Foundation Program (22ZR1439500), and the General Program of National Natural Science Foundation of China (81870107) to YL; the General Program of National Natural Science Foundation of China (82070137) to XW; the General Program of National Natural Science Foundation of China (81970127) to JD, and the General Program of National Natural Science Foundation of China (82070194) to XC., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

SARS-CoV-2 can induce vascular dysfunction and thrombotic events in patients with severe COVID-19; however, the cellular and molecular mechanisms behind these effects remain largely unknown. In this study, we used a combination of experimental and in silico approaches to investigate the role of PC in vascular and thrombotic events in COVID-19. Single-cell RNA-sequencing data from patients with COVID-19 and healthy subjects were obtained from the publicly available Gene Expression Omnibus (GEO) repository. In addition, HUVECs were treated with inactive protein C before exposure to SARS-CoV-2 infection or a severe COVID-19 serum. An RT-qPCR array containing 84 related genes was used, and the candidate genes obtained were evaluated. Activated protein C levels were measured using an ELISA kit. We identified at the single-cell level the expression of several pro-inflammatory and pro-coagulation genes in endothelial cells from the patients with COVID-19. Furthermore, we demonstrated that exposure to SARS-CoV-2 promoted transcriptional changes in HUVECs that were partly reversed by the activated protein C pretreatment. We also observed that the serum of severe COVID-19 had a significant amount of activated protein C that could protect endothelial cells from serum-induced activation. In conclusion, activated protein C protects endothelial cells from pro-inflammatory and pro-coagulant effects during exposure to the SARS-CoV-2 virus.

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., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background:  Protein C (PC) is a vitamin K-dependent anticoagulant serine protease zymogen which upon activation by the thrombin-thrombomodulin (TM) complex downregulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. We identified a thrombosis patient who carried a heterozygous mutation c.881G > A, p.Ser252Asn (S252N) in PROC . This mutation was originally described in a report of novel mutations in patients presenting with defective PC anticoagulant activity in Paris. The research identified PC-S252N (the "Paris" mutation) in a propositus and her family members and highlighted the critical role of Ser252 in the anticoagulation process of activated PC (APC)., Material and Methods:  We expressed the PC-S252N mutant in mammalian cells and characterized the properties in coagulation assays to decipher the molecular basis of anticoagulant defect of this mutation., Results:  We demonstrated that PC-S252N had a diminished ability to TM binding, which resulted in its impaired activation by the thrombin-TM complex. However, APC-S252N exhibited a slightly stronger cleavage capacity for the chromogenic substrate. Meanwhile, the catalytic activity of APC-S252N toward FVa was significantly reduced. Sequence analysis revealed that Ser252 to Asn substitution introduced a new potential N-linked glycosylation site ( 252 NTT 254 ) in the catalytic domain of PC, which adversely affected both the activation process of PC and anticoagulant activity of APC., Conclusion:  The new N-glycosylation site ( 252 NTT 254 ) resulting from the mutation of Ser252 to Asn252 in PROC affects the overall structure of the protease, thereby adversely affecting the anticoagulant function of protein C. This modification has a negative impact on both TM-promoted activation of protein C and APC cleavage of FVa, ultimately leading to thrombosis in the patient., Competing Interests: None declared., (Thieme. All rights reserved.)

Objectives: Protein C (PC) is an anticoagulant that is encoded by the PROC gene. Validation for the function of PC was carried out in mouse models., Methods: In this study, autosomal recessive PC deficiency (PCD) was selected as the target, and the specific mutation site was chromosome 2 2q13-q14, PROC c.1198G>A (p.Gly400Ser) which targets G399S (GGT to AGC) in mouse models. To investigate the role of hereditary PC in mice models, we used CRISPR/Cas9 gene editing technology to create a mouse model with a genetic PCD mutation., Results: The two F0 generation positive mice produced using the CRISPR/Cas9 gene editing technique were chimeras, and the mice in F1 and F2 generations were heterozygous. There was no phenotype of spontaneous bleeding or thrombosis in the heterozygous mice, but some of them were blind. Blood routine results showed no significant difference between the heterozygous mice and wild-type mice ( P  > 0.05). Prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin time (TT) were prolonged in the heterozygous mice, while the level of fibrinogen content (FIB) decreased, suggesting secondary consumptive coagulation disease. The protein C activity of heterozygous mice was significantly lower than that of wild-type mice ( P  < 0.001), but there was no significant difference in protein C antigen levels ( P  > 0.05). H&E staining showed steatosis and hydrodegeneration in the liver of heterozygous mice. Necrosis and exfoliated epithelial cells could be observed in renal tubule lumen, forming cell or granular tubules. Hemosiderin deposition was found in the spleen along with splenic hemorrhage. Immunohistochemistry demonstrated significant fibrin deposition in the liver, spleen, and kidney of heterozygous mice., Conclusion: In this study, heterozygotes of the mouse model with a PC mutation were obtained. The function of PC was then validated in a mouse model through genotype, phenotype, and PC function analysis., Competing Interests: The authors declare there are no competing interests., (©2024 Liu et al.)

Protein C (PC) is a vitamin K-dependent factor that plays a crucial role in controlling anticoagulant processes and acts as a cytoprotective agent to promote cell survival. Several mutations in human PC are associated with decreased protein production or altered protein structure, resulting in PC deficiency. In this study, we conducted a comprehensive analysis of nonsynonymous single nucleotide polymorphisms in human PC to prioritize and confirm the most high-risk mutations predicted to cause disease. Of the 340 missense mutations obtained from the NCBI database, only 26 were classified as high-risk mutations using various bioinformatic tools. Among these, we identified that 12 mutations reduced the stability of protein, and thereby had the greatest potential to disturb protein structure and function. Molecular dynamics simulations revealed moderate alterations in the structural stability, flexibility, and secondary structural organization of the serine protease domain of human PC for five missense mutations (L305R, W342C, G403R, V420E, and W444C) when compared to the native structure that could maybe influence its interaction with other molecules. Protein-protein interaction analyses demonstrated that the occurrence of these five mutations can affect the regular interaction between PC and activated factor V. Therefore, our findings assume that these mutants can be used in the identification and development of therapeutics for diseases associated with PC dysfunction, although assessment the effect of these mutations need to be proofed in in-vitro., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Farajzadeh-Dehkordi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total., Methods: Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes., Results: Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels ( GCKR , BAZ1B , and HP-TXNL4B ) and 1 with PS levels ( ORM1 - ORM2 ). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level ( FCGRT ), 1 with PC ( GOLM2 ), and 1 with PS ( MYL7 ). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR , SNX17 , and HP genes in antithrombin regulation., Conclusions: The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels., Competing Interests: Disclosures None.

