Prothrombotic Phenotype of a Protein S Mutant Lacking Anticoagulant Activity

Protein S is a vitamin K-dependent protein that plays an important role in balancing pro- and anti-thrombotic responses to vascular injury. On one hand, it circulates bound to activated protein C, functioning as an anticoagulant complex that downregulates the activities of coagulation factors V and VIII. On the other hand, protein S is a ligand for platelet TYRO3 and MERTK, which, along with the third paralog AXL, constitute the TAM family of receptor tyrosine kinases that functions to potentiate the action of platelet-activating agonists, ultimately resulting in activation of the α _IIbβ3 integrin. The relative importance of these two activities in vivo, however, is not known. To better understand the importance of the TYRO3/MERTK-stimulating ability of protein S and thus gain additional insight into its role in platelet activation, we used CRISPR/Cas9 technology to generate mice with a D136A mutant form of protein S that lacks the ability to bind protein C and function as an anticoagulant, while retaining its ability to bind platelet-activating TAM receptors - hereafter termed Pros1 ^D136A mice. Since homozygosity for this variant was embryonically lethal, all assays were carried out in wild-type animals and their heterozygous littermates comprising a wild-type Pros1 allele and an allele encoding Pros1 ^D136A. Though there was no significant difference between Pros1 ^D136A mice and their wild-type littermates in either a collagen/epinephrine-induced pulmonary embolism model or in tail vein bleeding times, platelets from Pros1 ^D136A mice accumulated at the injury site to a significantly greater degree following in vivo laser injury to the cremaster muscle microvasculature. Taken together with the embryonic phenotype of Pros1 ^D136A homozygous mice, these data support the notion that protein S can function to augment platelet responsiveness. Further studies on this and other ligands for the TAM family of receptor tyrosine kinases should provide additional insights into their roles in physiological platelet activation.