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.

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.

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.

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

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

The clinical and genetic landscape of early-onset thrombophilia in Japan.

OBJECTIVES: To determine the optimal management for early-onset thrombophilia (EOT), the genetic and clinical features of protein C (PC)-, protein S (PS)-, or antithrombin (AT)-deficient patients of ≤20 years of age were studied in Japan. METHODS/RESULTS: Clinical and genetic information of all genetically diagnosed cases was collected through the prospective, retrospective study, and literature review. One-hundred-one patients had PC (n = 55), PS (n = 29), or AT deficiency (n = 18). One overlapping case had PC- and PS-monoallelic variant. Fifty-five PC-deficient patients (54%) had 26 monoallelic or 29 biallelic variant(s), and 29 (29%) PS-deficient patients had 20 monoallelic or nine biallelic variant(s). None of the patients had AT-biallelic variants. The frequent low-risk allele p.K193del (PC-Tottori) was found in five patients with monoallelic (19%) but not 29 with biallelic variant(s). The most common low-risk allele p.K196E (PS-Tokushima) was found in five with monoallelic (25%) and six with biallelic variant(s) (67%). One exceptional de novo PC variant was found in 32 families with EOT. Only five parents had a history of thromboembolism. Thrombosis concurrently developed in three mother-newborn pairs (two PC deficiency and one AT deficiency). The prospective cohort revealed the outcomes of 35 patients: three deaths with PC deficiency and 20 complication-free survivors. Neurological complications were more frequently found in patients with PC-biallelic variants than those with PC-, PS-, or AT-monoallelic variants (73% vs. 24%, p = .019). CONCLUSIONS: We demonstrate the need for elective screening for EOT targeting PC deficiency in Japan. Early prenatal diagnosis of PC deficiency in mother-infant pairs may prevent perinatal thrombosis in them.

Computational analysis of the functional and structural impact of the most deleterious missense mutations in the human Protein C.

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.

Evaluation of acquired and hereditary risk factors for the development of thromboembolism in patients with systemic lupus erythematosus.

Although the contribution of antiphospholipid antibodies (aPL) to thrombolembolism in systemic lupus erythematosus (SLE) is well known, there is not enough data on the contribution of various hereditary thrombophilic factors. In this study, we aimed to determine acquired and hereditary thrombophilic factors in adult patients with SLE. A total of 93 SLE patients (87 women and 6 men) were included. Data on clinical, demographic and laboratory characteristics, and disease activity scores (SLEDAI) of the patients were evaluated. The patients were analyzed with a screen, including lupus anticoagulant, anticardiolipin antibodies (aCL), antithrombin III, protein C, protein S, and homocysteine levels; factor V Leiden ( FVL ), methylenetetrahydrofolate reductase ( MTHFR ) and prothrombin G20210A gene mutations. A total of 23 thromboembolic events were reported in 17 (18.3%) of the patients. The frequency of pregnancy complications and SLEDAI scores were significantly higher in SLE patients who had a thromboembolism event ( P  < 0.05). Thromboembolism was detected in 12 (32.4%) of 37 patients with positive aPL antibody and 5 (8.9%) of 56 patients with negative aPL antibody ( P  = 0.006). In addition, thromboembolism developed in 11 (32.3%) of 34 lupus anticoagulant-positive patients and 6 (10.1%) of 59 lupus anticoagulant-negative patients ( P  = 0.012). Moreover, protein C levels were significantly lower in patients who developed thromboembolism ( P  < 0.05). Patients with and without thromboembolism were similar in terms of genetic thrombophilia factors ( MTHFR A1298C, MTHFR C677T, FVL and Prothrombin G20210A ) ( P  > 0.05). In conclusion, in the current study, some acquired (aPL, lupus anticoagulant and cCL IGG) and hereditary (protein C deficiency) thrombophilic factors were shown to be associated with the development of thrombosis in SLE patients. However, the effect of other hereditary factors on the development of thromboembolism could not be demonstrated. According to the data of this study, genetic screening seems inappropriate in terms of the risk of thromboembolism in patients with SLE.

