PC consists of two polypeptide chains of multi-structural domain glycoproteins, and the light chain of PC contains a γ-carboxyglutamic acid (Gla) structural domain and two EGF structural domains. The heavy chain comprises the activating peptide and the trypsin-like serine protease structural domain [9]. The Gla structural domain at the N-terminus is the region that binds Ca2+, and it contributes to APC and PS binding [10]. Acidic residues within the activation peptide protect against proteolysis by endogenous proteases [11]. The C-terminal trypsin-like serine protease structural domain is the active region of PC [10].
In our study, we found two mutations of the PROC gene, including p.Arg440Cys and p.Trp444Arg.The heterozygous state of two mutations showed simultaneous reductions in PC:A and PC:Ag levels, which can be classified as type Ⅰ hereditary PC deficiency. Simultaneously, impaired anticoagulant function was observed in the two probands by thrombin generation curve, and the PC-deficient probands were tested by supplementation with exogenous sTM, which revealed that PC-deficient patients had a significantly reduced inhibitory capacity for thrombin generation. The results of this assay indicated that plasma APC activity was significantly reduced in the probands. Therefore, we believe that the missense mutations of c.1313C > T and c.1330T > C caused the decrease of PC:A and PC:Ag. The two sites being examined in our study are situated within the trypsin-like serine protease structural domain and contain the catalytic triad responsible for the proteolysis of APC substrates. Additionally, this domain encompasses substrate binding regions, where APC substrates must bind before being cleaved. Mutations occurring within this domain are likely to directly impact the catalytic efficiency and the binding of substrates to the protease. Conservation analysis revealed that Arg440 and Trp444 are highly conserved throughout biological evolution, indicating that these amino acid residues are irreplaceable for PC proteins. By constructing a structural model of the PC protein, it was found that the loss of the hydrogen bond between Arg440 and Glu383 is likely to cause a change in the electrostatic attraction between the mutant residue and the surrounding amino acid residues. Additionally, the mutated Cys440 contains a disulfide bond that can connect two amino acid residues, thereby enhancing the stability of the protein's spatial structure.
Trp444 is located in the alpha-helical region facing hydrophobic residues and is stabilized by hydrogen bonding with His448 and Tyr441. Huang et al. [12] found through their studies on various protein structures that the folding kinetics of proteins are significantly correlated with the hydrophobic properties of residues. As the number of hydrophobic amino acid residues increases, the folding rate of proteins decreases. In addition, amino acids with aromatic side chains may also act as inhibitors of protein folding reactions. Therefore, when the polar amino acid Arg replaces the non-polar amino acid Trp, the aromatic ring structure disappears, and the side chain becomes longer. This substitution can potentially affect the stability of the secondary structure of the PC as well as its protein folding. It has been found that the secretion of PC is dependent on the carboxy-terminal region located after the 28 amino acid loop (positions 398 to 426) [13]. As a result, both the p.Arg440Cys and the p.Trp444Arg mutation exhibit unstable expression and low secretion rates of PC. This may be the reason for the decrease of PC:Ag in p.Arg440Cys and p.Trp444Arg heterozygotes.
So far, 396 mutations have been identified in the world according to the HGMD, with the majority being missense or nonsense mutations [14]. Heterozygous individuals typically have around 50% of normal PC levels and are usually asymptomatic before adulthood [15]. Among them, patients with monohybrid have a significantly increased risk of thrombosis when exposed to prothrombotic factors such as smoking, pregnancy, obesity, trauma, and prolonged immobilization. The two probands in this study are heterozygous carriers of mutations in the PROC gene. The lower limb vein thrombosis in proband A may be associated with the p.Arg440Cys mutation and prolonged immobilization. Proband B experienced severe DVT and pulmonary embolism despite having a long history of smoking. The thrombotic events in this proband may be attributed to the combined effects of the p.Trp444Arg heterozygous missense mutation, long-term smoking, accidental falls, and advanced age as prothrombotic factors. Long-term anticoagulant therapy is required for both patients, with regular monitoring of anticoagulation levels and timely adjustment of medication dosage to prevent thrombosis while minimizing the risk of bleeding.
In conclusion, this study describes two mutations (p.Arg440Cys and p.Trp444Arg) in the PROC gene from two independent Chinese families. These two pathogenic mutations were responsible for the PC defect observed in both pedigrees. The possible molecular pathogenic mechanisms were preliminarily explored, which contributed to enriching the mutation database for hereditary PC deficiency. However, further in vitro experiments are needed to validate specific pathogenic mechanisms.