One goal of my research program is to define the assembly of receptor complexes and determine how they work together at the molecular level to mediate physiological responses. Since 2007, I have focused on the factors that determine the efficiency of thrombin signaling through protease activated receptor 4 (PAR4), which is one of the initiating steps of platelet activation. Our initial studies determined the molecular interactions between PAR4 and thrombin and how they influence the kinetics of PAR4 cleavage. More recently, we determined that PAR1-PAR4 heterodimers are required for efficient PAR4 cleavage by thrombin. We have now turned our attention to uncovering the structural basis for PAR4 activation using structural mass spectrometry approaches.
In addition to their role in hemostasis, platelets cooperate with neutrophils, monocytes and tumor cells to influence the behavior of these cells in a variety of physiological and pathophysiological conditions. A second focus of my research program is to identify the mechanisms by which platelets adhere to and communicate with these multiple cell types using both in vitro and in vivo approaches.
The underlying theme of my research program is that protease-activated receptor subtypes interact with one another to mediate the full range of thrombin signaling for activation of platelets and endothelial cells. Thrombin is a potent platelet agonist that signals through PAR1 and PAR4 to mediate adhesion and aggregation. PAR4 is associated with a prolonged stimulus as measured by intracellular Ca2+-mobilization and may be required for stable clot formation. The rate of PAR4 activation by thrombin is enhanced ~10 fold due to a thrombin-dependent association with PAR1. Heterodimerization may be a common theme for regulating signaling because three major platelet GPCRs (PAR1, PAR4, P2Y12) have agonist depending interactions. PAR4 is at the center of this group of receptors since it interacts with both PAR1 and P2Y12. Moreover, PAR1 and P2Y12 are the targets of vorapaxar and clopidogrel, respectively, which makes PAR4 the primary receptor for patients on these therapies. The increased reliance on PAR4 in this setting may have particular importance given the recent observation that there are race differences in PAR4 reactivity.
Nieman MT. PARtitioning protease signaling. Blood. 2015;125:1853-5
Mumaw MM, de la Fuente M, Arachiche A, Wahl III JK, Nieman MT. Development and characterization of monoclonal antibodies against protease activated receptor 4 (PAR4). Thromb Res. 2015;135:1165-71.
Mumaw M and Nieman MT. Race differences in platelet reactivity. Is PAR4 a predictor of response to therapy? Arterioscl. Thromb. Vasc. Biol. 2014;34:2524-36.
Edelstein LC, Simon LM, Lindsay CR, Kong X, Montoya RT, Tourdot BE, Chen ES, Ma L, Coughlin S, Nieman M, Holinstat M, Shaw C, Bray PF. Common variants in the human platelet PAR4 thrombin receptor alter platelet function and differ by race. Blood. 2014 Nov 27;124(23):3450-8. doi: 10.1182/blood-2014-04-572479. Epub 2014 Oct 7.
Mumaw M, de la Fuente M, Nobel DN, Nieman MT. Targeting the anionic region of human protease activated receptor 4 (PAR4) inhibits platelet aggregation and thrombosis without interfering with hemostasis. J Thromb Haemost. 2014;12:1331-41 PMCID: 4127092.
Arachiche A, de la Fuente M, Nieman MT. Platelet specific promoters are insufficient to express Protease Activated Receptor 1 (PAR1) transgene in mouse platelets. PLoS One. 2014;9:e97724 PMCID 4022678.
Arachiche A, Mumaw MM, de la Fuente M, Nieman MT. Protease-activated Receptor 1 (PAR1) and PAR4 Heterodimers Are Required for PAR1-enhanced Cleavage of PAR4 by alpha-Thrombin. J Biol Chem. 2013;288:32553-62. PMCID: 3820888.
Arachiche A, de la Fuente M, Nieman MT. Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets. PLoS One. 2013;8:e55740. PMCID: 3566007.
de la Fuente M, Noble DN, Verma S, Nieman MT. Mapping human protease-activated receptor 4 (PAR4) homodimer interface to transmembrane helix 4. J Biol Chem. 2012;287:10414-23. PMCID: 3322995.
Nieman MT. Protease-activated receptor 4 uses anionic residues to interact with alpha-thrombin in the absence or presence of protease-activated receptor 1. Biochemistry. 2008 Dec 16;47(50):13279-86. doi: 10.1021/bi801334s.