Protein C Research Articles

Endothelium-Dependent Protein C Activation in Hereditary Protein C Deficiency.

Protein C (PC) activation on endothelial cells is a critical antithrombotic mechanism. Hereditary PC deficiency (PCD), which is caused by mutations in the PROC gene, can predispose affected individuals to thrombophilia. Previous studies investigated activated protein C (APC) generation in PCD patients without including endothelial cells, which are essential for physiological PC activation. This study aimed to assess APC generation in PCD patients using a novel endothelial cell-based assay.Plasma samples from 21 patients with 19 heterozygous PROC mutations (median PC level 58%) and 24 healthy controls were analyzed. Endothelium-dependent APC generation was initiated by overlaying plasma on human umbilical vein endothelial cells (HUVECs) and adding tissue factor (1 pmol/L). APC levels were quantified using an oligonucleotide-based enzyme capture assay. The area under the curve (AUC) was calculated to monitor cumulative APC formation over time. A calibration curve generated from wild-type PC in PC-deficient plasma established reference ranges.Mean peak levels of APC were significantly lower in PCD patients than in healthy controls (0.75 vs. 1.83 nmol/L, p = 2 × 10-10). The AUC APC was below the reference range in 8 of 21 (38%) patient samples, indicating disproportionately severe impairment in APC generation. The observed variability in APC generation suggests that endothelial contributions may identify functional differences undetected by standard PC activity or antigen assays.This study introduces a novel endothelial cell-based APC generation assay, demonstrating the functional consequences of PROC mutations and providing insights into the regulation of APC generation, with potential applications in thrombosis risk assessment and personalized therapy.

Cryo-EM structure of coagulation factor Va bound to activated protein C.

Cryo-EM structure of coagulation factor Va bound to activated protein C.

Recessively inherited protein C deficiency in two sisters: intracerebral hemorrhage and coagulopathy complications.

Protein C (PROC) deficiency is a rare hematological disorder caused by mutations in the PROC gene, leading to severe coagulopathy in homozygous cases. This report highlights the hematological challenges in managing homozygous protein C deficiency, particularly its association with intracerebral hemorrhage and thrombosis. We describe two sisters with a homozygous PROC gene mutation (c.797A>G, p.Tyr266Cys), presenting with protein C activity <10%. The older sister (born 2020) developed neonatal intracerebral hemorrhage, followed by recurrent hemorrhage and deep vein thrombosis by 2025, managed with heparin, warfarin, and rivaroxaban. The younger sister (born 2022) also experienced neonatal hemorrhage and required anticoagulant therapy with heparin and warfarin. Both developed neurological complications, including epilepsy. Homozygous protein C deficiency can lead to severe neonatal coagulopathy, necessitating early diagnosis and aggressive anticoagulant therapy. This case underscores the importance of multidisciplinary management and genetic screening in affected families.

Asn384Ser Mutation in Protein C is Associated with Multiple-Site Thrombosis in a Young Heterozygous Male.

Protein C (PC) is an important physiological anticoagulant factor in humans. Activated protein C (APC) is generated from the PC zymogen through proteolytic activation by thrombin. APC inhibits thrombin generation by inactivating activated factors V and VIII via limited proteolysis. In addition to its anticoagulant function, APC also exhibits potent cytoprotective and anti-inflammatory properties. We have identified a young male with multiple-site thrombosis, who carries a heterozygous mutation c.1151A > G,p.Asn384Ser(N384S) in PC. Although this mutation has been previously documented, limited functional research has been conducted to elucidate its pathogenesis.To elucidate the functional alternations of the N384S mutant protein C and delineate the molecular mechanism underlying thrombosis in the patient carrying this mutation.We expressed the recombinant PC-N384S in mammalian cells and characterized its properties in established coagulation and anti-inflammatory assay systems.The expression level of the PC-N384S was reduced to approximately 7% of that observed for PC-WT. The activation of PC-N384S by thrombin or thrombin-thrombomodulin (TM) complex was significantly impaired, although the addition of TM exhibited a slight enhancement in the activation process. In terms of cleaving a chromogenic substrate, the catalytic efficiency reduced to approximately 50% of that observed in the wild type. In addition, in comparison with APC-WT, APC-N384S demonstrated a pronounced decline in amidolytic activity following an extended incubation period at 37°C. APC-N384S exhibited slightly impaired anticoagulant activity in either FVa inhibition assay or plasma-based assay systems. Furthermore, anti-inflammatory activity of APC-N384S was dramatically impaired as determined by evaluating the barrier-protective effect.The Asn384Ser mutation impairs both the anticoagulant and barrier-protective activities of protein C, thereby increasing the thrombosis risk in the heterozygous young male.