Severe Neonatal Interstitial Lung Disease Caused by a Rare Surfactant Protein C Mutation.

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.

Antithrombin, Protein C, and Protein S: Genome and Transcriptome-Wide Association Studies Identify 7 Novel Loci Regulating Plasma Levels.

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.

Laboratory Testing for Activated Protein C Resistance (APCR): An Update.

Activated protein C resistance (APCR) reflects a hemostatic state defined by a reduced ability of activated protein C (APC) to affect an anticoagulant response. This state of hemostatic imbalance is characterized by a heightened risk of venous thromboembolism. Protein C is an endogenous anticoagulant that is produced by the hepatocytes and undergoes proteolysis-mediated activation to APC. APC in turn degrades activated Factors V and VIII. APCR describes a state of resistance by activated Factors V and VIII to APC-mediated cleavage of these factors, thereby promoting amplified thrombin production and a potentially procoagulant state. This resistance of APC may be inherited or acquired. Mutations in Factor V are responsible for the most frequent form hereditary APCR. The predominant mutation, a G1691A missense mutation at Arginine 506, the so-called Factor V Leiden [FVL], causes a deletion of an APC-targeted cleavage site in Factor Va, thereby rendering it resistant to inactivation by APC. There are a variety of laboratory assays for APCR, but this chapter focuses on a procedure using a commercially available clotting assay that utilizes a snake venom and ACL TOP analyzers.

Evaluation of Activated Protein C Resistance Using Thrombin Generation Test.

Activated protein C (APC) resistance (APCR) has been identified as a risk factor of venous thromboembolism (VTE). A mutation at the level of factor (F) V has at first permitted the description of this phenotypic pattern and corresponded to a transition (guanine to adenine) at nucleotide 1691 in the gene coding for factor V, resulting in the replacement of arginine at position 506 by a glutamine. This confers to this mutated FV a resistance toward the proteolytic action of the complex formed by activated protein C with protein S. However, many other factors also lead to APCR, such as other F5 mutations (e.g., FV Hong Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy, and postpartum. All these conditions lead to the phenotypic expression of APCR and are associated with an increased risk of VTE. Considering the large population affected, the proper detection of this phenotype is a public health challenge. Currently, two types of tests are available: clotting time-based assays and their multiple variants and a thrombin generation-based assays and the endogenous thrombin potential (ETP)-based APCR assay. As APCR was thought to be uniquely related to the FV Leiden mutation, clotting time-based assays were specifically designed to detect this inherited condition. Nevertheless, other APCR conditions have been reported but were not captured by these clotting methods. Thus, the ETP-based APCR assay has been proposed as a global coagulation test able to these multiple APCR conditions, as it provides much more information, which makes it a potential candidate for screening coagulopathic conditions before therapeutic interventions. This chapter will describe the current method used for the realization of the ETP-based APC resistance assay.

Laboratory Evaluation of Antithrombin, Protein C, and Protein S.

Thrombophilia is a complex disease process, clinically manifesting in various forms of venous thromboembolism. Although both genetic and acquired (or environmental) risks factors have been reported, the presence of a genetic defect (antithrombin [AT], protein C [PC], protein S [PS]) is considered three of the major contributing factors of thrombophilia. The presence of each of these risk factors can be established by clinical laboratory analysis; however, the clinical provider and laboratory personnel must understand the testing limitations and shortcomings associated with the assays for these factors to be able to ensure an accurate diagnosis. This article will describe the major pre-analytical, analytical, and post-analytical issues associated with the various types of assays and discuss evidence-based algorithms for analyzing AT, PC, and PS in plasma.

Interstitial Lung Disease in Adulthood Associated with Surfactant Protein C Gene Mutation in a Patient with a History of Lipoid Pneumonia in Infancy.

