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Sublocalization of the human protein C gene on chromosome 2q13-q14
Abstract
The localization of human protein C gene on chromosome 2 was investigated by in situ hybridization using a partial cDNA for protein C. Silver-grain analysis indicates that the protein C gene is located on 2q13-q14.
... The proteins activated in this process include coagulation factors II, VII, IX, and X [107,114]. Vitamin K-dependent proteins also include protein C, whose task is to prevent blood coagulation [107,117,118]; protein S, which serves as a cofactor for activated protein C and participates in the inactivation of factors Va and VIIIa of the coagulation process The main sources of vitamin K 1 are green plants and vegetable oils [106,110,113,114]. Meat, cheese, and fermented soybean products contain a form of vitamin K 2 [106,108,110,114]. ...
... The proteins activated in this process include coagulation factors II, VII, IX, and X [107,114]. Vitamin K-dependent proteins also include protein C, whose task is to prevent blood coagulation [107,117,118]; protein S, which serves as a cofactor for activated protein C and participates in the inactivation of factors Va and VIIIa of the coagulation process [107,118,119]; and protein Z, which also belongs to the factors of the blood coagulation cascade and is involved in the degradation of factor Xa [107,118,120]. Gla proteins are found throughout the body and are a component of bones as osteocalcin, matrix Gla protein (MGP) of the skeleton, kidney Gla protein, growth arrestspecific 6 protein which is involved in the stimulation of cell proliferation, and Gla-rich protein involved in the mineralization of soft tissues [106,108,121,122]. ...
Ferroptosis is a recently discovered form of programmed cell death. It is characterized by the accumulation of iron and lipid hydroperoxides in cells. Vitamin K is known to have antioxidant properties and plays a role in reducing oxidative stress, particularly in lipid cell membranes. Vitamin K reduces the level of reactive oxygen species by modulating the expression of antioxidant enzymes. Additionally, vitamin K decreases inflammation and potentially prevents ferroptosis. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to coronavirus disease 2019 (COVID-19) is associated with oxidant–antioxidant imbalance. Studies have shown that intensified ferroptosis occurs in various tissues and cells affected by COVID-19. Vitamin K supplementation during SARS-CoV-2 infection may have a positive effect on reducing the severity of the disease. Preliminary research suggests that vitamin K may reduce lipid peroxidation and inhibit ferroptosis, potentially contributing to its therapeutic effects in COVID-19 patients. The links between ferroptosis, vitamin K, and SARS-CoV-2 infection require further investigation, particularly in the context of developing potential treatment strategies for COVID-19.
... Protein C deficiency is inherited in an autosomal pattern. The gene for protein C is located on chromosome 2 (2q13-14) [5]. Two major subtypes of protein C deficiency have been delineated. ...
... is a substantial contribution to review the manuscript, revising the intellectual content before final approval of the version to be published. 5. All co-authors will be notified about the results and conclusion. ...
... These functions are mediated by cell-specific and context-specific receptor complexes and intracellular signaling pathways, such as the endothelial protein C receptor (EPCR), protease-activated receptor1 (PAR1), and PAR3 [1,2]. The PC gene (PROC) is located on chromosome 2 at position q14-21, comprising nine exons, one non-translated exon, and eight introns [3]. The presence of a short untranslated exon (exon 1) can affect the high-level and liver-specific expression of PC and produce different splice patterns for the human PC gene [4][5][6][7]. ...
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.
... The amino acid sequence is known and contains 9 Gla residues and 1 -hydroxyaspartic acid. [11][12][13] The gene encoding protein C is located on chromosome 2 at q13-14 14,15 and is approximately 11 kb, containing 9 exons and 8 introns. 7 The majority of the introns separate functional domains in the protein in a manner similar to other vitamin K-dependent proteins. ...
Objective.—To review the current understanding of the pathophysiology of protein C deficiency and its role in congenital thrombophilia. Recommendations for diagnostic testing for protein C function and concentration, derived from the medical literature and consensus opinions of recognized experts in the field, are included, specifying whom, how, and when to test. The role of related proteins, such as thrombomodulin and endothelial protein C receptor, is also reviewed.
Data Sources.—Review of the published medical literature.
Data Extraction and Synthesis.—A summary of the medical literature and proposed testing recommendations were prepared and presented at the College of American Pathologists Conference XXXVI: Diagnostic Issues in Thrombophilia. After discussion at the conference, consensus recommendations presented in this manuscript were accepted after a two-thirds majority vote by the participants.