Childhood interstitial lung disease (chILD) is a collective term for a group of rare lung disorders of heterogeneous origin. Surfactant dysfunction disorders are a cause of chILD with onset during the neonatal period and infancy. Clinical signs of tachypnea and hypoxemia are nonspecific and usually caused by common conditions like lower respiratory tract infections. We report on a full-term male newborn who was readmitted to the hospital at 7 days of age with marked tachypnea and poor feeding during the respiratory syncytial virus season. After exclusion of infection and other, more common congenital disorders, chILD was diagnosed using chest computed tomography and genetic analysis. A likely pathogenic heterozygous variant of SFTPC (c.163C>T, L55F) was detected by whole exome sequencing. The patient received supplemental oxygen and noninvasive respiratory support and was treated with intravenous methylprednisolone pulses and hydroxychloroquine. Despite the treatment, his respiratory situation deteriorated continuously, leading to several hospitalizations and continuous escalation of noninvasive ventilatory support. At 6 months of age, the patient was listed for lung transplant and transplanted successfully aged 7 months., (Copyright © 2023 by the American Academy of Pediatrics.)

Dicentric chromosome assay (DCA) is the most accepted cytological technique for the purpose of biological dosimetry in radiological and nuclear accidents, however, it is not always easy to evaluate dicentric chromosomes because of the technical difficulty in identifying dicentric chromosomes on Giemsa-stained metaphase chromosome samples. Here, we applied an antibody recognizing centromere protein (CENP) C, CENP-C, whose antigenicity is resistant to the fixation with Carnoy's solution. Normal human diploid cells were irradiated with various doses of 137Cs γ rays at 1 Gy/ min, treated with hypotonic solution, fixed with Carnoy's fixative, and metaphase chromosome spreads were stained with anti-CENP-C antibody. Dose-dependent induction of dicentric chromosomes was confirmed between 1 and 10 Gy of γ rays, and the results were compatible with those obtained by the conventional Giemsa-stained chromosome samples. The CENP-C assay also uncovered the difference in the fluorescence from the sister centromeres on the same chromosome, which was more pronounced after radiation exposure. Although the underlying mechanism is still to be determined, the result suggests a novel effect of radiation on centromeres. The innovative protocol for CENP-C-based DCA, which enables ideal visualization of centromeres, is simple, effective and reliable. It does not require skilled examiners, so that it may be an alternative method, avoiding uneasiness of the current DCA using Giemsa-stained metaphase chromosome samples., (© 2023 by Radiation Research Society. All rights of reproduction in any form reserved.)

Activated protein C (APC) is an important anticoagulant protein that regulates thrombin generation through inactivation of factor V (FV) and activated factor V (FVa). The rate of APC inactivation of FV is slower compared to FVa, although proteolysis occurs at the same sites (Arg 306 , Arg 506 , and Arg 679 ). The molecular basis for FV resistance to APC is unknown. Further, there is no information about how FV-short, a physiologically relevant isoform of FV with a shortened B-domain, is regulated by APC. Here, we identify the molecular determinants which differentially regulate APC recognition of FV versus FVa and uncover how FV-short can be protected from this anticoagulant pathway. Using recombinant FV derivatives and B-domain fragments, we show that the conserved basic region (BR; 963-1008) within the central portion of the B-domain plays a major role in limiting APC cleavage at Arg 506 . Derivatives of FV lacking the BR, including FV-short, were subject to rapid cleavage at Arg 506 and were inactivated like FVa. The addition of a FV-BR fragment reversed this effect and delayed APC inactivation. We also found that anticoagulant glycoprotein TFPIα, which has a C-terminal BR homologous to FV-BR, protects FV-short from APC inactivation by delaying cleavage at Arg 506 . We conclude that the FV-BR plays a major role in protecting FV from APC inactivation. Using a similar mechanistic strategy, TFPIα also shields FV-short from APC. These findings clarify the resistance of FV to APC, advance our understanding of FV/FVa regulation, and establish a mechanistic framework for manipulating this reaction to alter coagulation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Pulmonary surfactant dysfunction disorders are caused by genetic defects that alter pulmonary surfactant metabolism. They are rare disorders and cause significant morbidity and mortality in the neonatal and pediatric populations., Objective: To describe the clinical, histopathological, and ultrastructural findings of the lamellar body that suggest surfactant protein C (SP-C) dysfunction, where confirmatory genetic studies are not available., Clinical Case: We report three pediatric cases of pul monary surfactant dysfunction disorders from a pediatric hospital in Peru. Video-assisted lung biop sy was performed in all cases. Ultrastructural studies of the lamellar body were compatible with type- C pulmonary surfactant dysfunction. The treatment used was methylprednisolone pulses monthly for six months, then every two months, varying the duration according to the clinical evolution. They also received daily hydroxychloroquine and azithromycin three times a week. Clinical evaluations, eye fundus, echocardiogram, electrocardiogram, and biochemistry were performed periodically. At follow-up, there was a good response to treatment and no adverse effects were observed. One case died despite the therapies received., Conclusions: In 3 patients with type-C surfactant dysfunction, treatment with corticosteroids, hydroxychloroquine, and azithromycin was successful in 2 of them. This is one of the first case series reported in Peru that contributes to the study of these diseases, es pecially in low- and medium-income countries.

Homozygous protein C deficiency is a rare hypercoagulability disorder. This study describes the ocular manifestations and the genetic background in a family with two affected children. This is a retrospective review of ophthalmic examinations, investigations, genetic testing, and blood work-up of two children with homozygous protein C deficiency from a single family. A family with a positive history of consanguineous marriage was found to have two affected children with homozygous protein C deficiency. Abnormal visual behavior was the presenting symptom. Both children had bilateral total tractional retinal detachments at presentation. Skin manifestations included episodes of discoloration and bruising. Laboratory work-up revealed absent protein C activity. Genetic testing confirmed the presence of a homozygous pathogenic mutation in protein C gene (NM&#95;000312.3: c.1297G>A: p.Gly433Ser). Homozygous protein C deficiency should be considered in the differential diagnosis of early-onset tractional retinal detachment in infancy. Although rare, the ophthalmologist may be the first to encounter the condition, and treatment with protein C replacement or anticoagulants may be life-saving. Examination under anesthesia with fluorescein angiography and laser treatment early in life may be warranted to preserve vision. [ Ophthalmic Surg Lasers Imaging Retina . 2022;53:293-296. ].

The ubiquitin ligase C-terminus of Hsc70 interacting protein (CHIP) is an important regulator of proteostasis. Despite playing an important role in maintaining proteostasis, little progress has been made in developing small molecules that regulate ubiquitin transfer by CHIP. Here we used differential scanning fluorimetry to identify compounds that bound CHIP. Compounds that bound CHIP were then analyzed by quantitative ubiquitination assays to identify those that altered CHIP function. One compound, MS.001, inhibited both the chaperone binding and ubiquitin ligase activity of CHIP at low micromolar concentrations. Interestingly, we found that MS.001 did not have activity against isolated U-box or tetratricopeptide (TPR) domains, but instead only inhibited full-length CHIP. Using in silico docking we identified a potential MS.001 binding site on the linker domain of CHIP and mutation of this site rendered CHIP resistant to MS.001. Together our data identify an inhibitor of the E3 ligase CHIP and provides insight into the development of compounds that regulate CHIP activity., (© 2022 Wiley-VCH GmbH.)