Clinical Phenotype and Genetic Analysis of a Family with Hereditary Antithrombin Deficiency Caused by SERPINC1 Gene Mutation.

Inherited deficiency of the antithrombin (hereditary antithrombin deficiency, AT deficiency, OMIM #613118) is a relatively rare (1:2,000-3,000) autosomal-dominant disorder with high risk of venous thromboembolism. The molecular basis of this condition has not yet fully understood, highlighting the need for further research to elucidate the underlying pathological mechanisms.This study aimed to investigate coagulation parameters and genetic phenotypes in a proband with hereditary antithrombin deficiency and her family members. Additionally, the investigation sought to provide preliminary insights for the molecular pathogenesis of this condition.Blood coagulation parameters, including plasma antithrombin activity (AT:A), antithrombin antigen (AT:Ag), protein C activity (PC:A), and protein S activity (PS:A) were measured in the peripheral blood of each family member by a Stago instrument. Peripheral blood was also extracted and sequenced to identify possible genetic mutation sites. The functional impact of variants on protein was subsequently analyzed by bioinformatics software.The proband, her mother, and brother all exhibited decreased activity and antigen of AT but normal PC and PS activity. The proband's father had normal activity and antigen levels of AT, PC, and PS. Sequencing revealed the proband's mother inherited the SERPINC1:c.661T > C,p.(Trp221Arg) heterozygous variant and her father harbored PROC:c.572_574del,p.(Lys193del) heterozygous variant while the proband as well as her brother carried both. Conservation analysis revealed that Trp221 is highly conserved across homologous species. Bioinformatics tools consistently classify the p.Trp221Arg mutation as "pathogenic" or "deleterious." Protein modeling indicated that the p.Trp221Arg variant does not alter the protein structure but may modify glycosylation sites to affect its function.The proband and family members exhibited varying degrees of decreased levels of AT and thrombosis, which were closely associated with inheritance of SERPINC1:c.661T > C,p.(Trp221Arg).

Investigation of antibacterial, antifungal, and antioxidant activities in novel poly(DEAEMA‐co‐EMA)/polyindole composites

AbstractIn the current study, in the first stage, amphiphilic poly(2‐diethylamino ethylmethacrylate‐co‐ethyl methacrylate) [Poly(DEAEMA‐co‐EMA)] was synthesized from the 2‐diethylamino ethylmethacrylate (DEAEMA) and the ethyl methacrylate (EMA) using the free radical polymerization method. In the second stage, five series of composites were prepared at different concentrations using the indole conductive monomer in the presence of iron(III) chloride (FeCl3) using the in situ oxidative polymerization technique in poly(DEAEMA‐co‐EMA) copolymer. Scanning electron microscope and Atomic force microscope images proved the homogeneous structure of the composites, and Fourier transform infrared spectroscopy analysis of the composite has shown that it has been produced successfully. The antimicrobial activity of the prepared poly(DEAEMA‐co‐EMA)/PIN composites was evaluated. Moreover, antioxidant studies of the composites were carried out. The results showed that the enhanced bioactivity of poly(DEAEMA‐co‐EMA)/PIN(90%) composite. In this study, all poly(DEAEMA‐co‐EMA)/PIN composites and polymers showed antibacterial activity against Streptococcus mutans. PCs had more antifungal activity against Aspergillus flavus than PIN, Poly(DEAEMA), and poly(DEAEMA‐co‐EMA). The antimicrobial activity of PCs was enhanced by increasing the permeability of the cell membrane with DEAEMA. It was determined that the antibacterial activity increased in direct proportion to the indole concentration. OSI values of PIN, Poly(DEAEMA), and poly(DEAEMA‐co‐EMA), and PCs ranged between 1.3513 and 0.1183.Highlights Conductive Polymer Composites are a special commercial polymer composite class due to their biological properties. Discusses morphological properties of poly(DEAEMA‐co‐EMA)/PIN composites. Summarizes antimicrobial and antioxidant activities of poly(DEAEMA‐co‐EMA)/PIN composites. Demonstrate the high antimicrobial activity of Poly(DEAEMA‐co‐EMA)/PIN composites against S. mutans. Determine that Poly(DEAEMA‐co‐EMA)/PIN composites have antioxidant capacity.