Mutations in the surfactant protein C gene (SFTPC) are responsible for hereditary interstitial lung disease (ILD), which is a rare disease. We herein report a patient with a clinical history of endogenous lipoid pneumonia in infancy who developed diffuse progressive pulmonary fibrosis in adulthood associated with SFTPC mutations. A surgical lung biopsy and genetic sequencing revealed fibrotic interstitial pneumonia and two SFTPC mutations (c.215G>A and c.578C>A). Based on these findings, we diagnosed the series of lung diseases as sporadic ILD caused by SFTPC mutations. Physicians should suggest genetic sequencing in patients with early-onset ILD.

Anti-CENP-C Antibody-Based Immunofluorescence Dicentric Assay: Radiation Dose-Response, Validation Studies, and Radiation Dose-Dependency on Sister Centromere Fluorescence.

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.

A missense mutation in lectin domain of thrombomodulin causing functional deficiency.

Thrombomodulin (TM) functions in coagulation, fibrinolysis and inflammation by its cofactor activity for protein C, thrombin-activatable fibrinolysis inhibitor (TAFI) activation and high mobility group box 1 (HMGB1) degradation induced by thrombin. It has been widely reported that mutations in TM are related to thromboembolic diseases but hardly in lectin domain. Here we report our findings about the functional deficiencies in TM caused by substitution of aspartate with tyrosine at residue 126. Three patients suffering from recurrent thromboembolic diseases were identified with this mutation and their plasma soluble TM levels were decreased. Transfected cells expressing wild-type TM or the variant and corresponding proteins were used to examine TM functions in vitro. The cofactor activity of the mutant for protein C, TAFI activation was reduced to approximately 50% and 60% respectively. Loss in anti-inflammation due to weakened HMGB1 degradation was also observed. And the study with thrombosis models of mice suggested the decreased inhibition of thrombus development of the mutant. Together the results showed deleterious changes on TM function caused by this mutation, which may explain the thrombophilia tendency of the patients. This work provided supportive evidence that mutation in lectin domain of TM might be related to thrombotic diseases and may help us better understand the physiological roles of TM.

Downregulation of the Protein C Signaling System Is Associated with COVID-19 Hypercoagulability-A Single-Cell Transcriptomics Analysis.

Because of the interface between coagulation and the immune response, it is expected that COVID-19-associated coagulopathy occurs via activated protein C signaling. The objective was to explore putative changes in the expression of the protein C signaling network in the liver, peripheral blood mononuclear cells, and nasal epithelium of patients with COVID-19. Single-cell RNA-sequencing data from patients with COVID-19 and healthy subjects were obtained from the COVID-19 Cell Atlas database. A functional protein-protein interaction network was constructed for the protein C gene. Patients with COVID-19 showed downregulation of protein C and components of the downstream protein C signaling cascade. The percentage of hepatocytes expressing protein C was lower. Part of the liver cell clusters expressing protein C presented increased expression of ACE2. In PBMC, there was increased ACE2, inflammatory, and pro-coagulation transcripts. In the nasal epithelium, PROC, ACE2, and PROS1 were expressed by the ciliated cell cluster, revealing co-expression of ACE-2 with transcripts encoding proteins belonging to the coagulation and immune system interface. Finally, there was upregulation of coagulation factor 3 transcript in the liver and PBMC. Protein C could play a mechanistic role in the hypercoagulability syndrome affecting patients with severe COVID-19.

[Analysis of PROC gene variant in a Chinese pedigree affected with hereditary protein C deficiency].

