Unraveling the Molecular Pathogenesis of Protein C Deficiency–Associated VTE: Insights from Protein C Mutations C238G and R189W in Thai Patients

Keywords

PC mutations - PC deficiency - venous thromboembolism - protein misfolding - ER stress

Introduction

Protein C (PC) is a vitamin K-dependent plasma glycoprotein that plays a critical role in the anticoagulation system.[1] PC is encoded by PROC gene located on the second chromosome (2q13-q14) and consists of nine exons.[2] PC is synthesized in the liver as a prepro-protein C (single chain). The mature PC is formed when a dipeptide of K198 and R199 is cleaved, resulting in the transformation to a heterodimer protein. Mature PC has 419 amino acids in multiple domains, consisting of one Gla domain (residues 43–88), two epidermal growth factor (EGF)-like domains (residues 97–132 and 136–176), the activation peptide (residues 200–211), and a trypsin-like serine protease domain (212–450). The light chain is composed of the Gla- and two EGF-like domains, whereas the heavy chain contains the protease domain and the activation peptide. Upon activation, the activation peptide must be removed, and the light chain is linked with the heavy chain by a single disulfide bond at C183 and C319. Activated PC (APC) exerts the anticoagulative effect by inactivating the cofactor activities of factor (F) VIIIa in the intrinsic tenase complex, and FVa in the prothrombinase complex.[3] The APC-mediated FVIIIa and FVa inactivation provides a specific regulation of human coagulation. The PC system is physiologically important as demonstrated in newborns with homozygous PC deficiency presenting with purpura fulminans, which is a severe thrombotic disease.[4] [5]

Deficiency of PC is commonly a consequence of mutations in the PROC gene in which 300 mutations have been reported.[6] PC deficiency is considered a common thrombophilic risk reported in Thai and other Asian populations.[7] A single novel mutation in exon 8 of PROC in three Thai pediatric patients who presented with thromboembolic events, including homozygotic twin boys and unrelated girl, has been reported.[8] The mutation analysis demonstrated a homozygous T > G substitution at the nucleotide position 7172 (7172 T > G), which resulted in a missense mutation causing substitution of cysteine with glycine at amino acid position 238 (C238G) in the prepro-sequence. The mutation point does not reside in the promoter region of PROC; however, it causes a severe PC deficiency as demonstrated by a significant decrease in both PC activity and plasma antigen level (2 and ∼2.5% compared with unaffected individuals, respectively).[8] The R189W mutation is a common mutation in Thai children[9] and Taiwanese populations with venous thromboembolism (VTE).[10] The mutation analysis revealed homozygous and heterozygous R > W substitution at the nucleotide position 6152 (6152 C > T) in exon 7, which resulted in a missense mutation causing substitution of arginine with tryptophan at amino acid position 189 (R189W) in the prepro-sequence. According to the study in Taiwanese population, in a group of 85 individuals serving as the control group, the levels of PC antigen were found to be similar to the functional levels, with antigen levels measuring 105.0% ± 19.6% and functional levels at 104.8% ± 21.5%. Conversely, in the 22 patients with R189W heterozygotes, both the average PC antigen and functional levels were considerably lower than those of the control group, with the antigen levels measuring 71.7% ± 12.4% and the functional levels at 59.8% ± 9.2%.[10] Additionally, in our study from 2018,[9] we examined a group of 184 patients aged 18 years or younger. Within this sample, 100 patients (54.3%) were found to have arterial ischemic stroke, 38 patients (20.6%) had VTE, and 23 patients (12.5%) were diagnosed with cerebral venous sinus thrombosis.[9] Also, the study demonstrated that the mean PC activity levels were 120.4% in wild type, 80.0% in heterozygous mutation, and 44.3% in homozygous mutation. It is also suggested that the compound heterozygote of R189W with other mutations in the PROC could result in severe PC deficiency since childhood.

The two mutations, C238G and R189W, which are the main focus of the study, have been reported in Thai patients with thromboembolic events. These specific mutations were selected for the study due to their clinical significance and novelty (C238G), and prevalence (R189W) in the Thai population. The C238G mutation, leading to a severe reduction in PC secretion, and the R189W mutation, associated with a moderate decrease in PC secretion, present interesting molecular mechanisms that contribute to PC deficiency and thrombotic risk. By studying these mutations, we aim to unravel the underlying pathogenic mechanisms of PC deficiency in patients with thromboembolism (TE), providing valuable insights into disease mechanisms and potential therapeutic targets.

