Mutation Ter462GlnextTer17 introduces a tail to C-terminus of protein C and causes venous thrombosis.

Protein C (PC), a vitamin K-dependent serine protease zymogen in plasma, can be activated by thrombin-thrombomodulin(TM) complex, resulting in the formation of activated protein C (APC). APC functions to downregulate thrombin generation by inactivating active coagulation factors V(FVa) and VIII(FVIIIa). Deficiency in PC increases the risk of venous thromboembolism (VTE). We have identified two unrelated VTE patients with the same heterozygous mutation (c.1384 T > C, p.Ter462GlnextTer17) in PROC. To comprehend the role of this mutation in VTE development, we expressed recombinant PC-Ter462GlnextTer17 in mammalian cells and evaluated its characteristics using established coagulation assay systems. Functional studies revealed a significant impairment in the activation of the mutant by thrombin or thrombin-TM complex. Furthermore, APC-Ter462GlnextTer17 demonstrated diminished hydrolytic activity towards the chromogenic substrate S2366. APTT and FVa degradation assays showed that both the anticoagulant activity of the mutant protein was markedly impaired, regardless of whether protein S was present or absent. These results were further supported by a thrombin generation assay conducted using purified and plasma-based systems. In conclusion, the Ter462GlnextTer17 mutation introduces a novel tail at the C-terminus of PC, leading to impaired activity in both PC zymogen activation and APC's anticoagulant function. This impairment contributes to thrombosis in individuals carrying this heterozygous mutation and represents a genetic risk factor for VTE.

Met343Val mutation disrupts the shuttling of Trp380 leading to a low-activity conformer of activated protein C and causes thrombosis.

BACKGROUND: Protein C (PC) pathway serves as a major defense mechanism against thrombosis by the activation of PC through the thrombin-thrombomodulin complex and subsequent inactivation of the activated factor (F)V (FVa) and FVIII (FVIIIa) with the assistance of protein S, thereby contributing to hemostatic balance. We identified 2 unrelated patients who suffered from recurrent thrombosis and carried the same heterozygous mutation c.1153A>G, p.Met343Val (M343V), in PROC gene. This mutation had not been previously reported. OBJECTIVES: To explore the molecular basis underlying the anticoagulant defect in patients carrying the M343V mutation in PROC. METHODS: We expressed PC-M343V variant in mammalian cells and characterized its properties through coagulation assays. RESULTS: Our findings demonstrated that while activation of mutant zymogen by thrombin-thrombomodulin complex was slightly affected, cleavage of chromogenic substrate by APC-M343V was significantly impaired. However, Ca2+ increased the cleavage efficiency by approximately 50%. Additionally, there was a severe reduction in affinity between APC-M343V and Na+. Furthermore, the inhibitory ability of APC-M343V toward FVa was markedly impaired. Structural and simulation analyses suggested that Val343 might disrupt the potential hydrogen bonds with Trp380 and cause Trp380 to orient closer to His211, potentially interfering with substrate binding and destabilizing the catalytic triad of APC. CONCLUSION: The M343V mutation in patients adversely affects the reactivity and/or folding of the active site as well as the binding of the physiological substrate to the protease, resulting in impaired protein C anticoagulant activity and ultimately leading to thrombosis.

Ser252Asn Mutation Introduces a New N-Linked Glycosylation Site and Causes Type IIb Protein C Deficiency.

BACKGROUND: Protein C (PC) is a vitamin K-dependent anticoagulant serine protease zymogen which upon activation by the thrombin-thrombomodulin (TM) complex downregulates the coagulation cascade by degrading cofactors Va and VIIIa by limited proteolysis. We identified a thrombosis patient who carried a heterozygous mutation c.881G > A, p.Ser252Asn (S252N) in PROC. This mutation was originally described in a report of novel mutations in patients presenting with defective PC anticoagulant activity in Paris. The research identified PC-S252N (the "Paris" mutation) in a propositus and her family members and highlighted the critical role of Ser252 in the anticoagulation process of activated PC (APC). MATERIAL AND METHODS: We expressed the PC-S252N mutant in mammalian cells and characterized the properties in coagulation assays to decipher the molecular basis of anticoagulant defect of this mutation. RESULTS: We demonstrated that PC-S252N had a diminished ability to TM binding, which resulted in its impaired activation by the thrombin-TM complex. However, APC-S252N exhibited a slightly stronger cleavage capacity for the chromogenic substrate. Meanwhile, the catalytic activity of APC-S252N toward FVa was significantly reduced. Sequence analysis revealed that Ser252 to Asn substitution introduced a new potential N-linked glycosylation site (252NTT254) in the catalytic domain of PC, which adversely affected both the activation process of PC and anticoagulant activity of APC. CONCLUSION: The new N-glycosylation site (252NTT254) resulting from the mutation of Ser252 to Asn252 in PROC affects the overall structure of the protease, thereby adversely affecting the anticoagulant function of protein C. This modification has a negative impact on both TM-promoted activation of protein C and APC cleavage of FVa, ultimately leading to thrombosis in the patient.

Using Finite Element and Eigenmode Expansion Methods to Investigate the Periodic and Spectral Characteristic of Superstructure Fiber Bragg Gratings.

In this study, a numerical simulation method was employed to investigate and analyze superstructure fiber Bragg gratings (SFBGs) with five duty cycles (50%, 33.33%, 14.28%, 12.5%, and 10%). This study focuses on demonstrating the relevance between design period and spectral characteristics of SFBGs (in the form of graphics) for SFBGs of all duty cycles. Compared with complicated and hard-to-learn conventional coupled-mode theory, the result of the present study may assist beginner and expert designers in understanding the basic application aspects, optical characteristics, and design techniques of SFBGs, thereby indirectly lowering the physical concepts and mathematical skills required for entering the design field. To effectively improve the accuracy of overall computational performance and numerical calculations and to shorten the gap between simulation results and actual production, this study integrated a perfectly matched layer (PML), perfectly reflecting boundary (PRB), object meshing method (OMM), and boundary meshing method (BMM) into the finite element method (FEM) and eigenmode expansion method (EEM). The integrated method enables designers to easily and flexibly design optical fiber communication systems that conform to the specific spectral characteristic by using the simulation data in this paper, which includes bandwidth, number of channels, and band gap size.