Apoptosis Signal-Regulating Kinase 1 (ASK1) Is a Novel Regulator of Heparin-Induced Thrombocytopenia and Thrombosis in Mice

Heparin-induced thrombocytopenia and thrombosis (HITT) is a life-threatening (20-30% mortality risk) disease in which IgG antibodies against the heparin-PF4 complex bind and activate platelets via FcγRIIA. Our laboratory has identified that the Apoptosis Signal-Regulating Kinase (ASK1), a MAP3K, is present in both human and murine platelets and potentiates many platelet functions. Given that ASK1 regulates platelet function, and that platelets are known to play a major role in the pathogenesis of HITT, we hypothesized that ASK1 is a novel regulator of HITT.

To establish if ASK1 is activated downstream of FcγRIIA, we first stimulated washed human platelets with anti-CD9 (700ng/mL). Anti-CD9 induces the activation of FcγRIIA. We found that anti-CD9 induced a robust activation of ASK1, as measured by phosphorylation of ASK1 Thr⁸⁴⁵ (a marker of ASK1's kinase activity). ASK1 exclusively activates p38 in platelets; therefore we also measured phosphorylation of p38 a marker of ASK1's signaling activity. We found that anti-CD9 also induced a robust phosphorylation of p38.

To determine the role ASK1 plays in platelet-FcγRIIA signaling and HITT, we crossed Ask1^-/- mice to FcγRIIA^+/+(hFcR) mice. We found that genetic ablation of Ask1 did not have any effect on anti-CD9-induced activation of PLCγ2 or Syk in hFcR/ Ask1^-/- (KO) platelets compared to hFcR/ Ask1^+/+ (WT). However, loss of Ask1 did result in the complete absence of p38 activation in KO platelets following activation of FcγRIIA by anti-CD9 (500ng/mL). Further, we observed that anti-CD9-induced integrin α_IIbβ₃ activation, α-granule secretion, and platelet aggregation were all significantly attenuated in KO platelets (P<0.01). These in vitro results strongly suggested that ASK1 plays a prominent role in FcγRIIA-mediated platelet activation.

To further investigate the role of ASK1 in HITT pathogenesis, WT and KO mice were subjected to an in vivo model of HITT by injecting anti-CD9 IgG. Platelet counts were measured in samples of whole blood collected at various time points post-injection, and compared to resting platelet counts taken before HITT was initiated. We found that KO mice were significantly (P<0.01) protected from thrombocytopenia compared to WT mice when injected with 250μg/kg anti-CD9. However, when injected with 500μg/kg anti-CD9, both WT and KO mice displayed the same level of thrombocytopenia. Despite not protecting from thrombocytopenia, when observed for signs of shock, we found that KO mice were significantly (P<0.01) protected from anti-CD9 induced shock compared to WT mice regardless of the dose.

Taken together these in vitro and in vivo data strongly suggest that ASK1 regulates FcγRIIA-mediated platelet activation, and that ASK1 plays a key role in the pathogenesis of HITT. Furthermore, these data highlight the much-overlooked ASK1/p38 signaling pathway as key mediator of platelet activation during HITT pathogenesis. ASK1 may be a potential target for the development of novel drugs to treat HITT.