interacts_with DB00054?
Redistribution of membrane glycoproteins in platelets activated under flow conditions . A reduction in the ability of GPIb to bind specific MoAbs or ligands ( P04275 ) has been reported in platelets exposed to thrombin in suspension . We have analyzed modifications in the presence of glycoproteins ( GPs ) on platelets activated under flow conditions in a system which allows limited thrombin and fibrin generation . Normal blood anticoagulated with low molecular weight heparin ( LMWH , DB06779 20 IU/ml ) was recirculated for up to 10 min at 800 s-1 through annular chambers containing denuded arterial segments . Aliquots of blood were removed from the reservoir at 0 , 1 , 5 and 10 min and immediately mixed with paraformaldehyde . Membrane glycoproteins : GPIb ( CD42b ) , P08514 -IIIa ( CD41a ) , P16671 ( P16671 ) ; and activation dependent antigens : P16109 ( CD62P ) and lysosomal glycoprortein ( P08962 ) , were detected in whole blood by dual color flow cytometry . Circulation of through the perfusion system resulted in platelet activated as demonstrated by the increased percentage of platelets positive for antigens CD62P and P08962 . A gradual increase in the binding of MoAbs directed against GPIb , P08514 -IIIa , and P16671 epitopes was noted during the entire perfusion period . Observed differences in mean fluorescence intensities at all the observation times were statistically significant ( P < 0.001 ) . Our results obtained on platelets in an experimental thrombosis system indicate that GPIb , P08514 -IIIa and P16671 remain on the surface of activated platelets and actually increase their expression . Alterations detected at the level of GPIb in platelets activated by thrombin in suspension may not take place under in vivo situations . Platelet-derived microparticle formation involves glycoprotein IIb-IIIa . Inhibition by RGDS and a Glanzmann 's thrombasthenia defect . While the physiologic role of platelet microparticles may include a stable , physical dispersion of concentrated surface procoagulant activity the mechanism(s) of platelet vesiculation remains unknown . We demonstrate using flow cytometric methods a central role for the beta 3 integrin glycoprotein ( GP ) IIb-IIIa complex and its ligand tetrapeptide DB00125 - DB00145 - DB00128 - DB00133 ( RGDS ) binding site in platelet vesiculation . Time- and calcium-dependent vesiculation of platelets in response to ADP , collagen , thrombin , phorbol myristate acetate , and the thrombin peptide SFLLRN were dramatically inhibited , in a concentration-dependent manner , by monoclonal antibodies to P08514 -IIIa ( A2A9 , DB00054 , PAC1 ) and RGDS . Complete inhibition with A2A9 and RGDS occurred at 7.5 micrograms/ml and 75 microM , respectively , while control antibodies and a mock peptide had no effect . Platelet vesiculation requires intact P08514 -IIIa and is fully supported by the intracellular pool of P08514 -IIIa alone since de-complexing of this heterodimer by calcium chelation completely abolished microparticle formation in response to collagen ( no alpha-granule release ) but not to thrombin or SFLLRN . A central role for P08514 -IIIa is supported by the near total inability of Glanzmann 's thrombasthenic ( type I ) platelets to vesiculate in response to thrombin , ADP , collagen , and phorbol 12-myristate 13-acetate . This extends the biologic roles of P08514 -IIIa to include platelet vesiculation and suggests that one or all of its binding ligands play a role . The disulfide-rich region of platelet glycoprotein ( GP ) IIIa contains hydrophilic peptide sequences that bind anti- P05106 autoantibodies from patients with immune thrombocytopenic purpura ( ITP ) . Immune thrombocytopenic purpura ( ITP ) is an autoimmune blood disease caused by autoantibody-mediated destruction of blood platelets . Platelet glycoprotein ( GP ) IIb/IIIa is a common target for antiplatelet autoantibodies . The present studies were undertaken (1). to confirm whether the disulfide rich repeat region of P05106 contains target epitopes for antiplatelet antibodies in patients with ITP ; (2). to determine whether these antigens were defined by peptide sequences in the absence of post-translational modification ; and (3). to correlate observed immunologic reactivity with the recently solved X-ray crystallographic structure of an analogous integrin complex , the vitronectin receptor , alpha(V)beta(3) . Recombinant fusion proteins of four P05106 extracellular sequences were prepared and purified . Immunoblotting results with purified recombinant peptides showed potent reactivity of 16 of 24 ITP patient serum anti- P08514 /IIIa antibodies with the fusion protein containing the P05106 sequence of residues from 468 to 691 . These results are consistent with a report by Kekomaki et al. that a 50 kDa chymotryptic digestion product of P05106 isolated from blood platelets contains target epitopes for serum antiplatelet antibodies in 16 of 33 ITP patients . Smaller peptides including residues 446-501 and residues 593-691 each reacted with only 5 of the 24 patient sera ; furthermore all but 3 of these interactions were very weak . Visualization of the conformation of the extracellular portion of alpha(V)beta(3) reveals the location of the 222-residue antigenic P05106 ( beta(3) ) peptide ' B ' at the immediately extracellular region of the protein that includes a beta-tail domain and several integrin- P01133 domains . In summary , predictions of hydrophilicity , surface accessibility and antigenicity and the three dimensional structure of the beta(3) integrin correlate with autoantibody binding to a recombinant P05106 peptide ' B ' containing residues 468-691 . The development of human megakaryocytes . II . P01730 expression occurs during haemopoietic differentiation and is an early step in megakaryocyte maturation . P01730 expression is not limited to T cells and monocytes . In both mouse and man the antigen has been detected on some early haemopoietic progenitors and we have shown that some mature megakaryocytes ( MK ) express P01730 , the surface molecule that serves as the high-affinity receptor for human immunodeficiency virus type-1 ( HIV-1 ) . Using a serum-free culture system in which sorted P28906 + haemopoietic progenitors are cultured with thrombopoietin ( P07202 ) , P08700 , P05231 and P21583 , we now show that P01730 expression is induced in virtually all developing haemopoietic cells . This phenomenon was