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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.

Hereditary thrombophilia (HT) is a genetic predisposition to thrombosis. Asian mutation spectrum of HT is different from Western ones. We investigated the incidence and clinical characteristics of HT in Korean patients with unprovoked venous thromboembolism (VTE). Among 369 consecutive patients with thromboembolic event who underwent thrombophilia tests, we enrolled 222 patients diagnosed with unprovoked VTE. The presence of HT was confirmed by DNA sequencing of the genes that cause deficits in natural anticoagulants (NAs). Median follow-up duration was 40±38 months. Among the 222 patients with unprovoked VTE, 66 (29.7%) demonstrated decreased NA level, and 33 (14.9%) were finally confirmed to have HT in a genetic molecular test. Antithrombin III deficiency (6.3%) was most frequently detected, followed by protein C deficiency (5.4%), protein S deficiency (1.8%), and dysplasminogenemia (1.4%). The HT group was significantly younger (37 [32-50] vs. 52 [43-65] years; P < 0.001) and had a higher proportion of male (69.7% vs. 47%; P = 0.013), more previous VTE events (57.6% vs. 31.7%; P = 0.004), and a greater family history of VTE (43.8% vs. 1.9%; P < 0.001) than the non-HT group. Age <45 years and a family history of VTE were independent predictors for unprovoked VTE with HT (odds ratio, 9.435 [2.45-36.35]; P = 0.001 and 92.667 [14.95-574.29]; P < 0.001). About 15% of patients with unprovoked VTE had HT. A positive family history of VTE and age <45 years were independent predictors for unprovoked VTE caused by HT.

Background: The early diagnosis of inherited thrombophilia in children is challenging because of the rarity and hemostatic maturation. Methods: We explored protein C (PC), protein S (PS), and antithrombin (AT) deficiencies in 306 thromboembolic patients aged ≤20 y using the screening of plasma activity and genetic analysis. Results: Reduced activities were determined in 122 patients (40%). Low PC patients were most frequently found in the lowest age group (0–2 y, 45%), while low PS or low AT patients were found in the highest age group (16–20 y; PS: 30% and AT: 20%). Genetic study was completed in 62 patients having no other causes of thromboembolism. Mutations were determined in 18 patients (8 PC, 8 PS, and 2 AT genes). Six of eight patients with PC gene mutation were found in age 0–2 y (75%), while six of eight patients with PS gene mutation were in 7–20 y. Two AT gene–mutated patients were older than 4 y. Four PC-deficient and two PS-deficient patients carried compound heterozygous mutations. All but one PC gene–mutated patient suffered from intracranial thromboembolism, while PS/AT gene–mutated patients mostly developed extracranial venous thromboembolism. Conclusion: Stroke in low PC infants and deep vein thrombosis in low PS/AT school age children could be targeted for genetic screening of pediatric thrombophilias.

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 (1012 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.

This study aims to investigate the clinical characterization and molecular pathogenic basis of hereditary protein C (PC) deficiency in two independent Chinese families, and conduct in vitro expression studies on the newly discovered p.Trp444Arg mutation. The PC activity (PC: A) was tested using the chromogenic substrate, and PC antigen (PC: Ag) was detected via enzyme-linked immunosorbent assay (ELISA). To identify the mutation sites, nine exons of the PROC gene were amplified by PCR, and the products were directly sequenced. The conservation and pathogenicity of the mutations, as well as changes in the spatial structure of PC proteins before and after mutations, were analyzed using ClustalX-2.1-win, online bioinformatics software, and PyMOL. The function of the mutant proteins was detected using the thrombin generation assay (TGA). Recombinant PC was ectopically expressed in HEK293T cells, with mRNA levels quantified by RT-qPCR. The recombinant protein was further characterized using Western blotting, ELISA, and immunofluorescence microscopy. Proband A and B, aged 39 and 63 respectively, are both diagnosed with deep vein thrombosis (DVT) in both lower limbs and pulmonary embolism (PE). Two missense mutations, p.Arg440Cys and p.Trp444Arg, were identified in the probands. Bioinformatics and protein modeling analyses revealed that the two mutations probably affected the normal function of PC. The thrombin generation assay revealed impaired thrombin generation capacity in both probands, with proband B showing more severe impairment. In vitro expression experiments demonstrated that p.Trp444Arg do not significantly affect mRNA expression levels of PC protein compared to wild-type, but result in lower PC: Ag content and protein expression in the supernatant and higher levels in the lysate. These two mutations may be the causes of reduced PC in two independent Chinese families. Notably, this is the first reported instance of the p.Trp444Arg mutation.

