Key Points Hemostatic plugs develop a regional architecture defined by the extent of platelet activation and packing density. The regional composition of a hemostatic plug is due to the interaction of local conditions with the platelet-signaling network.

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. ...

Hemostatic thrombi formed after a penetrating injury have a distinctive structure in which a core of highly activated, closely packed platelets is covered by a shell of less-activated, loosely packed platelets. We have shown that differences in intrathrombus molecular transport emerge in parallel with regional differences in platelet packing density and predicted that these differences affect thrombus growth and stability. Here we test that prediction in a mouse vascular injury model. The studies use a novel method for measuring thrombus contraction in vivo and a previously characterized mouse line with a defect in integrin αIIbβ3 outside-in signaling that affects clot retraction ex vivo. The results show that the mutant mice have a defect in thrombus consolidation following vascular injury, resulting in an increase in intrathrombus transport rates and, as predicted by computational modeling, a decrease in thrombin activity and platelet activation in the thrombus core. Collectively, these data (1) demonstrate that in addition to the activation state of individual platelets, the physical properties of the accumulated mass of adherent platelets is critical in determining intrathrombus agonist distribution and platelet activation and (2) define a novel role for integrin signaling in the regulation of intrathrombus transport rates and localization of thrombin activity.

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking cell surface receptors to intracellular signaling pathways. Gz is a member of the Gi family of G proteins that is prominently expressed in platelets and brain. Here, we show that deletion of the ␣ subunit of Gz in mice: (i) impairs platelet aggregation by preventing the inhibition of cAMP formation normally seen at physiologic concentrations of epinephrine, and (ii) causes the mice to be more resistant to fatal thromboembolism. Loss of Gz␣ also results in greatly exaggerated responses to cocaine, reduces the analgesic effects of morphine, and abolishes the effects of widely used antidepressant drugs that act as catecholamine reuptake inhibitors. These changes occur despite the presence of other Gi␣ family members in the same cells and are not accompanied by detectable compensatory changes in the level of expression of other G protein subunits. Therefore, these results provide insights into receptor selectivity among G proteins and a model for understanding platelet function and the effects of psychoactive drugs.H eterotrimeric guanine nucleotide binding proteins (G proteins) comprised of ␣, ␤, and ␥ subunits mediate diverse cellular responses by linking receptors on the cell surface to intracellular signaling pathways. At least 20 human genes are known to encode GTP-binding ␣ subunits. Half of these are members of the G i␣ family, including the ubiquitously expressed and nearly identical G i1␣ , G i2␣ , and G i3␣ , as well as several with restricted expression, such as G z␣ , G o␣ , and transducin. The best described effector for most G i family members is adenylyl cyclase. However, others exist as well, including cGMP phosphodiesterase, ion channels, phospholipase C, and Rap1GAP (1-5). One unresolved issue is the need for such a multiplicity of G i␣ family members, many of which are commonly expressed within the same cell. One approach to addressing this question has been the development of mice that lack individual family members. Of the three broadly expressed members of the family, mice lacking G i2␣ have the most striking phenotype with abnormalities of T cell function and thymocyte maturation, as well as an increased susceptibility to develop inflammatory bowel disease (6-8). Deletion of G i1␣ or G i3␣ , on the other hand, has not been reported to produce an obvious effect. Clearly, however, if there are functional differences among these and the other G i family members, then receptor selection among them is potentially a major determinant for cellular responses.Of the 10 known members of the G i␣ family, the sequence of G z␣ bears the least similarity to the others. G z␣ has a limited distribution in humans with prominent expression in blood platelets and selected areas of the brain. Like other members of the family, G z␣ has been shown to inhibit cAMP formation by adenylyl cyclase when over-expressed (9), but it is not known whether this is part of its role in vivo. Similarly, in over-expression systems, ...

Eph receptor tyrosine kinases and their ligands, ephrins, mediate neurodevelopmental processes such as boundary formation, axon guidance, vasculogenesis, and cell migration. We determined the expression profiles of the Eph family members in five glioma cell lines under migrating and nonmigrating conditions. EphB2 mRNA was overexpressed in all five during migration (1.2-2.8-fold). We found abundant EphB2 protein as well as strong phosphorylation of EphB2 in migrating U87 cells. Confocal imaging showed EphB2 localized in lamellipodia of motile U87 cells. Treatment with ephrin-B1/Fc chimera stimulated migration and invasion of U87, whereas treatment with a blocking EphB2 antibody significantly inhibited migration and invasion. Forced expression of EphB2 in U251 cells stimulated cell migration and invasion and diminished adhesion concomitant with the tyrosine phosphorylation of EphB2. U251 stably transfected with EphB2 showed more scattered and more pronounced invasive growth in an ex vivo rat brain slice. In human brain tumor specimens, EphB2 expression was higher in glioblastomas than in lowgrade astrocytomas or normal brain; patterns of phosphorylated EphB2 matched the expression levels. Laser capture microdissection of invading glioblastoma cells revealed elevated EphB2 mRNA (1.5-3.5-fold) in 7 of 7 biopsy specimens. Immunohistochemistry demonstrated EphB2 localization primarily in glioblastoma cells (56 of 62 cases) and not in normal brain. This is the first demonstration that migrating glioblastoma cells overexpress EphB2 in vitro and in vivo; glioma migration and invasion are promoted by activation of EphB2 or inhibited by blocking EphB2. Dysregulation of EphB2 expression or function may underlie glioma invasion.

