The platelet response to collagen is a primary event in hemostasis and thrombosis, but the precise roles of the numerous identified platelet collagen receptors remain incompletely defined. Attention has recently focused on glycoprotein VI (GPVI), a receptor that is expressed on platelets in association with a signaling adapter, the Fc receptor gamma chain (Fc R␥). Genetic and pharmacologic loss of GPVI function results in loss of collagen signaling in platelets, but studies to date have failed to demonstrate that GPVI-Fc R␥ expression is sufficient to confer collagen signaling responses. These results have led to the hypothesis that collagen responses mediated by GPVI-Fc R␥ may require the collagen-binding integrin ␣2␤1 as a co-receptor, but this model has not been supported by a recent study of mouse platelets lacking ␣2␤1. In the present study we have used a novel anti-GPVI monoclonal antibody to measure the level of GPVI on human platelets and to guide the development of GPVI-expressing cell lines to assess the role of GPVI in mediating platelet collagen responses. GPVI receptor density on human platelets appears tightly regulated, is independent from the level of ␣2␤1 expression, and significantly exceeds that on previously characterized GPVI-expressing RBL-2H3 cells. Using newly generated GPVI-expressing RBL-2H3 cells with receptor densities equivalent to that on human platelets, we demonstrate that GPVI expression confers both adhesive and signaling responses to collagen in a graded fashion that is proportional to the GPVI receptor density. These results resolve some of the conflicting data regarding GPVIcollagen interactions and demonstrate that 1) GPVI-Fc R␥ expression is sufficient to confer both adhesion and signaling responses to collagen, and 2) GPVI-mediated collagen responses are receptor density-dependent at the receptor levels expressed on human platelets.The response of platelets to vessel wall injury is a primary event in arterial thrombosis (1). Platelets respond to vessel wall injury via surface receptors that recognize exposed subendothelial matrix proteins, the most abundant of which is collagen. Collagen is unusual among platelet ligands because it mediates both platelet adhesion and platelet activation (2). Platelet adhesion to collagen initiates the thrombotic response, and platelet activation in response to collagen accelerates the thrombotic response through release of granule contents and activation of platelet integrins. The ability of collagen to mediate both of these steps suggests that the platelet response to collagen may be a key step in the regulation of arterial thrombosis. A molecular understanding of the platelet receptors that mediate collagen responses is therefore likely to shed light on the pathogenesis of common vascular diseases such as myocardial infarction and stroke.The receptors for collagen on the surface of platelets may be divided into those which interact indirectly through collagenbound von Willebrand factor (vWF), 1 including GPIb␣ and the integrin ␣ IIb ␤ 3 ...

The role of caspase-3 in photoreceptor degeneration was examined in a line of transgenic rats that carry a rhodopsin mutation S334ter. Photoreceptor degeneration in these animals is rapid. It is detected as early as postnatal day (PD) 8, and by PD 20, only one of the original 12 rows of nuclei remain in the outer nuclear layer. At PD 11 and 12, the number of photoreceptors dying per day reaches a peak of approximately 30% of the total photoreceptors in the retina. Coincident with this rapid degeneration is an increase in caspase-3-like activity as assessed by the cleavage of a fluorescent substrate N-acetyl-Asp-Glu-Val-Asp-aminomethylcoumarin and an increase in activated caspase-3 as determined by Western blot analysis for its 12 kDa subunit. Intraocular injection of an irreversible caspase-3 inhibitor N-benzyloxycarbonal-Asp(OMe)-Glu(OMe)-Val-Asp(Ome)-fluoromethyk etone partially protected photoreceptors from degeneration. These findings indicate that a caspase-3-dependent mechanism is operative in photoreceptor death in the transgenic rats under investigation.

Constant exposure to bright light induces photoreceptor degeneration and at the same time upregulates the expression of several neurotrophic factors in the retina. At issue is whether the induced neurotrophic factors protect photoreceptors. We used a preconditioning paradigm to show that animals preconditioned with bright light became resistant to subsequent light damage. The preconditioning consisted of a 12-48 hr preexposure, followed by a 48 hr "rest phase" of normal cyclic lighting. The greatest protection was achieved by a 12 hr preexposure. Preconditioning induces a prolonged increase in two endogenous neurotrophic factors: basic fibroblast growth factor (bFGF) and ciliary neurotrophic factor (CNTF). It also stimulates the phosphorylation of extracellular signal-regulated protein kinases (Erks) in both photoreceptors and Müller cells. These findings indicate that exposure to bright light initiates two opposing processes: a fast degenerative process that kills photoreceptors and a relatively slower process that leads to the protection of photoreceptors. The extent of light damage, therefore, depends on the interaction of the two processes. These results also suggest a role of endogenous bFGF and CNTF in photoreceptor protection and the importance of Erk activation in photoreceptor survival.