Conclusions.—Protein C deficiency is an uncommon genetic abnormality that may be a contributing cause of thrombophilia, often in conjunction with other genetic or acquired risk factors. When assay of protein C plasma levels is included in the laboratory evaluation of thrombophilia, a functional amidolytic protein C assay should be used for initial testing. The diagnosis of protein C deficiency should be established only after other acquired causes of protein C deficiency are excluded. A low protein C level should be confirmed with a subsequent assay on a new specimen. Antigenic protein C assays may be of benefit in subclassification of the type of protein C deficiency. The role of thrombomodulin and endothelial cell protein C receptor in thrombosis has yet to be clearly established, and diagnostic testing is not recommended at this time.
... 5 The PROC gene is 11.2 kb in size and contains a promoter region, 9 exons and 8 intronic regions. 6,7 Exon 1 is a noncoding sequence and the start codon is located in exon 2. 8 Exons 2 and 3 are pre-pro-peptides including the signal peptide and the pro-peptide sequence ( Figure 1). Exons 3 and 4 encode for the Gla domain. ...
Protein C (PC) deficiency is associated with an increased risk for venous thromboembolism (VTE). In daily practice, exclusion of a hereditary PC deficiency is often based on a single determination of PC activity, by either clotting time–based or mostly chromogenic assay. However, diagnosis of hereditary PC deficiency is challenging due to several laboratory and clinical limitations. We compared the potential of PC activity values measured by either chromogenic or clotting time–based assay to predict a variation in the PROC gene. One hundred one (35%) of 287 patients carried variations within the PROC gene, including 2 previously not published variations. In 20 (20%) patients with identified variation, PC activity, determined by chromogenic assay, was within the reference range. For prediction of an underlying genetic defect determined by chromogenic and clotting time–based assay, sensitivity was 80% versus 99%, specificity 75% versus 18%, positive predictive value 64% versus 39%, and negative predictive value (NPV) 88% versus 97%. The lower NPV of chromogenic versus clotting time–based PC assay can be mainly explained by the presence of PC deficiency type IIb. Following our proposed diagnostic algorithm, additional measurement of PC activity by clotting time–based assay in case of a positive VTE history improves detection of this subtype of PC deficiency. Considering potential therapeutic consequences for primary and especially for secondary VTE prophylaxis, genetic analysis is required not only for confirmation but also for clarification of PC deficiency.
Aims: Measurement of protein S (PS) activity in patients taking direct oral anticoagulants (DOACs) using reagents based on a clotting assay results in falsely high PS activity, thus masking inherited PS deficiency, which is most frequently seen in the Japanese population. In this study, we investigated the effect of factor Xa (FXa) inhibitors on PS activity using the reagent on the basis of the chromogenic assay, which was recently developed in Japan.
Methods: The study enrolled 152 patients (82 males and 70 females; the average age: 68.5±14.0 years) receiving three FXa inhibitors (rivaroxaban, edoxaban, and apixaban). PS activity was measured using the reagents on the basis of the clotting and chromogenic assays.
Results: PS activity measured by the clotting assay reagents exhibited falsely high values depending on the plasma concentrations of FXa inhibitors in patients taking either rivaroxaban or edoxaban. However, none of the three FXa inhibitors affected PS activity when measured using the chromogenic assay.
Conclusion: In patients taking rivaroxaban or edoxaban, inherited PS deficiency is likely missed because the levels of PS activity measured using the reagents based on the clotting assay are falsely high. However, we report that three FXa inhibitors do not affect PS activity measured by the chromogenic assay. When measuring the levels of PS activity in patients undergoing DOACs, the principles of each reagent should be understood. Furthermore, plasma samples must be collected at the time when plasma concentrations of DOACs are lowest or the DOAC-Stop reagent should be used.
Since trauma-induced coagulopathy (TIC) was first recognized as a clinical phenomenon, substantial effort has been dedicated to identifying its underlying mechanisms. The activated protein C (APC) pathway serves as the primary anticoagulant brake on normal hemostasis and plays a major cytoprotective role in inflammation. Through in vitro, animal, and clinical research over the last two decades, the APC pathway has been implicated as a central mechanistic driver of TIC. In the setting of significant tissue injury and shock, activation of protein C leads to the hypocoagulable state characteristic of TIC. Aberrant activation of this pathway might be susceptible to targeted therapeutic interventions to reverse or attenuate deranged coagulation after injury. In this chapter, we review the components and regulation of the APC pathway and its clinical role in the etiology of TIC.
