Background:The Protein C (PC) pathway is an essential negative regulator of blood coagulation. Activated Protein C (aPC) downregulates coagulation by proteolytically inactivating the FVa and FVIIIa, thereby diminishing additional thrombin generation. PC deficiency manifests as an increased risk for thrombosis and purpura fulminans, with complete PC deficiency being neonatal lethal (Proc-/-) in mice. The significant impact of perturbations of the PC pathway on blood coagulation make the PC pathway a promising therapeutic target for hemostatic disorders. Thus, more comprehensive investigations of PC regulation and its potential genetic interactors are warranted.

Aim:We used mouse genetic techniques to identify and characterize PC expression and phenotypic regulation.

Methods: To search for mutations in genes that restored survival to the homozygous lethal Proc-/- mice, we conducted a sensitized genome-wide ENU mutagenesis screen for dominant thrombosis suppressor genes. 99 Proc+/- male mice were treated with the potent mutagen ENU. Mutagenized Proc+/- mice were bred to Proc+/- female mice on the C57BL/6J genetic background to screen for live offspring carrying the Proc-/- genotype.

To investigate the RNA levels of the Proc gene in mouse strains, organ samples were collected from 129S1 (n=12), A/J (n=12), wildtype (B6, n=13), CAST (n=6), and DBA (n=12) mice. Relative Proc expression differences were investigated.

To investigate variation in plasma PC levels in mouse strains, plasma samples were collected from male and female 129S1/SvImJ (129S1), A/J, C57BL/6J (B6), CAST/EiJ (CAST), and DBA/2J (DBA) mice. Samples were assayed for PC levels using a mouse specific PC ELISA. To identify PC genetic regulatory loci, CAST mice were crossed with DBA mice in a two step process to generate genetically informative CASTD2F2 mice. Plasma PC levels from F2 (n=59) mice were obtained and corresponding tail DNA was subjected to whole genome genotyping using the MiniMUGA platform. Quantitative Trait Locus (QTL) analyses were performed for loci regulating plasma PC levels.

Results:The Proc ENU crossed produced a total of 962 weaning-age progeny, with no Proc-/- genotype mice produced. Based on this number and the calculated ENU mutation rate, this corresponded to approximately whole genome coverage.

Liver RT-qPCR analysis demonstrated a significant difference in Proc expression between A/J and CAST (p=0.0037), A/J and DBA (p=0.0228), and A/J and B6 (p=0.0319) mice. We also found age-related tissue-specific differences in Proc expression in the 129S1 strain. As the age of the mice increased from 9 weeks to 30 weeks, lung Proc expression levels decreased significantly in 30-week-old mice (p=0.0006).

Plasma analysis revealed that the 129S1 strain had the lowest PC levels, with significant differences between 129S1 and DBA (p=0.0264), and 129S1 and A/J (p=0.0029). We found a significant PC sex difference in B6 , CAST, DBA (p<0.0001), 129S1 (p=0.0137) and A/J (p=0.0028) with male mice having higher plasma PC in all cases. Initial QTL analysis revealed no significant or suggestive loci for controlling plasma PC. However, including sex as an additive covariate revealed a suggestive peak on Chr 7 (LOD 3.054308, suggestive threshold 2.80, p= 0.339). Mice homozygous for the CAST allele at this locus have a plasma higher trending PC level than those homozygous for the DBA allele.

Conclusions: Our results suggest that the neonatal lethal Proc-/- phenotype is not rescuable by dominantly inherited mutations, affirming its thrombotic notable severity. Our studies also revealed significant strain specific differences in liver Proc gene expression, as well as an age related increase in Proc expression in the lungs of 129S1 strain mice. Our findings indicate tissue-specific regulatory mechanisms that could be explored in future studies. Significant differences in plasma PC levels were observed between inbred strains, with a suggestive sex influenced genetic locus for controlling plasma PC identified in a small cohort of genetically informative F2 mice. Additional studies using larger numbers could reveal additional significant PC regulatory loci for this important antithrombotic molecule.

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2025
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