Identification of autophagy-related immune targets for enhancing immunotherapy in pancreatic cancer aggressiveness

BackgroundPancreatic cancer (PC) presents significant challenges in oncology, with metastasis critically affecting patient outcomes. Autophagy-related genes (ARGs)’s involvement in influencing immune activity and metastasis in PC remains inadequately understood.AimThis study seeks to identify and validate five ARGs that could serve as immune targets, enhancing enhancing Pancreatic cancer metastasis (PCM)’s prognostic models and informing immunotherapy strategies.MethodsARGs that were diffentially expressed were screened, followed by Cox regression and LASSO analyses to pinpoint five genes linked to overall survival (OS). A prognostic model was developed and validated using ROC curves. Functional analyses, including GO and KEGG, were performed to elucidate ARG mechanisms. Immune infiltration and TFs/microRNA/mRNA networks were assessed to understand ARG-immune cell interactions. Experimental validation employed real-time PCR, IHC, and Western blotting, supported by TCGA data. Functional assays explored RHEB’s role in PC, particularly its interaction with LC3.ResultsFive ARGs (CASP1, RHEB, CHMP2B, MYC, and HDAC6) were identified, contributing to a robust prognostic model where low-risk individuals showed significantly longer OS. The model demonstrated high AUC scores, indicating strong prognostic capability. CD8 T cells and Treg cells’ elevated levels were observed in metastatic subjects. RHEB knockdown suppressed cancer cell proliferation and invasion, with a negative correlation between RHEB and LC3, suggesting a role in autophagy-mediated modulation of PC metastasis.ConclusionThis study introduces a novel prognostic model incorporating five ARGs, highlighting their potential as immune targets for cancer immunotherapy. The negative correlation between RHEB and LC3 suggests a therapeutic pathway for PCM intervention, laying the groundwork for more effective anti-cancer strategies. These findings advance the identification of novel immune targets and signaling pathways, aligning with precision medicine goals in cancer treatment.

Protein C activator derived from snake venom protects human umbilical vein endothelial cells against hypoxia-reoxygenation injury by suppressing ROS via upregulating HIF-1α and BNIP3

To investigate the antioxidative mechanism of snake venom-derived protein C activator (PCA) in mitigating vascular endothelial cell injury. Human umbilical vein endothelial cells (HUVECs) were cultured in DMEM containing 1.0 g/L D-glucose and exposed to hypoxia (1% O2) for 6 h followed by reoxygenation for 2 h to establish a cell model of oxygen-glucose deprivation/reoxygenation (OGD/R). The cell model was treated with 2 μg/mL PCA alone or in combination with 2-ME2 (a HIF-1α inhibitor) or DMOG (a HIF-1α stabilizer), and intracellular production of reactive oxygen species (ROS) and protein expression levels of HIF-1α, BNIP3, and Beclin-1 were detected using DCFH-DA fluorescence probe, flow cytometry, and Western blotting. The OGD/R cell model was transfected with a BNIP3-specific siRNA or a scrambled control sequence prior to PCA treatment, and the changes in protein expressions of HIF-1α, BNIP3 and Beclin-1 and intracellular ROS production were examined. In the OGD/R cell model, PCA treatment significantly upregulated HIF-1α, BNIP3 and Beclin-1 expressions and reduced ROS production. The effects of PCA were obviously attenuated by co-treatment with 2-ME2 but augmented by treatment with DMOG (a HIF-1α stabilizer). In the cell model with BNIP3 knockdown, PCA treatment increased BNIP3 expression and decreased ROS production without causing significant changes in HIF-1α expression. Compared with HUVECs with PCA treatment only, the cells with BNIP3 knockdown prior to PCA treatment showed significantly lower Beclin-1 expression and higher ROS levels. Snake venom PCA alleviates OGD/R-induced endothelial cell injury by upregulating HIF-1α/BNIP3 signaling to suppress ROS generation, suggesting its potential as a therapeutic agent against oxidative stress in vascular pathologies.