Background: Genetics play a significant role in coagulation phenotype and venous thromboembolism risk. Resistance to the anticoagulant activated protein C (APC) is an established risk for thrombosis. Herein, we explored the genetic determinants of thrombin generation (TG) and thrombomodulin (TM)-modulated TG using plasma from the Human Functional Genomics Project., Methods: Calibrated TG was measured both in absence and presence of TM using tissue factor as trigger. Genetic determinants of TG parameters and protein C pathway function were assessed using genome-wide single-nucleotide polymorphism (SNP) genotyping. Plasma samples were supplemented with purified apolipoprotein A-IV, prekallikrein, or kallikrein to test their influence on the anticoagulant function of TM and APC in TG., Results: Thrombin generation data from 392 individuals were analyzed. Genotyping showed that the KLKB1 gene (top SNP: rs4241819) on chromosome 4 was associated with the normalized sensitivity ratio of endogenous thrombin potential to TM at genome-wide level (nETP-TMsr, P = 4.27 × 10 -8 ). In vitro supplementation of kallikrein, but not prekallikrein or apolipoprotein A-IV, into plasma dose-dependently augmented the anticoagulant effect of TM and APC in TG. Variations of rs4241819 was not associated with the plasma concentration of prekallikrein. Association between rs4241819 and nETP-TMsr was absent when TG was measured in presence of a contact pathway inhibitor corn trypsin inhibitor., Conclusions: Our results suggest that kallikrein plays a role in the regulation of the anticoagulant protein C pathway in TG, which may provide a novel mechanism for the previously observed association between the KLKB1 gene and venous thrombosis., (© 2021 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated protein C (aPC), a promising therapeutic, signals via protease-activated receptor-1 (PAR1) and mediates several cytoprotective responses, including endothelial barrier stabilization and anti-apoptotic responses. We showed that aPC-activated PAR1 signals preferentially via β-arrestin-2 (β-arr2) and dishevelled-2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti-apoptotic activities are not known. aPC/PAR1 cytoprotective responses also require coreceptors; however, it is not clear how coreceptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. Here, we define a β-arr2-mediated sphingosine kinase-1 (SphK1)-sphingosine-1-phosphate receptor-1 (S1PR1)-Akt signaling axis that confers aPC/PAR1-mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 coexist in caveolin-1 (Cav1)-rich microdomains and that S1PR1 coassociation with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates β-arr2-dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1-Akt signaling and protection against cell death. While aPC/PAR1-induced, extracellular signal-regulated kinase 1/2 (ERK1/2) activation is also dependent on β-arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1-β-arr2-mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete, β-arr2-driven signaling pathways in caveolae., Competing Interests: The authors declare no competing interest.

To explore the pathogenesis of protein C (PC) deficiency in two independent families by mutations detection and bioinformatics analysis. The PC activity (PC:A) and PC antigen (PC:Ag) were detected by chromogenic substrate and ELISA, respectively. The PROC sequencing was performed to identify the mutational sites. The molecular pathogenesis of the mutations were studied by the conservation, bioinformatics and model analysis. The PC:A and PC:Ag of the proband 1 were observably reduced at 35 and 44%, respectively. Gene sequencing analysis revealed the p.Leu278Pro derived from a heterozygous c.833T>C point mutations in exon 9 of PROC gene. For proband 2, the PC:A and PC:Ag were decreased to 40 and 51%, respectively, caused p.Ala178Pro missense mutation by a heterozygous point mismatch of c.532G>C in exon 5 of PROC gene. Bioinformatics and model analysis indicated that it was the Leu278Pro and Ala178Pro that caused clinical PC deficiency (PCD). The heterozygous mutations Leu278Pro and Ala178Pro were observed in two independent families. The Leu278Pro mutation in the PROC gene has not been described elsewhere. The two mutations can both lead to the type I hereditary PCD, and probably be the major causes of PCD in the families., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Activated protein C (APC) is an anticoagulant plasma serine protease which exhibits potent cytoprotective and anti-inflammatory activities. Here, we studied protective effects of APC on the proinflammatory function of polyinosinic:polycytidylic acid [poly(I:C)], a synthetic analog of viral double-stranded RNA, in cellular and animal models. Poly(I:C) induced histone H3 extranuclear translocation via interaction with toll-like receptor 3 in two established endothelial cell lines. Furthermore, poly(I:C) induced histone H3 extranuclear translocation in J774A.1 macrophages and human neutrophils and formation of macrophage and neutrophil extracellular traps (ETs). Mechanistically, poly(I:C) was found to upregulate expression of peptidylarginine deiminase 4 and enhance its interaction with histone H3, thereby leading to increased histone citrullination and neutrophil ET formation. Poly(I:C) elicited proinflammatory signaling responses by inducing nuclear factor kappa B activation and disrupting endothelial cell permeability. In vivo, poly(I:C) enhanced cell surface expression of Mac-1 on neutrophils in mice and facilitated their infiltration to lung tissues. Poly(I:C) also downregulated thrombomodulin expression in mouse tissues and reduced its circulating soluble level in plasma. We demonstrate in this study that APC and a signaling-selective mutant of APC effectively inhibit proinflammatory signaling effects of poly(I:C) in both cellular and animal models. We further demonstrate that unlike the requirement for endothelial protein C receptor on endothelial cells, the integrin Mac-1 is involved in the protease-activated receptor 1-dependent APC inhibition of macrophage ET formation in J774A.1 cells. Taken together, these results support a key role for APC signaling in inhibiting the viral mimetic-induced proinflammatory signaling responses and histone translocation-associated formation of ETs by innate immune cells., Competing Interests: None declared., (Thieme. All rights reserved.)

Ischemic stroke (IS) is a complex disease regarding its risk factors; among those factors, genetics has an important role. Protein C (PC) is an important antithrombotic enzyme which its genetic variations disrupt the normal cascade of blood coagulation, resulting in thrombosis and increases the chance of stroke. Therefore, we aimed to investigate three single-nucleotide polymorphisms (SNPs) located in the core promoter of PC in order to find their role in this condition in the Iranian population. Blood samples from IS patients (n = 249) and healthy volunteers (n = 203) were collected. Biochemical analysis was performed. Genotyping was conducted on the extracted DNA from blood samples via the HRM technique. Bioinformatic investigations were used to assess how these SNPs may be involved in the IS. Smoking, hypertension, low-density lipoprotein cholesterol, and fasting blood glucose were significantly different between healthy and IS groups. rs1799809 and rs1799810 SNPs were significantly more frequent among IS patients. Also, among four identified haplotypes, CGT was found associated with IS (p = 0.001). It was also found that these SNPs may interfere with the binding of transcription factors to alter the expression of PC. Our data predict that SNPs at the core promoter of PC can affect the binding affinity of transcription factors which in turn reduces the expression of PC and increases the risk of IS., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature.)

