Tryptase is a serine protease secreted by mast cells that able to activate other cells. In the present studies we have tested whether these responses could be mediated thrombin receptors or PAR-2, two G-protein-coupled are activated proteolysis. When added peptide corresponding N terminus of tryptase cleaved at activating site, but higher concentrations it also downstream, as did trypsin, known activator PAR-2. Thrombin, factor Xa, plasmin, urokinase, plasma kallikrein, and tissue kallikrein...

The chemokine receptor CXCR4 is required, together with CD4, for entry by some isolates of HIV-1, particularly those that emerge late in infection. use these viruses likely has profound effects on viral host range and correlates the evolution immunodeficiency. Stromal cell-derived factor-1 (SDF-1), ligand CXCR4, can inhibit infection CXCR4-dependent viruses. To understand mechanism this inhibition, we used a monoclonal antibody specific to analyze phorbol esters SDF-1 surface expression...

The 2014 NIH Physician-Scientist Workforce (PSW) Working Group report identified distressing trends among the small proportion of physicians who consider research to be their primary occupation. If unchecked, these will lead a steep decline in size workforce. They include high rates attrition young investigators, failure maintain robust and diverse pipeline, marked increase average age physician-scientists, as older investigators have chosen continue working too few younger entered workforce...

PAR-2 is a second member of novel family G-protein-coupled receptors characterized by proteolytic cleavage the amino terminus, thus exposing tethered peptide ligand that autoactivates receptor. The physiological and/or pathological role(s) are still unknown. This study provides tissue-specific cellular localization in normal human tissues immunohistochemical techniques. A polyclonal antibody, PAR-2C, was raised against corresponding to terminal sequence SLIGKVDGTSHVTGKGV PAR-2. Significant...

Thrombin Receptor Activationteins suggest a three-dimensional model for the interaction of receptor N terminus with remainder that is compatible observed effects amino acid substitutions on platelet activation by peptide. MATERIALS AND METHODSPlatelet Aggregation and PAC1 Binding-Blood was obtained from healthy donors anticoagulated citrate (1 part 3.8% sodium to 9 parts blood).Platelet-rich plasma prepared centrifugation at 1000 X g 3 min.Platelet aggregation measured in Chrono-Log...

The recent identification of two new thrombin receptors, PAR3 and PAR4, led us to re-examine the basis for endothelial cell responses thrombin. Human umbilical vein cells (HUVEC) are known express PAR1 trypsin/tryptase receptor, PAR2. Northern blots detected both those receptors and, a lesser extent, PAR3, but PAR4 message was undetectable there no response agonist peptides. To determine whether or any other receptor contributes signaling in HUVEC, cleavage blocked with selective antibodies...

Shortly after activation by either thrombin or the tethered ligand domain peptide SFLLRN, receptors undergo homologous desensitization, temporarily losing their ability to respond both agonists. We have examined role of receptor internalization and recycling in this process using receptor-directed antibodies as probes. The results show within 1 min > 85% approximately 200,000 on megakaryoblastic human erythroleukemia (HEL) CHRF-288 cells are sequestered into endosomes via coated pits, which...

Two of the earliest known events in platelet activation include formation inositol 1,4,5-triphosphate (IP,) and Caz+ release from dense tubular system.Although mechanism which triggers Ca" system is unknown, recent evidence suggests that IP3 plays a role.In present studies, human platelets permeabilized with saponin were used to examine Ca2+ movements granule secretion response IPS.At low concentrations, caused complete loss cytosolic enzyme lactate dehydrogenase without liberating markers...

We have used platelets permeabilized with saponin to examine the mechanism by which platelet activation causes exposure of surface receptors for fibrinogen.Receptor was detected using 1261-fibrinogen and '261-PAC1, a monoclonal antibody specific activated form fibrinogen receptor.The potential mediators that were studied included guanyl-S'-yl imidodiphosphate (Gpp(NH)p) guanosine 5'0-(thiotriphosphate) (GTPyS), cause G proteindependent phospholipase C in platelets; inositol...