particularly striking in the MK lineage , where P01730 expression began whilst the cells were still P28906 + but after they expressed CD41 ( P08514 /IIIa ) . P01730 expression and endomitotic polyploidization occur at the same time in MK development . In culture , maximum P01730 expression occurred 4-6 d after CD41 expression and lasted for a few days . Expression of P01730 declined gradually thereafter and most MK were P01730 - by the end of the culture period . The amount of P01730 on the surface of some MK , as measured by intensity of fluorescence staining , exceeded that of normal monocytes and approached the brightness of T cells . Appearance of the surface antigen correlated with the presence of mRNA for P01730 , as measured by RT-PCR . Inhibition of cholinergic signaling causes apoptosis in human bronchioalveolar carcinoma . Recent case-controlled clinical studies show that bronchioalveolar carcinomas ( BAC ) are correlated with smoking . DB00184 , the addictive component of cigarettes , accelerates cell proliferation through nicotinic acetylcholine receptors ( nAChR ) . In this study , we show that human BACs produce acetylcholine ( ACh ) and contain several cholinergic factors including acetylcholinesterase ( P22303 ) , choline acetyltransferase ( P28329 ) , choline transporter 1 ( Q9GZV3 , Q9GZV3 ) , vesicular acetylcholine transporter ( Q16572 , Q16572 ) , and nACh receptors ( AChRs , CHRNAs ) . DB00184 increased the production of ACh in human BACs , and ACh acts as a growth factor for these cells . DB00184 -induced ACh production was mediated by α7- , α3β2- , and β3-nAChRs , P28329 and Q16572 pathways . We observed that nicotine upregulated P28329 and Q16572 . Therefore , we conjectured that Q16572 antagonists , such as vesamicol , may suppress the growth of human BACs . Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models . Vesamicol did not have any effect on P01133 or insulin-like growth factor-II-induced growth of human BACs. siRNA-mediated attenuation of Q16572 reversed the apoptotic activity of vesamicol . We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol . Taken together , our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy . Chronic daily tadalafil prevents the corporal fibrosis and veno-occlusive dysfunction that occurs after cavernosal nerve resection . OBJECTIVES : To determine whether a long-term single daily oral dose of a longer half-life phosphodiesterase-5 ( O76074 ) inhibitor , tadalafil , has a similar effect to that of the shorter half-life O76074 inhibitors sildenafil and vardenafil , and can prevent the fibrosis and resultant corporal veno-occlusive dysfunction ( CVOD ) occurring after cavernosal nerve ( CN ) injury . MATERIALS AND METHODS : Male rats ( 10 per group ) had either a sham operation , unilateral CN resection ( P21554 ) or bilateral P21554 , and were left untreated or given retrolingually 5 mg/kg per day of tadalafil . After 45 days , CVOD was assessed via cavernosometry , and the underlying corporal tissue changes were examined by immunohistochemistry and histochemistry ( followed by quantitative image analysis ) , Western blots , and ad hoc methods . RESULTS : DB00820 treatment normalized the low response to papaverine and high drop rate in the intracavernosal pressure measured by cavernosometry after P21554 compared with sham-operated rats . DB00820 also normalized the increase in penile shaft collagen content , and the reduction in corporal smooth muscle cell ( SMC ) content , SMC/collagen , and replication index , and improved the lower collagen III/I ratio and the increase in apoptotic index , caused by P21554 , compared with sham operation . There were no effects of tadalafil on increased transforming growth factor beta1 , inducible nitric oxide synthase and xanthine oxidoreductase levels . CONCLUSIONS : A long-term single daily dose of tadalafil prevented CVOD and the underlying corporal fibrosis in the rat caused by CN damage , as effectively as the previously reported continuous treatment with vardenafil or sildenafil , through a cGMP-related mechanism that appears to be independent of inducible nitric oxide synthase induction . Purification and characterization of platelet aggregation inhibitors from snake venoms . Proteins that inhibit glycoprotein ( GP ) IIb/IIIa mediated platelet aggregation have been purified from the venom of two snake species . A small platelet aggregation inhibitor ( p1.AI ) , multisquamatin ( Mr = 5,700 ) , was purified from Echis multisquamatus venom by hydrophobic interaction HPLC and two steps on C18 reverse phase HPLC . A larger p1.AI , contortrostatin ( Mr = 15,000 ) , was purified by a similar HPLC procedure from the venom of Agkistrodon contortrix contortrix . Both p1.AIs inhibit ADP-induced human , canine and rabbit platelet aggregation using platelet rich plasma ( PRP ) . Multisquamatin has an IC50 of 97 nM , 281 nM and 333 nM for human , canine and rabbit PRP , respectively . Contortrostatin has an IC50 of 49 nM , 120 nM and 1,150 nM for human , canine and rabbit PRP , respectively . In a competitive binding assay using 125I- DB00054 ( a monoclonal antibody to P08514 /IIIa that inhibits platelet aggregation ) both contortrostatin and multisquamatin demonstrated P08514 /IIIa specific binding to human and canine platelets . The IC50 for contortrostatin displacement of 7E3 binding to human and canine P08514 -/IIIa is 27 nM and 16 nM , respectively and for multisquamatin it is 3 nM and 63 nM , respectively . Our results indicate that both p1.AIs inhibit platelet aggregation by binding with high affinity to P08514 /IIIa . A novel mutation in P30518 causing congenital nephrogenic diabetes insipidus with complete resistance to antidiuretic hormone . A 6-month-old male infant presented with failure to thrive . Hypernatraemia and elevated serum osmolality in the presence of low urine sodium and osmolality led to the diagnosis of diabetes insipidus . Administration of DB00035 ( dDAVP ) neither decreased urine volume nor increased urine osmolality indicating congenital nephrogenic diabetes insipidus . Molecular analysis in the arginine-vasopressin receptor-2 gene ( P30518 ) located on chromosome Xq28 demonstrated a novel 5-base pair deletion ( c.962-966delACCCC ; g.1429-1433delACCCC ) leading to a shift of the reading frame ( p.Asn321fs ) and a premature termination codon implying an absent or non-functional protein . Treatment with hydrochlorothiazide , amiloride