Murine Platelets Retain High Amounts of Alpha Granule Proteins on Their Surface upon Stimulation with Multiple Agonists.

Abstract

Human platelets exposed to two strong agonists have recently been noted to form a unique subpopulation characterized by increased amounts of bound alpha granule constituents, including fibrinogen and factor V. We have identified and characterized a similar population of platelets in the mouse utilizing thrombin and the GPVI agonist, convulxin, as dual stimulants. We demonstrate that these platelets are analogous to those previously characterized in humans and examine their formation in both Fc Receptor γ-chain (FcRγ) and Factor XIIIa null mice. C57/Bl6 mouse platelets were exposed to convulxin, thrombin, and ionomycin singly or in combination at concentrations previously demonstrated to give maximal aggregation. Platelets were then examined by flow cytometry. Upon stimulation by any of the agonists, all platelets exhibited moderate amounts of fibrinogen binding and equivalent P-selectin exposure. Approximately 70% of convulxin and thrombin stimulated platelets (CoaT platelets) and 10% of thrombin stimulated platelets retained high amounts of alpha granule proteins, including fibrinogen and vWF. Interestingly, despite their high amount of bound fibrinogen, CoaT platelets demonstrated decreased binding of the GPIIb/IIIa activated state-specific antibody, JON/A, relative to single agonist stimulated platelets. Further characterization identified this platelet population as having high phosphatidylserine exposure (increased annexin V binding) and increased ability to bind the actin specific marker, phalloidin. All of these characteristics were present within two minutes of agonist stimulation. CoaT platelets could not be generated in the FcRγ-chain knockout mouse demonstrating the importance of this signaling pathway in their formation. Since human CoaT platelet formation is inhibited both by transglutaminase inhibitors and a monoclonal antibody to Factor XIIIa, CoaT platelet formation was examined in the FXIIIa null mouse. The absence of FXIIIa did not affect CoaT platelet formation, aggregation, or phosphatidylserine exposure. These findings demonstrate the presence of a unique subpopulation of platelets in the mouse formed on exposure to dual agonists analogous to the previously described CoaT platelets. This population is characterized by high amounts of bound alpha granule proteins, decreased binding of a GPIIb/IIIa activation specific mAb, and increased phosphatidylserine exposure; and in the mouse is not dependent on FXIIIa.