Regulation of the early events of platelet activation Lawrence F. Brass, MD PhD Platelets are essential for normal hemostasis, but can also contribute to thrombosis and to the evolution and consequences of common diseases of the vessel wall including arteriosclerosis. Although a great deal is known about platelet activation, much less is known about the events within platelets that modulate responsiveness, limiting platelet activation when it is not needed and ensuring that the response to injury halts bleeding without causing vascular occlusion. The focus of this proposal is on the events of platelet activation that occur immediately downstream of agonists such as thrombin, ADP and TxA2, all of whose receptors are coupled to heterotrimeric G proteins. Our hypothesis is that dysregulation of G protein dependent events is prothrombotic and potentially contributes to vascular disease progression. This hypothesis will be tested in studies with human platelets and selected mouse models. In our preliminary studies, we have identified a previously-undescribed regulatory complex in resting platelets in which at least two RGS (regulators of G protein signaling) proteins and the tyrosine phosphatase, SHP-1, are bound to the 130 kDa scaffold protein, spinophilin (SPL), which in resting platelets is tyrosine phosphorylated. Platelet activation causes SHP-1- dependent dephosphorylation of spinophilin and agonist-selective dissociation of the SPL/RGS/SHP-1 complex. These events can be recapitulated in transfected CHO cells. Based on these observations, we propose that spinophilin first sequesters RGS proteins, allowing signaling to begin, and then releases them in order to limit signaling magnitude and duration. Support for this model is drawn from our studies on SPL(-/-) mice and mice expressing Gi2a(G184S), a mutation that renders the a subunit of the G protein, Gi2, resistant to inactivation by RGS proteins. Those studies show that 1) loss of spinophilin impairs platelet responses to agonists, 2) this defect is limited to agonists that can cause dissociation of the SPL/RGS/SHP-1 complex, and 3) blocking RGS-dependent negative feedback on Gi2 produces, as the model would predict, a gain of platelet function in vitro and in vivo. The proposed studies are divided into three specific aims.
Aim 1 will test our hypothesis that RGS proteins help to regulate platelet responsiveness by limiting the duration of G protein signaling during platelet activation.
Aim 2 will test our hypothesis that the decay of the SPL/RGS/SHP-1 complex provides a timed brake on G protein signaling and identify the mechanisms involved. Finally, Aim 3 will focus on the role of RGS proteins and the SPL/RGS/SHP-1 complex in regulating the conversion of adherent platelets to a fully activated state and in avoiding the adverse consequences of a chronic increase in platelet reactivity.
Platelet activation occurring at the wrong place and at the wrong time remains a major cause of cardiovascular and cerebrovascular morbidity in the United States. In the proposed studies we will examine novel mechanisms that we believe helps to regulate the extent of platelet responses to injury, ensuring that the response is neither inadequate nor so robust that the health of the surrounding tissues is endangered. A better understanding of this critical, but under-explored aspect of platelet biology is key to understanding and preventing what goes wrong when people have heart and strokes.
|Ma, Peisong; Foote, Darci C; Sinnamon, Andrew J et al. (2015) Dissociation of SHP-1 from spinophilin during platelet activation exposes an inhibitory binding site for protein phosphatase-1 (PP1). PLoS One 10:e0119496|
|Ma, Peisong; Ou, Kristy; Sinnamon, Andrew J et al. (2015) Modulating platelet reactivity through control of RGS18 availability. Blood 126:2611-20|
|Brass, Lawrence F; Tomaiuolo, Maurizio; Stalker, Timothy J (2013) Harnessing the platelet signaling network to produce an optimal hemostatic response. Hematol Oncol Clin North Am 27:381-409|
|Stalker, Timothy J; Newman, Debra K; Ma, Peisong et al. (2012) Platelet signaling. Handb Exp Pharmacol :59-85|
|Brass, Lawrence F; Stalker, Timothy J (2012) Minding the gaps--and the junctions, too. Circulation 125:2414-6|
|Brass, Lawrence F; Ma, Peisong (2012) Applying the brakes to platelet activation. Blood 119:3651-2|
|Ma, Peisong; Cierniewska, Aleksandra; Signarvic, Rachel et al. (2012) A newly identified complex of spinophilin and the tyrosine phosphatase, SHP-1, modulates platelet activation by regulating G protein-dependent signaling. Blood 119:1935-45|
|Brass, L F; Wannemacher, K M; Ma, P et al. (2011) Regulating thrombus growth and stability to achieve an optimal response to injury. J Thromb Haemost 9 Suppl 1:66-75|