Materials and Methods

DNA Mutagenesis and Plasmid Construction

Human PC complementary DNA (cDNA) for wild-type (WT; reference sequences: BC034377.1, DQ890791.2), C238G, and R189W (GenScript, New Jersey, United States) was synthesized as a fragment DNA, and subsequently ligated into pcDNA3.1(+) plasmid (Invitrogen, California, United States). The constructs containing WT, C238G, and R189W were verified by full-length sequencing of the cDNA (Macrogen Inc., Seoul, Korea).

Cell Culture Condition

Human embryonic kidney 293T (HEK293T) cells were used for transient transfection and PC protein expression. Cells were cultured in Dulbecco's Modified Eagle Medium/high glucose medium (HyClone, Utah, United States) supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin (HyClone, Utah, United States), 2 mM L-glutamine (HyClone, Utah, United States), and 10% fetal bovine serum (FBS; Gibco, New York, United States). Cells were grown in 37°C with 5% CO₂ and 95% humidity.

Expression of PC Molecules in Mammalian Cells

HEK293T cells were grown to 80% confluence in six-well plates 24 hours prior to the transfection. HEK293T cells expressing human PC WT, C238G, and R189W were constructed via transient transfection with plasmids containing WT, C238G, and R189W cDNA using FuGENE6 transfection reagent (Promega, Madison, Wisconsin, United States) for 48 hours. After transfection, the transfected cells were cultured in serum-free media supplemented with 10 μg/mL vitamin K and 1× insulin–transferrin–selenium for 24 hours at 37°C. Cell culture supernatants were collected and stored in aliquots at −80°C. The transfected cells were lysed in lysis buffer containing protease inhibitor cocktail (Bio-rad, California, United States). PC protein expression levels were measured in both cell lysates and culture supernatants using Western blot (WB) analysis and enzyme-linked immunosorbent assay (ELISA), respectively.

Measurement of PC Protein Levels in Culture Media

The amount of secreted PC in the culture medium was measured by ELISA using the Human Protein C ELISA Kit (Abcam, Cambridge, United Kingdom) according to the manufacturer's instruction. A standard curve was constructed by twofold serial dilutions to generate eight standard points of human PC standard protein (0–200 ng/mL). The absorbance was measured in the Synergy HT multi-detection microplate reader (Biotek, California, United States) at a wavelength of 450 nm and compared with the standard curve.

Western Blot Analysis

The expression of PC was determined by WB analysis. Protein samples were linearized under the reducing condition, separated by 12% SDS-PAGE, and transferred from gels to PVDF membranes (Thermo Fisher Scientific, Massachusetts, United States). Membranes were blocked with 5% skimmed milk in TBS-T (10 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4) overnight at 4°C, incubated with the rabbit polyclonal anti-human PC (1:3,000; Abcam, Cambridge, United Kingdom) for 3 hours at room temperature (RT), and probed with the peroxidase-conjugated goat anti-rabbit IgG H&L (1:10,000; Abcam, Cambridge, United Kingdom) for 1 hour at RT. Immunoreactive proteins were detected by the ECL detection substrate (Bio-rad, California, United States) and visualized using the ChemiDoc MP Imaging System (Bio-rad, California, United States).

Immunofluorescence Microscopy

The transfected cells (2 × 10⁵ cells/well) were plated on coverslips precoated with poly-D-lysine in a 6-well plate 24 hours prior to the immunofluorescence staining. Cells were fixed with 4% paraformaldehyde for 10 minutes at RT, and blocked in 10% FBS for 30 minutes at RT. Cells were co-incubated with primary antibodies: rabbit polyclonal anti-human PC (1:1,000; Abcam, Cambridge, United Kingdom) and mouse monoclonal anti-protein disulfide isomerase (PDI; 1:100; Thermo Fisher Scientific, Massachusetts, United States), or mouse polyclonal anti-GM130 (1:250; Abcam, Cambridge, United Kingdom) for 2 hours at RT. Cells were co-incubated with secondary antibodies: Alexa Fluor 488 goat anti-rabbit (1:1,000; Abcam, Cambridge, United Kingdom) and Cy3 goat anti-mouse (1:500; Abcam, Cambridge, United Kingdom) for 1 hour at RT. Cells were mounted in the mounting medium with DAPI (Abcam, Cambridge, United Kingdom), then observed and imaged using the Eclipse Ts2R inverted research microscope with Nikon NIS-Elements D version 5.21.00 software (Nikon Instruments Inc., New York, United States).