Background Hereditary protein C deficiency (PCD) increases thrombotic risk, but the molecular mechanisms of distinct missense variants remain incompletely defined. Two siblings from a family with recurrent deep vein thrombosis and a strong family history of thrombotic events were found to carry compound heterozygous PROC variants. Objectives To elucidate the biosynthetic and functional consequences of two PROC missense variants, c.632G > A (p.Arg211Gln) and c.1099G > A (p.Val367Met), and their combined impact on anticoagulant capacity. Methods Two siblings with recurrent deep vein thrombosis were analyzed. Family segregation analysis was performed on multiple heterozygous carriers to support genotype-phenotype correlations. Recombinant protein C variants were expressed in HEK293T cells to assess expression efficiency and intracellular accumulation. Zymogen activation by thrombin-thrombomodulin and APC anticoagulant activity were evaluated using FVa degradation, clotting, and thrombin generation assays. Structural modeling and Na⁺-dependent enzymatic assays were performed to explore variant-specific mechanistic defects. Results Both variants exhibited partial expression defects, with p.Val367Met showing significantly increased intracellular accumulation and reduced secretion efficiency. p.Arg211Gln selectively impaired zymogen activation without affecting APC function (type IIa deficiency), whereas p.Val367Met preserved activation but reduced Na⁺-dependent catalytic efficiency (type IIb deficiency). Structural modeling revealed that Arg211 substitution disrupts activation peptide conformation, while Val367Met perturbs the Na⁺-binding loop adjacent to the S1 pocket. Co-inheritance of both resulted in synergistic anticoagulant impairment, leading to markedly elevated thrombin generation. Conclusions These findings demonstrate that PCD pathophysiology can arise from combined biosynthetic and functional defects, with distinct structural perturbations differentially affecting zymogen processing and protease activity. Integrated genetic, biochemical, and structural analyses, in the context of family history and clinical phenotype, are essential for accurate variant interpretation. Understanding the specific molecular mechanisms of PROC variants can inform clinical decision-making, guide personalized anticoagulant therapy, and help prevent thrombotic events in affected individuals. Therapeutic strategies targeting zymogen activation or proteostasis may offer potential avenues to mitigate thrombotic risk in hereditary PCD.

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

A female newborn was transferred to the hospital due to progressive necrotic skin lesions. She was born full-term with an average birth weight and no prenatal or perinatal problems. She developed the first purplish skin lesion on her left buttock since postnatal day 1. She was initially treated with antibiotics under the impression of early-onset neonatal sepsis; however, her lesions worsened and rapidly progressed. The protein C chromogenic activity was remarkably decreased at below 10%, similar to a protein C antigen level below 1%, highly suggestive of severe congenital protein C deficiency. Molecular analysis revealed compound heterozygous likely pathogenic variants in the protein C gene. After the diagnosis, fresh frozen plasma (FFP) transfusions every 8 hours until the patient was stabilized and therapeutic low-molecular-weight heparin was started. She developed complications related to FFP administration, such as acute kidney injury, hypertension and proteinuria, during the interim period, awaiting the initiation of protein C concentrate replacement therapy.

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. 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. 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 (  > 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 (  < 0.001), but there was no significant difference in protein C antigen levels (  > 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. 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.

Objectives: Protein C (PC) deficiency is an inherited thrombophilia with a prevalence of 0.5% in the general population and 3% in subjects with a first-time deep vein thrombosis (DVT). Here we report a series of 14 PC-deficient Polish patients with comprehensive clinical and molecular characteristics, including long-term follow-up data and a deep mutational analysis of the PROC gene. Patients and Methods: Fourteen unrelated probands (mean ± SD age 43.8 ± 13.0 years) with suspicion of PC deficiency, who experienced thromboembolic events and a majority of whom received anticoagulants (92.8%), were screened for PROC mutations by sequencing the nine PROC exons and their flanking intron regions. Results: Ten probands (71.4%) had missense mutations, two patients (14.3%) carried nonsense variants, and the other two subjects (14.3%) had splice-site mutations, the latter including the c.401-1G>A variant, reported here for the very first time. The proband carrying the c.401-1A allele had a hepatic artery aneurysm with a highly positive family history of aneurysms and the absence of any mutations known to predispose to this vascular anomaly. Conclusion: A novel detrimental PROC mutation was identified in a family with aneurysms, which might suggest yet unclear links of thrombophilia to vascular anomalies, including aneurysms at atypical locations in women. The present case series also supports data indicating that novel oral anticoagulants (NOACs) are effective in PC deficient patients.