Risk of First Venous Thrombosis by Comparing Different Thrombin Generation Assay Conditions: Results from the MEGA Case-control Study.

Hypercoagulability is a risk factor for venous thromboembolism (VTE). Thrombin generation (TG) is a global coagulation assay that measures an individual's clotting tendency. We hypothesise that slow-onset TG (achieved by using a low procoagulant stimulus or an inhibitor of coagulation) is the optimal responsive TG method for detecting hypercoagulability.This study aimed to compare different TG assay conditions with respect to VTE risk and assess the risk of the first VTE. Basal TG at low tissue factor (TF) concentration and high TF concentration in the presence and absence of activated protein C (APC) were measured in plasma samples from 2,081 patients with first VTE and 2,908 healthy controls from the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA) study. TG parameters and normalised activated protein C sensitivity ratio (nAPCsr) were categorised into quartiles as measured in the controls. We calculated odds ratios (ORs) with 95% confidence intervals (CIs) of the first VTE for different TG categories. Under all assay conditions the thrombin peak height was associated with VTE risk: peak height of >75th percentile, at low TF OR 6.8 (95% CI 5.5-8.3), at high TF, OR 3.0 (95% CI 2.5-3.6), and at high TF + APC, OR 3.8 (95% CI 3.2-4.5), all compared with a peak height of <25th percentile obtained in controls. An increased nAPCsr (higher resistance to APC) was also associated with VTE risk, OR 3.4 (95% CI 2.8-4.1). Increased TG is associated with the risk of first VTE, particularly when triggered with a low procoagulant stimulus.

Analysis of two cases of hereditary protein C deficiency causing venous thrombosis

Objective: To investigate the molecular pathogenic mechanism of venous thrombosis caused by heterozygous missense mutations in two protein C (PROC) genes through laboratory phenotype analysis, genetic mutation analysis, and in vitro expression experiments. Methods: Two probands presented with venous thromboembolism at the First Affiliated Hospital of Wenzhou Medical University. Clinical data and blood samples were collected from the probands and their family members to evaluate the plasma protein C (PC) activity (PC∶A), PC antigen (PC∶Ag) levels, and other relevant coagulation parameters. The anticoagulant capacity was assessed using the thrombin generation test (TGT). The mutation sites of the PROC gene were identified using direct DNA sequencing. Bioinformatics software was used to analyze the conservation and pathogenicity of the mutated gene. PyMOL software was used for the analysis of the protein three-dimensional models and interactions between mutated amino acids. Wild-type and two mutant expression vectors were constructed and HEK293T cells were transiently transfected. Total cellular RNA was extracted from positively transfected cells to investigate the transcriptional levels of the mutant PROC gene. Enzyme-linked immunosorbent assay, Western blot, and cellular immunofluorescence assays were used to investigate the translation levels of the mutant PROC protein. Results: Probands 1 and 2 exhibited PC∶A levels of 35% and 40% and PC∶Ag levels of 44% and 39%, with increasing D-dimer levels to 4.42 mg/L and 0.83 mg/L, respectively. Meanwhile, other coagulation parameters revealed no significant abnormalities. TGT demonstrated impaired anticoagulant function in both proband witnesses and their familial PC carriers. Sequencing analysis revealed heterozygous missense mutations c. 833T>C (p. Leu278Pro) in proband 1 and c. 1330T>C (p. Trp444Arg) in proband 2 within exon 9 of the PROC gene. Conservation analysis revealed that Leu278 and Trp444 were highly conserved across homologous species. Pathogenicity analysis indicated that both p. Leu278Pro and p. Trp444Arg mutations are deleterious. Protein modeling analysis demonstrated that both mutations induce structural alterations in the protein. In vitro expression experiments revealed that compared with the wild-type, both p. Leu278Pro and p. Trp444Arg mutations showed no significant differences in the mRNA expression level of the PC protein. However, both mutations caused significantly lower PC∶Ag content and protein expression levels in the cell culture supernatant compared with the wild-type, whereas higher levels were observed in the cell culture lysate. This indicates the association of both mutations with the secretion function of the PC protein. Conclusion: The heterozygous missense mutations p. Leu278Pro and p. Trp444Arg in exon 9 of the PROC gene in both probands are associated with decreased PC levels.

