High Mobility Group Box 1 enhances ADP-mediated platelet activation by increasing platelet surface P2Y12 localization

Thrombosis and inflammation are intimately linked and synergistically contribute to the pathogenesis of a number of vascular diseases. On a cellular level, while the platelet is central to thrombus formation as well as an active mediator of inflammation, the molecular mechanisms of cross-talk between thrombosis and inflammation remain elusive. High-Mobility Group Box 1 protein (HMGB1) is an inflammatory regulator that also stimulates platelet activation through its interaction with toll-like receptor 4 (TLR4). However, it remains unclear whether cross-talk between HMGB1 and traditional thrombotic agonists exists to modulate platelet activation. Using isolated human platelets, we tested whether HMGB1 treatment affects platelet activation mediated by traditional agonists. We found that HMGB1 enhances ADP-mediated platelet activation, but not platelet activation stimulated by thrombin or collagen. Further, inhibition of the canonical ADP purinergic P2Y12 receptor attenuates HMGB1-dependent platelet activation. Mechanistically, we discovered that HMGB1 activates platelet surface TLR4 to release ADP from the platelet and concomitantly increase the localization of P2Y12 on the platelet membrane. These data demonstrate that ADP-dependent P2Y12 activation contributes to HMGB1 mediated platelet activation, while HMGB1 primes platelets for an enhanced activation response to ADP. These novel findings further our understanding of thrombo-inflammatory signaling and provide new insight for therapeutic P2Y12 inhibition. Key PointsO_LIHMGB1 enhances ADP-mediated platelet activation but not platelet activation stimulated by collagen or thrombin. C_LIO_LIHMGB1 stimulates platelet ADP release and increases platelet surface localization of P2y12 receptors via TLR4-dependent mechanism(s). C_LI Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=163 HEIGHT=200 SRC="FIGDIR/small/436776v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@17a31c0org.highwire.dtl.DTLVardef@158391corg.highwire.dtl.DTLVardef@10d78a0org.highwire.dtl.DTLVardef@16a6a8f_HPS_FORMAT_FIGEXP M_FIG Caption: HMGB1 activates TLR4 to activate platelets, release platelet ADP, and upregulate P2Y12 at the platelet surface. C_FIG