The deficiency of protein C (PROC) can be partly rescued by Rex shunt through restoring portal blood flow in children with extra-hepatic portal venous obstruction (EHPVO). However, the decline of PROC is still found in some patients with a normal portal blood flow after Rex shunt. The aim of this study was to identify the candidate miRNAs involving in the decline of PROC and their mechanism. The protein level of PROC was detected by the ELISA assay, and was compared between sick and healthy groups. The expressions of miRNAs and PROC mRNA were measured using qRT-PCR, and were compared between sick and healthy groups. The correlation between PROC and candidate miRNAs was analysed by a Pearson correlation analysis to identify the most significant miRNAs. The expression of PROC mRNA was detected by qRT-PCR in HL-7702 and LX-2 cells tansfected with miRNAs mimics or inhibitors and negative control (NC) mimics, which was compared among the different groups. The rates of liver cells' proliferation and apoptosis were detected in HL-7702 and LX-2 cells tansfected with miRNAs mimics or inhibitors or with overexpressing PROC and negative control mimics by CKK8 assay and flow cytometry, which were compared among the different groups. The expressions of COX-2 and VEGF were measured by qRT-PCR, and were compared between the miRNAs groups and NC group. Western blot was assayed for detecting the protein levels of PROC, COX-2, VEGF, Bcl-2 and Bax, which were compared between the miRNAs groups and NC group. The expression of PROC mRNA was lower, and the expressions of miR-506-3p and miR-124-3p were higher in children with EHPVO than healthy group. PROC mRNA was negatively correlated with the expression of miR-506-3p and miR-124-3p. Compared to the NC group, the transcription activity of PROC was lower after exposure of miR-506 and miR-124 mimics in HL-7702 and LX-2 cells, but this phenomenon was reversed after inhibiting miR-506 and miR-124. The rate of cell proliferation was lower after exposure of miR-506 and miR-124 than the NC group, which was increased after inhibiting miR-506 and miR-124 in HL-7702 cells and overexpressing PROC in LX-2 cells. The apoptotic rate was higher after exposure of miR-506 and miR-124 than the NC group, which was decreased after inhibiting miR-506 and miR-124 in HL-7702 cells and overexpressing PROC in LX-2 cells. The mRNA levels of COX-2 and VEGF were significantly higher after exposure of miR-506 and miR-124 mimics than those in the NC group. The protein levels of PROC and Bcl-2 were down-regulated, and the levels of COX-2, Bax and VEGF were up-regulated after exposure of miR-506 and miR-124 in HL-7702 cells, but this phenomenon was reversed after inhibiting miR-506 and miR-124. MiR-506-3p and miR-124-3p may involve in the decline of PROC in protein and transcriptional level, in which the anti-proliferation and pro-apoptosis role of miR-506-3p and miR-124-3p for liver cells may involve in this mechanism.

We previously demonstrated that heterozygous Gly197 to Arg mutation in PROC is associated with venous thrombosis due to the mutation abrogating both zymogenic and enzymatic activities of protein C and activated protein C (APC). In this study, we investigated the role of Gly197 on the structure and function of protein C by replacing it with Ala, Lys and Glu in separate constructs. Characterization of protein C mutants indicated their activation by thrombin is improved ~5-20-fold with the order of PC-G197K > PC-G197E > PC-G197A > PC-WT. Interestingly, the cofactor function of thrombomodulin (TM) in promoting the activation of zymogens by thrombin followed the reverse order of PC-WT > PC-G197A > PC-G197E > PC-G197K. The thrombin-generation inhibitory profiles of zymogens in a tissue factor-mediated thrombin generation assay using protein C-deficient plasma with or without supplementation with TM followed the same order of zymogen activation in the purified system. Evaluation of anticoagulant activities of APC derivatives by prothrombinase and aPTT assays revealed a normal activity for APC-G197A but dramatically impaired activity for the other two mutants. In the endothelial cell permeability assay, APC-G197A exhibited normal antiinflammatory activity, but the other two mutants were nearly inactive. These results suggest that Gly197 plays a key role in TM cofactor-dependent protein C activation by thrombin. It facilitates the recognition of protein C by thrombin in the presence of TM but impedes it in the absence of the cofactor. In APC, a small residue at this position is required for the proper folding/reactivity of the active-site pocket of the protease, a hypothesis supported by structural modeling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mechanisms thought to regulate activated factor VIII (FVIIIa) cofactor function include A2-domain dissociation and activated protein C (APC) cleavage. Unlike A2-domain dissociation, there is no known phenotype associated with altered APC cleavage of FVIII, and biochemical studies have suggested APC plays a marginal role in FVIIIa regulation. However, the in vivo contribution of FVIIIa inactivation by APC is unexplored. Here we compared wild-type B-domainless FVIII (FVIII-WT) recombinant protein with an APC-resistant FVIII variant (FVIII-R336Q/R562Q; FVIII-QQ). FVIII-QQ demonstrated expected APC resistance without other changes in procoagulant function or A2-domain dissociation. In plasma-based studies, FVIII-WT/FVIIIa-WT demonstrated dose-dependent sensitivity to APC with or without protein S, whereas FVIII-QQ/FVIIIa-QQ did not. Importantly, FVIII-QQ demonstrated approximately fivefold increased procoagulant function relative to FVIII-WT in the tail clip and ferric chloride injury models in hemophilia A (HA) mice. To minimize the contribution of FV inactivation by APC in vivo, a tail clip assay was performed in homozygous HA/FV Leiden (FVL) mice infused with FVIII-QQ or FVIII-WT in the presence or absence of monoclonal antibody 1609, an antibody that blocks murine PC/APC hemostatic function. FVIII-QQ again demonstrated enhanced hemostatic function in HA/FVL mice; however, FVIII-QQ and FVIII-WT performed analogously in the presence of the PC/APC inhibitory antibody, indicating the increased hemostatic effect of FVIII-QQ was APC specific. Our data demonstrate APC contributes to the in vivo regulation of FVIIIa, which has the potential to be exploited to develop novel HA therapeutics., (© 2021 by The American Society of Hematology.)

Introduction: Genetic mutations of PROC and PROS1 are well-known risk factors for deep venous thrombosis (DVT) in the Asian population. However, the genetic profile of Vietnamese patients with DVT remains elusive. This study aimed to investigate the spectrum of genetic mutations of these two genes in Vietnamese patients diagnosed with idiopathic DVT., Materials and Methods: A total of 50 Vietnamese patients diagnosed with idiopathic DVT were recruited in this study. The entire coding regions of the protein C and protein S genes were amplified and directly sequenced to determine genetic alterations., Results: Four and six genetic mutations were detected in protein C and protein S genes, respectively, in 24 Vietnamese DVT patients. PROC c.565C > T (p.R189W) was the most common mutation found in 13 out of 50 patients, while the mutations of PROS1 comprised three missense and three nonsense variants which diffuse along the gene., Conclusions: This study shows that mutations of protein C and protein S genes are prevalent in Vietnamese patients diagnosed with idiopathic DVT, and PROC c.565C > T (p.R189W) was the most common genetic alteration., (© 2020 John Wiley & Sons Ltd.)

