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Department of Molecular Medicine, Cornell University, Ithaca NY 14853-6401
SUMMARY
Tryptophan hydroxylase 1 (Tph1) is essential for the production of serotonin. Tph1–/– mice have increased risk of thromboembolism, although there appears to be no observable change to the ultrastructure of the platelets in the knockout mice. The platelets are deficient in their ability to adhere to wounded surfaces and this arises from a decrement in secreted adhesive granular proteins. Surprisingly, the defective function of the platelets can be traced back to an impairment in the transamidation of Rab4 and RhoA by serotonin. During the normal process of platelet activation and granular protein secretion, these GTPases are activated; however, in the knockout mice, lack of serotonin translates into lack of platelet activation and adhesion, leading to increased likelihood of thrombosis.
Platelets, small cell fragments derived from the cortical cytoplasm of megakaryocytes, circulate in an inactive form and adhere specifically to the endothelial cell lining of damaged blood vessels, where they rapidly become activated. Platelet activation is accompanied by dramatic morphological changes and the release of internal contents from several types of secretory granules. Release of these intracellular stores serves to further recruit and activate more platelets at the site of injury. Platelet activation ultimately results in the formation of a fibrin-stabilized platelet plug, which prevents excessive bleeding. These reactions are an essential response to bleeding; however, inappropriate activation of platelets produces clots in blood vessels, causing thrombosis. Therefore, the mechanisms underlying platelet adhesion and activation are of major biomedical interest.
Platelets can be purified in large quantities, making them an excellent experimental system; their use was instrumental in the characterization of one of the first identified mitogens: platelet-derived growth factor. Because platelets are relatively simple cells, lacking a nucleus, they also provide a great system to study signal transduction events that are not governed by, or dependent on, alterations in gene expression. Platelet aggregation involves a complex set of pathways that require the coordinated activity of a variety of receptors, including integrins, leucine-rich repeat adhesion proteins, and GPCRs; aggregation also engages regulatory feedback loops and interplay between signal transduction pathways and a multitude of agonists and antagonists. Delineating the specific contribution of each factor in this complex cascade is a major challenge for the field. Although pharmacological and genetic approaches have uncovered certain pathways critical to platelet activation, it is also clear that our knowledge of platelet physiology is far from comprehensive (1). The availability of genetically modified mice offers a powerful tool to elucidate the impact of different players in the pathway of platelet activation and their role in platelet physiology (2, 3). Walther and colleagues have made terrific use of this approach to examine the role of serotonin (5-HT) in platelet activation using mice lacking the gene for tryptophan hydroxylase 1 (Tph1), the rate-limiting enzyme for 5-HT biosynthesis. Tph1–/– mice contain normal levels of 5-HT in the brain, but are depleted of the monoamine outside the CNS and so are excellent models for studies of the diverse effects that have been ascribed to 5-HT in peripheral tissues (4, 5). Platelets express a cell-surface receptor for 5-HT (5-HT2AR) (6) and a specific serotonin uptake receptor (SERT) (7), in addition to a monoamine transporter (8) that allows platelets to accumulate high concentrations of 5-HT (~ 65 mM) in dense granules (9). Dense granule release is an early event of platelet activation and is thought to occur prior to the exocytosis of the larger 
-granules, which contain a variety of proaggregatory proteins, including von Willebrand factor (vWf) (10, 11).
Platelets derived from Tph1–/– mice clearly demonstrate a requirement for intracellular 5-HT together with Ca2+ for the release of -granules during platelet activation. But what is the mechanism of action of 5-HT?
On the exterior of platelets, 5-HT is a substrate for Factor XIIIa, an enzyme with transglutaminase (TGase) activity (12). TGs are a family of enzymes of wide physiological importance (13), and catalyze the formation of isopeptide bonds between specific glutamine residues (acyl-donor), and either lysine or simple amine-containing compounds (amino-donor), in a Ca2+-dependent manner. In addition to serotonin, many other biologically active compounds containing primary amines, such as histamine and catecholamine can be utilized as substrates by TGases (14). Platelet Factor XIIIa uses 5-HT to transamidate ("serotonylation") vWf and other proaggregatory proteins released by platelets from -granules, enhancing their adhesive properties on the exterior of the platelet. Because platelets also contain high levels of intracellular TG (15), Walther and coworkers looked for substrates of intracellular TG that were transamidated with 5-HT. They focused their attention on small GTPases of the Ras superfamily because these are known substrates for bacterial TGases (16–18). Incubation of activated platelets with radiolabelled 5-HT revealed both high molecular mass targets of TG action, and low molecular mass proteins including the small GTPases RhoA and Rab4––members of the Ras superfamily that have been implicated in cellular responses involving the actin-mediated cytoskeleton and membrane trafficking, respectively (19).
Walther et al. found that intracellular platelet TGase covalently modifies RhoA and Rab4 with 5-HT at a position in the phosphate-binding site that is conserved among the sequences of all small Ras-related GTPases (20). A glutamic acid residue that is required for hydrolysis of the bound nucleotide occupies this site. Replacement or blockage of this active-site residue results in a mutant with diminished intrinsic GTPase activity. Such a mutation will stabilize the protein in the activated GTP-bound form of the molecule, which potently enhances its biological impact (19). Thus, with a single step, the platelet-activated response cascade activates both a Rho family member involved in mediating cytoskeletal rearrrangements, and a Rab family member involved in mediating granule exocytosis (21, 22).
One has to admire the elegance of this mechanism (Figure 1
), utilizing the commonality of GTPase activation mechanisms to coordinate their diverse downstream functions, in addition to utilizing the same signal transduction effector, TGase, to coordinate intra- and extracellular events.
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References
-granule secretion in platelets.
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