and indomethacin led to a favourable clinical course . Dynamics of P08514 /IIIa-mediated platelet-platelet interactions in platelet adhesion/thrombus formation on collagen in vitro as revealed by videomicroscopy . The conventional description of platelet interactions with collagen-coated surfaces in vitro , based on serial static measurements , is that platelets first adhere and spread to form a monolayer and then recruit additional layers of platelets . To obtain dynamic information , we studied gravity-driven platelet deposition in vitro on purified type 1 collagen by video phase-contrast microscopy at 22 degrees C . With untreated human and wild-type mouse platelets , soon after the initial adhesion of a small number of " vanguard " platelets , " follower " platelets attached to the spread-out vanguard platelets . Follower platelets then adhered to and spread onto nearby collagen or over the vanguard platelets . Thus , thrombi formed as a concerted process rather than as sequential processes . Treatment of human platelets with monoclonal antibody ( mAb ) DB00054 ( anti- P08514 /IIIa ( alphaIIbbeta3 ) + alphaVbeta3 ) or tirofiban ( anti- P08514 /IIIa ) did not prevent platelet adhesion but nearly eliminated the deposition of follower platelets onto vanguard platelets and platelet thrombi . Similar results were obtained with Glanzmann thrombasthenia platelets . Wild-type mouse platelets in the presence of mAb 1B5 ( anti- P08514 /IIIa ) and platelets from beta3-null mice behaved like human platelets in the presence of 7E3 or tirofiban . Deposition patterns of untreated human and wild-type mouse platelets were consistent with random distributions under a Poisson model , but those obtained with 7E3- and tirofiban-treated human platelets , 1B5-treated mouse platelets , or beta3-null platelets demonstrated a more uniform deposition than predicted . Thus , in this model system , absence or blockade of P08514 /IIIa receptors interferes with thrombus formation and alters the pattern of platelet deposition . Rapid and sustained coronary artery recanalization with combined bolus injection of recombinant tissue-type plasminogen activator and monoclonal antiplatelet P08514 /IIIa antibody in a canine preparation . The effects of bolus injections of recombinant single-chain tissue-type plasminogen activator ( rt-PA ) and of F(ab')2 fragments of a murine monoclonal antibody ( DB00054 ) against the human platelet P08514 /IIIa receptor [ 7E3-F(ab')2 ] on coronary arterial thrombolysis and reocclusion was studied in a canine preparation of coronary artery thrombosis superimposed on high-grade stenosis . Bolus intravenous injections of rt-PA at a dose of 0.45 mg/kg , repeated at 15 min intervals until reperfusion occurred ( maximum of four injections ) caused reperfusion in five of seven dogs within 100 min ( 33 +/- 15 min , mean +/- SD ) . Reperfusion was rapidly followed ( generally within 10 min ) by reocclusion and then by periods of cyclical reflow and reocclusion . A single intravenous injection of 7E3-F(ab')2 alone at 0.8 mg/kg caused reperfusion within 100 min in two of six dogs ( 19 and 37 min ) without subsequent reocclusion . Single bolus injections of different amounts ( 0.1 to 0.8 mg/kg ) of 7E3-F(ab')2 were then combined with bolus injections of 0.45 mg/kg of rt-PA . Stable reperfusion without reocclusion was accomplished with 0.8 or 0.6 mg/kg 7E3-F(ab')2 and a single injection of 0.45 mg/kg rt-PA within 6 +/- 3 min ( n = 6 , p less than .01 ) and 8 +/- 5 min ( n = 5 , p less than .02 ) , respectively . None of these animals suffered reocclusion of the coronary artery . Lower doses ( 0.1 to 0.2 mg/kg ) of 7E3-F(ab')2 did not significantly shorten the time to reperfusion and did not prevent reocclusion. ( ABSTRACT TRUNCATED AT 250 WORDS ) [ P08514 -IIIa inhibitors ] . Therapy involving the use of anti- P08514 -IIIa inhibitors has progressively evolved in recent years for patients undergoing percutaneous coronary intervention or with acute coronary syndromes . Patients receiving anti-GP IIb-IIIa therapy have a lower risk of death or myocardial infarction than those receiving the classic anti-agregant , aspirin , alone . Two classes of products have been used in clinic , the chimeric monoclonal antibody Fab fragment , c7E3 or abciximab ( ReoPro ) , which has been the pioneer , and synthetic peptides or peptidomimetics such as eptifibatide ( Integrilin ) or tirofiban ( Agrastat ) . DB00054 is a long-acting , high-affinity receptor blocker , whereas eptifibatide and tirofiban have much shorter biological half-lives . Another property that differentiates these compounds is that the peptides bind exclusively to GP IIb-IIIa whereas c7E3 also binds to alpha v beta 3 , the vitronectin receptor . The potent inhibitory effect of these compounds increases the risk of bleeding . By carefully controlling the levels of heparin and by removing the sheath as early as possible , the hemorrhagic problems may be limited . Another potential complication is the rapid development of thrombocytopenia . The cause has yet to be found and for c7E3 no correlation with the development of HACA ( human anti-chimeric antibodies ) has been observed . Because of the chronic nature of coronary artery disease , evaluation of the readministration of c7E3 to the same patient two or even more times is under investigation . The first results do not show major problems . The best biological way to investigate the efficiency of anti- P08514 -IIIa has to be determined . Interestingly , a new point-of-care test has been proposed , while monoclonal antibodies are available that differentiate between nonoccupied and occupied P08514 -IIIa complexes . Conjugation and evaluation of 7E3 x P4B6 , a chemically cross-linked bispecific F(ab')2 antibody which inhibits platelet aggregation and localizes tissue plasminogen activator to the platelet surface . A bispecific F(ab')2 monoclonal antibody which recognizes both the platelet P08514 /IIIa receptor and human tissue plasminogen activator was produced to target tPA to platelets for enhancement of thrombolysis . A stable , thioether-cross-linked bispecific F(ab')2 ( 7E3 X P4B6 ) combining the P08514 /IIIa-specific monoclonal antibody DB00054 , which inhibits platelet aggregation , and a nonneutralizing anti-tPA monoclonal antibody ( P4B6 ) was produced . This was performed by coupling each of the parental Fab ' moieties with the homobifunctional cross-linker bis ( maleimido methyl ) ether ( BMME ) . 