Validation for the function of protein C in mouse models.

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.

The natural anticoagulant protein S; hemostatic functions and deficiency.

Protein S (PS) is a vital endogenous anticoagulant. It plays a crucial role in regulating coagulation by acting as a cofactor for the activated protein C (APC) and tissue factor pathway inhibitor (TFPI) pathways. Additionally, it possesses direct anticoagulant properties by impeding the intrinsic tenase and prothrombinase complexes. Protein S oversees the coagulation process in both the initiation and propagation stages through these roles. The significance of protein S in regulating blood clotting can be inferred from the significant correlation between deficits in protein S and an elevated susceptibility to venous thrombosis. This is likely because activated protein C and tissue factor pathway inhibitor exhibit low efficacy as anticoagulants when no cofactors exist. The precise biochemical mechanisms underlying the roles of protein S cofactors have yet to be fully elucidated. Nevertheless, recent scientific breakthroughs have significantly enhanced comprehension findings for these functions. The diagnosis of protein S deficiency, both from a technical and genetic standpoint, is still a subject of debate due to the complex structural characteristics of the condition. This paper will provide an in-depth review of the molecular structure of protein S and its hemostatic effects. Furthermore, we shall address the insufficiency of protein S and its methods of diagnosis and treatment.

What is the purpose of this summary? To provide an in-depth review of the molecular structure of protein S and its hemostatic effects.To address the deficiency of protein S and its methods of diagnosis and treatment.What is known? Protein S operates as an anticoagulant through its roles as a cofactor for APC, TFPI, and an inhibitor of FIXa.Protein S deficiency can be either inherited or acquired.What is new? Plasma protein S and platelet-derived protein S contribute to regulating coagulation and maintaining hemostasis. Protein S can be used as a potential promising treatment target for persons diagnosed with hemophilia.

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

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

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases.

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 µg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

A potential mechanism for the cytoprotective effects of activated protein C-released endothelial extracellular vesicles.

ABSTRACT: Activated protein C (APC) was shown to release extracellular vesicles (EVs). APC bound to the EVs was thought to be responsible for cytoprotection. Our study demonstrates that the cytoprotective effects of APC-released EVs are independent of APC. APC-released EVs carry anti-inflammatory microRNAs in their cargo.

N-Terminal Fragment of Cardiac Myosin Binding Protein C Modulates Cooperative Mechanisms of Thin Filament Activation in Atria and Ventricles.

Cardiac myosin binding protein C (cMyBP-C) is one of the essential control components of the myosin cross-bridge cycle. The C-terminal part of cMyBP-C is located on the surface of the thick filament, and its N-terminal part interacts with actin, myosin, and tropomyosin, affecting both kinetics of the ATP hydrolysis cycle and lifetime of the cross-bridge, as well as calcium regulation of the actin-myosin interaction, thereby modulating contractile function of myocardium. The role of cMyBP-C in atrial contraction has not been practically studied. We examined effect of the N-terminal C0-C1-m-C2 (C0-C2) fragment of cMyBP-C on actin-myosin interaction using ventricular and atrial myosin in an in vitro motility assay. The C0-C2 fragment of cMyBP-C significantly reduced the maximum sliding velocity of thin filaments on both myosin isoforms and increased the calcium sensitivity of the actin-myosin interaction. The C0-C2 fragment had different effects on the kinetics of ATP and ADP exchange, increasing the affinity of ventricular myosin for ADP and decreasing the affinity of atrial myosin. The effect of the C0-C2 fragment on the activation of the thin filament depended on the myosin isoforms. Atrial myosin activates the thin filament less than ventricular myosin, and the C0-C2 fragment makes these differences in the myosin isoforms more pronounced.

The Protein S Erlangen Mutation PROS1c.1904T>C (F635S) Suppresses Secretion.