A nomogram based on hematological parameters for prediction of spontaneous abortion risk in pregnancies

BackgroundPregnancy loss significantly affects physical and mental health. A nomogram for predicting spontaneous abortion risk was developed to improve pregnancy outcomes.MethodsA total of 1346 pregnant women were enrolled from The Third Affiliated Hospital of Wenzhou Medical University (May 2020 - May 2022). The training set included 941 participants, and the validation set had 405. Feature selection was optimized using a random forest model, and a predictive model was constructed via multivariable logistic regression. The nomogram’s performance was assessed with receiver operator characteristic (ROC), Hosmer-Lemeshow test, calibration curve, and clinical impact curve (CIC). Discrimination and clinical utility were compared between the nomogram and its individual variables.ResultsAntithrombin III (AT-III), homocysteine (Hcy), complement component 3 (C3), protein C (PC), and anti-β2 glycoprotein I antibody (anti-β2GP1) were identified as risk factors. The nomogram demonstrated satisfactory discrimination (Training AUC: 0.813, 95% CI: 0.790–0.842; Validation AUC: 0.792, 95% CI: 0.741–0.838). The Hosmer-Lemeshow test (P = 0.331) indicated a good fit, and the CIC showed clinical net benefit. The nomogram outperformed individual variables in discrimination (AUC: 0.804, 95% CI: 0.779–0.829).ConclusionThe developed nomogram, incorporating AT-III, Hcy, C3, PC, and anti-β2GP1, aids clinicians in identifying pregnant women at high risk for spontaneous abortion.

Discovery of New Inhibitors of Human Activated Protein C to Treat Hemophilia

Discovery of New Inhibitors of Human Activated Protein C to Treat Hemophilia

Endothelium Modulates the Prothrombotic Phenotype of Factor V Leiden: Evidence From an Ex Vivo Model.

Clinical expressivity of the thrombophilic factor V Leiden (FVL) mutation is highly variable. Recently, we demonstrated an increased APC (activated protein C) response in asymptomatic FVL carriers compared with FVL carriers with a history of venous thromboembolism (VTE) after in vivo coagulation activation. Here, we further explored this association using a recently developed ex vivo model based on patient-specific endothelial colony-forming cells (ECFCs). ECFCs and citrated plasma were obtained from FVL carriers with previous VTE (VTE+, n=9), FVL carriers without previous VTE (VTE-, n=8), and 7 healthy controls. Coagulation was activated by TF (tissue factor) in defibrinated recalcified plasma added to confluent cell cultures. Thrombin and APC concentration were measured over time, and the respective areas under the curve (AUCs) were calculated. Additionally, inhibition kinetics of exogenously added APC, APC inhibitor levels, and APC sensitivity ratios were measured in plasma. Expression of TM (thrombomodulin) and EPCR (endothelial protein C receptor) on ECFCs was assessed using cell-based ELISAs. In autologous plasma on ECFCs, the APC response (AUC APC/AUC thrombin) was higher in FVL VTE- than in FVL VTE+ patients (0.138 versus 0.028; P=0.026). APC inhibitor levels, APC inactivation kinetics, and APC sensitivity ratios did not differ between cohorts. Crossover experiments, which combined pooled plasma from FVL VTE- patients with FVL VTE+ ECFCs in the ex vivo model, and vice versa, showed increased APC response rates when FVL VTE- ECFCs were used, regardless of the plasma component. In cell-based ELISAs, TM and EPCR expression showed no significant difference. Although the FVL gene product induces an almost identical APC resistance phenotype in plasma, the endothelial cell-dependent APC response rates differ significantly, with a higher APC response in asymptomatic FVL carriers. Further studies are warranted to elucidate the disease-modulating role of the endothelium in FVL carriers at the molecular level.

