Signaling Mechanisms of the Glycoprotein Ib-IX-V Complex and Role of LIM kinase 1 in Platelet Activation
thesisposted on 18.10.2016, 00:00 authored by Brian Estevez
Upon blood vessel injury, the platelet glycoprotein Ib-IX-V (GPIb-IX-V) complex mediates initial platelet adhesion to immobilized von Willebrand factor (VWF), it also elicits signaling that is critical for platelet activation and thrombosis. However, the downstream events upon GPIb-IX-V ligation are unclear. By isolating platelets from LIMK1-/- mice we were able to characterize the function of LIMK1 in various platelet activation pathways. We discovered that LIMK1 plays an important role in promoting VWF/GPIb-IX-mediated platelet activation. LIMK1-/- platelets displayed a defective second wave of VWF/botrocetin-induced platelet aggregation, and were defective in ATP release following VWF/botrocetin stimulation. We show that LIMK1 is activated by VWF, downstream of MAPKs, and that LIMK1 promotes cPLA2 phosphorylation. Using in vivo experiments, we found that although deletion of LIMK1 in mice does not affect hemostasis, LIMK1-/- mice display a prolonged time to vessel occlusion in the FeCl3 carotid artery injury thrombosis model. Thus, we have discovered a role for LIMK1 in platelet function and have identified a GPIb-IX-specific signaling pathway leading to TXA2 production. To better understand GPIb-IX signaling, we also investigated the thrombin receptor function of GPIb-IX. We discovered that GPIb-IX does transduce signaling that is important for platelet activation at low doses of thrombin, but this signaling mechanism requires PAR activation. By reconstituting thrombin signaling in cells we were able to investigate the individual contributions of GPIb-IX and PAR1 in thrombin signaling. Expression of WT GPIb-IX complex enhanced cellular response to thrombin. Similar to VWF/GPIb-IX-dependent signaling we found that deletion of the high affinity binding site or cytoplasmic C-terminal 14-3-3 binding motif on GPIb abolished GPIb-IX-dependent enhancement of thrombin response. Our studies demonstrate that LIMK1 plays differential roles in response to different platelet agonists, and we used this to dissect the thrombin receptor function of GPIb-IX, which has been the center of much debate in platelet biology. In GPIb-IX dependent pathways (VWF and thrombin) LIMK1 plays a stimulatory role and in GPIb-IX independent/GPCR pathways (TXA2) LIMK1 plays a negative role. Taken together, our studies indicate that thrombin- and VWF-induced GPIb-IX signaling require a C-terminal cytoplasmic domain interaction with cytosolic adaptor protein 14-3-3, and involve a Rac1 and LIMK1-dependent signaling pathway that promotes platelet activation.