Abstract 051: Common Single Nucleotide Polymorphisms in the Coagulation Factor XII Gene (F12) are Associated with Endogenous Thrombin Potential via In Situ Activation of the Intrinsic System of Coagulation: The Cardiovascular Health Study
Authors: Olson Nels C , Butenas Saulius , Lange Ethan M , Lange Leslie A , Jenny Nancy S , Walston Jeremy , Cushman Mary , Tracy Russell P , Reiner Alex P 
Published: July 2021

Background:
Currently, a majority of evidence suggests that the tissue factor-mediated extrinsic coagulation pathway is most important in generating thrombin
in vivo
, although accumulating evidence implicates the coagulation factor XII/factor XI-mediated intrinsic system as well. The endogenous thrombin potential (ETP), a measure of thrombin generation, is gaining importance in assessing risk of hemorrhage and thrombosis, but genetic determinants of ETP have not been investigated in population-based studies.

Methods:
Total ETP (ETP
T
) was measured in citrated plasma by TechnoClone TGA assay in a random subset of Cardiovascular Health Study (CHS) participants (
n
=1,000). Extrinsic pathway ETP (ETP
EX
) was measured by the addition of anti-FXIa antibody to the assay; Intrinsic pathway ETP (ETP
IN
) was calculated by subtracting ETP
EX
from ETP
T
. Genotyping was performed in the NHLBI’s Candidate-gene Association Resource (CARe) Study on 49,320 SNPs using Illumina’s custom IBCv2 genotyping array in 866 participants. Associations of SNPs with age- and sex-adjusted ETP phenotypes were evaluated by linear regression using PLINK; results were considered statistically significant if p≤ 2x 10
-6
.

Results:
Two SNPs on the IBC array were significantly associated with lower ETP
T
: rs2545801 (β=-45.87 nM ± 7.3 nM; p=5.7x10
-10
; minor allele frequency (MAF) =0.24), and rs1801020 (β=-45.76 nM ± 7.3 nM; p=7.4x10
-10
; MAF= 0.23). Both SNPs are located in the coagulation factor XII gene (
F12
); rs2545801is located in a 5’ region upstream of the transcriptional start site, and rs1801020 in a 5’ untranslated region; the SNPs are in strong linkage disequilibrium (LD) (r
2
= 0.92). Each SNP had a significant effect on ETP thrombin peak height. The mean (SD) thrombin peak heights were 408 nM (152nM) vs.523 nM (123 nM) and 404 nM (147 nM) vs. 523 nM (123 nM) for those homozygous for the minor and major alleles of rs2545801 and rs1801020, respectively. Addition of corn trypsin inhibitor (CTI; an inhibitor of activated FXII (FXIIa)) had no effect on ETP, suggesting that the
F12
SNPs may affect the FXII-dependent generation of activated FXI (FXIa)
in vivo.
Therefore, we inhibited FXIa by the addition of anti-FXIa antibody, which resulted in a large and variable effect on ETP thrombin peak height. Consistent with these observations, analyses of SNPs associated with ETP
IN
revealed increased significance of the p-values for the
F12
SNPs (p=1.40 x 10
-15
and p=1.60 x 10
-14
for rs1801020 and rs254580, respectively).

Conclusion:
These data support the importance of
F12
SNPs on thrombin generation, and suggest that these SNPs affect the amount of FXIa generated
in vivo
. Additional work is needed to confirm this hypothesis, and to examine relationships of these biomarkers and SNPs with thrombotic diseases.