Quantitative Real-Time PCR Analysis

Total RNA was extracted from transfected cells using FavorPrep Blood/Cultured Cell Total RNA Purification Mini Kit (Favorgen, Ping-Tung, Taiwan). The RNA was quantified using a NanoDrop One (Thermo Fisher Scientific, Delaware, United States). One microgram of RNA was converted into cDNA using iScript Reverse Transcription Supermix (Bio-rad, California, United States). ER stress-related gene expression profiles were quantified on an ABI7500 real-time polymerase chain reaction (PCR) system (Applied Biosystems, California, United States) with cycling conditions of 95°C for 3 minutes, followed by 40 cycles at 95°C for 10 seconds, 61°C for 20 seconds, and 72°C for 30 seconds. The specific primers of ER stress-related genes (ATF4, ATF6, BiP, CHOP, XBP1) used in this study were previously described.[11] GAPDH was used as an internal reference gene. Data were determined using the 2^−ΔΔCt method and presented as the relative gene expression levels.

Multiple Sequence Alignment

The human PC sequence (P04070) was retrieved from UniProt along with PC sequences from other species with a sequence similarity of at least 50%. Fragments of the full sequences were excluded, and only sequences with similar lengths were included in the alignment. To avoid redundancy, only one sequence from each species was included. In total, 74 sequences from 74 species were included in the multiple sequence alignment ([Supplementary Table S1], available in the online version). The entropy scores indicating the degree of variation or conservation of the amino acid at each position were calculated.

Statistical Analysis

Data were expressed as mean ± standard error of the mean values (n = 3). The statistical difference analysis was performed using one-way analysis of variance followed by the Tukey's multiple comparison test. p-Values of <0.05 was considered statistically significant. All figures and statistical analyses were performed with GraphPad Prism 5.0 (GraphPad Software Inc, La Jolla, California, United States).

Results

Transfected Cells Expressed the Zymogen Form of PC

After transient transfection, the forms of secreted PC proteins in the culture supernatant were determined by WB analysis under reducing conditions. PC was not detectable in nontransfected PC cells (negative control) and cells transfected with C238G. HEK293T transfected with WT and R189W expressed the zymogen form of PC ([Fig. 1A]) as the appearance of bands corresponding with the full-length zymogen (molecular weight [MW] ∼ 62 kDa) and the light chain of PC (MW ∼ 21 kDa). HepG2 cell lysate protein (positive control) showed the zymogen form of PC (MW ∼ 62 kDa).

Fig. 1 Expression of human PC. WB analysis of PC in culture supernatant (A). One microgram of total protein was applied to each lane. Nontransfected HEK293T (1 μg of total protein) was used as a negative control. HepG2 cell lysate protein (100 μg of total protein) was used as a positive control. The zymogen PC and the light chain of PC appeared as a single band at 62 and 21 kDa, respectively. Secreted PC protein levels in culture supernatant (B). PC protein levels were determined by ELISA. Intracellular PC expression (C). After transient transfection, the expression of PC in cell lysates was determined using WB. PC mRNA levels (D). PC mRNA levels were examined by qRT-PCR. Data were reported as mean ± SEM (n = 3). ***p < 0.0001 in comparison between two groups. mRNA, messenger RNA; NS, not significant; PC, protein C; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean; WB, Western blot.

Secreted PC Levels Were Reduced in Cells Expressing PC Variants

To investigate the effect of C238G and R189W on PC secretion, the secreted levels of PC in culture supernatant from the transfected cells were quantified using ELISA. Among the HEK293T cells transfected with the PC constructs, WT secreted the highest amount of PC (906.67 ± 10.14 ng/mL). The significant reduction of secreted PC levels by C238G (37.80 ± 1.53 ng/mL) was observed ([Fig. 1B]) compared with that of WT (p = 0.0001). PC levels by R189W was significantly decreased (640.00 ± 23.09 ng/mL) compared with that of WT (p = 0.0001). This result indicated that the mutations impaired the secretory function of the transfected cells. To determine whether the reduced PC secretion from the mutants was not due to the differences of mRNA levels, the mRNA expression levels of PC were determined by quantitative real-time PCR (qRT-PCR). No differences in the PC mRNA levels were observed between WT and both variants (C238G and R189W) in transfected cells ([Fig. 1D]).