7E3 X P4B6 was sequentially purified using gel-filtration chromatography and hydrophobic interaction ( HIC ) HPLC . HIC was shown to completely resolve each of the parental F(ab')2 species from the bispecific one . 7E3 X P4B6 was shown to retain completely each of the parental immunoreactivities in P08514 /IIIa and tPA binding EIA 's . The bispecific antibody inhibited platelet aggregation in vitro at levels comparable to those for 7E3 Fab . Recruitment of tPA activity to washed human platelets was demonstrated using the S-2251 chromogenic substrate assay . 7E3 X P4B6 recruited 12-fold more tPA to the washed platelets than a mixture of the parental F(ab')2 molecules used as controls . Time course of the effects of a single bolus injection of F(ab')2 fragments of the antiplatelet P08514 /IIIa antibody 7E3 on arterial eversion graft occlusion , platelet aggregation , and bleeding time in dogs . The time course of the effects of a single intravenous bolus injection of 10 mg/kg aspirin or 0.8 mg/kg F(ab')2 fragments of the monoclonal antiplatelet glycoprotein IIb/IIIa receptor antibody DB00054 [ 7E3-F(ab')2 ] on arterial occlusion , platelet aggregation , and bleeding time was studied in 30 dogs with an everted ( inside out ) carotid arterial segment inserted into the femoral artery . In the absence of an antiplatelet agent , the eversion grafts occluded spontaneously with platelet-rich thrombus within 30 minutes . With aspirin , arterial occlusion persisting for 2 hours occurred in 5 of 10 dogs and cyclic occlusion and reflow in 4 animals ; arterial occlusion was observed in all dogs at 24 hours . With 7E3-F(ab')2 , arterial patency persisted throughout a 2-hour observation period in all of 10 dogs and for 24 hours in 4 of the 10 dogs . Contralateral eversion grafting 24 hours after aspirin or 7E3-F(ab')2 injection was associated with graft patency for 2 hours in 1 of 5 aspirin dogs and in 3 of 5 7E3-F(ab')2 dogs ; patency persisted for 24 hours . In dogs grafted 48 hours after aspirin or 7E3-F(ab')2 injection , patency at 24 hours was seen in 0 of 5 dogs given aspirin and 3 of 5 dogs given 7E3-F(ab')2 . The overall frequencies of arterial graft patency at 2 , 24 , 48 , and 72 hours after study drug injection were significantly higher in the 7E3-F(ab')2 groups than in the aspirin groups ( P < .0005 , n = 10 in each group ; P < .05 , n = 15 ; P < .005 , n = 15 ; and P = .05 , n = 5 , respectively ) . ( ABSTRACT TRUNCATED AT 250 WORDS ) A chimeric murine/human antibody Fab fragment directed against the platelet P08514 /IIIa receptor enhances and sustains arterial thrombolysis with recombinant tissue-type plasminogen activator in baboons . Inhibition of the platelet glycoprotein ( GP ) IIb/IIIa receptor with the murine monoclonal antibody 7E3 abolishes ex vivo platelet aggregation , reduces thrombogenicity , and sustains arterial recanalization with recombinant tissue-type plasminogen activator ( rt-PA ) . A chimeric murine/human Fab fragment of DB00054 ( c7E3-Fab ) has a markedly reduced immunogenicity , but its potency as an adjunct for thrombolysis with rt-PA has not been evaluated . The effects of a single intravenous bolus injection of aspirin ( 17 mg/kg ) or c7E3-Fab ( 0.45 mg/kg ) on thrombolysis and reocclusion induced with rt-PA were studied in groups of six baboons with femoral arterial thrombosis and superimposed high-grade stenosis . This dose of c7E3-Fab blocked 96 +/- 1 % of the platelet P08514 /IIIa receptors and abolished ADP-induced platelet aggregation . Bolus intravenous injections of rt-PA ( 0.25 mg/kg ) were repeated at 15-minute intervals until reperfusion occurred ( maximum of four injections ) . In the aspirin group , reperfusion was obtained within 51 +/- 16 minutes ( mean +/- SD ) but was rapidly followed by reocclusion within 6 +/- 9 minutes and by cyclic reflow and reocclusion . In the c7E3-Fab group , reperfusion was obtained within 25 +/- 8 minutes ( P < .01 versus aspirin group ) and was associated with a delayed reocclusion of 63 +/- 63 minutes ( P < .05 versus aspirin group ) . Template bleeding times remained unchanged in the aspirin/rt-PA group but were markedly prolonged ( to > 30 minutes ) in the c7E3-Fab/rt-PA group. ( ABSTRACT TRUNCATED AT 250 WORDS ) Neonatal platelets are less reactive than adult platelets to physiological agonists in whole blood . Previous studies have reported that the platelets of healthy term neonates have either diminished or normal reactivity compared to the platelets of adults . To circumvent the methodologic problems of previous studies , we used a whole blood flow cytometric method to study neonatal platelet reactivity to thrombin , a combination of ADP and epinephrine , and U46619 ( a stable thromboxane A2 analogue ) . Inclusion in the assay of the peptide GPRP ( an inhibitor of fibrin polymerization ) enabled us to study platelet reactivity to human alpha-thrombin in whole blood . Umbilical cord blood and day 1 peripheral blood were collected from 30 healthy term neonates and compared to peripheral blood from 20 normal adults . In whole blood samples without added agonist , there were no significant differences between neonates and adults in the platelet binding of monoclonal antibodies 6D1 ( GPIb-specific ) or DB00054 ( P08514 -IIIa complex-specific ) . As determined by P28222 ( a P16109 -specific monoclonal antibody ) , neither neonates nor adults had circulating degranulated platelets . However , in both cord and peripheral whole blood samples , neonatal platelets were significantly less reactive than adult platelets to thrombin , ADP/epinephrine , and U46619 , as determined by the extent of increase in the platelet surface expression of P16109 and the P08514 -IIIa complex , and the extent of decrease in the platelet surface expression of the GPIb-IX complex. ( ABSTRACT TRUNCATED AT 250 WORDS ) Prevention of rethrombosis after coronary thrombolysis in a chronic canine model . I . Adjunctive therapy with monoclonal antibody 7E3 F(ab')2 fragment . We examined the efficacy of the monoclonal antibody ( MoAb ) 7E3 F(ab')2 fragment , an inhibitor of the platelet glycoprotein (GP)IIb/IIIa receptor , to prevent coronary artery rethrombosis after successful thrombolysis with rt-PA . The circumflex coronary artery of anesthetized dogs was instrumented with a flow probe , an electrode , and a stenosis . After recovery from the surgical procedure , the animals were reanesthetized