OBJECTIVE: To explore the molecular pathogenesis of a Chinese pedigree affected with inherited protein C (PC) deficiency. METHODS: The protein C activity (PC:A) and protein C antigen (PC:Ag) of the proband and his family members were determined by a chromogenic substrate method and enzyme-linked immunosorbent assay, respectively. The proband was subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing of other members of the pedigree. RESULTS: The PC:A and PC:Ag of proband were reduced to 15% and 11%, respectively. The above parameters of his parents and elder sister were also decreased to approximately 50% of reference values. Next generation sequencing has revealed that the proband has harbored a heterozygous c.572_574delAGA (p.Glu191_Lys192delinsGlu) variant in exon 7 and a missense c.752C>T (p.Ala251Val) variant in exon 8 of the PROC gene. His father was heterozygous for the c.572_574delAGA variant, while his mother and elder sister were heterozygous for the c.752C>T variant. According to the American College of Medical Genetics and Genomics Standards and Guidelines, the c.572_574delAGA (p.Glu191_Lys192 delinsGlu) variant was predicted to be likely pathogenic (PS1+PM4+PP3). c.752 C>T (p.Ala251Val) variant was also likely pathogenic (PS1+PM1+PP3). CONCLUSION: The deletional variant of c.572_574delAGA (p.Glu191_Lys192delinsGlu) in exon 7 and missense variant c.752C>T (p.Ala251Val) in exon 8 of the PROC gene probably underlay the inherited protein C (PC) deficiency in this pedigree. Above finding has enriched the spectrum of PROC gene variants and provided a basis for genetic counseling for this pedigree.

Factor V Leiden, Factor II, Protein C, Protein S, and Antithrombin and Ischemic Strokes in Young Adults: A Meta-Analysis.

Ischemic strokes are one of the leading causes of death worldwide. The aim of this meta-analysis is to elaborate on the role of inherited predisposition to thrombophilia in the etiology of ischemic strokes in young adults. The keywords factor V Leiden (FVL), factor II, prothrombin (PT), protein C (PC), protein S (PS), antithrombin (AT), ischemic stroke, and young were used to search different databases. We selected studies with participants who were between 18 and 65 years. A total of 104 studies were eligible for inclusion in the meta-analysis. All the studied genetic markers were risk factors for ischemic stroke according to our results (FVL OR = 1.74; PT OR = 1.95; PC OR = 10.20; PS OR = 1.74; AT OR = 3.47; p < 0.05). There was moderate heterogeneity for most of the results, and subgroup analyses were conducted by dividing the studies according to the geographic location, gender ratio, and selection criteria of the performed study. There were no significant differences between the groups, but different geographic location was a probable source of heterogeneity. All of the studied markers-FVL, prothrombin, PC, PS, and AT-were significantly associated with increased risk of ischemic stroke in young adults and, if tested, could improve the quality of care.

Laboratory Testing for the Evaluation of Phenotypic Activated Protein C Resistance.

Activated protein C (APC) resistance (APCR) is considered a risk factor of venous thromboembolism (VTE). The most common genetic disorder conferring APCR is a factor (F) V Leiden mutation, but many other factors are also implicated, such as other F5 mutations (e.g., FV Hong-Kong and FV Cambridge), protein S deficiency, elevated factor VIII, exogenous hormone use, pregnancy and postpartum, depending on how APCR is defined. Considering the large population affected, the detection of this phenotype is crucial. Two types of tests are currently available: clotting time-based assays (with several versions) and thrombin generation-based assays with the endogenous thrombin potential (ETP)-based assay. The purpose of this review is therefore to discuss the performances of these tests and the cases in which it would be appropriate to use one over the other. Initially, as APCR was thought to be solely related to the FV Leiden mutation, the objective was to obtain a 100% specific assay. Clotting-time based assays were thus specifically designed to detect this inherited condition. Later on, an APCR condition without a FV Leiden mutation was identified and highlighted as an independent risk factor of VTE. Therefore, the development of a less specific assay was needed and a global coagulation test was proposed, known as the ETP-based APCR assay. In light of the above, these tests should not be used for the same purpose. Clotting time-based assays should only be recommended as a screening test for the detection of FV mutations prior to confirmation by genetic testing. On the other hand, the ETP-based APC resistance assay, in addition to being able to detect any type of APCR, could be proposed as a global screening test as it assesses the entire coagulation process.