Investigating the predictive role of serum amyloid A and its association with immunological and coagulation biomarkers in recurrent pregnancy loss

ABSTRACT To evaluate the predictive role of serum amyloid A (SAA) levels and their association with antiphospholipid antibodies (APA) and coagulation markers such as lupus anticoagulants (LA), anti-cardiolipin (ACA), protein C (PC) deficiency, protein S (PS) deficiency, and antithrombin III (ATIII) deficiency in recurrent pregnancy loss (RPL). This prospective case-control study comprised two groups: the study group (n = 88) included women with recurrent pregnancy loss at Mansoura University Hospital between January 2019 and December 2020, and the control group (n = 52) included women without obstetric or medical complications. Demographic, clinical, and laboratory data, including serum samples collected at 10 weeks of gestation, were collected from all participants. The study measured SAA levels, lupus anticoagulants, anti-cardiolipin, protein C, protein S, and antithrombin III levels. The SAA level was significantly elevated in the recurrent pregnancy loss group compared to that in the control group. Lupus anticoagulant positive, anti-cardiolipin positive Immunoglobulin M (IgM), and deficiencies in protein C, protein S, and antithrombin III were significantly observed in patients with RPL (p < 0.05). The SAA levels were significantly elevated in both LA-positive and ACA-positive IgM patients. The receiver operating characteristic (ROC) curve analysis demonstrated that at SAA > 24.8 for the prediction of recurrent pregnancy loss, sensitivity was 98.86%, and specificity was 92.31%. Positive and negative predictive values were 95.6% and 98.0%, respectively. The area under the curve = 0.971 (0.927–0.992). SAA is associated with recurrent pregnancy loss and may therefore serve as a potential predictor of this condition. The observed elevation in SAA levels could be primary or secondary to the inflammatory response that promotes thrombotic activity in RPL patients at risk of APA, Protein S, Protein C, and ATIII deficiencies. Implementing SAA screening during pregnancy may facilitate the identification of individuals who could potentially benefit from novel treatment strategies.

Age- and sex-dependency of thrombin generation parameters in the general Italian population: the Moli-sani study.

Recent developments have made the thrombin generation (TG) test accessible to the clinical laboratory. Therefore, the clinical interpretation of TG parameters has become of increasing interest, and reference values are required. Age and sex have been shown to affect TG parameters, but no consensus has been reached on the subject. We investigated the effect of age and sex on TG parameters to determine the need for age and sex specific reference values for TG. TG was measured in 22,014 individuals of the Moli-sani study, a cross-sectional cohort of the general adult Italian population. TG was measured using Calibrated Automated Thrombinography in platelet poor plasma (PPP) using PPP reagent Low and PPP reagent, in the presence and absence of thrombomodulin (TM). Individuals were grouped into 6 age categories: 35-44 years of age (n = 5,073), 45-54 (n = 6,448), 55-64 (n = 5,516), 65-74 (n = 3,539), 75-84 (n = 1,261), and 85 years of age and older (n = 106). Men and women were distributed evenly in the age categories. Smoking was more common at younger age, whereas cardiovascular diseases, hypertension, hypercholesterolemia, and diabetes were more common at older age (p < 0.001). Lag time and velocity index increased with age, whereas the endogenous thrombin potential (ETP) and time-to-peak decreased. The inhibitory effect of TM was reduced at higher age (p < 0.001). The TG lag time was shorter in women than men at younger age (6%-7% lower in women), and the ETP was lower in women. The activated protein C (APC) pathway was desensitized in women and older individuals. The TG profile becomes more "procoagulant" at older age, especially in women. The sensitivity of the APC pathway to TM is reduced with increasing age in men and women. Therefore, age and sex appropriate reference values for TG parameters would be of interest for the ongoing clinical implementation of the TG assay.

Enhanced cerebellar climbing fiber-Purkinje cell synaptic transmission via corticotropin-releasing factor receptor 2 during the chronic phase of spinal cord injury mice.