In platelets activated by thrombin, the hydrolysis of phosphatidylinositol 4,5-bisphosphate phospholipase C produces inositol 1,4,5-triphosphate (IP3) and diacylglycerol, metabolites which are known to cause Ca2+ release from platelet dense tubular system granule secretion. Previous studies suggest that activation is coupled thrombin receptors a guanine nucleotide-binding protein or G protein. The present examine contribution this thrombin-induced compare its properties with those Gi,...

The interaction of the chemokine stromal cell-derived factor 1 (SDF-1) with its receptor CXCR4 is vital for cell trafficking during development, capable inhibiting human immunodeficiency virus type (HIV-1) utilization as a coreceptor, and has been implicated in delaying disease progression to AIDS vivo. Because importance this chemokine-chemokine pair both development disease, we investigated molecular basis between ligands SDF-1 HIV-1 envelope. Using chimeras mutants, determined that...

Abstract The effects of the pleiotropic serine protease thrombin on tumor cells are commonly thought to be mediated by receptor protease-activated 1 (PAR1). We demonstrate here that PAR1 activation has a role in experimental metastasis using anti-PAR1 antibodies ATAP2 and WEDE15, which block cleavage activation. Thrombin also stimulates chemokinesis human melanoma toward fibroblast conditioned media soluble matrix proteins. Thrombin-enhanced migration is abolished antibodies, demonstrating...

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 activation was explored here. Two isoforms of Akt were detected mouse platelets, with expression Akt2 being greater than Akt1. Deletion gene encoding impaired aggregation, fibrinogen binding, and granule secretion, especially response low concentrations agonists activate Gq-coupled receptors...

MD-PhD training programs provide an integrated approach for physician-scientists. The goal of this study was to characterize the career path taken by program alumni during past 40 years and identify trends that affect their success.In 2007-early 2008, 24 enrolling 43% current trainees representing half National Institutes Health-funded submitted anonymous data on 5,969 former trainees.The average enrolled 90 trainees, required 8.0 complete, had attrition rate 10%. Nearly all (95%) those who...

Heterotrimeric G proteins mediate the earliest step in cell responses to external events by linking surface receptors intracellular signaling pathways. z is a member of i family that prominently expressed platelets and brain. Here, we show deletion α subunit mice: ( ) impairs platelet aggregation preventing inhibition cAMP formation normally seen at physiologic concentrations epinephrine, ii causes mice be more resistant fatal thromboembolism. Loss zα also results greatly exaggerated...

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 activation was explored here. Two isoforms of Akt were detected mouse platelets, with expression Akt2 being greater than Akt1. Deletion gene encoding impaired aggregation, fibrinogen binding, and granule secretion, especially response low concentrations agonists activate Gq-coupled receptors...

Based upon its recently cloned nucleotide sequence, the human platelet thrombin receptor is thought to be formed by a single polypeptide chain with seven transmembrane domains and an extracellular N terminus that can cleaved thrombin. As yet, however, little known from studies of protein itself. To obtain such information, we have prepared monoclonal antibodies against peptide corresponding residues Ser42 through Phe55, domain immediately distal site cleavage By flow cytometry, all reacted...

Semaphorin 4D (sema4D; CD100) is an integral membrane protein and the ligand for two receptors, CD72 plexin-B1. Soluble sema4D has been shown to evoke angiogenic responses from endothelial cells impair monocyte migration, but origin of soluble sema4D, particularly at sites vascular injury, unclear. Here we show that platelets express both its receptors provide evidence these molecules promote thrombus formation. We also surface expression increases during platelet activation, followed by...

Preincubation of human platelets with activators protein kinase C such as phorbol 12-myristate 13-acetate (PMA) has been shown previously to attenuate the ability agonists both suppress formation cAMP and stimulate hydrolysis phosphoinositides. In present study, we have examined whether attenuation caused by PMA can be attributed phosphorylation alpha subunit(s) Gi, a GTP-binding regulatory implicated in several pathways signal transduction. was found promote proteins within...