Background: As hematopoietic cells, platelets express Janus kinase (JAK) and signal transducer and activator of transcription (STAT) proteins; however, roles for JAK/STAT and associated innate immunity signaling pathways in platelet function remain unclear. Recent phosphoproteomics studies from our group found activation of a JAK/STAT5 pathway in platelets following stimulation with collagen-related peptide (CRP)-XL, which signals through the glycoprotein VI (GPVI) receptor, suggesting a role for JAK/STAT5 in GPVI-mediated platelet function.

Aims: To determine roles for the JAK/STAT5 axis in platelet function induced by GPVI activation in vitro.

Methods: Washed platelets prepared from healthy human volunteers were pretreated with five therapeutic JAK inhibitors, or “jakinibs” (ruxolitinib, oclacitinib, upadacitinib, baricitinib, tofacitinib) before stimulation with CRP-XL. Platelet functional responses were analyzed with biochemical, microscopy, flow cytometry and aggregometry assays.

Results: Ruxolitinib and baricitinib significantly reduced GPVI-mediated platelet aggregation, adhesion, and α-granule secretion. JAK inhibitors also inhibited platelet cytoskeletal changes, as shown by a reduction in F-actin formation and decreased spreading on fibrinogen. In contrast, platelet responses to the G protein-coupled receptor agonist thrombin were unaffected by jakinibs. Western blot analyses of platelet lysates probed with phosphorylation site-specific antisera found that platelet JAK2 and STAT5 were activated in response CRP-XL and inhibited by preincubation with JAK inhibitors. In addition, all the inhibitors impaired Akt pathways activation downstream of GPVI and demonstrated specific effects on downstream mediators such as dual adaptor of phosphotyrosine and 3-phosphoinositides 1 (DAPP1) and p21 activated kinase 1 (PAK1). Pretreatment of platelets with a STAT5 inhibitor also demonstrated a reduction in integrin activation and α-granule secretion in response to CRP-XL as well as spreading on collagen and fibrinogen.

Conclusions: The JAK inhibitors ruxolitinib and baricitinib and a STAT5 inhibitor impaired GPVI-mediated platelet adhesion, secretion, and aggregation, suggesting a role for JAK/STAT innate immunity signaling pathways in platelet hemostatic responses.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/a-rrle-for-the-jak-stat5-axis-in-gpvi-mediated-platelet-function/