Spinal cord injury (SCI) causes interruption of external information input from the spinal cord to the cerebellum. We here investigated the effect of SCI on mouse cerebellar climbing fiber-Purkinje cell (CF-PC) synaptic transmission. The SCI was caused at T10 using 6-week-old ICR mice. Mice recovered 4 weeks after surgery, the spontaneous complex spike (CS) activity of PC was recorded using cell-attached recording and whole-cell recording method in urethane-anesthetized mice cerebellar Crus II. The CF-PC excitatory postsynaptic currents (EPSCs) were evoked by paired electrical stimulation of CF in cerebellar slices to evaluate the changes of CF-PC synaptic transmission and paired-pulse ratio (PPR). The results showed that the number of spikelets, duration of spontaneous CS, and pause of simple spike firing were significantly increased in SCI than that in sham group. Application of a nonselective corticotropin-releasing factor receptor (CRF-R) antagonist significantly decreased spontaneous CS activity in SCI group but not in sham group. The enhanced CS activity in SCI mice was significantly decreased by a selective CRF-R2 antagonist but not a specific CRF-R1 antagonist. The amplitude of CF-PC EPSC1 was large accompanied by a lower PPR in SCI group than that in sham group. Blockade of CRF-R2 antagonist significantly decreased the amplitude of EPSC1 and increased PPR in SCI group. SCI induces enhancement of the spontaneous CS activity and CF-PC synaptic transmission via CRF-R2 in mouse cerebellar cortex, which suggests that remodeling of CF-PC synaptic transmission occurred in cerebellar cortex after SCI.

The novel protein C variant p.C101F results in early intracellular degradation that drives type I protein C deficiency.

Hereditary protein C (PC) deficiency is an inherited thrombophilic disorder caused by variants in the PC gene (PROC). We identified a novel PROC variant, c.302G>T, p.Cys101Phe (C101F), in a patient with type I PC deficiency. We analyzed the intracellular dynamics of the C101F variant of PC (PC-C101F) to elucidate the pathogenic mechanism underlying this condition. Wild-type PC (PC-WT) and PC-C101F were transiently expressed in HEK293 cells for expression and functional analyses. The PC antigen levels in the cell lysate and culture supernatant of PC-C101F-expressing cells were significantly lower than those of PC-WT-expressing cells. In cycloheximide (CHX) chase experiments, the intracellular PC antigen level gradually decreased in PC-C101F-expressing cells, but remained stable at 0 and 6h in the presence of CHX/MG132. No significant difference in co-localization with the endoplasmic reticulum was observed between PC-C101F and PC-WT. 101Cys forms a disulfide bond with 106Cys, which is crucial for maintaining the conformation of PC. PC-C101F likely results in protein misfolding and proteasomal degradation, leading to type I PC deficiency. These findings highlight the importance of cysteine residues in the three-dimensional structure of PC and provide insight into the mechanism of type I PC deficiency.

Comparison of natural anticoagulant deficiency in cerebral venous thrombosis with deep venous thrombosis.

The impact of a deficiency in natural anticoagulants on the occurrence of cerebral venous thrombosis (CVT) is controversial, as well as whether there is a difference between CVT and deep venous thrombosis (DVT). The present study aimed to evaluate the association between a deficiency in natural anticoagulants and the occurrence of CVT vs. DVT. For this purpose, 274 patients newly diagnosed with venous thromboembolism (VTE), including 114 patients with DVT (41.6%), 81 patients with CVT (29.6%) and 79 patients (28.8%) with another type of VTE were retrospectively analyzed. In addition, 219 patients without thrombosis were used as the control group. Protein C (PC), protein S (PS) and antithrombin III (AT III) assays were performed prior to commencing treatment. The rates of PC, PS, AT III deficiency in the VTE group were 23.7, 28.8 and 14.2%, respectively. The rates of PC, PS, AT III deficiency in the CVT group were 21, 29.6 and 7.4%, respectively. The rates of PC, PS, AT III deficiency in the DVT group were 28, 34.2 and 15.8%, respectively. There was no significant difference between the DVT and CVT groups. Univariable and multivariable regression analysis revealed that PS deficiency was associated with the occurrence of all VTE types, DVT and CVT with odds ratios of 1.895, 2.330 and 2.052, respectively. On the whole, the present study demonstrates that PS deficiency is associated with the occurrence of CVT. No marked differences were noted between the deficiency in natural anticoagulants and CVT and DVT. These results may prove to be useful in deciding whether to perform natural anticoagulants testing in patients with CVT.