BACKGROUND: The recently identified PROS1 mutation Protein S Erlangen c.1904T>C, resulting in amino acid exchange F635S, is associated with severe quantitative protein S (PS) deficiency and clinical thrombosis. It was hypothesized that this deficiency is due to a secretion defect [1]. This report aims to further elucidate the potential secretion defect of PS Erlangen. METHODS: Coding sequences (CDS) of wild type (WT) PROS1 (encoding PS) and mutated PROS1c.1904T>C (encoding PSF635S) were cloned in front of the CDS of green fluorescent protein (GFP), and the respective plasmids were introduced into HEK293T cells. PROS1-GFP and PROS1c.1904T>C-GFP expressing HEK293T cell lines were analyzed by confocal laser scanning microscopy and western blot for cellular proteins and proteins secreted to the growth medium. RESULTS: Western blot analysis revealed a significantly reduced secretion of PSF635S compared to WT PS. This observation was confirmed by the detection of mutant PSF635S-GFP fusion exclusively in the endoplasmic reticulum (ER), while PS-GFP passed through the entire secretory pathway, as indicated by the localization within both the ER and Golgi apparatus. CONCLUSIONS: The Protein S Erlangen mutation results in type I PS deficiency caused by a secretion defect.

Characteristics of factor V and protein C based on results from Korean testing centers.

OBJECTIVE: The global incidence of thrombosis is increasing. However, research on thrombosis in the context of Korea is scarce. We aimed to analyze the relationship between factor V and protein C test results and thrombosis in Koreans through a domestic commissioned testing institution conducting mass examinations. METHODS: Results of factor V and protein C tests of 1386 individuals referred simultaneously to EONE Laboratories (Incheon, Republic of Korea) from January 2017 to July 2023 were analyzed retrospectively to identify the association with thrombotic disease. The tests were performed using a STAR MAX (Diagnostica Stago, Asnieres, France) automatic blood coagulation analyzer. The results were analyzed by age and sex. RESULTS: The inspection rate increased gradually from 2017 to 2022. Women (70.0%) demonstrated a higher test rate than did men (30.0%). Young women reported high test rates; the test rate and age distribution differed by sex. Women aged between 20 and 49 years reported lower factor V and higher protein C concentrations than did men between 20 and 49 years of age. CONCLUSIONS: The tests were more commonly performed in women than in men. Women aged between 20 and 49 years reported lower factor V concentrations and higher protein C concentrations than men between 20 and 49 years of age. This study will facilitate recognizing and preventing thrombotic diseases in women.

Single-Cell RNA-seq reveals transcriptomic modulation of Alzheimer's disease by activated protein C.

Single-Cell RNA sequencing reveals changes in cell population in Alzheimer's disease (AD) model 5xFAD (5x Familial AD mutation) versus wild type (WT) mice. The returned sequencing data was processed through the 10x Genomics CellRanger platform to perform alignment and form corresponding matrix to perform bioinformatic analysis. Alterations in glial cells occurred in 5xFAD versus WT, especially increases in microglia proliferation were profound in 5xFAD. Differential expression testing of glial cells in 5xFAD versus WT revealed gene regulation. Globally, the critical genes implicated in AD progression are upregulated such as Apoe, Ctsb, Trem2, and Tyrobp. Using this differential expression data, GO term enrichment was completed to observe possible biological processes impacted by AD progression. Utilizing anti-inflammatory and cyto-protective recombinant Activated Protein C (APC), we uncover inflammatory processes to be downregulated by APC treatment in addition to recuperation of nervous system processes. Moreover, animal studies demonstrated that administration of recombinant APC significantly attenuated Aß burden and improved cognitive function of 5xFAD mice. The downregulation of highly expressed AD biomarkers in 5xFAD could provide insight into the mechanisms by which APC administration benefits AD.

Efficient production of peptidylglycine α-hydroxylating monooxygenase in yeast for protein C-terminal functionalization.

Peptidylglycine α-hydroxylating monooxygenase (PHM) is pivotal for C-terminal amidation of bioactive peptides in animals, offering substantial potential for customized protein synthesis. However, efficient PHM production has been hindered by the complexity of animal cell culture and the absence of glycosylation in bacterial hosts. Here, we demonstrate the recombinant expression of Caenorhabditis elegans PHM in the yeast Pichia pastoris, achieving a remarkable space-time yield of 28.8 U/L/day. This breakthrough surpasses prior PHM production rates and eliminates the need for specialized cultivation equipment or complex transfection steps. Mass spectrometry revealed N-glycosylation at residue N182 of recombinant CePHM, which impacts the enzyme's activity as indicated by biochemical experiments. To showcase the utility of CePHM, we performed C-terminal amidation on ubiquitin at a substrate loading of 30 g/L, a concentration meeting the requirements for pharmaceutical peptide production. Overall, this work establishes an efficient PHM production method, promising advancements in scalable manufacturing of C-terminally modified bioactive peptides and probe proteins.

