Departments of Medicine and Program in Genetics, State University of New York, Stony Brook, New York, USA wbahou@notes.cc.sunysb.edu
Thrombin leads to blood clotting through activation of specialized G protein−coupled receptors. In mice, small peptides call pepducins inhibit thrombin receptors and prevent blood clotting (pages 1161−1165).
Blood clotting is controlled by a tightly regulated cascade of proteases and their cofactors that sequentially leads to generation of a gelatinous meshwork composed of a protein called fibrin. This coagulation cascade ensures the normal cessation of blood flow that occurs during physiologic processes such as menstruation, or during recovery from injury, and is termed hemostasis. Exaggerated (or pathological) hemostasis can lead to formation of a blood clot (or thrombus) with devastating clinical results such as heart attack or stroke. Interventions designed to minimize thrombotic risks target not only proteins of the coagulation cascade, but also the cellular network known to provide the second arm of the hemostatic mechanism, which includes blood platelets and cells of the vessel walls, including vascular endothelial cells.
The coupling of these two regulatory arms is linked by serine proteases such as -thrombin1, which is generated from its inactive precursor (prothrombin) at sites of vascular damage. -Thrombin regulates coagulation by cleavage of the fibrin precursor fibrinogen, and it also potently activates platelets and vascular endothelial cells by stimulating G protein−coupled receptors on their surface. In this issue, Covic et al.2, present a novel means of blocking thrombin-induced, G protein−mediated cellular activation. The strategy is of potential relevance not only for heart disease and stroke, but for the broader category of diseases regulated by G protein−coupled receptors.
-Thrombin activates an unusual class of G protein−coupled receptors termed proteolytically activated receptors (PARs)(Fig. 1). As suggested by their name, PARs are activated through proteolytic cleavage, as opposed to receptor−ligand binding. Otherwise, PARs resemble most other G protein−coupled receptors, displaying the characteristic seven-transmembrane serpentine structure comprising three alternating extracellular and intracellular loops. Conformational changes that occur after activation facilitate receptor interaction with GTP-bound G-subunits, often involving the third intracellular loop (i3)3. Four PARs have been identified in humans, three of which mediate thrombin cellular responses (PAR1, PAR3 and PAR4). The cellular distribution of thrombin-responsive PARs is distinct, as are their dose-response curves to thrombin. Human platelets express PAR1 and PAR4, although murine platelets use a PAR3/PAR4 dual receptor system, with evidence that murine PAR3 functions as a cofactor for PAR4 responsiveness4. Vascular endothelial cells remain less well characterized. It is known that they express PAR1 and PAR3, but there is less information on functional roles for PAR3 and PAR4 in thrombin-responsiveness in these cells5.
Figure 1. Regulation of blood clotting by the serine protease -Thrombin.
-Thrombin is generated from its zymogen precursor, prothrombin, at sites of vascular injury. -Thrombin regulates coagulation by directly cleaving fibrinogen to form a fibrin meshwork, while also acting as a potent activator of human platelets. Thrombin's cellular effects are mediated by proteolytically activated receptors (PARs), highly evolved G protein−coupled receptors activated by proteolytic cleavage of their N-terminal extensions. Vascular endothelial cells express PAR1 and PAR3, and subsets may express PAR4. Human platelet thrombin responses are mediated by PAR1 and PAR4. In quiescent platelets, PARs are associated with heterotrimeric G proteins, with the -subunit maintained in the inactive (GDP-bound) state. Thrombin stimulation promotes the release of GDP with replacement by cytosolic GTP, with coupling to G proteins (members of the G12/13, Gq and Gi families). Downstream effector events result in platelet activation and aggregation. Pepducins targeting the i3 cytoplasmic loop apparently block the interaction of the GTP-bound G protein with PAR1 and PAR4, minimally blocking the Gq pathway, intracellular calcium mobilization and platelet activation.
Given this unique mechanism of activation, progress in the development of thrombin receptor inhibitors has been slow, and limited to strategies aimed at blocking the extracellular regions6,
7. Now, Covic et al.2 have generated peptides corresponding to human PAR1 and PAR4 i3 loops as novel antagonists of thrombin signaling. The researchers chemically linked the peptides to a hydrophobic palmitoyl group, which ferries the peptides across the lipid bilayer and simultaneously serves as an anchor that embeds the peptides into the intracellular lipid interface. These palmitoylated peptides are called pepducins, and have the potential to display either agonist or antagonist properties, as previously shown for a limited number of G protein−coupled receptors8.