Rationale: Hereditary Protein C (PC) deficiency is a rare genetic disorder caused by PROC gene mutation. In this article, we report a case of PC deficiency in a Chinese family due to a novel PROC gene mutation., Study Subject: The proband presented with recurrent cerebral infarction over the course of the previous 3 years. He was admitted to the hospital due to signs of mental retardation., Diagnoses: Physical examination, laboratory tests, and magnetic resonance imaging demonstrated that the proband had a manifestation of PC deficiency that included acute cerebral infarction. DNA sequencing analysis revealed a missense variant, c.1015G > A (p.V339 M from valine to methionine) in exon 9 of the PROC gene. In addition, Sanger sequencing confirmed that the proband's son was heterozygous for the same variant. Therefore, the PROC gene mutation was transmitted in an autosomal dominant inheritance manner., Interventions: The patient was treated with a daily dosage of Warfarin (3.5 mg) and was scheduled to undergo regular blood coagulation tests., Outcomes: At the 3-month follow-up appointment, the patient showed improvements in his overall health condition., Lessons: We identified a novel missense mutation in the PROC gene in a Chinese family which caused a decrease in the PC antigen level and recurrent cerebral infarction., Competing Interests: The authors have no conflicts of interest to disclose., (Copyright © 2021 the Author(s). Published by Wolters Kluwer Health, Inc.)

Bartonellae are Gram-negative facultative-intracellular pathogens that use a type-IV-secretion system (T4SS) to translocate a cocktail of Bartonella effector proteins (Beps) into host cells to modulate diverse cellular functions. BepC was initially reported to act in concert with BepF in triggering major actin cytoskeletal rearrangements that result in the internalization of a large bacterial aggregate by the so-called 'invasome'. Later, infection studies with bepC deletion mutants and ectopic expression of BepC have implicated this effector in triggering an actin-dependent cell contractility phenotype characterized by fragmentation of migrating cells due to deficient rear detachment at the trailing edge, and BepE was shown to counterbalance this remarkable phenotype. However, the molecular mechanism of how BepC triggers cytoskeletal changes and the host factors involved remained elusive. Using infection assays, we show here that T4SS-mediated transfer of BepC is sufficient to trigger stress fiber formation in non-migrating epithelial cells and additionally cell fragmentation in migrating endothelial cells. Interactomic analysis revealed binding of BepC to a complex of the Rho guanine nucleotide exchange factor GEF-H1 and the serine/threonine-protein kinase MRCKα. Knock-out cell lines revealed that only GEF-H1 is required for mediating BepC-triggered stress fiber formation and inhibitor studies implicated activation of the RhoA/ROCK pathway downstream of GEF-H1. Ectopic co-expression of tagged versions of GEF-H1 and BepC truncations revealed that the C-terminal 'Bep intracellular delivery' (BID) domain facilitated anchorage of BepC to the plasma membrane, whereas the N-terminal 'filamentation induced by cAMP' (FIC) domain facilitated binding of GEF-H1. While FIC domains typically mediate post-translational modifications, most prominently AMPylation, a mutant with quadruple amino acid exchanges in the putative active site indicated that the BepC FIC domain acts in a non-catalytic manner to activate GEF-H1. Our data support a model in which BepC activates the RhoA/ROCK pathway by re-localization of GEF-H1 from microtubules to the plasma membrane., Competing Interests: No

Combined oral contraceptives (COCs) induce several changes in the levels of coagulation factors. The levels of procoagulant factors are often increased, while levels of anticoagulant factors are decreased. Fibrinolysis is also affected, even if the effect seems to be more counterbalanced by opposite regulation of profibrinolytic and antifibrinolytic factors. These effects on hemostasis are more pronounced with third- or fourth-generation COC compared with second-generation COC. Venous thromboembolism (VTE) risk increases when multiple risk factors, including genetic and environmental, are present simultaneously. COC use causes changes in coagulation that modify the prothrombotic state induced by preexisting hemostatic alterations in a supra-additive manner. Therefore, testing appears to be of importance not only before implementing COC but also to monitor any potential thrombogenicity induced by COC therapy. Inherited genetic factors, such as factor V Leiden, G20210A prothrombin mutation, antithrombin, protein C or protein S deficiencies, non-O blood group, as well as CYP2C9*2 and the rs4379368 mutations, have all been identified as genetic predictive risk factors of VTE in women. Nevertheless, the screening of these genetic biomarkers is not capable of assessing the phenotypic expression of the risk. This review will focus on the different options for screening the thrombogenic status in this population. Specific attention will be given to the endogenous thrombin potential-based activated protein C resistance, a test aiming at assessing the thrombogenicity induced by hormonal therapies and inherited or acquired thrombophilia., Competing Interests: Dr. Douxfils reports personal fees from QUALIblood, during the conduct of the study; personal fees from Stago, Roche, Roche Diagnostics, Daiichi-Sankyo, and Portola, outside the submitted work. Dr. Bouvy and Mrs. Morimont report personal fees from QUALIblood, during the conduct of the study., (Thieme. All rights reserved.)

Objective: To analyze the phenotype and genetic variants of a pedigree affected with hereditary protein C (PC) deficiency., Methods: The protein C activity (PC:A) of the proband and her family members (a four-generation pedigree including 11 individuals) were tested by chromogenic substrate method, and the protein C antigen (PC:Ag) was detected with an enzyme-linked immunosorbent assay(ELISA). The 9 exons and flanking sequences of the protein C (PROC) gene were amplified by PCR and directly sequenced. Suspected mutation was validated by clone sequencing and in other members of the family. MutationTaster and ClustalX-2.1-win was used to analyze the pathogenicity and conservation of the mutation site,respectively. Three-dimentional protein model and amino acids interaction were analyzed with Swiss-PdbViewer software., Results: The PC: A and PC: Ag of the proband were decreased to 46% (reference range: 70%-130%) and 50% (referencerange:70%-140%), respectively. Her grandmother,aunt, cousin and younger brother also showed declined PC:A and PC:Ag by approximately 50%. Genetic analysis revealed that the above individuals have all carried a deletional mutation c.1212-1212delG (p.Met364TrpfsX15) in exon 9 of the PROC gene which can cause replacement of Methionine at position 364 by Tryptophan and alteration of 15 downstream amino acids, and produce a premature stop codon at position 378. The score of MutationTaster was 1.000, indicating that the variant is pathogenic. Conservative analysis showed that the 15 altered amino acids are located in a conserved region across nine homologous species. Protein model analysis showed that the mutation has disrupted a catalytic domain of protein C thereby affected its function., Conclusion: The heterozygous c.1212-1212delG deletional mutation in exon 9 of the PROC gene, which was unreported previously,probably accounts for the decrease of PC:A and PC:Ag in this pedigree.