Activated protein C and free protein S in patients with mild to moderate bleeding disorders.

BACKGROUND: Underlying mechanisms for bleeding and impaired thrombin generation (TG) and plasma clot formation (PCF) in patients with mild to moderate bleeding disorders (MBDs) are still to be elucidated, especially in bleeding disorder of unknown cause (BDUC). The role of the natural anticoagulants activated protein C (APC) and free protein S (PS) has not yet been investigated in this patient population. AIMS: To analyze antigen levels of APC and PS in patients with MBDs and BDUC and investigate associations to clinical bleeding phenotype and severity as well as and hemostatic capacity. METHODS: Antigen levels of APC and free PS were measured in 262 patients from the Vienna Bleeding Biobank (VIBB), a single-center cohort study, by ELISA and compared to 61 healthy controls (HC). RESULTS: Antigen levels of APC were higher in MBD patients than in HC when adjusted for age, sex and BMI (median (IQR) 33.1 (20.6-52.6) and 28.6 (16.4-47.2) ng/mL). This was most pronounced in patients with BDUC (35.3 (21.7-54.3) ng/mL). No differences in PS antigen levels between patients and HC were seen overall, or according to specific diagnoses. Further, no association between APC or PS and bleeding severity or global tests of hemostasis or TG were identified, while paradoxically APC weakly correlated with shorter lag time and time to peak of PCF in BDUC. CONCLUSION: Our data demonstrate increased antigen levels of APC in BDUC, which might contribute to the bleeding tendency in some patients and could be a future therapeutic target in BDUC.

The link between high factor VIII to protein C ratio values and poor liver function after major hepatectomy.

Our goal was to assess the coagulation profile in the immediate postoperative time after major liver surgery and its association with the liver function. Our hypothesis is that a decreased synthesis of the coagulation factor levels reflects an impaired liver synthesis following hepatic resection and will be associated with poor outcomes. This is a prospective, observational study recruiting consecutive patients scheduled for major liver resection in a tertiary hospital. Coagulation profile was assessed by conventional assays, viscoelastic assays and coagulation factor levels preoperatively and, on postoperative days 1, 2 and 6. Factor VIII to protein C (FVIII/PC) ratio has been used as a surrogate marker of hemostatic imbalance. Liver function was measured with conventional and indocyanine green (ICG) clearance tests, which were obtained preoperatively and on postoperative days 1 and 2. Sixty patients were recruited and 51 were included in the study. There is a clear increase in FVIII/PC ratio after surgery, which was significantly associated with low liver function, being more pronounced beyond postoperative day 2 and in patients with poorer liver function ( P  < 0.001). High FVIII/PC ratio values were significantly associated with higher postoperative morbidity, prolonged ICU and hospital stay and less survival ( P  < 0.05). High FVIII/PC ratio on postoperative day 2 was found to be predictor of posthepatectomy liver failure (PHLF; area under the ROC curve = 0.8129). Early postoperative high FVIII/PC ratio values are associated with low liver function, PHLF and poorer outcomes in patients undergoing major hepatic resection.

Hydrocortisone plus fludrocortisone for community acquired pneumonia-related septic shock: a subgroup analysis of the APROCCHSS phase 3 randomised trial.