The authors demonstrated that PAR1-based pepducins blocked platelet aggregation in response to low-dose thrombin. They did not work in the presence of high-dose thrombin—as predicted because of persistent PAR4 activation. Somewhat unexpectedly, however, PAR4-based pepducins displayed platelet inhibitory effects to fully saturating concentrations of thrombin—concentrations capable of activating both PAR1 and PAR4. These initial in vitro results were subsequently confirmed in vivo. In mice, preinfusion of the PAR4 pepducin effectively prolonged the bleeding time, while partially inhibiting systemic platelet activation.
What is the general mechanism of action of pepducins? And why should PAR4-based pepducins have such global effects on thrombin activation? On the simplest level, pepducins may function as dominant-negative PAR inhibitors by blocking interaction of PAR1/PAR4 i3 loops with their cognate G proteins. This could occur through a functional interaction of the pepducin with its homologous segment within the intact i3 loop. Alternatively, the micromolar excess of intracellular pepducins may simply function as 'sinks' that efficiently bind effector G proteins, obviating a need for direct receptor interaction to block downstream effector proteins. Both models imply a direct interaction of pepducins with G proteins.
The cross-inhibitory effects of PAR4 pepducins on PAR1 activation may provide clues to alternative molecular mechanisms. The authors note that a homologous stretch of basic residues is found within both PAR1 and PAR4 i3 loops that would be predicted to function in G protein coupling. Although this would explain why a PAR4-based pepducin might simultaneously inhibit PAR1 signaling, other intracellular segments also contain key recognition sequences for G protein coupling3,
9, suggesting that the mechanism may be more complex. More speculative mechanisms could incorporate functional cooperativity between PARs, possibly by physical interactions such as heterodimerization10. The latter explanations are intriguing possibilities, given prior evidence for PAR3's function as a cofactor in murine PAR4 signaling4, and evidence that PAR1 and PAR2 are functionally coupled in vascular endothelial cells11. Finally, although C-terminal domains have been implicated in the formation of receptor heterodimers12, no evidence exists that the i3 loop may participate in comparable functions (as might be suggested by this study). Given the potential importance of pepducins in drug development, it is likely that continued research in this area will provide a clearer explanation for their functional effects.
If further developed, it is possible that selective thrombin receptor inhibitors such as PAR1/PAR4 pepducins may be used as adjunctive therapies in the future to prevent thrombus formation. The rationale for the approach includes evidence that an antibody against PAR1 can inhibit experimental arterial thrombosis in non-human primates13. Other research suggests that clot-bound -thrombin may remain active for up to two weeks, as a continual stimulus for platelet activation.
Nonetheless, a number of challenges persist. The optimal therapy should effectively inhibit both PAR1 and PAR4. As PAR1-based pepducins have minimal efficacy in inhibiting PAR4 activation, PAR4-optimized pepducins (which appear to affect thrombin signaling through both receptors) may be superior agents. Although the PAR4 pepducin inhibited systemic platelet activation in this study, the pepducin awaits testing in a more formalized model of arterial thrombosis. Secondly, the PAR3/PAR4−thrombin receptor system in murine platelets seems to have a mechanism of activation distinct from that of human platelets in which PAR1 is the predominant thrombin receptor, with no evidence for PAR4 cooperativity. Thus, in vivo efficacy of PAR4-based pepducins as antiplatelet agents in humans remains unestablished. One additional limitation could be the widespread distribution of thrombin receptors in humans. PAR1 is expressed in many human cells and tissues (including vascular endothelial cells), and its inhibition in these cells may be associated with unwanted side effects such as blocking the release of nitric oxide—a potent inhibitor of platelet activation.
More generally, however, pepducins represent a novel means for characterizing G protein−coupled receptors, a major target for drug development. Nearly 5% of the human genome encodes receptors, with G protein−coupled receptors representing the largest subcategory14. Given the general importance of these receptors in a wide-spectrum of cellular functions and human diseases, high-throughput strategies are the most effective approaches for determining function. Pepducins may provide unique tools to facilitate these studies.
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-thrombin
-subunits, often involving the third intracellular loop (i3)
-Thrombin.
G proteins, with the 
