Page 255 - Haematologica - Vol. 105 n. 6 - June 2020

APC-I73N variant
complex.1,5 Through its multi-domain structural feature, APC can function as an allosteric enzyme and its prote- olytic activity is modulated by protein and metal ion co- factors binding to different domains of the protein.6-8 Protein S functions as a co-factor to promote the anticoag- ulant function of APC by binding to the light chain of the protease (primarily to Gla and EGF1 domains),9-12 thereby stabilizing the active conformation of APC on negatively charged membrane surfaces in a topographical orientation in which the catalytic triad of the protease is aligned with scissile bonds of target co-factors Va and VIIIa for optimal cleavage.13-15 Similarly, binding of both monovalent and divalent cations to specific sites of APC on both light and heavy chains allosterically modulates the catalytic and anticoagulant function of APC on membrane surfaces.4,6-10
Protein C deficiency exhibits autosomal dominant pat- tern of inheritance. The heterozygous deficiency of pro- tein C increases risk of venous thromboembolism (VTE) and its homozygous deficiency is associated with purpu- ra fulminans, which may be fatal if not treated by protein C replacement therapy.16,17 The complete protein C defi- ciency in knockout mice is lethal.18 There are two com- mon types of protein C deficiency: type-I deficiency is characterized by both low antigen and activity levels, and type-II deficiency is characterized by only a lower activi- ty level for APC.19,20 Protein C database (http://www.hgmd.cf.ac.uk/ac/gene.php?gene=PROC) analy- sis suggests that the mutations are scattered on both light and heavy chains and involve all functional domains of the protein (Gla, EGF1, EGF2 and catalytic domains). In this study, we have identified a compound heterozygous protein C deficient VTE patient whose plasma antigen (PC:Ag) level is 63% of the normal and activity (PC:A) levels are 23% and 44% of the normal as monitored by both clotting and chromogenic activity assays, respective- ly. Genetic analysis revealed that the proband carries compound heterozygous mutations (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC, inheriting the I73N mutation from her mother and R352Q mutation from her father. The first mutation is located on the N-terminal EGF1 and the second mutation is located on the catalytic domain of protein C. We individually expressed both pro- tein C variants in mammalian cells and characterized their properties in established coagulation assays. We demonstrate that thrombin-TM activates both protein C variants normally and anticoagulant and amidolytic activ- ities of the R352Q variant are normal; however, the anti- coagulant activity of the I73N variant has been specifical- ly and significantly impaired in the presence of protein S. Further analysis revealed that the basis for the protein S- dependent defect is that the Ile to Asn substitution intro- duces a novel N-linked glycosylation site on EGF1 of pro- tein C, thereby interfering with interaction of the APC mutant with its co-factor.
Methods
Construction, expression, and purification of recombinant protein C derivatives
Expression, purification and activation of recombinant human wild-type protein (WT) C and the Ile73 to Asn (I73N) and Arg352 to Gln (R352Q; R187Q in chymotrypsin numbering)21 derivatives in human embryonic kidney cells have been described previously.22-24
A complete list and sources of reagents, together with details of the experimental methods used, are provided in the Online Supplementary Appendix.
Analysis of thrombin generation in plasma
Thrombin generation (TG) assay was performed with Thrombinoscope (Fluoroskan Ascent (Thermo Fisher Scientific, Waltham, MA, USA) using citrated human normal or protein C- deficient plasma reconstituted with either WT protein C or mutant protein C derivatives (60nM) as described.25
Anticoagulant assays
Anticoagulant activities of APC derivatives in the absence and presence of different concentrations of protein S and/or different concentrations of PC/PS vesicles were monitored both in purified and plasma-based assay systems as described.24,25
Endothelial cell permeability
Intracellular signaling activity of APC derivatives was evaluated in a permeability assay using EA.hy926 endothelial cells as described.24 Cell permeability in response to thrombin [10nM for 10 minutes (min)] following treatment with APC derivatives [25nM for 3 hours (h)] was quantitated by spectrophotometric measurement of the flux of Evans blue-bound albumin across functional cell monolayers using a modified 2-compartment chamber model as described.24
Statistical analysis
Data are expressed as mean±standard deviation from three or more experiments. Data were analyzed by Student t-test. P<0.05 was considered statistically significant.
Results
Case presentation
The proband is a 36-year old female (II-2) who was referred to the hematology clinic because of recurrent deep vein thrombosis (DVT) of the left lower limb (Figure 1A). Plasma levels of the proband’s protein C revealed a combined type-I/type-II deficiency as evidenced by a moderately lower protein C antigen level based on the ELISA, but a significantly lower activity level based on the chromogenic assay, and an even more pronounced lower activity level based on aPTT (Figure 1C). Results of other routine coagulation and thrombophilia screening assays were normal (data not shown). Genetic analysis identified a compound heterozygous missense mutation (c.344T>A, p.I73N and c.1181G>A, p.R352Q) in PROC (Figure 1B). Further genetic analysis using blood samples from the proband’s parents revealed that she has inherited I73N mutation from her mother and R352Q mutation from her father (Figure 1B). Both I73N and R352Q are novel muta- tions in PROC, though a thrombosis patient with type-II protein C deficiency carrying a R352W mutation has been reported previously.19 To understand the molecular defect causing recurrent thrombosis in this patient, we expressed both I73N and R352Q protein C variants in mammalian cells for further characterization.
Characterization of recombinant protein C mutants
Following expression, protein C derivatives were puri- fied by a combination of immunoaffinity and ion exchange chromatography as described.22 Recombinant zymogens were activated by thrombin and APC deriva-
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