C238G Variant Were Retained in the Endoplasmic Reticulum

Because cells expressing C238G demonstrated severely decreased PC secretion levels, we speculated that C238G-PC could, potentially, be intracellularly retained. Therefore, PC in the cell lysates and intracellular localization of the PC variants in transiently transfected cells were performed by WB analysis and immunofluorescence staining, respectively. PC in the cell lysates appeared in all cells transfected with the PC constructs (WT, C238G, and R189W) as revealed by the appearance of dense band intensity corresponding with the zymogen PC (MW ∼ 62 kDa; [Fig. 1C]). The intracellular localization study revealed that both WT and the two PC mutants colocalized with the endoplasmic reticulum (ER) marker, PDI, ([Fig. 2A]) and the Golgi marker, GM130, ([Fig. 2B]). However, the C238G-PC predominately colocalized with PDI compared with WT and R189W ([Fig. 2A]). These results indicated that the C238G-PC was primarily retained in the ER and led to impaired PC secretion.

Fig. 2 Intracellular localization of PC. Intracellular localization of PC in the ER (A) and the Golgi (B). The markers of cell organelles (ER and Golgi) were stained in red. PC was stained in green. Colocalization of PC with a cell organelle marker (ER or Golgi) is shown in yellow. Scale bars: 10 μm. ER, endoplasmic reticulum; PC, protein C.

ER Stress-Related Genes Were Upregulated in Cells Expressing PC Variants

Because C238G-PC retained in the ER, we postulated that it might cause the ER stress. Therefore, the mRNA levels of the downstream target genes of the unfolded protein response (UPR) pathway in ER stress, including ATF4, ATF6, BiP, CHOP, and XBP1, were determined in cells expressing PC variants by qRT-PCR. The highest messenger RNA (mRNA) expression levels of ER stress-related genes (ATF4, ATF6, CHOP, and XBP1) were observed in cells expressing C238G compared with those of cells expressing WT by 1.25 ± 0.10, 2.36 ± 0.26, 1.25 ± 0.12, and 2.44 ± 0.08 folds, respectively ([Fig. 3A–C, E]). Also, the mRNA levels of ER stress-related genes (ATF6, BiP, and XBP1) were significantly increased in cells expressing R189W compared with those of cells expressing WT by 1.40 ± 0.12, 1.45 ± 0.11, and 1.50 ± 0.12 folds, respectively ([Fig. 3B, C, E]). The data indicated that the C238G-PC caused the ER stress in the C238G-expressing cells.

Fig. 3 Expression of ER stress-related genes. ATF4 (A), ATF6 (B), BiP (C), CHOP (D), and XBP1 (E) were performed in each PC variant–transfected HEK293T by qRT-PCR. Data were reported as mean ± SEM (n = 3). ***p < 0.001; **p < 0.01; *p < 0.05 in comparison between two groups. NS, not significant. PC, protein C; qRT-PCR, quantitative real-time polymerase chain reaction; SEM, standard error of the mean.

Multiple Sequence Alignment

Multiple sequence alignment of PC from 74 species deposited revealed that residues R189 and C238 are conserved across 74 species but not at the same level ([Figs. 4] and [5]). The entropy scores of the catalytic triad, conserved disulfide-forming cysteine residues, proteolytic cleavage sites upon PC activation, and R189 were compared to analyze the degree of conservation and the importance of these residues through the evolution. The entropy score of the catalytic triads were 0.071, while the entropy scores of the conserved disulfide-forming cysteine residues were between 0.071 and 0.141. The entropies of K198 and R199, the cleavage site upon PC activation, were 0.334 and 0.631. On the other hand, the entropy score for R189 was calculated to be 0.773, indicating a moderate conservation compared with the former groups. Variants at this position in other species included lysine, glutamine, and proline. Glutamine and proline were predominantly in the species with lower sequence identities to human PC, such as 63% in Camelus dromedarius, 62% in Equus asinus asinus, and 68% in Galemys pyrenaicus. Additionally, variations were observed in PC sequences from species with different length of activation peptides. Notably, tryptophan was not present at position 189 in any of the 74 species examined. The stabilizing effect of consensus sequences, where amino acids with higher frequency at a specific position are more likely to provide stability, has been reported.[12] On the other hand, C238 is highly conserved with sequence entropy scoring of 0.141. It directly forms the disulfide bond with C254 in the heavy chain, which is also highly conserved in all species (data not shown).

