The Thrombosis Phenotype of GPVI-Knockout Mice Is Dependent upon Modifier Gene Loci.
Authors: Cheli Yann , Laipply Heidi , Ruggeri Zaverio M. , Ware Jerry , Kunicki Thomas J. 
Published: April 2020

Abstract
Targeted disruption of murine platelet GPVI leads to defects in collagen-induced platelet responses, reflecting the important contribution of this receptor to hemostasis. Nonetheless, we have observed a reproducible and profound variation between GPVI-null littermates that is reflected in extreme differences in tail bleeding times and collagen-induced aggregation. The thrombotic phenotype of individual GPVI-null mice was defined by the tail bleeding time, determined by immersion of the severed tail in warm saline, and individual mouse tail bleeding times were repeated on two occasions yielding comparable results. Mice with a HIGH thrombotic phenotype had bleeding times consistently ≤250 seconds; those with a LOW thrombotic phenotype had elevated times, consistently ≥400 seconds, with several terminated at an arbitrary upper limit of 720 seconds. For platelet aggregation assays, it was necessary to use pooled blood (PRP) from four mice with similar thrombotic phenotypes. Collagen-induced aggregation of HIGH mice (2 μg/ml purified human Type I collagen) was roughly 50% that of wild-type littermates, while aggregation of LOW mice was more dramatically reduced (maximal aggregation ≤15% of controls). In either case, lag time to onset of aggregation was not prolonged. These differences in collagen-induced aggregation cannot be attributed to changes in the surface expression of other platelet adhesion receptors. By FACS in whole blood, there was no difference between levels of platelet GPIb or integrins αIIbβ3, α2β1, α5β1 or α6β1, in HIGH versus LOW mice. Similarly, no difference was observed in platelet aggregation induced by ADP or thrombin, eliminating differences in these receptors as a likely explanation. The thrombosis phenotype is inherited in a Mendelian fashion among littermates of breeding pairs with defined phenotypes. For genetic studies, breeding pairs were selected that exhibited extreme phenotypes (LOW, 720 seconds; HIGH, ≤250 seconds). A gene dosage effect was apparent, such that obligatory heterozygotes exhibited an intermediate functional response (bleeding times ranging from 300 to 450 seconds). Our results establish that variation in the thrombotic phenotype of GPVI-null mice is genetically controlled. The gene loci that are responsible for this difference are evidently not related to GPVI or other major platelet receptors. These functional differences may reflect strain-related differences in the background of C57BL/6J and 129X1/SvJ mice that are used to generate the KO mice, although such differences appear to be masked in the parental strains. The phenotype only becomes obvious upon elimination of the targeted gene and may be influenced by a compensatory increase or decrease in the expression of other genes that remain to be identified. We are initiating genome wide linkage analyses to directly address this question. Whatever the explanation, this variation in thrombosis phenotype of GPVI-null mice indicates that there is (are) yet to be identified genes that influence thrombosis in general and platelet responses to collagens in particular. In addition, our findings add weight to the argument raised in other studies that complex phenotypes, such as thrombosis and/or hemostasis, that are affected by targeted gene disruptions in mice are influenced by modifier gene loci and cannot always be attributed exclusively to the targeted gene disruption.