Accumulating evidence reveals that genetic variants play pivotal roles in familial atrial fibrillation (AF). However, the molecular defects in most patients with AF remain to be identified. Here, we report on three novel KCNA5 mutations that were identified in 4 of 120 unrelated AF families. Among them, T527M was found in two AF families, and A576V and E610K in two other AF families, respectively. The mutations T527M and A576V were also detected in 2 and 1 of 256 patients with idiopathic AF, respectively. The same mutations were not observed in 200 secondary AF patients and 500 controls. Functional analyses revealed consistent loss-of-function effects of mutant KCNA5 proteins on the ultrarapidly activating delayed rectifier potassium currents. These findings expand the spectrum of mutations in KCNA5 linked to AF and provide new insight into the molecular mechanism involved in AF.

Summary Background In resting platelets, endothelial cell specific adhesion molecule (ESAM) is located in alpha granules, increasing its cell surface expression following platelet activation. However, the function of ESAM on platelets is unknown. Objective To determine whether ESAM has a role in thrombus formation. Methods and results We found that following platelet activation ESAM localizes to the junctions between adjacent platelets, suggesting a role for this protein in contact-dependent events that regulate thrombus formation. To test this hypothesis we examined the effect of ESAM deletion on platelet function. In vivo, ESAM−/− mice achieved more stable hemostasis than wild-type mice following tail transection, and developed larger thrombi following laser injury of cremaster muscle arterioles. In vitro, ESAM−/− platelets aggregated at lower concentrations of G protein-dependent agonists than wild-type platelets, and were more resistant to disaggregation. In contrast, agonist-induced calcium mobilization, αIIbβ3 activation, alpha-granule secretion and platelet spreading, were normal in ESAM-deficient platelets. To understand the molecular mechanism by which ESAM regulates platelet activity, we utilized a PDZ domain array to identify the scaffold protein NHERF-1 as an ESAM binding protein, and further demonstrated that it associates with ESAM in both resting and activated platelets. Conclusions These findings support a model in which ESAM localizes to platelet contacts following platelet activation in order to limit thrombus growth and stability so that the optimal hemostatic response occurs following vascular injury.

Platelet responses at sites of vascular injury are regulated by intracellular cAMP levels, which rise rapidly when prostacyclin (PGI 2 ) is released from endothelial cells. Platelet agonists such as ADP and epinephrine suppress PGI 2 -stimulated cAMP formation by activating receptors coupled to G i family members, four of which are present in platelets. To address questions about the specificity of receptor:G protein coupling, the regulation of cAMP formation in vivo and the contribution of G i -mediated pathways that do not involve adenylyl cyclase, we studied platelets from mice that lacked the ␣ subunits of one or more of the three most abundantly expressed G i family members and compared the results with platelets from mice that lacked the PGI 2 receptor, IP. As reported previously, loss of G i2␣ or G z␣ inhibited aggregation in response to ADP and epinephrine, respectively, producing defects that could not be reversed by adding an adenylyl cyclase inhibitor. Platelets that lacked both G i2␣ and G z␣ showed impaired responses to both agonists, but the impairment was no greater than in the individual knockouts. Loss of G i3␣ had no effect either alone or in combination with G z␣ . Loss of either G z␣ or G i2␣ impaired the ability of ADP and epinephrine to inhibit PGI 2 -stimulated adenylyl cyclase activity and caused a 40%-50% rise in basal cAMP levels, whereas loss of G i3␣ did not. Conversely, deletion of IP abolished responses to PGI 2 and caused cAMP levels to fall by 30%, effects that did not translate into enhanced responsiveness to agonists ex vivo. From these results we conclude that 1) cAMP levels in circulating platelets reflect ongoing signaling through G i2 , G z , and IP, but not G i3 ; 2) platelet epinephrine (␣ 2A -adrenergic) and ADP (P2Y12) receptors display strong preferences among G i family members with little evidence of redundancy; and 3) these receptor preferences do not extend to G i3 . Finally, the failure of ADP and epinephrine to inhibit basal, as opposed to PGI 2 -stimulated, cAMP formation highlights the need during platelet activation for G i signaling pathways that involve effectors other than adenylyl cyclase.