Fig. 4 Part of the multiple sequence alignment of PC from 74 species, highlighting the conservation at position R189. The number in the top row indicates the amino acid positions in the alignment, including gaps. The right-hand side shows the position of the last amino acid in the selected segment along the primary sequence. R189 on human PC is shown on the bottom row and is highlighted in yellow. PC, protein C.

Fig. 5 Part of the multiple sequence alignment of PC from 74 species, highlighting the conservation at position C238. The number in the top row indicates the amino acid positions in the alignment, including gaps. The right-hand side shows the position of the last amino acid in the selected segment along the primary sequence. C238 on human PC is shown on the bottom row and is highlighted in yellow. PC, protein C.

Discussion

In this study, we aimed to better understand the pathogenesis of PC deficiency previously observed in Thai pediatric patients by transient transfection of HEK293T cells with cDNAs encoding the WT, C238G, and R189W mutants. The C238G demonstrated a 95% reduction in PC secretion compared with WT, whereas the R189W showed a 30% reduction in PC secretion. The reduced levels of secreted PC variants in our study were consistent with the observed plasma PC antigen (PC:Ag) levels in PC deficiency patients with C238G and R189W who had a reduction in PC:Ag in the range of <1 to 2.7% and 32 to 64%, respectively.[8]

Although the secretions of C238G-PC and R189W-PC were significantly impaired, there were no differences in PC mRNA levels among WT and the two PC mutants. At this point, we hypothesized that the C238G-PC could be retained in the cells. As expected, WB analysis revealed that the C238G-PC was detected in the cell lysates as well as in cells expressing WT and R189W. Moreover, immunofluorescence staining showed that the C238G-PC was predominantly visualized in the ER. Our result was consistent with the previous study showing that the majority of mutated A267T PC was likely located in the ER of the CHO-K1 cells, while WT was observed in both ER and Golgi, indicating a rapid process and secretion of WT.[13] Moreover, several studies demonstrated that mutations of FVII, which is one of the vitamin K-dependent coagulant proteins, impaired FVII secretion due to the ER protein retention.[14] [15] [16] [17] Expression of mutant proteins altered the accurate folding pathway or proper conformation of proteins, preventing them to form their native structure.[18] [19] As a result, the mutant proteins were mainly localized into the ER, and inefficiently transported to the Golgi, suggesting the abnormal trafficking due to protein misfolding.[20] A single-point mutation can alter an amino acid that affects the three-dimensional structure of protein, potentially resulting in misfolding and unfolding of proteins.[21] [22] The aberrancy in protein folding can lead to various diseases such as Huntington's disease[23] and diabetes.[24] ER stress induced by the accumulation of misfolded or unfolded proteins within the ER can be alleviated by the activation of UPR.[25]

The UPR is sensed by three distinct receptors: IRE1, PERK, and ATF6 located in the ER membrane.[26] Under unstressed conditions, these receptors are maintained in the inactive state by binding to the ER chaperone called binding immunoglobulin protein (BiP).[27] Upon accumulation of misfolded or unfolded proteins in the ER, BiP dissociates from the three ER stress sensors (IRE1, PERK, and ATF6) and preferentially binds to misfolded proteins. Activated PERK reduces protein translation by phosphorylating eIF2α. Subsequently, the phosphorylated eIF2α increases the transcription of ATF4 and CHOP to increase cytoprotective gene transcription and induce apoptosis, respectively.[28] ATF6 is activated by translocating to the Golgi where it is cleaved by proteases. Activated ATF6 then increases the production of BiP.[29] ER stress also triggers IRE1 to activate its endoribonuclease function, which results in the production of spliced XBP1, leading to the production of BiP and the promotion of ER-associated degradation of misfolded proteins.[30] In this study, the highest upregulation of all ER stress-related genes was detected in cells expressing C238G, consistent with the immunofluorescence results showing that the C238G-PC was predominantly localized in the ER. Moreover, activation of some ER stress-related genes (ATF6, BiP, and XBP1) was observed in cells expressing R189W. We postulated that R189W may also result in some extents of protein misfolding as observed by a 30% decrease in R189W-PC secretion, resulting in modest activation of ER stress compared with those in C238G-expressing cells. Taken together, these results indicated that the drastically reduced secretion of the C238G-PC was due to the intracellular retention within the ER, subsequently induced the ER stress and the upregulation of UPR genes.

