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Lawrence F Brass

Publications and source records attributed to Lawrence F Brass.

18 recordsLinked to original sources
PubMed2007

An RNAi-based approach identifies molecules required for glutamatergic and GABAergic synapse development.

We report the results of a genetic screen to identify molecules important for synapse formation and/or maintenance. siRNAs were used to decrease the expression of candidate genes in neurons, and synapse development was assessed. We surveyed 22 cadherin family members and demonstrated distinct roles for cadherin-11 and cadherin-13 in synapse development. Our screen also revealed roles for the class 4 Semaphorins Sema4B and Sema4D in the development of glutamatergic and/or GABAergic synapses. We found that Sema4D affects the formation of GABAergic, but not glutamatergic, synapses. Our screen also identified the activity-regulated small GTPase Rem2 as a regulator of synapse development. A known calcium channel modulator, Rem2 may function as part of a homeostatic mechanism that controls synapse number. These experiments establish the feasibility of RNAi screens to characterize the mechanisms that control mammalian neuronal development and to identify components of the genetic program that regulate synapse formation and/or maintenance.

Animals↗
PubMed2006

Contact-dependent signaling events that promote thrombus formation.

There is increasing evidence that formation of a stable hemostatic plug requires adhesive and signaling events that continue beyond the onset of platelet aggregation. These events are facilitated and, in some cases, made possible, by the persistent close contacts between platelets that can only occur when platelets begin to aggregate. Participants include integrins and other cell adhesion molecules, secreted agonists, receptor tyrosine kinases, and protein fragments that are shed from the surface of activated platelets. Collectively, these molecules promote the continued growth and stability of the hemostatic plug.

Blood Platelets↗
PubMed2005

Eph kinases and ephrins support thrombus growth and stability by regulating integrin outside-in signaling in platelets.

The ability of activated platelets to adhere to each other at sites of vascular injury depends on the integrin alpha(IIb)beta(3). However, as aggregation continues, other signaling and adhesion molecules can contribute as well. We have previously shown that human platelets express on their surface the Eph receptor kinases EphA4 and EphB1 and the Eph kinase ligand ephrinB1. We now show that EphA4 is physically associated with alpha(IIb)beta(3) in resting platelets, increases its surface expression when platelets are activated, and colocalizes with alpha(IIb)beta(3) at sites of contact between platelets. We also show that Eph/ephrin interactions can support the stable accumulation of platelets on collagen under flow and contribute to postengagement "outside-in" signaling through alpha(IIb)beta(3) by stabilizing platelet aggregates and facilitating tyrosine phosphorylation of the beta(3) cytoplasmic domain. beta(3) phosphorylation allows myosin to bind to alpha(IIb)beta(3) and clot retraction to occur. The data support a model in which the onset of aggregation permits Eph/ephrin interactions to occur, after which signaling downstream from ephrinB1 and its receptors favors continued growth and stability of the thrombus by several mechanisms, including positive effects on outside-in signaling through alpha(IIb)beta(3).

Ephrin-B1↗
PubMed2005

Spontaneous fetal loss caused by placental thrombosis in estrogen sulfotransferase-deficient mice.

Estrogen sulfotransferase (EST, encoded by SULT1E1) catalyzes the sulfoconjugation and inactivation of estrogens. Despite decades of biochemical study and the recognition that high levels of estrogen sulfates circulate in the blood of pregnant and nonpregnant women, the physiological role of estrogen sulfation remains poorly understood. Here we show that ablation of the mouse Sult1e1 gene caused placental thrombosis and spontaneous fetal loss. This phenotype was associated with elevated free estrogen levels systemically and in the amniotic fluid, increased tissue factor expression in the placenta and heightened platelet sensitivity to agonist-induced activation ex vivo. Treatment of pregnant Sult1e1-null mice with either an anticoagulant or antiestrogen prevented the fetal loss phenotype. Our results thus identify Est as a critical estrogen modulator in the placenta and suggest a link between estrogen excess and thrombotic fetal loss. These findings may have implications for understanding and treating human pregnancy failure and intrauterine growth retardation.

Animals↗
PubMed2005

Minding the gaps to promote thrombus growth and stability.

Efforts to understand the role of platelets in hemostasis and thrombosis have largely focused on the earliest events of platelet activation, those that lead to aggregation. Although much remains to be learned about those early events, this Review examines a later series of events: the interactions between platelets that can only occur once aggregation has begun, bringing platelets into close contact with each other, creating a protected environment in the gaps between aggregated platelets, and fostering the continued growth and stability of the hemostatic plug.

