C‐terminal residues of activated protein C light chain contribute to its anticoagulant and cytoprotective activities

Abstract

Background: Activated protein C (APC) is an important homeostatic blood coagulation protease that conveys anticoagulant and cytoprotective activities. Proteolytic inactivation of factors Va and VIIIa facilitated by cofactor protein S is responsible for APC's anticoagulant effects, whereas cytoprotective effects of APC involve primarily the endothelial protein C receptor (EPCR), protease activated receptor (PAR)1 and PAR3.Objective: To date, several binding exosites in the protease domain of APC have been identified that contribute to APC's interaction with its substrates but potential contributions of the C-terminus of the light chain have not been studied in detail. Methods:Site-directed Ala-scanning mutagenesis of six positively charged residues within G142-L155 was used to characterize their contributions to APC's anticoagulant and cytoprotective activities.Results and Conclusions: K151 was involved in protein S dependent-anticoagulant activity of APC with some contribution of K150. 3D structural analysis supported that these two residues were exposed in an extended protein S binding site on one face of APC. Both K150 and K151 were important for PAR1 and PAR3 cleavage by APC, suggesting that this region may also mediate interactions with PARs. Accordingly, APC's cytoprotective activity as determined by endothelial barrier protection was impaired by Ala substitutions of these residues. Thus, both K150 and K151 are involved in APC's anticoagulant and cytoprotective activities. The differential contribution of K150 relative to K151 for protein S-dependent anticoagulant activity and PAR cleavage highlights that binding exosites for protein S binding and for PAR cleavage in the C-terminal region of APC's light chain overlap. K E Y W O R D S activated protein C, anticoagulant activity, cytoprotective activity, protease activated receptor, protein S S U PP O RTI N G I N FO R M ATI O N Additional supporting information may be found online in the Supporting Information section.

Supplementary research on K150del variant of activated protein C

“…We reported reduced anti-inflammatory of the APC mutant, as demonstrated by enhanced leukocyte recruitment and adhesion, higher endothelial permeability, and altered gene expression profiles. These results corroborate with the previous findings that not only the reduced anticoagulant activity but also impaired cytoprotective effect of the variant promote thrombogenesis [ 14 , 15 ]. Furthermore, we also demonstrated impaired anti-apoptotic capacity of the K150del variant, which played an important role for nerve repair in IS [ 7 ].…”

“…Diminished anticoagulant activity and cytoprotective effects of certain APC variants can greatly increase the risk of thrombosis, especially when the vascular endothelium is inflamed or mechanically damaged [ 24 ]. For instance, the K150del variant is associated with an increased risk of venous thromboembolism and IS due to impaired anticoagulant activity and endothelial barrier protection effect [ 12 – 15 ]. We reported reduced anti-inflammatory of the APC mutant, as demonstrated by enhanced leukocyte recruitment and adhesion, higher endothelial permeability, and altered gene expression profiles.…”

“…This variant was first identified in Japanese patients with venous thrombosis and then as a common genetic risk factor for venous thrombosis and IS in the Chinese population [ 13 ]. The affected individuals had different thrombotic tendencies, manifested as poor recognition of Arg506 cleavage site of FV due to impaired interaction of APC with protein S. Furthermore, the APC variant was reported to be associated with impaired cytoprotective effects and reduced cleavage of PAR1 and PAR3 [ 14 , 15 ]. We studied the cytoprotective activity of the K150del variant and defined its molecular mechanisms for clinical phenotypes that could assist the clinicians to evaluate the disease risks of carriers, assess the risk score of patients, and make optimal therapeutic decisions.…”

Evaluation of prothrombotic risk of two PROC hotspot mutations (Arg189Trp and Lys193del) in Chinese population: a retrospective study

“…Our previous study showed that R189W of PC had normal amidolytic and proteolytic activities in the absence of cofactors but exhibited ~ 3 times lower affinity for binding to endothelial protein C receptor, while K193del of PC had normal amidolytic and proteolytic activities in the absence of cofactors but exhibited ~ 2–3-fold impaired anticoagulant activity in the presence of PS in vitro [ 17 ]. In 2020, Yamashita et al stressed the functional importance of six residues within G184-L197 of PC (R185, K188, R189, K192, K193 and R194) on constituting activated protein C (APC)’s anticoagulant and cytoprotective activities [ 25 ]. According to their study, mutations at residue K192 or K193 exhibited 50% reduced APC’s anticoagulant activity with a decreased sensitivity for PS and reduced cytoprotective activities with an impaired ability to cleave PAR cleavage, while other residues including R189 presented similar APC’s anticoagulant activity and cytoprotective activities to wide-type APC [ 25 ].…”

The structural basis of the EPCR-APC complex induced biased PAR1 signaling

“…After cleavage, before tethered ligand binding to PAR1 to activate it, the PAR1 N-terminus interacts with the light chain of APC in two regions: C-terminus including Lys-150 and Lys-151 (59), and EGF-like domain 1 of APC (60). We modeled these interactions using several approaches: in the first approach, the PAR1 N- terminal peptide fragments were docked to APC using the flexible peptide docking method (29); the second approach included folding of PAR1 and EPCR-APC as heterotrimer with AlphaFold software; and the third approach was MD simulation of the system containing APC and PAR1 N-terminus.…”