Semaphorin 4D (sema4D; CD100) is an integral membrane protein and the ligand for two receptors, CD72 and plexin-B1. Soluble sema4D has been shown to evoke angiogenic responses from endothelial cells and impair monocyte migration, but the origin of soluble sema4D, particularly at sites of vascular injury, has been unclear. Here we show that platelets express sema4D and both of its receptors and provide evidence that these molecules promote thrombus formation. We also show that the surface expression of sema4D and CD72 increases during platelet activation, followed by the gradual shedding of the sema4D extracellular domain. Shedding is blocked by metalloprotease inhibitors and abolished in mouse platelets that lack the metalloprotease ADAM17 (TACE). Mice that lack sema4D exhibit delayed arterial occlusion after vascular injury in vivo, and their platelets show impaired collagen responses in vitro. In resting platelets, as in B lymphocytes, CD72 is associated with the protein tyrosine phosphatase SHP-1. Platelet activation causes dissociation of the complex, as does the addition of soluble sema4D. These findings suggest a dual role for sema4D in vascular responses to injury. As thrombus formation begins, platelet-associated sema4D can bind to its receptors on nearby platelets, promoting thrombus formation. As thrombus formation continues, sema4D is shed from the platelet surface and becomes available to interact with receptors on endothelial cells and monocytes, as well as continuing to interact with platelets.signaling ͉ thrombosis ͉ metalloprotease ͉ CD72 ͉ plexin-B1 P latelet activation typically begins with the exposure of collagen within a damaged vessel wall or the local generation of thrombin, but the establishment of a stable thrombus requires the recruitment of additional platelets and the development of stable contacts between platelets (1). Platelet activation also results in the release from platelets of molecules that can affect nearby cells, including endothelial cells and leukocytes as well as other platelets. In a continuing search for molecules that might contribute to contact-dependent events during thrombus formation, we screened human platelets for members of the semaphorin family. Although sempahorins are best known as regulators of neurite outgrowth and vascular development, individual family members have been shown to participate in a variety of events. Class IV semaphorin [semaphorin 4D (sema4D; CD100)] is a type I integral membrane protein first reported on T cells where it supports B cell development by binding to CD72 (2-4). However, sema4D receptors are not limited to B cells. Prior work has shown that a soluble sema4D extracellular domain fragment can activate endothelial cells by its other known receptor, plexin-B1. This causes endothelial migration, actin rearrangement, and the formation of tube-like structures in vitro, responses that are relevant for wound healing and angiogenesis (5-11). Soluble sema4D has also been shown to inhibit monocyte (12) and dendritic cell (13) migration. ...

Platelets are essential for normal hemostasis, but close regulation is required to avoid the destructive effects of either inappropriate platelet activation or excessive responses to injury . Here, we describe a novel complex comprising the scaffold protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1, and show that it can modulate platelet activation by sequestering RGS10 and RGS18, 2 members of the regulator of G protein signaling family. We also show that SPL/ RGS/SHP1 complexes are present in resting platelets where constitutive phosphorylation of SPL(Y398) creates an atypical binding site for SHP-1. Activation of the SHP-1 occurs on agonist-induced phosphorylation of SHP-1(Y536), triggering dephosphorylation and decay of the SPL/ RGS/SHP1 complex. Preventing SHP-1 activation blocks decay of the complex and produces a gain of function. Conversely, deleting spinophilin in mice inhibits platelet activation. It also attenuates the rise in platelet cAMP normally caused by endothelial prostacyclin (PGI 2 ). Thus, we propose that the role of the SPL/RGS/ SHP1 complex in platelets is time and context dependent. Before injury, the complex helps maintain the quiescence of circulating platelets by maximizing the impact of PGI 2 . After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs. IntroductionPlatelet responses to most agonists are mediated by G proteincoupled receptors, giving rise to the intracellular events that trigger platelet aggregation and granule exocytosis. 1 It has been known for some time that signaling by G proteins in platelets is subject to regulation by extrinsic factors arising from endothelial cells, especially nitric oxide and prostacyclin (PGI 2 ). 2 However, intrinsic modulators of platelet activation also exist, including members of the RGS (regulator of G protein signaling) family, 3 proteins that suppress G protein signaling by accelerating the hydrolysis of GTP bound to active G ␣ . 4,5 In contrast to nitric oxide and PGI 2 , RGS proteins are thought to have their effect once activation has begun; hence, the gain of function that we observed when an RGSinsensitive variant of G i2␣ was introduced into platelets. 3 This inhibitory role for RGS proteins produces a potential conundrum: although preventing unwarranted platelet activation is desirable, preventing the rapid onset of the hemostatic response to injury is not. We have, therefore, sought the means by which the onset of signal suppression by RGS proteins can be delayed, allowing signaling to begin. That search brought us to spinophilin (SPL or neurabin-II), a 130-kDa scaffold protein originally identified in screens for brain proteins that can bind to the serine/ threonine phosphatase, PP1, 6 and F-actin, 7 and subsequently found to associate with other proteins as well, 8 including a limited set of G protein-coupled receptors and RGS proteins. [9][10][11][12] Prior evidence suggests that one reg...