Animals↗
PubMed2005

Did dinosaurs have megakaryocytes? New ideas about platelets and their progenitors.

Biological evolution has struggled to produce mechanisms that can limit blood loss following injury. In humans and other mammals, control of blood loss (hemostasis) is achieved through a combination of plasma proteins, most of which are made in the liver, and platelets, anucleate blood cells that are produced in the bone marrow by megakaryocytes. Much has been learned about the underlying mechanisms, but much remains to be determined. The articles in this series review current ideas about the production of megakaryocytes from undifferentiated hematopoietic precursors, the steps by which megakaryocytes produce platelets, and the molecular mechanisms within platelets that make hemostasis possible. The underlying theme that connects the articles is the intense investigation of a complex system that keeps humans from bleeding to death, but at the same time exposes us to increased risk of thrombosis and vascular disease.

Animals↗
PubMed2004

Boundary events: contact-dependent and contact-facilitated signaling between platelets.

The theme of this review is that formation of a stable hemostatic plug requires adhesive interactions and signaling events that continue beyond the initial phases of platelet aggregation. These interactions and events are facilitated and, in some cases made possible, by the persistent close contacts between platelets that can only occur after the onset of aggregation. The molecules that are involved include integrins, cell adhesion molecules, receptor tyrosine kinases, and ligands that are either attached to or shed from the surface of activated platelets. The picture that emerges is one in which events after aggregation are nearly as complex as those that precede aggregation and the initiation of platelet plug formation.

Animals↗
PubMed2004

Defects in secretion, aggregation, and thrombus formation in platelets from mice lacking Akt2.

Prior studies have shown that PI3Ks play a necessary but incompletely defined role in platelet activation. One potential effector for PI3K is the serine/threonine kinase, Akt, whose contribution to platelet activation was explored here. Two isoforms of Akt were detected in mouse platelets, with expression of Akt2 being greater than Akt1. Deletion of the gene encoding Akt2 impaired platelet aggregation, fibrinogen binding, and granule secretion, especially in response to low concentrations of agonists that activate the G(q)-coupled receptors for thrombin and thromboxane A(2). Loss of Akt2 also impaired arterial thrombus formation and stability in vivo, despite having little effect on platelet responses to collagen and ADP. In contrast, reducing Akt1 expression had no effect except when Akt2 was also deleted. Activation of Akt by thrombin was abolished by deletion of Galpha(q) but was relatively unaffected by deletion of Galpha(i2), which abolished Akt activation by ADP. From these results we conclude that Akt2 is a necessary component of PI3K-dependent signaling downstream of G(q)-coupled receptors, promoting thrombus growth and stability in part by supporting secretion. The contribution of Akt1 is less evident except in the setting in which Akt2 is absent.

Animals↗
PubMed2004

Protease-activated receptors (PAR1 and PAR2) contribute to tumor cell motility and metastasis.

The effects of the pleiotropic serine protease thrombin on tumor cells are commonly thought to be mediated by the thrombin receptor protease-activated receptor 1 (PAR1). We demonstrate here that PAR1 activation has a role in experimental metastasis using the anti-PAR1 antibodies ATAP2 and WEDE15, which block PAR1 cleavage and activation. Thrombin also stimulates chemokinesis of human melanoma cells toward fibroblast conditioned media and soluble matrix proteins. Thrombin-enhanced migration is abolished by anti-PAR1 antibodies, demonstrating that PAR1 cleavage and activation are required. The PAR1-specific agonist peptide TFLLRNPNDK, however, does not stimulate migration, indicating that PAR1 activation is not sufficient. In contrast, a combination of TFLLRNPNDK and the PAR2 agonist peptide SLIGRL mimics the thrombin effect on migration, whereas PAR2 agonist alone has no effect. Agonist peptides for the thrombin receptors PAR3 and PAR4 used alone or with PAR1 agonist also have no effect. Similarly, activation of PAR1 and PAR2 also enhances chemokinesis of prostate cancer cells. Desensitization with PAR2 agonist abolishes thrombin-enhanced cell motility, demonstrating that thrombin acts through PAR2. PAR2 is cleaved by proteases with trypsin-like specificity but not by thrombin. Thrombin enhances migration in the presence of a cleavage-blocking anti-PAR2 antibody, suggesting that thrombin activates PAR2 indirectly and independent of receptor cleavage. Treatment of melanoma cells with trypsin or PAR2 agonist peptide enhances experimental metastasis. Together, these data confirm a role for PAR1 in migration and metastasis and demonstrate an unexpected role for PAR2 in thrombin-dependent tumor cell migration and in metastasis.