Analysis of the crystal structure of human APC (PDB ID: 1AUT[31]) revealed a disorder in the C-terminal region of the light chain starting from K188, as indicated by high b-factor values. Since the crystal structure of the full-length PC is not available, the predicted full-length PC structure obtained from AlphaFold was utilized to analyze the structure of the activation peptide, and the potential role of R189 ([Fig. 6A, B]). It was suggested that R189 is part of a hydrophilic loop in the full-length PC, with its side chain exposed to the aqueous environment and not form the hydrogen bond with other amino acids. Upon proteolytic cleavage of the activation peptide, the region from K188 becomes disordered. The hydrophilicity at this position is likely to be essential as illustrated in the multiple sequence alignment where the amino acid variation at this position included lysine, glutamine, and proline. Substitution of R189 with tryptophan resulted in the loss of a positively charged amino acid, and subsequently introduced the bulky and hydrophobic amino acid to the aqueous environment. This can potentially lead to protein misfolding, as indicated by the increase in ER retention. The mutation in PC could lead to the loss of amidolytic activity or protein misfolding leading to impaired secretion, low plasma level, and poor interaction with protein S (PS). Since multiple mechanisms may be caused by mutations, data from previously reported patients should be considered to further elucidate the mechanism of R189W on PC deficiency.

Fig. 6 The location of R189 on human PC in the crystal structure. (A) The structure with b-factor was shown to illustrate the flexible region near the C-term in the activated PC. (B) The surface potential of PC was shown to illustrate that the amino acids from W187 onward are exposed to the solvent. The activation peptide is in red. K198 is in blue stick. The structure of full-length PC was predicted by AlphaFold (available from https://alphafold.ebi.ac.uk/entry/P04070) and was superimposed with the crystal structure of protein C (PDB ID: 1AUT[31]). PC, protein C.

The previous study on R189W mutation showed that 1 in 5 patients with R189W had normal plasma PC activity, while the other 4 showed less than 75% plasma PC activity.[32] However, all the patients exhibited thrombotic phenotypes. Another study in the Korean population indicated that patients with the double mutation R189W/K193Del had PC deficiency.[33] Furthermore, a study of Japanese patients suggested that K193del showed normal plasma levels of PC.[34] Moreover, the other study indicated that K193del mutation did not result in a significant decrease of the amidolytic activity of PC.[35] Therefore, it is suggested that the R189W would contribute to PC deficiency and TE. However, R189W did not seem to lose the amidolytic activity.[36] All the clinical reports are in agreement with our finding that the R189W mutation is likely to cause PC deficiency through a poor secretion, resulting in the low PC levels in plasma. However, the possibility that R189W does not affect PC–PS complex formation cannot be excluded. Further investigation is warranted to fully understand the role of R189 and its molecular pathogenesis in TE.

Residue C238 in human PC plays a critical role in maintaining the structural integrity and functionality of the protein. This cysteine residue forms a disulfide bond with C254 in the heavy chain, a bonding event of paramount importance ([Fig. 7]). This disulfide bond not only lies in proximity to the active site but is also indispensable for upholding the three-dimensional (3D) structure of PC. The conservation of this cysteine residue across all species underscores its evolutionary significance. The disruption of this residue could prove detrimental, impacting both the structural stability and functional capacity of PC. Given its role in a crucial disulfide bond, any alteration to C238 has the potential to compromise the overall integrity of the protein and, subsequently, its biological activity. This analysis highlights the dual importance of C238—not only as a structural element contributing to the 3D conformation of PC but also as a conserved residue critical for the preservation of its functional attributes.

