Distinct Genetic Loci Are Associated with Platelet Aggregation Responses to Collagen and Collagen Synthetic Related Peptide (SRP) in Mice

There is substantial inter-individual variation in responsiveness of human platelets to different agonists in platelet aggregation studies. Much of this variation is likely heritable, and some of this variation can be attributed to genetic variants in platelet agonist receptors. However, beyond variants in known candidate receptor genes, the genetic factors underlying variation in platelet aggregation are undefined. In this study, we are taking a genome wide approach to identify genetic factors associated with platelet aggregation responses to different agonists in mice. We developed a method to quantify platelet aggregation responses to multiple agonists in single mouse donors and tested platelets from C57BL6/J (B6) and FVB/NJ (FVB) inbred mouse strains for their patterns of response to collagen (type I), collagen synthetic reactive peptide (SRP), the thromboxane A2 (TxA₂) mimetic U44069, and PAR-4 activating peptide (AYPGKF) (B6 and FVB, n=9 for each strain). The platelet aggregation response was then quantified as an area under the aggregation curve (AUC) for each agonist. There was a significant difference between B6 and FVB in platelet responsiveness to collagen (9.06 x 10³ ± 1.44 x 10³ vs 3.7 x 10³ ± 1.77 x 10³. p<0.018), collagen SRP (2.04 x 10³ ± 1.31 x 10³ vs 1.25 x 10⁴ ± 1.55 x 10³. p<5 x 10^-5), and U44069 (1.2 x 10⁴ ± 2.19 x 10³ vs 6.47 x 10³ ± 1.85 x 10³. p<0.05), respectively. No difference was observed in response to PAR-4 AP (7.07 x 10³ ± 6.92 x 10² vs 8.24 x 10³ ± 1.25 x 10³. p=0.2). We then performed a B6 x FVB F2 intercross which generated 152 mice which were phenotyped for platelet aggregation responsiveness to collagen, collagen SRP, and U44069, using AYPGKF as a positive control. Thus far, we have performed genome-wide association studies in 54 mice using a 1449 SNP Illumina BeadChip platform. Quantitative trait loci (QTL) analysis was performed to identify QTLs associated with platelet responsiveness to collagen, SRP, and U44069. In this interim analysis, we have identified several significant QTL for platelet aggregation response to collagen and SRP. A single highly significant QTL for collagen SRP was identified on proximal chromosome 7 (LOD = 8.26) that spans ~23 Mb (5’ end to rs6239372), termed Plsrp1. Additionally, statistically significant but broad QTLs were identified for collagen on chromosome 14 (Plcoll1 and Plcoll2, peak LOD scores 3.78 and 3.87, respectively), with additional suggestive regions on chromosome 10 and chromosome 7. Intriguingly, the QTL with the strongest associations for collagen on chromosome 14 (Plcoll1 and Plcoll2) are necessarily distinct from the single collagen SRP QTL (Plsrp1) on chromosome 7. Interestingly, Plcoll1 and Plcoll2 would have been suspected from previous studies of collagen responsiveness in mice as chromosome 14 does not harbor the major candidate genes thought to interact with collagen, in contrast to Plsrp1 which contains Gp6. These findings indicate distinct and previously unsuspected genetic elements underlie the differences in platelet responsiveness to collagen and collagen SRP in mice.