BACKGROUND: Glucocorticoids probably improve outcomes in patients hospitalised for community acquired pneumonia (CAP). In this a priori planned exploratory subgroup analysis of the phase 3 randomised controlled Activated Protein C and Corticosteroids for Human Septic Shock (APROCCHSS) trial, we aimed to investigate responses to hydrocortisone plus fludrocortisone between CAP and non-CAP related septic shock. METHODS: APROCCHSS was a randomised controlled trial that investigated the effects of hydrocortisone plus fludrocortisone, drotrecogin-alfa (activated), or both on mortality in septic shock in a two-by-two factorial design; after drotrecogin-alfa was withdrawn on October 2011, from the market, the trial continued on two parallel groups. It was conducted in 34 centres in France. In this subgroup study, patients with CAP were a preselected subgroup for an exploratory secondary analysis of the APROCCHSS trial of hydrocortisone plus fludrocortisone in septic shock. Adults with septic shock were randomised 1:1 to receive, in a double-blind manner, a 7-day treatment with daily administration of intravenous hydrocortisone 50 mg bolus every 6h and a tablet of 50 µg of fludrocortisone via the nasogastric tube, or their placebos. The primary outcome was 90-day all-cause mortality. Secondary outcomes included all-cause mortality at intensive care unit (ICU) and hospital discharge, 28-day and 180-day mortality, the number of days alive and free of vasopressors, mechanical ventilation, or organ failure, and ICU and hospital free-days to 90-days. Analysis was done in the intention-to-treat population. The trial was registered at ClinicalTrials.gov (NCT00625209). FINDINGS: Of 1241 patients included in the APROCCHSS trial, CAP could not be ruled in or out in 31 patients, 562 had a diagnosis of CAP (279 in the placebo group and 283 in the corticosteroid group), and 648 patients did not have CAP (329 in the placebo group and 319 in the corticosteroid group). In patients with CAP, there were 109 (39%) deaths of 283 patients at day 90 with hydrocortisone plus fludrocortisone and 143 (51%) of 279 patients receiving placebo (odds ratio [OR] 0·60, 95% CI 0·43-0·83). In patients without CAP, there were 148 (46%) deaths of 319 patients at day 90 in the hydrocortisone and fludrocortisone group and 157 (48%) of 329 patients in the placebo group (OR 0·95, 95% CI 0·70-1·29). There was significant heterogeneity in corticosteroid effects on 90-day mortality across subgroups with CAP and without CAP (p=0·046 for both multiplicative and additive interaction tests; moderate credibility). Of 1241 patients included in the APROCCHSS trial, 648 (52%) had ARDS (328 in the placebo group and 320 in the corticosteroid group). There were 155 (48%) deaths of 320 patients at day 90 in the corticosteroid group and 186 (57%) of 328 patients in the placebo group. The OR for death at day 90 was 0·72 (95% CI 0·53-0·98) in patients with ARDS and 0·85 (0·61-1·20) in patients without ARDS (p=0·45 for multiplicative interaction and p=0·42 for additive interaction). The OR for observing at least one serious adverse event (corticosteroid group vs placebo) within 180 days post randomisation was 0·64 (95% CI 0·46-0·89) in the CAP subgroup and 1·02 (0·75-1·39) in the non-CAP subgroup (p=0·044 for multiplicative interaction and p=0·042 for additive interaction). INTERPRETATION: In a pre-specified subgroup analysis of the APROCCHSS trial of patients with CAP and septic shock, hydrocortisone plus fludrocortisone reduced mortality as compared with placebo. Although a large proportion of patients with CAP also met criteria for ARDS, the subgroup analysis was underpowered to fully discriminate between ARDS and CAP modifying effects on mortality reduction with corticosteroids. There was no evidence of a significant treatment effect of corticosteroids in the non-CAP subgroup. FUNDING: Programme Hospitalier de Recherche Clinique of the French Ministry of Health, by Programme d'Investissements d'Avenir, France 2030, and IAHU-ANR-0004.

Thrombomodulin: a multifunctional receptor modulating the endothelial quiescence.

Thrombomodulin (TM) is a type 1 receptor best known for its function as an anticoagulant cofactor for thrombin activation of protein C on the surface of vascular endothelial cells. In addition to its anticoagulant cofactor function, TM also regulates fibrinolysis, complement, and inflammatory pathways. TM is a multidomain receptor protein with a lectin-like domain at its N-terminus that has been shown to exhibit direct anti-inflammatory functions. This domain is followed by 6 epidermal growth factor-like domains that support the interaction of TM with thrombin. The interaction inhibits the procoagulant function of thrombin and enables the protease to regulate the anticoagulant and fibrinolytic pathways by activating protein C and thrombin-activatable fibrinolysis inhibitor. TM has a Thr/Ser-rich region immediately above the membrane surface that harbors chondroitin sulfate glycosaminoglycans, and this region is followed by a single-spanning transmembrane and a C-terminal cytoplasmic domain. The structure and physiological function of the extracellular domains of TM have been extensively studied, and numerous excellent review articles have been published. However, the physiological function of the cytoplasmic domain of TM has remained poorly understood. Recent data from our laboratory suggest that intracellular signaling by the cytoplasmic domain of TM plays key roles in maintaining quiescence by modulating phosphatase and tensin homolog signaling in endothelial cells. This article briefly reviews the structure and function of extracellular domains of TM and focuses on the mechanism and possible physiological importance of the cytoplasmic domain of TM in modulating phosphatase and tensin homolog signaling in endothelial cells.