Fig. 7 The active site of human PC and the disulfide bond between C238 and C254. The crystal structure was retrieved from PDB ID: 3F6U.[37] The catalytic residues are in blue. The peptide inhibitor is in orange. The cysteine residues forming disulfide bonds are in green. The heavy chain is in gray. PC, protein C.

In summary, our research unraveled the molecular mechanisms behind the PC mutations (C238G and R189W) that contribute to PC deficiency. The findings showed that the R189W mutation triggered a mild activation of the UPR genes, suggesting a relatively minor disruption in protein folding and secretion. In contrast, the C238G mutation significantly impaired PC secretion due to protein misfolding, leading to increased retention in the ER and subsequent ER stress. The mild activation of UPR genes in R189W mutation suggests that modulating UPR pathways might be a potential therapeutic strategy for mitigating the thrombotic risk associated with PC deficiency. Future studies could investigate the efficacy of UPR modulators in reducing thrombotic risk in PC deficiency.

• References

• 1 Dahlbäck B. Blood coagulation. Lancet 2000; 355 (9215) 1627-1632
• 2 Plutzky J, Hoskins JA, Long GL, Crabtree GR. Evolution and organization of the human protein C gene. Proc Natl Acad Sci U S A 1986; 83 (03) 546-550
• 3 Dahlbäck B, Villoutreix BO. Molecular recognition in the protein C anticoagulant pathway. J Thromb Haemost 2003; 1 (07) 1525-1534
• 4 Price VE, Ledingham DL, Krümpel A, Chan AK. Diagnosis and management of neonatal purpura fulminans. Semin Fetal Neonatal Med 2011; 16 (06) 318-322
• 5 Mukherjee D, Pal P, Kundu R. Purpura fulminans due to acquired protein C deficiency. Indian J Dermatol 2015; 60 (06) 637
• 6 D'Ursi P, Marino F, Caprera A, Milanesi L, Faioni EM, Rovida E. ProCMD: a database and 3D web resource for protein C mutants. BMC Bioinformatics 2007; 8 (Suppl 1, Suppl 1): S11
• 7 Angchaisuksiri P, Atichartakarn V, Aryurachai K. et al. Risk factors of venous thromboembolism in thai patients. Int J Hematol 2007; 86 (05) 397-402
• 8 Sirachainan N, Sasanakul W, Visudibhan A, Chuansumrit A, Wongwerawattanakoon P, Parapakpenjune S. Protein C deficiency in Thai children with thromboembolism: a report of clinical presentations and mutation analysis. Thromb Res 2010; 125 (02) 200-202
• 9 Sirachainan N, Chuansumrit A, Sasanakul W. et al. R147W in PROC gene is a risk factor of thromboembolism in Thai children. Clin Appl Thromb Hemost 2018; 24 (02) 263-267
• 10 Tsay W, Shen MC. R147W mutation of PROC gene is common in venous thrombotic patients in Taiwanese Chinese. Am J Hematol 2004; 76 (01) 8-13
• 11 Xiang P, Liu RY, Li C, Gao P, Cui XY, Ma LQ. Effects of organophosphorus flame retardant TDCPP on normal human corneal epithelial cells: implications for human health. Environ Pollut 2017; 230: 22-30
• 12 Lehmann M, Loch C, Middendorf A. et al. The consensus concept for thermostability engineering of proteins: further proof of concept. Protein Eng 2002; 15 (05) 403-411
• 13 Tjeldhorn L, Iversen N, Sandvig K, Bergan J, Sandset PM, Skretting G. Functional characterization of the protein C A267T mutation: evidence for impaired secretion due to defective intracellular transport. BMC Cell Biol 2010; 11: 67
• 14 Tanaka R, Nakashima D, Suzuki A. et al. Impaired secretion of carboxyl-terminal truncated factor VII due to an F7 nonsense mutation associated with FVII deficiency. Thromb Res 2010; 125 (03) 262-266
• 15 Hunault M, Arbini AA, Carew JA, Peyvandi F, Bauer KA. Characterization of two naturally occurring mutations in the second epidermal growth factor-like domain of factor VII. Blood 1999; 93 (04) 1237-1244