Activated protein C (APC) is a serine protease with anticoagulant and cytoprotective activities. Nonanticoagulant APC mutants show beneficial effects as cytoprotective agents. To study, if such biased APC signaling can be achieved by APC-binding ligands, the aptamer technology has been used. A G-quadruplex-containing aptamer, G-NB3, has been selected that binds to the basic exosite of APC with a K D of 0.2 nM and shows no binding to APC-related serine proteases or the zymogen protein C. G-NB3 inhibits the inactivation of activated cofactors V and VIII with IC 50 values of 11.6 and 13.1 nM, respectively, without inhibiting the cytoprotective and anti-inflammatory functions of APC as tested using a staurosporine-induced apoptosis assay and a vascular barrier protection assay. In addition, G-NB3 prolongs the plasma half-life of APC through inhibition of APC-serine protease inhibitor complex formation. These physicochemical and functional characteristics qualify G-NB3 as a promising therapeutic agent usable to enhance the cytoprotective functions of APC without increasing the risk of APC-related hemorrhage.

Inherited protein C (PC) deficiency caused by mutations in the PROC gene is a well-known risk factor for venous thromboembolism. Few studies have investigated the relationship between PROC genotype and plasma or clinical phenotypes. We addressed this issue in a large retrospective cohort of 1,115 heterozygous carriers of 226 PROC pathogenic or likely pathogenic mutations. Mutations were classified in three categories according to their observed or presumed association with type I, type IIa, or type IIb PC deficiency. The study population comprised 876 carriers of type I category mutations, 55 carriers of type IIa category mutations, and 184 carriers of type IIb category mutations. PC anticoagulant activity significantly influenced risk of first venous thrombosis ( p trend < 10 -4 ). No influence of mutation category on risk of whole or unprovoked thrombotic events was observed. Both PC anticoagulant activity and genotype significantly influenced risk of venous thrombosis. Effect of detrimental mutations on plasma phenotype was ambiguous in several carriers, whatever the mutation category. Altogether, our findings confirm that diagnosing PC inherited deficiency based on plasma measurement may be difficult but show that diagnosis can be improved by PROC genotyping., Competing Interests: None declared., (Thieme. All rights reserved.)

Upon activation, neutrophils release their content through different mechanisms like degranulation and NETosis, thus prompting thrombosis. The natural anticoagulant activated protein C (APC) inhibits neutrophil NETosis and, consequently, this may lower the levels of neutrophil activation markers in plasma, further diminishing the thrombotic risk exerted by this anticoagulant. We aimed to describe the status of markers of neutrophil activation in plasma of patients with venous thrombosis, their association with the thrombotic risk and the potential contribution of APC. We quantified three markers of neutrophil activation (cell-free DNA, calprotectin, and myeloperoxidase) in 253 patients with venous thromboembolism (VTE) in a stable phase (192 lower extremity VTE and 61 splanchnic vein thrombosis) and in 249 healthy controls. In them, we also quantified plasma APC, soluble endothelial protein C receptor (EPCR), and soluble thrombomodulin (TM), and we genotyped two genetic regulators of APC: the EPCR gene ( PROCR ) haplotypes (H) and the TM gene ( THBD ) c.1418C>T polymorphism. We found a significant increase in plasma cell-free DNA ( p < 0.0001), calprotectin ( p = 0.0001) and myeloperoxidase ( p = 0.005) in VTE patients compared to controls. Furthermore, all three neutrophil activation markers were associated with an increase in the thrombotic risk. Cell-free DNA and calprotectin plasma levels were significantly correlated (Spearman r = 0.28; p < 0.0001). As expected, the natural anticoagulant APC was significantly decreased in VTE patients ( p < 0.0001) compared to controls, what was mediated by its genetic regulators PROCR -H1, PROCR -H3, and THBD -c.1418T, and inversely correlated with cell-free DNA levels. This is the largest case-control study that demonstrates the increase in markers of neutrophil activation in vivo in VTE patients and their association with an increased thrombotic risk. This increase could be mediated by low APC levels and its genetic regulators, which could also increase NETosis, further enhancing thrombosis and inflammation.

Protein C is a natural anticoagulant activated by thrombin in a reaction accelerated by the cofactor thrombomodulin. The zymogen to protease conversion of protein C involves removal of a short activation peptide that, relative to the analogous sequence present in other vitamin K-dependent proteins, contains a disproportionately high number of acidic residues. Through a combination of bioinformatic, mutagenesis and kinetic approaches we demonstrate that the peculiar clustering of acidic residues increases the intrinsic disorder propensity of the activation peptide and adversely affects the rate of activation. Charge neutralization of the acidic residues in the activation peptide through Ala mutagenesis results in a mutant activated by thrombin significantly faster than wild type. Importantly, the mutant is also activated effectively by other coagulation factors, suggesting that the acidic cluster serves a protective role against unwanted proteolysis by endogenous proteases. We have also identified an important H-bond between residues T176 and Y226 that is critical to transduce the inhibitory effect of Ca 2+ and the stimulatory effect of thrombomodulin on the rate of zymogen activation. These findings offer new insights on the role of the activation peptide in the function of protein C.

Background: Activated protein C (APC) downregulates thrombin generation by inactivating procoagulant cofactors Va and VIIIa by limited proteolysis. We identified two protein C-deficient patients both of whom carry a heterozygous Gly197 to Arg (G197R) mutation in PROC and experience venous thrombosis., Objective: The objective of this study was to determine the molecular basis of the clotting defect in patients carrying the G197R mutation., Methods: We expressed protein C-G197R in mammalian cells and characterized its properties in established coagulation and anti-inflammatory assay systems., Results: The activation of protein C-G197R by thrombin was improved ~10-fold; however, its activation by thrombin was not promoted by thrombomodulin (TM). In a tissue factor-mediated thrombin generation assay, the addition of soluble TM to protein C-deficient plasma, supplemented with protein C-G197R, did not have a significant inhibitory effect on thrombin generation parameters. APC-G197R did not exhibit a significant anticoagulant activity in either purified or plasma-based assay systems. APC-G197R was essentially inactive because it showed no activity in an aPTT assay. Anti-inflammatory activity of APC-G197R was also dramatically impaired as determined by an endothelial cell permeability assay. Structural modeling predicted that the side-chain of Arg cannot be accommodated at this site of APC without a major distortion of the local structure that appears to propagate and adversely affect the reactivity/folding of the catalytic pocket., Conclusion: The G197R mutation in patients appears to be functionally equivalent to a heterozygous protein C knockout with half of the protein having no significant activity and thus causing thrombosis., (© 2020 International Society on Thrombosis and Haemostasis.)