The intracellular ability of the "10 -23" DNAzyme to efficiently inhibit expression of targeted proteins has been evidenced by in vitro and in vivo studies. However, standard conditions for kinetic measurements of the DNAzyme catalytic activity in vitro include 25 mM Mg 2؉ , a concentration that is very unlikely to be achieved intracellularly. To study this discrepancy, we analyzed the folding transitions of the 10 -23 DNAzyme induced by Mg 2؉ . For this purpose, spectroscopic analyzes such as fluorescence resonance energy transfer, fluorescence anisotropy, circular dichroism, and surface plasmon resonance measurements were performed. The global geometry of the DNAzyme in the absence of added Mg 2؉ seems to be essentially extended, has no catalytic activity, and shows a very low binding affinity to its RNA substrate. The folding of the DNAzyme induced by binding of Mg 2؉ may occur in several distinct stages. The first stage, observed at 0.5 mM Mg 2؉ , corresponds to the formation of a compact structure with limited binding properties and without catalytic activity. Then, at 5 mM Mg 2؉ , flanking arms are projected at right position and angles to bind RNA. In such a state, DNAzyme shows substantial binding to its substrate and significant catalytic activity. Finally, the transition occurring at 15 mM Mg 2؉ leads to the formation of the catalytic domain, and DNAzyme shows high binding affinity toward substrate and efficient catalytic activity. Under conditions simulating intracellular conditions, the DNAzyme was only partially folded, did not bind to its substrate, and showed only residual catalytic activity, suggesting that it may be inactive in the transfected cells and behave like antisense oligodeoxynucleotide.The typical DNAzyme, 1 known as the "10 -23" model, has tremendous potential in gene suppression for both target validation and therapeutic applications (1). It is capable of cleaving single-stranded RNA at specific sites under simulated physiological conditions and can be used to control even complex biological processes such as tumor angiogenesis. For example, DNAzymes to ␤1 and ␤3 mRNA reduced expression of targeted integrin subunits in endothelial cells and blocked proliferation, migration, and network formation in a fibrin and Matrigel™ matrix (2). In a cell culture system, a 10 -23 deoxyribozyme designed against 12-lipoxygenase mRNA specifically down-regulated expression of this protein and its metabolites, which are known to play a crucial role in tumor angiogenesis (3). Similarly, the DNAzyme to VEGFR2 mRNA cleaved its substrate efficiently and inhibited the proliferation of endothelial cells with a concomitant reduction of VEGFR2 mRNA and blocked tumor growth in vivo (4).The origins of the DNAzyme catalytic activity are not yet fully understood, but the observed rate enhancements probably are generated by a number of factors, including metal ion and nucleobase catalysis and local stereochemical effects. The 10 -23 DNAzyme has been developed using an in vitro selection strategy on the basis of it...

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