Endothelial Protein C Receptor and 3K3A-Activated Protein C Protect Mice from Allergic Contact Dermatitis in a Contact Hypersensitivity Model.

Endothelial protein C receptor (EPCR) is a receptor for the natural anti-coagulant activated protein C (aPC). It mediates the anti-inflammatory and barrier-protective functions of aPC through the cleavage of protease-activated receptor (PAR)1/2. Allergic contact dermatitis is a common skin disease characterized by inflammation and defective skin barrier. This study investigated the effect of EPCR and 3K3A-aPC on allergic contact dermatitis using a contact hypersensitivity (CHS) model. CHS was induced using 1-Fluoro-2,4-dinitrobenzene in EPCR-deficient (KO) and matched wild-type mice and mice treated with 3K3A-aPC, a mutant form of aPC with diminished anti-coagulant activity. Changes in clinical and histological features, cytokines, and immune cells were examined. EPCRKO mice displayed more severe CHS, with increased immune cell infiltration in the skin and higher levels of inflammatory cytokines and IgE than wild-type mice. EPCR, aPC, and PAR1/2 were expressed by the skin epidermis, with EPCR presenting almost exclusively in the basal layer. EPCRKO increased the epidermal expression of aPC and PAR1, whereas in CHS, their expression was reduced compared to wild-type mice. 3K3A-aPC reduced CHS severity in wild-type and EPCRKO mice by suppressing immune cell infiltration/activation and inflammatory cytokines. In summary, EPCRKO exacerbated CHS, whereas 3K3A-aPC could reduce the severity of CHS in both EPCRKO and wild-type mice.

Analysis of laboratory parameters before the occurrence of hepatic sinusoidal obstruction syndrome in children, adolescents, and young adults after hematopoietic stem cell transplantation.

PURPOSE: Hepatic sinusoidal obstruction syndrome (SOS) is a serious complication following hematopoietic stem cell transplantation (HSCT) in which early diagnosis improves patient outcome. The aim of our study was to detect laboratory parameters following HSCT that can predict the occurrence of SOS. METHODS: This retrospective study included 182 children, adolescents, and young adults who underwent allogeneic or autologous HSCT for the first time (median age 7.2 years). The diagnosis of SOS was based on the pediatric criteria of European Society for Blood and Marrow Transplantation (EBMT). We investigated 15 laboratory parameters after HSCT before the onset of SOS. RESULTS: The overall incidence of SOS was 14.8%. SOS developed in 24 of 126 allogeneic (19.1%) and in 3 of 56 autologous (5.4%) HSCT patients at a median time of 13 days after HSCT. We observed a low SOS mortality rate of 11.1% within 100 days after HSCT. International normalized ratio (INR) ≥ 1.3, activated partial thromboplastin time (aPTT) ≥ 40 s, reptilase time ≥ 18.3 s, factor VIII ≤ 80%, antithrombin III ≤ 75%, protein C ≤ 48%, D-dimer ≥ 315 µg/L, bilirubin ≥ 9 µmol/L, and ferritin ≥ 3100 µg/L showed significant associations with the onset of SOS in the univariate analyses. In the multivariate analysis, INR ≥ 1.3 [odds ratio (OR) = 8.104, p = 0.006], aPTT ≥ 40 s (OR = 10.174, p = 0.001), protein C ≤ 48% (OR = 5.215, p = 0.014), and ferritin ≥ 3100 µg/L (OR = 7.472, p = 0.004) could be confirmed as independent risk factors after HSCT before SOS. If three of the four significant cut-off values were present, the probability of developing SOS was more than 70%. The probability of SOS was 96%, if all four laboratory parameters were changed according to the cut-off values. The values of factor XIII, von Willebrand factor (vWF), von Willebrand factor activity (vWF activity), protein S, fibrinogen, and alanine aminotransferase (ALT) were not relevant for the occurrence of SOS. CONCLUSION: In summary, the laboratory parameters INR, aPTT, protein C, and ferritin were very useful to predict the occurrence of SOS. In addition, this is the first report on a significant association between SOS and high values of INR and aPTT after HSCT before SOS.