The mechanism of signal transduction underlying the activation of platelets by collagen has been actively investigated for over 30 years, but the receptors involved remain incompletely understood. Studies of human platelets, which are unresponsive to collagen, mouse knockout models, and platelet biochemical studies support the hypothesis that the recently cloned platelet surface protein GPVI functions as a signaling receptor for collagen. To directly test this hypothesis, we have expressed wild-type and mutant forms of GPVI in RBL-2H3 cells, which express the Fc⑀ receptor ␥-chain (Fc R␥), the putative signaling co-receptor for GPVI in platelets, but lack GPVI itself. Expression of GPVI in RBL-2H3 cells confers strong adhesive and signaling responses to convulxin (a snake venom protein that directly binds GPVI) and weak responsiveness to collagenrelated peptide but no responsiveness to collagen. To elucidate the mechanism of GPVI intracellular signaling, mutations were introduced in the receptor's transmembrane domain and C-terminal tail. Unlike reported studies of other Fc R␥ partners, these studies reveal that both the GPVI transmembrane arginine and intracellular C-tail are necessary for coupling to Fc R␥ and for signal transduction. To our knowledge, these studies are the first to demonstrate a direct signaling role for GPVI and the first to directly test the role of GPVI as a collagen receptor. Our results suggest that GPVI may be necessary but not sufficient for collagen signaling and that a distinct ligand-binding collagen receptor such as the ␣ 2 ␤ 1 integrin is likely to play a necessary role for collagen signaling as well as adhesion in platelets.Platelet activation is essential for both normal hemostasis and arterial thrombosis that occurs in the setting of vascular diseases such as stroke and myocardial infarction. One of the earliest steps in arterial thrombosis is the adhesion of circulating platelets to areas of injured vessel wall and the activation of adherent platelets, which recruits additional platelets to form a hemostatic plug. Activation of platelets at sites of vascular injury occurs in response to exposed subendothelial matrix proteins, the most important of which is collagen.Exposed collagen initiates two essential platelet functions: the adhesion of circulating platelets to the site of injury and the activation of platelet signaling, which stimulates thrombus growth. Platelet adhesion to collagen has been shown to occur both indirectly, via interaction of platelet GPIb with plasma von Willebrand's factor bound to exposed collagen (1), and directly, via collagen interaction with the platelet integrin ␣ 2 ␤ 1 (2). In contrast, although the activation of platelets by collagen has been observed for over 30 years (3), the receptors and signaling pathways that mediate platelet activation by collagen are only beginning to be fully understood. Indirect evidence suggests that both ␣ 2 ␤ 1 and GPIb can initiate signaling when bound to collagen (1, 4, 5). However, this signaling does not appear to...

The platelet collagen receptor glycoprotein VI (GPVI) couples to the immune receptor adaptor Fc receptor ␥-chain (FcR␥) and signals using many of the same intracellular signaling molecules as immune receptors. Studies of immune receptor signaling have revealed a critical role for specialized areas of the cell membrane known as lipid rafts, which are enriched in essential signaling molecules. However, the role of lipid rafts in signaling in nonimmune cells such as platelets remains poorly defined. This study shows that GPVI-FcR␥ does not constitutively associate with rafts, but is recruited to lipid rafts following receptor stimulation in both GPVI-expressing RBL-2H3 cells and human platelets. FcR␥ is required for GPVI association with lipid rafts, as mutant GPVI receptors that do not couple to FcR␥ were unable to associate with lipid rafts after receptor clustering. Following GPVI stimulation in platelets, virtually all phosphorylated FcR␥ was found in lipid rafts, but inhibition of FcR␥ phosphorylation did not block receptor association with lipid rafts. This work demonstrates that lipid rafts orchestrate GPVI receptor signaling in platelets in a manner analogous to immune cell receptors and supports a model of GPVI signaling in which FcR␥ phosphorylation is controlled by ligand-dependent association with lipid rafts. Glycoprotein VI (GPVI)1 activates platelets through many of the same downstream kinases, adaptors, and effector molecules as Fc, T-cell, and B-cell receptors (1, 2). Like these immune receptors, GPVI is a multisubunit receptor in which the ligand-binding subunit (GPVI) is noncovalently associated with a signaling subunit (Fc receptor ␥-chain (FcR␥)) that contains an immunoreceptor tyrosine activation motif (3, 4). Cellular signaling by multisubunit immune receptors is initiated by receptor clustering (5), and platelet activation by GPVI is also believed to result from receptor clustering initiated by interaction with collagen (6) or the GPVI-specific ligand convulxin (CVX) (7). Precisely how clustering of immune receptors initiates signal transduction is not well understood, but one proposed mechanism is through receptor association with specialized areas of the cell membrane known as lipid rafts, which are enriched in signaling proteins such as Src family kinases and the transmembrane adaptor LAT (reviewed in Ref. 8).Lipid rafts, also known as detergent-resistant/insoluble membranes or glycolipid-enriched membranes, are areas of the cell membrane that are enriched in glycosphingolipids, saturated or near-saturated phospholipids, and intercalating cholesterol (9, 10). Lipid rafts are too small to be detected by standard microscopy, but they are resistant to solubilization at low temperature by nonionic detergents and have been isolated using density gradients (11, 12). Lipid rafts form distinct membrane compartments that exclude most membrane-associated proteins, but are enriched for some, including acylated Src family kinases such as Lyn and Fyn and palmitoylated adaptor proteins such as LAT (9,1...