Identification of genetic loci regulating plasma and platelet factor v expression in mice

Abstract

Abstract Background:Factor V (FV) is a protein in the coagulation cascade found in two circulating compartments: 80% in plasma and 20% in platelet alpha-granules. Human FV is made by liver hepatocytes, contributing to the FV in the plasma, and plasma FV is endocytosed by megakaryocytes and stored in platelet alpha granules. In mice, hepatocytes produce plasma FV, and megakaryocytes directly produce platelet FV. FV deficiency causes bleeding of the mucosal lining as well as post-trauma. Complete absence of FV in humans results in increased bleeding. In mice, FV deficiency is incompatible with life. Currently, little is known about the genetic regulation of this important protein at the center of coagulation. Hypothesis:Inbred mouse strains have significant differences in their FV antigen levels in the platelets and the plasma. We can leverage these differences to identify genetic loci responsible for regulating FV in inbred mice. Methods:We isolated platelets and plasma from inbred mouse strains: C57BL/6J (B6), DBA/2J (DBA), A/J, CAST/EiJ (CAST), and 129S1/SvImJ (129S1). FV levels were measured in these strains via a mouse specific FV ELISA. To generate genetically informative mice for further analysis, we crossed the CAST and DBA strains to make the CASTD2F1 (F1) generation. We crossed the F1 mice to produce 146 CASTD2F2 (F2) mice, which were genetically informative due to meiotic recombination. We isolated platelets (n=85) and plasma (n=61) from F2 mice to measure the FV antigen levels. Tail biopsies were taken from 146 F2 mice, two DBA females, and two CAST males and sent for whole genome genotyping using TransnetYX MiniMUGA. The genotyping and phenotyping data were used to run two independent quantitative trait loci (QTL) analyses, for plasma and platelet FV expression levels, to investigate potential genomic loci controlling FV expression. Results:In platelets, significant FV expression differences were observed between DBA and CAST (p=0.0033), with no sex differences within the strains. There was a significant difference between male CAST and male DBA mice (p=0.0336). In plasma, CAST and DBA mice had no significant differences in their antigen levels when sexes were grouped. We found a significant difference between female CAST and female DBA mice (p=0.0044) and sex differences within the two strains (CAST p=0.0236, DBA p=0.0011). Based on these findings, we performed a DBA x CAST intercross to produce F2 offspring. We analyzed 85 F2 mice for platelet FV levels via QTL to identify genetic loci controlling platelet FV, with no significant or suggestive peaks in our initial run. Adding sex as an interactive covariate led us to identify one significant peak on Chromosome (Chr) 14 (LOD 3.45, significance threshold 2.32, p=0.00641) and one suggestive peak on Chr15 (LOD 1.64, suggestive 20% threshold 1.58, p=0.17844). With sex as an additive covariate, we found a suggestive peak on Chr 2 (LOD 3.66, suggestive threshold 2.78, p=0.113). Tissue Factor Pathway Inhibitor (TFPI) and a modifier of FV PLXDC2 are located on Chr 2, a significant distance from our mapped region, suggesting that potential effects of these loci are due to higher order interactions. Analysis of 61 F2 mice for plasma FV levels via QTL analysis identified a significant peak on Chr 1 (LOD 5.24, significance threshold 4.21, p=0.00641). Accounting for sex as both an interactive and additive covariate showed an increased LOD score at Chr 1 (LOD 6.79, significance threshold 6.34, p=0.0246). The mouse FV gene is located on Chr 1 within our minimally significant recombinant interval, but it is a substantial distance from the main significance peak. Within our peak region, we identified a serpin cluster that includes Serpinb2 (PAI-2), a known member of the coagulation cascade. Presently, there are no known links between serpins and FV levels, but it is possible that alterations in PAI-2 or other serpins could affect FV expression. Conclusions:Our findings support our hypothesis that strain-dependent differences in FV platelet and plasma levels exist in inbred mouse strains. We identified loci responsible for regulating FV levels in the CAST and DBA mice. Future work of identifying the specific regulatory elements controlling platelet and plasma FV expression will increase our understanding of cell-type specific gene regulation. This may reveal novel targets or strategies for modulating FV in humans, which could be used to treat hemostatic disease.