on post-operative day 9 , and vessel wall injury was induced with current applied to the intimal surface of the circumflex coronary artery . The resulting occlusive thrombus was aged for 30 min , and recombinant tissue plasminogen activator ( rt-PA ) was administered . The animals were allocated to receive either placebo or a single dose of DB00054 [ 0.8 mg/kg intravenous ( i.v. ) bolus ] as the sole adjunctive agent . Ex vivo platelet function and coronary artery blood flow velocity were recorded on each of 5 consecutive days . Reocclusion and mortality were reduced significantly in animals treated with 7E3 as compared with the placebo-treated group . Significant inhibition of ex vivo platelet aggregation persisted for 48 h after a single injection of DB00054 . The MoAb 7E3 F(ab')2 fragment is effective as the sole adjunctive agent with rt-PA for prevention of rethrombosis . The present study is unique in that it examined the efficacy of P08514 /IIIa inhibition in an experimental model for an extended time , demonstrating the duration of antiplatelet therapy required to prevent rethrombosis after thrombolysis . DB00054 pharmacodynamics are unaffected by antecedent therapy with other P08514 /IIIa antagonists in non-human primates . BACKGROUND : Tirofiban and eptifibatide are currently approved for the medical stabilization of non-ST segment elevation acute coronary syndromes . In patients undergoing percutaneous coronary intervention ( P05154 ) during infusion of these drugs , conversion to abciximab , which has long term proven clinical efficacy and cost-effectiveness , following P05154 may be desirable . The purpose of this study was to determine if the binding or pharmacodynamics of abciximab is affected by a prior infusion of either tirofiban or eptifibatide . METHODS : In vitro binding experiments were performed to determine if prior exposure to tirofiban or eptifibatide altered the affinity and extent of binding of abciximab to P08514 /IIIa . For in vivo experiments , cynomolgus monkeys were pretreated with a bolus and 18 hour infusion of saline , tirofiban , or eptifibatide . At the end of the initial treatment , a bolus and 12 hr infusion of abciximab was started without delay . Inhibition of platelet aggregation , P08514 /IIIa receptor blockade and abciximab pharmacokinetics were measured during and after both infusions . RESULTS : Equilibrium binding of abciximab in vitro was unaffected by tirofiban or eptifibatide . The extent and duration of abciximab inhibition of ex vivo platelet aggregation , receptor blockade , and abciximab pharmacokinetics in monkeys during and after the abciximab infusion were not affected by prior infusion of the animals with tirofiban or eptifibatide . CONCLUSIONS : In vitro and in vivo studies revealed that the molecular interaction of abciximab with the platelet P08514 /IIIa receptor is not altered by immediate prior exposure of platelets to small molecule P08514 /IIIa antagonists . These preclinical studies suggest that the efficacy of abciximab should not be impaired if it is initiated following termination of therapy with small molecule P08514 /IIIa antagonists . Contribution of platelets and the vessel wall to the antithrombotic effects of a single bolus injection of Fab fragments of the antiplatelet P08514 /IIIa antibody 7E3 in a canine arterial eversion graft preparation . The contribution of platelets and the vessel wall to the antithrombotic effects of a single intravenous bolus injection of 0.8 mg/kg Fab fragments of the monoclonal antiplatelet glycoprotein IIb/IIIa receptor antibody DB00054 ( 7E3-Fab ) , combined with continuous heparin anticoagulation ( 100 U/kg bolus and 50 U/kg per hour ) , was studied in a canine preparation consisting of an everted ( inside out ) carotid arterial segment that had been inserted into a transected femoral artery . In all 6 control dogs without antibody , persistent or transient eversion graft occlusion occurred during an initial 2-hour observation period , and 5 of the 6 grafts were occluded at 24 hours . In 6 dogs given 7E3-Fab 24 hours before receiving an everted carotid artery segment from a donor dog , cyclic occlusion and reflow occurred in all dogs , whereas the grafts were patent at the end of a 2-hour observation period in 5 of the 6 dogs ( P = .056 versus control ) . When transferred back to the donor dogs , the patient eversion segments showed brief periods of cyclic occlusion and reflow within 2 hours in 3 of 5 dogs ( P = .034 versus control ) , whereas all of the 5 eversion segments were patent at 24 hours ( P < .005 versus control ) . ( ABSTRACT TRUNCATED AT 250 WORDS ) Investigation of interaction of human platelet membrane components with anticoagulant drugs DB00054 and DB00063 . DB00054 ( Abci ) and eptifibatide ( Epti ) are antiaggregate drugs which may reduce thrombotic complications in acute coronary syndromes . The aim of this work was the investigation of the interaction between the phospholipid- P08514 /IIIa glycoprotein complex and Abci or Epti , and the influence of these drugs on the phospholipid ratio in the platelet membrane . The interaction between the phospholipid- P08514 /IIIa glycoprotein complex and antiaggregate drugs were investigated using the Surface Plasmon Resonance Imaging technique ( SPRI ) . Phospholipids phosphatidylinositol ( PI ) , phosphatidylserine ( PS ) , phosphatidylethanolamine ( PE ) , phosphatidylcholine ( PC ) and sphingomyelin ( SM ) were first immobilized onto the gold chip surface . The phospholipid ratio in the platelet membrane was determined by the HPLC . Only PI , PS , PE and PC were determined . Human platelets treated ' in vitro ' with Abci or Epti exhibit changes in the phospholipid ratio in the platelet membrane . The ratio of PS decreases and PC rises . The SPRI distinctly shows interactions between phospholipids and glycoprotein P08514 /IIIa , and between the phospholipid-glycoprotein P08514 /IIIa complex and Abci or Epti . The interaction between phospholipids and glycoprotein P08514 /IIIa is growing in the sequence : PI << SM < PE < PC < PS . The interaction between phospholipid-glycoprotein P08514 /IIIa complex and Abci/Epti is growing in the sequence : PS < PI < PC < PE < SM . SPRI was proved to be excellent tool for observation of such interactions . DB00054 as an adjunctive therapy for patients undergoing percutaneous coronary interventions . INTRODUCTION : Platelets play a central role in the pathophysiology