• 16 Andersen E, Chollet ME, Myklebust CF. et al. Activation of endoplasmic reticulum stress and unfolded protein response in congenital factor VII deficiency. Thromb Haemost 2018; 118 (04) 664-675
• 17 Seanoon K, Payongsri P, Vivithanaporn P. et al. Mutations of TFPI-binding exosites on factor VII cause bleeding phenotypes in factor VII deficiency. Blood Adv 2022; 6 (22) 5887-5897
• 18 Schröder M, Kaufman RJ. ER stress and the unfolded protein response. Mutat Res 2005; 569 (1–2): 29-63
• 19 Chambers JE, Marciniak SJ. Cellular mechanisms of endoplasmic reticulum stress signaling in health and disease. 2. Protein misfolding and ER stress. Am J Physiol Cell Physiol 2014; 307 (08) C657-C670
• 20 Chollet ME, Andersen E, Skarpen E. et al. Factor VII deficiency: unveiling the cellular and molecular mechanisms underlying three model alterations of the enzyme catalytic domain. Biochim Biophys Acta Mol Basis Dis 2018; 1864 (03) 660-667
• 21 Valastyan JS, Lindquist S. Mechanisms of protein-folding diseases at a glance. Dis Model Mech 2014; 7 (01) 9-14
• 22 Gomes CM. Protein misfolding in disease and small molecule therapies. Curr Top Med Chem 2012; 12 (22) 2460-2469
• 23 Shacham T, Sharma N, Lederkremer GZ. Protein misfolding and ER stress in Huntington's disease. Front Mol Biosci 2019; 6: 20
• 24 Wu J, Kaufman RJ. From acute ER stress to physiological roles of the unfolded protein response. Cell Death Differ 2006; 13 (03) 374-384
• 25 Rutkowski DT, Kaufman RJ. A trip to the ER: coping with stress. Trends Cell Biol 2004; 14 (01) 20-28
• 26 Haeri M, Knox BE. Endoplasmic reticulum stress and unfolded protein response pathways: potential for treating age-related retinal degeneration. J Ophthalmic Vis Res 2012; 7 (01) 45-59
• 27 Todd DJ, Lee AH, Glimcher LH. The endoplasmic reticulum stress response in immunity and autoimmunity. Nat Rev Immunol 2008; 8 (09) 663-674
• 28 Oyadomari S, Mori M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell Death Differ 2004; 11 (04) 381-389
• 29 Mei Y, Thompson MD, Cohen RA, Tong X. Endoplasmic reticulum stress and related pathological processes. J Pharmacol Biomed Anal 2013; 1 (02) 1000107
• 30 Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman JS, Walter P. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 2000; 101 (03) 249-258
• 31 Mather T, Oganessyan V, Hof P. et al. The 2.8 a crystal structure of Gla-domainless activated protein C. EMBO J 1996; 15 (24) 6822-6831
• 32 Gu Y, Shen W, Zhang L, Zhang J, Ying C. Deficiency of antithrombin and protein C gene in 202 Chinese venous thromboembolism patients. Int J Lab Hematol 2014; 36 (02) 151-155
• 33 Kim HJ, Seo JY, Lee KO. et al. Distinct frequencies and mutation spectrums of genetic thrombophilia in Korea in comparison with other Asian countries both in patients with thromboembolism and in the general population. Haematologica 2014; 99 (03) 561-569
• 34 Miyata T, Sakata T, Yasumuro Y. et al. Genetic analysis of protein C deficiency in nineteen Japanese families: five recurrent defects can explain half of the deficiencies. Thromb Res 1998; 92 (04) 181-187
• 35 Tang L, Lu X, Yu JM. et al. PROC c.574_576del polymorphism: a common genetic risk factor for venous thrombosis in the Chinese population. J Thromb Haemost 2012; 10 (10) 2019-2026
• 36 Ding Q, Yang L, Hassanian SM, Rezaie AR. Expression and functional characterisation of natural R147W and K150del variants of protein C in the Chinese population. Thromb Haemost 2013; 109 (04) 614-624
• 37 Schmidt AE, Padmanabhan K, Underwood MC, Bode W, Mather T, Bajaj SP. Thermodynamic linkage between the S1 site, the Na+ site, and the Ca2+ site in the protease domain of human activated protein C (APC). Sodium ion in the APC crystal structure is coordinated to four carbonyl groups from two separate loops. J Biol Chem 2002; 277 (32) 28987-28995