Background: Rare coding mutations underlying deficiencies of antithrombin and proteins C and S contribute to familial venous thromboembolism (VTE). It is uncertain whether rare variants play a role in the etiology of VTE in the general population., Objectives: We conducted a deep whole-exome sequencing (WES) study to investigate the associations between rare coding variants and the risk of VTE in two population-based prospective cohorts., Patients/methods: Whole-exome sequencing was performed in the Longitudinal Investigation of Thromboembolism Etiology (LITE), which combines the Atherosclerosis Risk in Communities (ARIC) study (316 incident VTE events among 3159 African Americans [AAs] and 458 incident VTEs among 7772 European Americans [EAs]) and the Cardiovascular Healthy Study (CHS; 60 incident VTEs among 1751 EAs). We performed gene-based tests of rare variants (allele frequency < 1%, exome-wide significance P < 1.47 × 10 -6 ) separately in each study and ancestry group, and meta-analyzed the results for the EAs in ARIC and CHS., Results: In the meta-analysis of EAs, we identified one gene, PROC, in which the burden of rare, coding variants was significantly associated with increased risk of VTE (HR = 5.42 [3.11, 9.42] for carriers versus non-carriers, P = 2.27 × 10 -9 ). In ARIC EAs, carriers of the PROC rare variants had on average 0.75 standard deviation (SD) lower concentrations of plasma protein C and 0.28 SD higher D-dimer (P < .05) than non-carriers. Adjustment for low protein C status did not eliminate the association of PROC burden with VTE. In AAs, rare coding PROC variants were not associated with VTE., Conclusions: Rare coding variants in PROC contribute to increased VTE risk in EAs in this general population sample., (© 2019 International Society on Thrombosis and Haemostasis.)

Introduction: Protein C deficiency is a heritable thrombophilia caused by numerous different genetic alterations in the protein C (PROC) gene. We aimed to identify variants causing protein C deficiency in a Danish population., Material and Methods: Sanger sequencing of the PROC gene was performed in 20 probands and 26 relatives. In total, 30participants were previously diagnosed with protein C deficiency. Protein C activity was measured by a chromogenic substrate method (N = 40) and antigen level by an enzyme-linked immunosorbent assay (N = 26)., Results: Ten different single nucleotide variants were detected in 13 probands (65%) and in seven of the relatives previously diagnosed with protein C deficiency. Five variants were novel. The median protein C activity level was lower in participants with an identified variant (50% (range: 38-75%)) than in protein C deficient participants without a variant (65% (range: 36-73%); P = 0.18). A protein C activity of 57% resulted in the highest detection rate (12/13 (92%)). Likewise, the median antigen level was lower in participants with detectable variants than in participants without (49% (range: 35-99%) vs 70% (range: 41-101%); P = 0.09). No difference was found in venous thromboembolism (VTE) prevalence comparing participants with (12/20 (60%)) and without (7/10 (70%)) a variant (P = 0.59)., Conclusion: In a Danish population, a PROC gene variant was identified in 67% of participants previously diagnosed with protein C deficiency. Five variants were novel. The study confirmed an association between biochemical severity and the presence of a PROC gene variant. The VTE risk did not seem to differ between protein C deficient participants with and without a variant., (Copyright © 2019. Published by Elsevier Ltd.)

: Protein S Tokushima (p.Lys196Glu) and two protein C gene variants (p.Arg189Trp, p.Lys193del) are hereditary thrombophilia in Japanese and Chinese populations, respectively; however, their diagnosis by plasma analyses is difficult because of the type II deficiency phenotype. Three gene variant genotypes were examined in young Japanese women (n = 231). Plasma total protein S activity and total protein S antigen levels were measured using a total protein S assay system, protein C and protein S activities by clot-based methods, and protein C and free protein S antigen levels by latex agglutination methods. protein S Tokushima (p.Lys196Glu) and protein C p.Lys193del variants were prevalent among participants with allele frequencies of 1.08 and 0.86%, respectively, whereas any carrier of protein C p.Arg189Trp variant was not identified. The plasma phenotype of the type II deficiency of protein S Tokushima heterozygotes was demonstrated by decreased total protein S activity with a normal total protein S antigen level; however, the protein C activities of protein C p.Lys193del heterozygotes were within reference intervals, whereas their protein C antigen levels were elevated. We compared the diagnostic accuracy of the total protein S activity/total protein S antigen ratio for identifying protein S Tokushima heterozygotes with that of the clot-based protein S activity/free protein S antigen ratio and found that sensitivity and specificity of 100% each was only achieved by the former. Protein S Tokushima and protein C p.Lys193del are prevalent among young Japanese women, and a plasma analysis using the total protein S assay system is more accurate than the clot-based protein S activity/free protein S antigen ratio for diagnosing protein S Tokushima carriers.

Statins are potent lipid-lowering drugs. Large prospective clinical trials have shown the anti-thrombotic effect of statins, e.g., preventing deep vein thrombosis. However, the mechanism underlying the beneficial effect of statins in reducing thrombus formation remains to be established. We, thus, conduct this study to investigate the potential molecular mechanisms. The cultured human hepatoma cells (HepG2) were used as the in vitro model. The human protein C gene promoter was cloned into the luciferase reporter to study the transcriptional regulation of human protein C gene. Wistar rats fed with simvastatin (5 mg/kg day) were used as the in vivo model. We found that simvastatin increased the expression of protein C in hepatocytes (361 ± 64% and 313 ± 59% after 2 h and 6 h of stimulation, respectively, both p < 0.01). In the animal study, the serum protein C levels were increased in the simvastatin-treated group (7 ± 2.2 unit/ml vs 23.4 ± 19.3 unit/ml and 23.4 ± 18.2 unit/ml and 1 and 2 weeks of treatment, respectively, both p < 0.05). Regarding the possible molecular mechanism, we found that the level of hepatocyte nuclear factor 1α (HNF1α) was also increased in both the in vivo and in vitro models. We found that the protein C promoter activity was increased by simvastatin, and this effect was inhibited by HNF1α knockdown and constitutively active Rac1. Therefore, stains may modulate protein C expression through small GTPase Rac 1 and HNF1α.