Effect of deficiency of natural anticoagulants on clinical heterogeneity of rheumatoid arthritis.

Aim: To investigate the concentration of natural anticoagulants protein C (PC) and tissue plasminogen activator (t-PA) in patients with rheumatoid arthritis (RA) and to evaluate the effect of their concentration on the course of the disease. Materials and Methods: We examined 74 patients with RA. There were 15 men and 59 women and control group consisted of 27 subjects. The average age of the patients was 47.3±1.12 years. Laboratory (PC, t-PA, C-reactive protein, TNF-alpha, von Willebrand factor, and lipid profile) and instrumental (ultrasonography and Doppler) examinations were performed. Results: PC deficiency was significantly more common among men with RA (26.6%), but did not depend on the age and the duration of the disease (p<0.05). PC deficiency correlates with the degree of activity of the inflammatory process (r = -0.27) and TNF-alpha (r = -0.37). The levels of PC and t-PA were associated with the level of cholesterol (r = -0.25), LDL-C (r = -0.31), and HDL-C (r = 0.31). In patients with PC and t-PA deficiency, significantly higher (by 19% and 12%, respectively) serum levels of Von Willebrand factor were recorded than in patients with normal levels (p<0.05). Conclusions: The study of PС and t-PA revealed a deficiency of important natural anticoagulants, that helps us to expand the understanding of the mutually aggravating effect of the deficiency of these compounds and changes in known biologically active substances on the course of RA, and to supplement the pathogenetic picture of RA with certain links.

DOP070 Activated protein C mitigates thrombo-inflammatory CD4+ T cell activity present in Inflammatory Bowel Disease.

Abstract Background IBD patients exhibit up to 6-fold increased risk of venous thromboembolism (VTE) compared to the general population, although the mechanistic basis for this increased risk is unknown. Pre-clinical studies suggest an important role for diminished protein C (PC) pathway activity in IBD pathophysiology. Aims To evaluate the procoagulant activity of colitogenic T cells and assess the impact of activated protein C (APC) signalling in regulating T cell immunothrombotic activity. Methods The pro-thrombotic potential of T cells was determined using bespoke T-cell-dependent calibrated automated thrombinography assays, flow cytometry and microscopy. Meta-transcriptomic and gene expression analysis were used to assess the dysregulated expression of coagulation-associated genes in IBD patient gut biopsies. To elucidate the potential anti-thromboinflammatory effect of APC on T cells, ex vivo functional assays were established using paediatric IBD patients or donor PBMC-derived T cells treated with APC variants. Results We identified the presence of tissue factor (TF)+CD4+CD3+ T cells in the colons of both colitogenic mice and paediatric IBD patients during active disease (Fig 1.A-E). TF is best known as the predominant cellular initiator of blood coagulation, and subsequent ex vivo functional studies revealed that pro-inflammatory CD4+ T cells upregulate TF expression in response to T cell receptor ligation, resulting in rapid TF-dependent plasma thrombin generation and clot formation. TF is typically expressed in an ‘encrypted’ state and requires decryption for optimal procoagulant activity. Notably, activated and inflammatory CD4+ T cells were found to express significantly increased levels of acid sphingomyelinase and protein disulfide isomerase, critical mediators for TF decryption, on their cell membrane compared to naïve T cells. To understand how this process might be regulated, we performed meta-transcriptomic and gene expression analysis of IBD patient gut biopsy tissue, identifying dysregulated expression of genes involved in the regulation of coagulation, including PC (PROC) and its receptor (EPCR; PROCR) in IBD patients (Fig 1.F), revealing an environment permissive to, but deficient in, PC pathway anti-inflammatory activity. Subsequent functional studies revealed that activated protein C (APC) anti-inflammatory signalling reduced colitogenic T cell generation and activity, potently impaired TF decryption and significantly reduced T cell-mediated thrombin generation and clot formation (Fig 1.G-K). Conclusion These data implicate activated T cell thrombogenicity in IBD and demonstrate a prominent role for APC signalling in regulating colitogenic T cell thrombo-inflammatory activity.