of acute coronary syndromes ( ACS ) and activation of platelet glycoprotein ( GP ) IIb/IIIa receptor is critical to platelet aggregation . DB00054 , a human murine chimeric antibody to the P08514 /IIIa receptor , is an important biological therapy in the management of patients presenting with ACS . AREAS COVERED : The objective of this review is to define the role of abciximab in the management of ACS by interpreting the available data from randomized clinical trials using abciximab in various clinical scenarios , particularly in percutaneous coronary intervention ( P05154 ) . We also review different modes of delivery and describe the adverse effects of abciximab including thrombocytopenia . Where possible , we attempt to compare abciximab to the other available P08514 /IIIa inhibitors . We hope the reader will gain a better understanding of the benefits and risks of abciximab and the important role it has in the management of cardiology patients . EXPERT OPINION : DB00054 was a breakthrough drug in the management of high risk ACS patients undergoing P05154 . However , with newer available therapies and improvement in P05154 technology , dose and delivery of this drug have evolved as we try to extract maximum benefit while minimizing the adverse effects associated with it . O15554 channels participate in the EMT induced by O75365 in colorectal cancer . Studies have shown that phosphatase of regenerating liver-3 ( O75365 ) promotes the invasion , migration , and metastasis of human tumor cells by facilitating an epithelial-mesenchymal transition ( EMT ) . However , the mechanism by which O75365 induces tumor cell EMT is unknown . Our previous research revealed that O75365 promotes LoVo cell proliferation by up-regulating O15554 channels . In the current study , we explored the mechanism by which O75365 mediates EMT . We demonstrated that O75365 induced the expression of O15554 channels , leading to EMT and the down-regulation of P12830 . Further studies revealed that O15554 channels increased intracellular calcium levels and activated components of cell signaling downstream of calcium , including P62158 -kinase II and glycogen synthase kinase-3 beta ( P49841 ) , which increased Snail expression . Inhibiting O15554 with siRNA and TRAM-34 , a specific inhibitor , restored P12830 expression and inhibited Snail expression . These results implicated the up-regulation of O15554 channels in the O75365 -mediated induction of EMT and promotion of cancer metastasis . A new clinical evidence-based gene-environment interaction model of depression . In our current understanding of mood disorders , the role of genes is diverse including the mediation of the effects of provoking and protective factors . Different or partially overlapping gene sets play a major role in the development of personality traits including also affective temperaments , in the mediation of the effects of environmental factors , and in the interaction of these elements in the development of depression . Certain genes are associated with personality traits and temperaments including e.g. , neuroticism , impulsivity , openness , rumination and extroversion . Environmental factors consist of external ( early and provoking life events , seasonal changes , social support etc. ) and internal factors ( hormones , biological rhythm generators , comorbid disorders etc ) . Some of these environmental factors , such as early life events and some prenatal events directly influence the development of personality traits and temperaments . In the NEWMOOD cohort polymorphisms of the genes of the serotonin transporter , P08908 , P28222 and 5- Q13049 and endocannabinoid P21554 receptors , tryptophan hydroxylase , P16220 , P23560 and GIRK provide evidence for the involvement of these genes in the development of depression . Based on their role in this process they could be assigned to different gene sets . The role of certain genes , such as promoter polymorphisms of the serotonin transporter ( 5-HTTLPR ) and P21554 receptor has been shown in more than one of the above factors . Furthermore , gene-gene interactions of these promoters associated with anxiety suggest the application of these polymorphisms in personalized medicine . In this review we introduce a new model including environmental factors , genes , trait and temperament markers based on human genetic studies . Matrix metalloproteinases are differentially expressed in adipose tissue during obesity and modulate adipocyte differentiation . Matrix metalloproteinases ( MMPs ) are essential for proper extracellular matrix remodeling , a process that takes place during obesity-mediated adipose tissue formation . Here , we examine expression profiles and the potential role of MMPs and their tissue inhibitors ( TIMPs ) in adipose tissue remodeling during obesity . Expression patterns are studied by Northern blot and real-time PCR in two genetic models of obesity ( ob/ob and db/db mice ) and in a diet-induced model of obesity ( AKR mice ) . Of the MMPs and TIMPs studied , mRNA levels for P08253 , P08254 , P39900 , P50281 , Q99542 , and P01033 are strongly induced in obese adipose tissues compared with lean tissues . In contrast , P09237 and P35625 mRNAs are markedly decreased in obesity . Interestingly , enzymatic activities of P39900 and of a new identified adipocyte-derived 30-kDa metalloproteinase are enhanced in obese adipose tissue fractions , demonstrating that MMP/ P01033 balance is shifted toward increased matrix degradation in obesity . Finally , we analyze the modulation of P08253 , Q99542 , and P01033 during 3T3- Q9NUQ9 preadipocyte differentiation , and we explore the effect of inhibition of MMP activity on in vitro adipogenesis . We find that the synthetic MMP inhibitor BB-94 ( DB03880 ) decreases adipose conversion of 3T3- Q9NUQ9 and primary rat preadipocytes . BB-94 represses differentiation without affecting mitotic clonal expansion but prevents the early expression of P17676 , a transcription factor that is thought to play a major role in the adipogenic program . Such findings support a role for the MMP/ P01033 system in the control of proteolytic events and adipogenesis during obesity-mediated fat mass development . Glycoprotein IIb/IIIa antagonists induce apoptosis in rat cardiomyocytes by caspase-3 activation . The platelet integrin glycoprotein ( GP ) IIb/IIIa , which mediates platelet aggregation , has been the target for novel antiplatelet agents , the P08514 /IIIa antagonists . Several P08514 /IIIa antagonists have been developed based on the peptide RGDS present in adhesion proteins , including the principle ligand fibrinogen . The apoptosis enzyme , procaspase-3 , contains an RGD-recognition sequence and is activated by RGDS . We examined the effects of RGDS and several P08514 /IIIa antagonists on cell death and procaspase-3 activation in rat neonatal cardiomyocytes . These antagonists do not recognize rat integrins , yet RGDS , orbofiban , and xemilofiban induced dose-dependent apoptosis and procaspase-3 activation in cardiomyocytes over 72 h , particularly under hypoxic conditions . Scrambled peptide , the monoclonal antibody 7E3 or integrelin ( a peptide containing a KGD sequence ) , had little or no effect . Immunoprecipitation of procaspase-3 followed by treatment with the compounds showed that procaspase-3 was activated directly by RGDS , orbofiban , xemilofiban , and by monoclonal DB00054 , the latter demonstrating that compounds must enter cells to induce apoptosis through caspase activation . DB00063 had no effect . Binding studies with (3)H-SC52012B , a P08514 /IIIa antagonist analogue of orbofiban , showed no specific binding to cardiomyocytes , but the radioligand accumulated intracellularly over 72 h . (3)H-SC52012B also bound directly to human recombinant caspase-3 ( K(d) , 59 +/- 2 nm ) , and this was prevented by orbofiban , xemilofiban , and the monoclonal DB00054 but not by integrelin . Finally confocal microscopy showed that RGDS co-localized with caspase-3 inside the cell . These data show that RGDS and its mimetics induce cardiomyocyte apoptosis by direct activation of procaspase-3 . Potential future clinical applications for the P08514 /IIIa antagonist , abciximab in thrombosis , vascular and oncological indications . DB00054 ( ReoPro ) is a mouse-human chimeric monoclonal antibody Fab fragment of the parent murine monoclonal antibody DB00054 , and was the first of these agents approved for use as adjunct therapy for the prevention of cardiac ischemic complications in patients undergoing percutaneous coronary intervention ( P05154 ) . DB00054 binds with high avidity to both the non-activated and activated form of the P08514 /IIIa receptor of platelets , the major adhesion receptor involved in aggregation . Additional cardiovascular indications for abciximab are unstable angina , carotid stenting , ischemic stroke and peripheral vascular diseases . DB00054 also interacts with two other integrin receptors ; the a av b b3 receptor , which is present in low numbers on platelets but in high density on activated endothelial and smooth muscle cells , and a aMb b2 integrin which is present on activated leukocytes . Cell types that express integrins P08514 /IIIa and a av b b3 such as platelets , endothelial and tumor cells have been implicated in angiogenesis , tumor growth and metastasis . Since abciximab interacts with high avidity to integrins P08514 /IIIa and a av b b3 , it is reasonable to assume that it may possess anti-angiogenic properties in angiogenesis-related diseases , as well as anti-metastastatic properties in case of disseminating tumors expressing the target integrin receptors . Neutrophil P16109 -glycoprotein-ligand-1 binding to platelet P16109 enhances metalloproteinase 2 secretion and platelet-neutrophil aggregation . Platelets and neutrophils constitute a high source of metalloproteinases ( MMPs ) , and their interactions via P16109 and P16109 -glycoprotein-ligand-1 ( Q14242 ) are involved in thrombosis , vascular remodelling , and restenosis . We investigated the impact of these interactions on platelet P08253 secretion and function in platelet and neutrophil aggregation . The secretion of P08253 from human platelets was significantly increased three-fold after thrombin activation , and enhanced two-fold in the presence of neutrophils . Neutrophil supernatant had no effect on platelet P08253 secretion . While no P08253 was detected in the supernatant of neutrophils , a high amount of P14780 was released by neutrophils , and remained unchanged upon thrombin activation or in the presence of platelets . Platelet P16109 , which increased significantly after activation , triggered platelet binding to neutrophils that was completely inhibited by P16109 or Q14242 antagonists , and was reduced by 50 % with a P08514 / IIIa antagonist . P16109 or Q14242 antagonism abolished the enhanced secretion of platelet P08253 in the presence of neutrophils and reduced platelet-neutrophil aggregation . Platelet activation and binding to neutrophils enhance the secretion of platelet P08253 via an adhesive interaction between P16109 and Q14242 , which contribute to increase platelet-neutrophil aggregation . Comparative studies of a humanized anti-glycoprotein IIb/IIIa monoclonal antibody , YM337 , and abciximab on in vitro antiplatelet effect and binding properties . The effects of YM337 , the Fab fragment of a humanized anti-glycoprotein IIb/IIIa ( P08514 /IIIa ) monoclonal antibody C4G1 , on in vitro platelet function and binding properties were compared with those of abciximab , the Fab fragment of the human/murine chimeric anti- P08514 /IIIa monoclonal antibody DB00054 . Both agents completely inhibited platelet aggregation caused by all agonists tested except ristocetin . Further , both inhibited human platelet adhesion to P04275 , fibrinogen , fibronectin and subendothelial matrix with similar potency . DB09222 binding to washed platelets was dose-dependently inhibited by both agents . In binding assay using 125I-YM337 and 125I-abciximab , Kd values determined with platelet-rich plasma were 6.74 +/- 0.56 nM for YM337 and 6.65 +/- 1.45 nM for abciximab , and the number of binding sites were 42,700 +/- 3,000 for YM337 and 76,000 +/- 5,400 for abciximab . P08514 /IIIa was precipitated from the solubilized fraction of platelets by both agents . In contrast , integrin alphavbeta3 was precipitated from the solubilized fraction of human umbilical vein endothelial cells by abciximab but not by YM337 . DB09222 binding to purified P08514 /IIIa was dose-dependently inhibited by both agents . In contrast , vitronectin binding to purified integrin alphavbeta3 was dose-dependently inhibited by abciximab but not by YM337 , supporting the idea that abciximab reacts to integrin alphavbeta3 . Therefore , YM337 was suggested to bind to a different epitope of P08514 /IIIa from abciximab . These results