Pathogenic mutation of protein C (PROC) gene results into the deficiency of PROC activity. This study aimed to identify the pathogenic genetic variants and to explore the functional consequence in Chinese familial venous thrombosis (VTE). Whole exome sequencing was performed to identify the pathogenic variants of anticoagulant factors. Serum coagulation and anti-coagulation factors activity were assayed to evaluate the genetic association. Functional study of PROC antigen secretion deficiency was conducted in VTE subjects and in vitro cell lines. One rare pathogenic variant (p.Ala178Pro) was identified in the four VTE subjects but not in the normal subjects from the family. An inframeshift variant (rs199469469) was also identified in a paediatric subject of the pedigree. Further evaluation of serum PROC activity levels in p.Ala178Pro variants VTE carriers showed significantly lower PROC activity compared to non-carriers. Furthermore, in vitro study showed that the p.Ala178Pro mutant cells had a consistent reduction in concentration of PROC antigen. In conclusions, our study demonstrated the pathogenic variant (p.Ala178Pro) contributed to PROC type I activity deficiency, which may be due to decreased secretion of PROC., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Presently, no data on the molecular basis of hereditary protein C (PC) deficiency in Spain is available. We analyzed the PC gene ( PROC ) in 109 patients with symptomatic PC deficiency and in 342 relatives by sequencing the 9 PROC exons and their flanking intron regions. In 93 probands, we found 58 different mutations (26 novel). Thirty-seven consisted of a nucleotide change, mainly missense mutations, 1 was a 6-nucleotide insertion causing the duplication of 2 amino acids, and 4 were deletions of 1, 3, 4, and 16 nucleotides. Nine mutations caused type II deficiencies, with the presence of normal antigen levels but reduced anticoagulant activity. Using a PC level of 70% as lowest normal limit, we found no mutations in 16 probands and 25 relatives with PC levels ≤ 70%. On the contrary, 4 probands and 12 relatives with PC levels > 70% carried the mutation identified in the proband. The spectrum of recurrent mutations in Spain is different from that found in the Netherlands, where the most frequent mutations were p.Gln174* and p.Arg272Cys, and is more similar to that found in France, where the most frequent were p.Arg220Gln and p.Pro210Leu. In our study, p.Val339Met (9 families), p.Tyr166Cys (7), p.Arg220Gln (6), and p.Glu58Lys (5) were the most prevalent. This study confirms the considerable heterogeneity of the genetic abnormality in PC deficiencies, and allowed genetic counseling to those individuals whose PC levels were close to the lower limit of the normal reference range., Competing Interests: F.E. reports grants from Spanish Society of Thrombosis and Haemostasis, during the conduct of the study; P.M. reports grants from Spanish Society of Thrombosis and Haemostasis, during the conduct of the study; S.N. reports grants from Spanish Society of Thrombosis and Haemostasis, during the conduct of the study. Other authors report no conflict of interest., (Georg Thieme Verlag KG Stuttgart · New York.)

Objective: The aim of this study was to assess the association of single nucleotide polymorphisms (SNPs) in protein C (PROC) and protein S (PROS1) genes with deep venous thrombosis (DVT) in a thrombophilia family., Methods: DNA were extracted from blood of participants. Five PROC SNPs and 11 PROS1 SNPs were selected from the Hapmap and 1000 Genomes databases. The minor allele frequencies (MAFs) of SNPs in the thrombophilia family (Group I) and healthy controls (Group II) were detected. SNPs were analysed by Chi-square, logistic regression and linkage disequilibrium patterns., Results: MAFs for all 16 SNPs were greater than 0.05. Chi-square analysis showed significant differences between Group I and Group II in the frequency of mutant alleles of rs1799808, rs5936, rs6123, rs12634349, rs6441600 and rs13062355 SNPs (P < .05). Logistic regression analysis found that mutant alleles of rs1799808, rs6441600 and rs13062355 SNPs may contribute to DVT in this family (OR > 1, L95 > 1). Linkage disequilibrium was found among 15 of the PROC and PROS SNPs., Conclusions: Single nucleotide polymorphisms (SNPs) of PROC and PROS1 may be closely associated with DVT in this thrombophilia family. Mutant alleles of rs1799808, rs6441600 and rs13062355 SNPs may contribute to DVT, whereas mutant alleles of rs1799810, rs6123 and rs12634349 may protect individuals from DVT. With the exception of rs9681204, there was linkage disequilibrium between PROC and PROS1 SNPs. We found that 12 haplotypes were in linkage disequilibrium, but linkage disequilibrium was strong in only eight of these (frequency >5%)., (© 2019 John Wiley & Sons Ltd.)

Background The most common cause of activated protein C (aPC) resistance is a missense substitution (Arg506Gln), known as Factor V Leiden (FVL). Due to its low cost, many laboratories use the aPC ratio as a primary test with a unisex cut-off. However, the association between the aPC ratio and FVL including any relation to gender has been sparsely investigated. Methods Results of the aPC ratio and FVL analyses from 1081 patients referred to the Thrombophilia Clinic at Odense University Hospital were compared. Results In 153 FVL positive patients, the mean aPC ratio was 2.1 ± 0.3, which differed from 2.7 ± 0.4 in FVL negative individuals (p < 0.01). The receiver operating characteristics (ROC), with area under the curve (AUC) of 0.93, indicated the optimal aPC cut-off at 2.3-2.4, with sensitivity 89%-94%, specificity 71%-84%, positive predictive value 35%-48% and negative predictive value 98%-99%. In FVL positive females, the mean aPC ratio was 2.0 ± 0.3, which differed from males (2.1 ± 0.3, p < 0.05). In FVL negative females, the mean aPC ratio was 2.6 ± 0.4, also different from males (2.8 ± 0.5, p < 0.01). Of note, 35% of FVL negative females had an aPC ratio ≤2.4 against 18% in males (p < 0.01). Conclusions Our results indicate that the aPC ratio is lower in females than in males. Due to a high negative predictive value the aPC ratio can be used as a first line test for FVL, but those found positive must be confirmed with a DNA test.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease with diverse etiologies. Therefore, the identification of common disease mechanisms and therapeutics targeting these mechanisms could dramatically improve clinical outcomes. To this end, we developed induced motor neuron (iMN) models from C9ORF72 and sporadic ALS (sALS) patients to identify targets that are effective against these types of cases, which together comprise ~90% of patients. We find that iMNs from C9ORF72 and several sporadic ALS patients share two common defects - impaired autophagosome formation and the aberrant accumulation of glutamate receptors. Moreover, we show that an anticoagulation-deficient form of activated protein C, 3K3A-APC, rescues these defects in both C9ORF72 and sporadic ALS iMNs. As a result, 3K3A-APC treatment lowers C9ORF72 dipeptide repeat protein (DPR) levels, restores nuclear TDP-43 localization, and rescues the survival of both C9ORF72 and sporadic ALS iMNs. Importantly, 3K3A-APC also lowers glutamate receptor levels and rescues proteostasis in vivo in C9ORF72 gain- and loss-of-function mouse models. Thus, motor neurons from C9ORF72 and at least a subset of sporadic ALS patients share common, early defects in autophagosome formation and glutamate receptor homeostasis and a single therapeutic approach may be efficacious against these disease processes.

: Hypodysfibrinogenemia and protein C deficiency are coagulopathies and in this report, we describe a young patient with both defects confirmed by molecular genetic tests. The patient was a 24-year-old woman referred for recurrent thrombophlebitis and finally deep venous thrombosis. Routine coagulation studies revealed mild decrease of protein C (0.49 IU, reference values 0.7-1.40 IU) and hypodysfibrinogenemia (0.88 g/l and 1.83 g/l for activity and antigen, respectively, reference values 2.0-4.0 g/l). Direct sequencing analyses were performed on FGA, FGB, and FGG genes to confirm hypodysfibrinogenemia and on the protein C gene to confirm protein C deficiency. As a result, the patient was shown to be heterozygous p.Ala82Gly in the FGG gene (Fibrinogen Dunedin) and for compound heterozygous missense mutation in protein C gene. To our knowledge, this is the first report on a case of combined dysfibrinogenemia and protein C deficiency confirmed by molecular genetic tests.