Protein C and protein S are essential factors in the thrombomodulin/protein C/protein S pathway, which plays a fundamental role in the regulation of the coagulation/fibrinolysis system, inflammation and cell survival. The conversion of protein C to its active form, activated protein C, occurs on the phospholipid-rich surface of cell membranes in the presence of calcium, thrombomodulin and its receptor endothelial protein C receptor. Activated protein C suppresses coagulation activation by inhibiting activated factors V and VIII, the inflammatory response by inhibiting cytokine secretion and improves cell survival through its receptors protease-activated receptor-1 and endothelial protein C receptor. Protein S inhibits coagulation independently or by enhancing the anticoagulant function of activated protein C. Protein S also inhibits the inflammatory response and cell apoptosis by binding to its (Tyro, Axl, Mer) tyrosine kinase receptors. Chronic inflammatory disorders of the lung are associated with decreased levels of protein S and activated protein C. Activated protein C and protein S effectively ameliorate lung injury, pulmonary fibrosis and allergic bronchial asthma in experimental models. Both activated protein C and protein S therefore may be useful for the treatment of incurable inflammatory disorders of the lung.
By studying the protein C gene of 121 consecutive patients with symptomatic type I protein C deficiency, we detected 55 different candidate mutations in 90 cases. The mutations, 76% of which were missense changes, were distributed throughout the gene. More than half the missense mutations involved Cys, Phe, Pro, or Gly, amino acids known to affect the structure of the polypeptide chain by various mechanisms. Thus, 40% of protein C deficiencies may be caused by polypeptide chain instability rather than a lack of expression of the mutated allele; this may also account for phenotypic heterogeneity. Seventeen of the 55 different mutations were found in apparently unrelated families. Half the French families we studied bore one of these 17 mutations. The wide variety of mutations suggests that both sporadic cases and a founder effect contribute to the spectrum of protein C mutations in a given population. The differences in both unique and recurrent mutations in French and Dutch populations-the only large population samples so far studied-support this hypothesis.
In a series of 40 patients with symptomatic protein C deficiency, we identified two sporadic cases with novel mutations that probably affect gene expression. The mutations, a 5-bp deletion of the donor splice site of intron f (nucleotides 3455 to 3459) and a mutation of nucleotide 8523 in exon IX leading to the substitution of Ser 270 by Pro, were not found in the protein C gene of the patients' parents. Transmission of the paternal and maternal protein C alleles was apparently normal on the basis of frequent polymorphisms in exons I, VI, and VIII. We also checked the transmission of the chromosomal material by analyzing the beta-globin gene frameworks and three variable number of tandem repeats (VNTRs). By combining the results of intragenic polymorphism, VNTR and beta-globin gene framework analyses, we were able to exclude nonpaternity and confirm the de novo origin of the mutation.
We report a case of a 5-month-old female with sporadic monolateral retinoblastoma (RB) with a constitutional de novo complex autosomal translocation involving chromosomes 8, 13 and 15 resulting in a deletion of chromosome 13q14 confirmed by esterase D assay. The translocation of the terminal portion of chromosome 8 has been observed by in situ hybridization with c-myc and thyroglobulin probes.
The effects of bovine activated protein C (APC) on the fibrinolytic activity of cultured bovine aortic endothelial cells were investigated. Confluent monolayers were incubated with purified APC under various conditions and changes in total fibrinolytic activity and in the level of plasminogen activator and plasminogen activator inhibitor (antiactivator) were monitored. The addition of APC to the cells in the absence of other blood or plasma components led to a rapid, dose-dependent increase of fibrinolytic activity both in the media and in cellular extracts. For example, 3.4 micrograms of APC per ml resulted in a 15-fold increase of fibrinolytic activity in the medium within 1 hour. The enhanced fibrinolytic activity reflected increases in both the urokinase-related and tissue-type plasminogen activators produced by these cells. Interestingly, treatment of cells with APC also caused a rapid, dose-dependent decrease in antiactivator activity. Diisopropyl fluorophosphate-inactivated APC did not decrease antiactivator or increase plasminogen activator. Although a small but significant direct (i.e., cell-independent) effect of APC on both fibrinolytic activity and antiactivator activity could be demonstrated, the major portion of these changes appeared to be cell-mediated. These observations indicate that the fibrinolytic potential of cultured endothelial cells is increased by APC and that the enzyme active site is essential for this change. Moreover, the results suggest that one of the primary mechanisms for this stimulation of endothelial cell fibrinolytic activity involves an APC-mediated decrease in antiactivator.
