PB0948 - Met343Val mutation attenuates flexibility of residue Trp380 and impairs anticoagulant function of protein C

Monday, June 24, 2024

13:45 – 14:45 ICT

Room: Exhibition Hall

Presenting Author(s)

Shijie Zhou, MA

postgraduate
ruijin hospital
Shanghai, Shanghai, China (People's Republic)

Co-Author(s)

FL

Fang Li

Ph.D
School of life sciences and biotechnology, Shanghai Jiaotong University School of Medicine
Shanghai, Shanghai, China (People's Republic)
zhe lai, MA

postgraduate
shanghai ruijin hospital in China
上海市市辖区, Shanghai, China (People's Republic)
Xi Wu, MD

Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China
Shanghai, Shanghai, China (People's Republic)
XW

Xuefeng Wang

Professor
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
Shanghai, Shanghai, China (People's Republic)
QX

Qin Xu

Ph.D
School of life sciences and biotechnology, Shanghai Jiaotong University School of Medicine
Shanghai, Shanghai, China (People's Republic)
JD

Jing Dai

Professor
Department of Laboratory Medicine, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China
Shanghai, Shanghai, China (People's Republic)
YL

Yeling Lu

Thesis Advisor
shanghai ruijin hospital in China
shanghai, Shanghai, China (People's Republic)

Background: Protein C (PC) deficiency is associated with a high risk of venous thrombosis. We identified two protein C-deficient patients both of whom carried a novel heterozygous Met343 to Val (M343V) mutation in PROC and experienced venous thrombosis.

Aims: The aim of this study was to determine the molecular basis of the anticoagulant defect in patients carrying the M343V mutation.

Methods: Plasma PC antigen was measured using enzyme-linked immunosorbent assay. PC activity was measured using chromogenic assay and clotting-based assay. Whole-exon sequencing was utilized to detect mutations in the probands’ gene. Protein C-M343V was expressed in mammalian cells and the properties was characterized in established coagulation test systems.

Results: Thrombin generation assays showed that TM-dependent anticoagulant activity was significantly impaired. Genetic analysis revealed that the patients carried the same heterozygous mutation c.1153A>G, p. Met343Val in PROC (Figure 1A), which had never been reported before. Coagulation tests demonstrated that activation of mutant zymogen by thrombin-TM was slightly affected. However, the cleavage of chromogenic substrate by activated protein C (APC)-M343V was significantly impaired (Figure 1B), whereas Ca2+ increased the cleavage efficiency by ~50%. The affinity of APC-M343V for Na+ was severely diminished (Figure 1C). The inhibitory ability of APC-M343V for activated factor V (FVa) was also markedly impaired (Figure 1D). Structural analysis revealed that the Met343 to Val substitution resulted in the loss of hydrogen bonding between this site and Trp380, as well as the formation of a new hydrogen bond between Trp380 and His211 (Figure 2).

Conclusion(s): The mutation from Met343 to Val343 in APC affected the interaction of residue 343 with Trp380, which contributed to block the active site and stabilized the protease in a biased E* conformation, leading to severely impaired amide hydrolysis and anticoagulant activity of mutant APC, and ultimately causing venous thrombosis in the patients.