Cell Line, Tumor↗
PubMed2003

Signaling by ephrinB1 and Eph kinases in platelets promotes Rap1 activation, platelet adhesion, and aggregation via effector pathways that do not require phosphorylation of ephrinB1.

We have previously shown that platelets express 2 receptor tyrosine kinases, EphA4 and EphB1, and the Eph kinase ligand, ephrinB1, and proposed that transcellular Eph/ephrin interactions made possible by the onset of platelet aggregation promote the further growth and stability of the hemostatic plug. The present study examines how this might occur. The results show that clustering of either ephrinB1 or EphA4 causes platelets to adhere to immobilized fibrinogen via alpha(IIb)beta(3). Adhesion occurs more slowly than with adenosine diphosphate (ADP) and requires phosphatidylinositol 3 (PI3)-kinase and protein kinase C activity but not ephrinB1 phosphorylation. By itself, Eph and ephrin signaling is insufficient to cause aggregation or the binding of soluble fibrinogen, but it can potentiate aggregation initiated by a Ca(++) ionophore or by agonists for thrombin and thromboxane receptors. It also enhances Rap1 activation without requiring ADP secretion, ephrinB1 phosphorylation, or the activation of PI3-kinase and Src. From this we conclude that (1) Eph/ephrin signaling enhances the ability of platelet agonists to cause aggregation provided that those agonists can increase cytosolic Ca(++); (2) this is accomplished in part by activating Rap1; and (3) these effects require oligomerization of ephrinB1 but not phosphotyrosine-based interactions with the ephrinB1 cytoplasmic domain.

Blood Platelets↗
PubMed2003

Thrombin receptors and protease-activated receptor-2 in human placentation: receptor activation mediates extravillous trophoblast invasion in vitro.

Proteolysis of the thrombin receptor, protease activated receptor-1 (PAR1), may enhance normal and pathological cellular invasion, and indirect evidence suggests that activation of PAR1 expressed by invasive extravillous trophoblasts (EVTs) influences human placentation. Here we describe PAR1, PAR2, and PAR3 protein distribution in the developing human placenta and implicate PAR1 and PAR2 activation in functions central to EVT invasion. PAR1, PAR2, and PAR3 are expressed in cultured 8- to 13-week-old EVTs, and in situ in 18- to 20-week-old placental syncytiotrophoblasts and invasive trophoblasts. Thrombin, but not the PAR2 agonist peptide SLIGKV, inhibited proliferation in cultured EVTs, although both agonists stimulated phosphoinositide hydrolysis and EVT invasion through Matrigel barriers. Thrombin-induced phosphoinositide hydrolysis was completely inhibited and the thrombin effect on proliferation was prevented when PAR1 cleavage was first blocked with specific monoclonal antibodies, indicating that PAR1 is the predominant thrombin receptor on EVTs. Together these results support a role for PAR1, and potentially PAR2 and PAR3 in the invasive phase of human placentation.

Cell Division↗
PubMed2003

Thrombin and platelet activation.

The accumulation of thrombin at sites of vascular injury provides one of the chief means for recruiting platelets into a growing hemostatic plug. Studies completed over the past 10 years show that platelet responses to thrombin are mediated by a subset of G protein-coupled receptors known as protease-activated receptors. These receptors are activated on cleavage by thrombin, initiating the intracellular signaling events needed to transform mobile, nonadhesive platelets into cells that can participate in the growth of an immobile hemostatic plug. How this is accomplished is the subject of this review.

Blood Platelets↗
PubMed2002

Signaling through Gi family members in platelets. Redundancy and specificity in the regulation of adenylyl cyclase and other effectors.

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 alpha 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)alpha or G(z)alpha 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)alpha and G(z)alpha showed impaired responses to both agonists, but the impairment was no greater than in the individual knockouts. Loss of G(i3)alpha had no effect either alone or in combination with G(z)alpha. Loss of either G(z)alpha or G(i2)alpha 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)alpha 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 (alpha(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.

Adenylyl Cyclases↗
PubMed2002

Interactions between Eph kinases and ephrins provide a mechanism to support platelet aggregation once cell-to-cell contact has occurred.