Protein C is a vitamin K dependent glycoprotein which exists in bovine plasma as a precursor of a serine enzyme. Incubation of this plasma protein with α-thrombin at an enzyme-to-substrate weight ratio of 1:50 resulted in the cleavage of an Arg-Ile bond between residues 14 and 15 of the heavy chain of the molecule and the formation of activated protein C. The heavy chain of this serine enzyme contains the active-site sequence of -Leu-Cys-Ala-Gly-Ile-Leu-Gly-Asp-Pro-Arg-Asp-Ala-Cys-Gln-Gly-Asp-SER-Gly which is homologous with the corresponding regions of a number of plasma serine proteases. Activated protein C markedly prolongs the kaolin-cephalin clotting time of bovine plasma but not that of human plasma. The anticoagulant effect was totally obviated by prior incubation of the enzyme with diisopropyl phosphorofluoridate or phenylmethanesulfonyl fluoride. Incubation of activated protein C with bovine factor V resulted in a time-and temperature-dependent inactivation of this clotting factor, and this reaction was dependent on the presence of phospholipid and calcium ions. Activated protein C had no effect on the coagulant activity of factor XII, factor XI, factor X, factor IX, factor VII, or prothrombin. These data provide evidence for a mechanism for the activation of protein C in plasma and a potential role for this enzyme in blood coagulation and hemostasis.
A human genomic DNA library was screened for the gene for protein C by using a cDNA probe coding for the human protein. Three different overlapping lambda Charon 4A phage were isolated that contain inserts for the gene for protein C. The complete sequence of the gene was determined by the dideoxy method and shown to span about 11 kilobases of DNA. The coding and 3' noncoding portion of the gene consists of eight exons and seven introns. The eight exons code for a preproleader sequence of 42 amino acids, a light chain of 155 amino acids, a connecting dipeptide of Lys-Arg, and a heavy chain of 262 amino acids. The preproleader sequence and the connecting dipeptide are removed during processing, resulting in the mature protein composed of a heavy and a light chain held together by a disulfide bond. The heavy chain also contains the catalytic region for the serine protease. Two Alu sequences and two homologous repeats of about 160 nucleotides were found in intron E. The seven introns in the gene for protein C are located in essentially the same positions in the amino acid sequence as the seven introns in the gene for human factor IX, while the first three introns in protein C are located in the same positions as the first three in the gene for human prothrombin.
Human liver cDNA coding for protein C has been synthesized, cloned and sequenced. The abundance of protein C message is approximately
0.02% of total mRNA. Three overlapping clones contain 1,798 nucleotides of contiguous sequence, which approximates the size
of the protein's mRNA, based upon Northern hybridization. The cDNA sequence consists of 73 5′-noncoding bases, coding sequence
for a 461 amino acid nascent polypeptide precursor, a TAA termination codon, 296 3′-noncoding bases, and a 38 base polyadenylation
segment. The nascent protein consists of a 33 amino acid “signal”, a 9 amino acid propeptide, a 155 amino acid “light” chain,
a Lys-Arg connecting dipeptide, and a 262 amino acid “heavy” chain. Human protein C and Factor IX and X precursors possess
about one third identical amino acids (59% in the γ-carboxyglutamate domain), including two forty-six amino acid segments
homologous to epidermal growth factor. Human protein C also has similar homology with prothrombin in the “leader”, γ-carboxyglutamate
and serine protease domains, but lacks the two “kringle” domains found in prothrombin.
The previously unassigned gene coding for the anti-coagulatory protein C has been mapped on chromosome 2 using a cDNA probe and genomic blots from a human-hamster somatic cell hybrid panel. The assignments of the genes coding for the coagulation factor X to chromosome 13, and for alpha 1-acid glycoprotein to chromosome 9 have been confirmed using a similar direct approach.
There are now 201 loci assigned to chromosome 1, 48 being confirmed, 149 provisional and 4 tentative. Seventy-seven of these loci are defined by anonymous DNA fragments, a detailed list of which can be found in the Report of the DNA Committee. One hundred thirty two loci have been assigned to chromosome 2 of which 33 are confirmed, 99 provisional. Of these loci, 50 are defined by anonymous DNA fragments.
We have isolated overlapping phage genomic clones covering an area of 21 kilobases that encodes the human protein C gene. The gene is at least 11.2 kilobases long and is made up of nine exons and eight introns. Two regions homologous to epidermal growth factor and transforming growth factor are encoded by amino acids 46-91 and 92-136 and are precisely delimited by introns, as is a similar sequence in the genes for coagulation factor IX and tissue plasminogen activator. When homologous amino acids of factor IX and protein C are aligned, the positions of all eight introns correspond precisely, suggesting that these genes are the product of a relatively recent gene duplication. Nevertheless, the two genes are sufficiently distantly related that no nucleic acid homology remains in the intronic regions and that the size of the introns varies dramatically between the two genes. The similarity of the genes for factor IX and protein C suggests that they may be the most closely related members of the serine protease gene family involved in coagulation and fibrinolysis.