PB0857 - IFN𝛾 augments aggregation and calcium flux in murine platelets

Background: Platelets possess immune receptors, enabling them to respond to inflammation. While interferon gamma (IFN𝛾) is crucial in host defense, excessive IFN𝛾 fosters pathological inflammation, often associated with heightened platelet reactivity and an increased risk of thrombosis. Although both human and murine megakaryocytes (MKs) respond to IFN𝛾, it remains unclear whether platelets inherit a functional IFN𝛾 receptor (IFNGR).

Aims: We sought to explore the effect of IFN𝛾 on murine platelets.

Methods: We incubated washed murine platelets (C56BL/6J, female/male) with IFN𝛾 (100 ng/ml) or vehicle, followed by stimulation with ADP (0.1-25 µM), thrombin (0.01-1 U/ml), and convulxin (0.2-200 ng/ml). Subsequently, we evaluated IFNGR surface expression, granule release, integrin 𝛼IIb𝛽III activation, phosphatidylserine exposure, calcium flux, and aggregation, using flow cytometry and a microplate reader.

Results: Murine platelets exhibited surface expression of the IFNGR subunits 1 and 2 (IFNGR1/2). Thrombin and convulxin enhanced the IFNGR1/2 surface availability, while ADP or IFN𝛾 itself had no effect.
IFN𝛾 did not alter the expression of CD62P, CD40L, LAMP-1, and phosphatidylserine in resting platelets or following stimulation by ADP, thrombin, or convulxin, indicating unchanged release of 𝛼-granules, lysosomal granules, and phosphatidylserine exposure. However, IFN𝛾 combined with thrombin or convulxin increased integrin 𝛼IIb𝛽III activation.
Interestingly, IFN𝛾 potentiated aggregation induced by thrombin and convulxin, while IFN𝛾 itself did not cause aggregation. This effect was negated by antibodies blocking IFNGR1 or IFN𝛾, highlighting specificity.
Similarly, in the presence of calcium, IFN𝛾 did not prompt calcium flux independently but augmented calcium flux when combined with thrombin, convulxin, or the SERCA inhibitor thapsigargin. IFN𝛾 also increased calcium flux induced by thrombin or convulxin in the presence of EDTA, suggesting that IFN𝛾 amplified calcium release from internal stores.

Conclusion(s): Our findings identify IFN𝛾 as a novel cytokine that modulates platelet function. Further exploration of functional implications in IFN𝛾-driven inflammatory conditions in mice and humans will be of interest.