Anticoagulant effects of protein C, protein S, and antithrombin levels on the protein C pathway in young children.

The protein C (PC) pathway involves physiological anticoagulant factors (PC, protein S [PS], and factor V) and performs major anticoagulant functions in adults. Variations in overall PC pathway function due to dynamic changes in PC and PS in early childhood are poorly understood. We aimed to evaluate the contributions of PC pathway function during early childhood by measuring changes in plasma thrombin generation (TG) after administration of the PC activator protac. We evaluated correlations between anticoagulant factors and percentage of protac-induced coagulation inhibition (PiCi%). Before protac addition, TG in newborns (n = 35), infants (n = 42), young children (n = 35), and adults (n = 20) were 525 ± 74, 720 ± 96, 785 ± 53, and 802 ± 64 mOD/min, and PiCi% were 42.1 ± 9.9, 69.8 ± 11.0, 82.9 ± 4.4, and 86.9 ± 3.4%, respectively. The distribution of PiCi% on the two axes of TG (with or without protac) changed continuously with age and differed from that of warfarin-treated plasma and adult PC- or PS-deficient plasma. PiCi% increased dynamically during infancy and correlated with PS levels in newborns and PC levels in young children. Addition of PC or fresh frozen plasma equivalent to approximately 25% PC to PC-deficient plasma improved PiCi%. This automatic measurement requires only a small sample volume and is useful for analysis of developmental hemostasis.

Structural vulnerability in EPCR suggests functional modulation.

The endothelial protein C receptor (EPCR) is a fundamental component of the vascular system in mammals due to its contribution in maintaining blood in a non-prothrombotic state, which is crucial for overall life development. It accomplishes this by enhancing the conversion of protein C (PC) into the anticoagulant activated protein C (APC), with this property being dependent on a known EPCR conformation that enables direct interaction with PC/APC. In this study, we report a previously unidentified conformation of EPCR whereby Tyr154, critical for PC/APC binding, shows a striking non-canonical configuration. This unconventional form is incompatible with PC/APC binding, and reveals, for the first time, a region of structural vulnerability and potential modulation in EPCR. The identification of this malleability enhances our understanding of this receptor, prompting inquiries into the interplay between its plasticity and function, as well as its significance within the broader framework of EPCR's biology, which extends to immune conditions.

Hemostatic balance between pro- and anticoagulant is maintained during glucocorticoid treatment.

BACKGROUND: Glucocorticoids are associated with an increased risk of venous thrombosis. Glucocorticoid treatment increases coagulation factor and anticoagulant levels; however, its effect on hemostatic function remains unclear. This study aimed to investigate the changes in comprehensive coagulation profiles after glucocorticoid treatment in noninflammatory diseases to elucidate the direct contribution of glucocorticoids to hemostatic function. PROCEDURE: Patients diagnosed with primary immune thrombocytopenia requiring glucocorticoid treatment were prospectively enrolled in this study. Changes in coagulation factors and anticoagulants during glucocorticoid treatment and changes in thrombin generation potential were determined in the absence and presence of soluble thrombomodulin (sTM). RESULTS: Seven treatment cases (four for steroid pulse therapy and three for oral glucocorticoid therapy) in six patients with immune thrombocytopenia were examined. After glucocorticoid treatment, activated partial thromboplastin time significantly shortened, and activities of factor VIII, IX, XI, and XII significantly increased, except for von Willebrand factor antigen. Moreover, antithrombin and protein C (PC) activities significantly increased after glucocorticoid treatment. Two major parameters of thrombin generation potential, endogenous thrombin potential (ETP) and peak thrombin (Peak), significantly increased in the absence of sTM after glucocorticoid treatment. However, no significant increases in either parameter were observed in the presence of sTM. ETP-TM and Peak-TM ratios, which represent resistance to the anticoagulant effect of the PC pathway, significantly decreased after glucocorticoid treatment, suggesting that anticoagulant function via the PC pathway is elevated after glucocorticoid treatment. CONCLUSIONS: As glucocorticoids increase intrinsic coagulation factor and anticoagulant levels, hemostatic balance between pro- and anticoagulant functions is maintained.