Eph kinases are receptor tyrosine kinases whose ligands, the ephrins, are also expressed on the surface of cells. Interactions between Eph kinases and ephrins on adjacent cells play a central role in neuronal patterning and vasculogenesis. Here we examine the expression of ephrins and Eph kinases on human blood platelets and explore their role in the formation of the hemostatic plug. The results show that human platelets express EphA4 and EphB1, and the ligand, ephrinB1. Forced clustering of EphA4 or ephrinB1 led to cytoskeletal reorganization, adhesion to fibrinogen, and alpha-granule secretion. Clustering of ephrinB1 also caused activation of the Ras family member, Rap1B. In platelets that had been activated by ADP and allowed to aggregate, EphA4 formed complexes with two tyrosine kinases, Fyn and Lyn, and the cell adhesion molecule, L1. Blockade of Eph/ephrin interactions prevented the formation of these complexes and caused platelet aggregation at low ADP concentrations to become more readily reversible. We propose that when sustained contacts between platelets have occurred in response to agonists such as collagen, ADP, and thrombin, the binding of ephrins to Eph kinases on adjacent platelets provides a mechanism to perpetuate signaling and promote stable platelet aggregation.

Adenosine Diphosphate↗
PubMed2002

Activation of Rap1B by G(i) family members in platelets.

It has become increasingly appreciated that receptors coupled to G(alpha)(i) family members can stimulate platelet aggregation, but the mechanism for this has remained unclear. One possible mediator is the small GTPase, Rap1, which has been shown to contribute to integrin activation in several cell lines and to be activated by a calcium-dependent mechanism in platelets. Here, we demonstrate that Rap1 is also activated by G(alpha)(i) family members in platelets. First, we show that platelets from mice lacking the G(alpha)(i) family member G(alpha)(z) (which couples to the alpha(2A) adrenergic receptor) are deficient in epinephrine-stimulated Rap1 activation. We also show that platelets from mice lacking G(alpha)(i2), which couples to the ADP receptor, P2Y12, exhibit reduced Rap1 activation in response to ADP. In contrast, platelets from mice that lack G(alpha)(q) show no decrease in the ability to activate Rap1 in response to epinephrine but show a partial reduction in ADP-stimulated Rap1 activation. This result, combined with studies of human platelets treated with ADP receptor-selective inhibitors, indicates that ADP-stimulated Rap1 activation in human platelets is dependent on both the G(alpha)(i)-coupled P2Y12 receptor and the G(alpha)(q)-coupled P2Y1 receptor. G(alpha)(i)-dependent activation of Rap1 in platelets does not appear to be mediated by enhanced intracellular calcium release because no increase in intracellular calcium concentration was detected in response to epinephrine and because the calcium response to ADP was not diminished in platelets from the G(alpha)(i2)-/- mouse. Finally, using human platelets treated with selective inhibitors of phosphatidylinositol 3-kinase (PI3K) and mouse platelets selectively lacking the G(beta)(gamma)-activated form of his enzyme (PI3Kgamma), we show that G(i)-mediated Rap1 activation is PI3K-dependent. In summary, activation of Rap1 can be stimulated by G(alpha)(i)- and PI3K-dependent mechanisms in platelets and by G(q)- and Ca(2+)-dependent mechanisms, both of which may play a role in promoting platelet activation.

Adenosine Diphosphate↗
PubMed2002

Extracellular signal-regulated kinases and g protein-coupled receptors in megakaryocytic human erythroleukemia cells: selective activation, differential regulation, and dissociation from mitogenesis.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are a group of kinases that play an important role in proliferation and differentiation. In megakaryocyte-like human erythroleukemia (HEL) cells, ERK2 was found to be predominantly expressed and strongly activated by prostaglandin (PG) E(2), thrombin, and epinephrine. On the other hand, adenosine, ADP, ATP, and UTP did not significantly increase ERK1/2 phosphorylation. However, of the agonists tested, only ADP was able to stimulate thymidine uptake. Pretreatment with pertussis toxin abolished the PGE(2) response but had less of an effect on thrombin. PGE(2)- and thrombin-induced ERK1/2 activation was mimicked by 4-beta-phorbol-12-myristate-13-acetate and ionomycin and blocked by mitogen-activated protein kinase kinase inhibitor 1,4 diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene but displayed differential sensitivity to protein kinase C inhibitor bisindolylmaleimide I and Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Analogs of cAMP or agents that stimulate cAMP production were either weak or ineffective activators. Further studies indicate that the effect of thrombin was blocked by the phosphoinositide 3-kinase inhibitor wortmannin but not by agents inhibiting tyrosine kinase activity. On the contrary, herbimycin, but not wortmannin, attenuated the effect of PGE(2). Collectively, these results indicate that ERK1/2 are selectively activated by G protein-coupled receptors and not functionally associated with proliferation in HEL cells. ERK1/2 activation in response to PGE(2) and thrombin is mediated by distinctive types of G proteins and is differentially regulated by multiple pathways, including calcium mobilization, protein kinase C, phosphoinositide 3-kinase, and tyrosine kinases.

Calcium↗