suggest that YM337 specifically acts on platelet P08514 /IIIa receptors and has similar inhibitory properties on platelet aggregation and platelet adhesion to abciximab . Glycoprotein IIb/IIIa inhibition attenuates platelet-activating factor-induced platelet activation by reducing protein kinase C activity . Glycoprotein (GP)IIb/IIIa inhibition may abolish activated leukocyte-induced platelet activation , in which leukocyte-released platelet-activating factor ( Q15004 ) is a major mediator . The present study thus investigated if and how P08514 /IIIa inhibitors interfere with Q15004 -induced platelet activation . Platelet and leukocyte activation were monitored by flow cytometry and immunoblotting . P08514 /IIIa inhibitors ( c7E3 , non-peptide SR121566 , and MAb RFGP56 ) attenuated Q15004 -induced , but not adenosine diphosphate ( ADP ) - or thrombin receptor activating peptide ( TRAP ) -induced platelet P16109 expression in whole blood . P08514 /IIIa blockade enhanced ADP- or TRAP-induced leukocyte CD11b expression , but not the response to Q15004 . P08514 /IIIa blockade attenuated Q15004 -induced , but enhanced ADP- or TRAP-induced platelet-leukocyte aggregation . Under the present experimental conditions , thromboxane A2 receptor antagonism did not significantly influence Q15004 -induced platelet activation , and P08514 /IIIa inhibition did not interfere with calcium mobilization/influx in platelets . Protein kinase C ( PKC ) blockade inhibited Q15004 -induced platelet P16109 expression , and Q15004 -induced PKC activity was reduced by P08514 /IIIa inhibition . Q15004 ( =1 micro m ) did not induce Q02750 /2 or P29323 1/2 phosphorylation , whilst thrombin induced marked responses , which were enhanced by P08514 /IIIa blockade . Thus , P08514 /IIIa inhibition attenuates Q15004 -induced platelet activation via inhibiting PKC activity . P08514 /IIIa blockade enhances thrombin-induced platelet Q02750 /2 and P29323 1/2 activation , and augments ADP- and TRAP-induced leukocyte activation by enhancing platelet-leukocyte aggregation . Q13444 is an adhesion receptor for platelet P08514 -IIIa and induces platelet activation . Cell adhesion and proteolytic matrix degradation are central processes in atherosclerosis . Being a member of the family of ADAMs ( " a disintegrin and metalloproteinase " ) , metargidin ( Q13444 ) combines a metalloproteinase domain and an RGD aminoacid sequence . We studied the potential role of Q13444 as an adhesion receptor on endothelial cells and interactions between platelets and Q13444 with respect to platelet adhesion , activation and thrombus formation . Q13444 was found to be expressed on cultured endothelial cells ( HUVEC ) . Platelet adhesion to immobilized recombinant Q13444 was effectively enhanced under both static and high shear rate conditions reaching the maximum level of adhesion to fibrinogen . Consistently , platelet adhesion onto Q13444 overexpressing endothelial cells was significantly increased . Adhesion to Q13444 was reduced by blockade of P08514 -IIIa using neutralizing anti-alpha(IIb)beta3 mAbs ( DB00054 , 2G12 ) , but not by anti-alpha(v)beta3 ( LM609 ) . Soluble Q13444 binds to activated but not to resting P08514 -IIIa . Moreover , platelets adherent to Q13444 additionally attracted platelets under high shear rates indicating an initial role of platelet- Q13444 interactions for thrombus formation . Furthermore , incubation of platelets with soluble Q13444 showed a dose-dependent increase in secretion of CD62P and P29965 . Q13444 is expressed on endothelial cells and can serve as an adhesion receptor for platelets via P08514 -IIIa binding . Platelet adhesion to Q13444 leads to platelet activation , secretion and promotes thrombus formation . Thus , Q13444 may represent a novel target for antithrombotic strategies in cardiovascular pathologies . Antenatal maternally-administered phosphodiesterase type 5 inhibitors normalize P29474 expression in the fetal lamb model of congenital diaphragmatic hernia . PURPOSE : Pulmonary hypertension ( pHTN ) , a main determinant of survival in congenital diaphragmatic hernia ( Q8NE62 ) , results from in utero vascular remodeling . Phosphodiesterase type 5 ( O76074 ) inhibitors have never been used antenatally to treat pHTN . The purpose of this study is to determine if antenatal O76074 inhibitors can prevent pHTN in the fetal lamb model of Q8NE62 . METHODS : Q8NE62 was created in pregnant ewes . Postoperatively , pregnant ewes received oral placebo or tadalafil , a O76074 inhibitor , until delivery . Near term gestation , lambs underwent resuscitations , and lung tissue was snap frozen for protein analysis . RESULTS : Mean cGMP levels were 0.53±0.11 in placebo-treated fetal lambs and 1.73±0.21 in tadalafil-treated fetal lambs ( p=0.002 ) . Normalized expression of P29474 was 82 % ±12 % in Normal-Placebo , 61 % ±5 % in Q8NE62 -Placebo , 116 % ±6 % in Normal- DB00820 , and 86 % ±8 % in Q8NE62 - DB00820 lambs . Normalized expression of β-sGC was 105 % ±15 % in Normal-Placebo , 82 % ±3 % in Q8NE62 -Placebo , 158 % ±16 % in Normal- DB00820 , and 86 % ±8 % in Q8NE62 - DB00820 lambs . P29474 and β-sGC were significantly decreased in Q8NE62 ( p=0.0007 and 0.01 for P29474 and β-sGC , respectively ) , and tadalafil significantly increased P29474 expression ( p=0.0002 ) . CONCLUSIONS : O76074 inhibitors can cross the placental barrier . β-sGC and P29474 are downregulated in fetal lambs with Q8NE62 . Antenatal O76074 inhibitors normalize P29474 and may prevent in utero vascular remodeling in Q8NE62 . Glycoprotein IIb/IIIa inhibition enhances platelet nitric oxide release . INTRODUCTION : Platelet aggregates form by fibrinogen binding to the membrane receptor glycoprotein IIb/IIIa ( P08514 /IIIa ) . While P08514 /IIIa inhibitors block fibrinogen-platelet binding , stimulation of other functionally important platelet receptors may still occur . Blocking the P08514 /IIIa receptor prevents platelet aggregation but not activation and the subsequent effect on other platelet pathways is largely unknown . MATERIALS AND METHODS : As activated platelets release reactive oxygen species that may influence thrombosis or vascular function , the effect of P08514 /IIIa inhibitors on the platelet release of nitric oxide ( NO ) and superoxide was determined using an electrochemical detector and luminescence , respectively . Location of relevant platelet proteins and the interaction between platelets and leukocytes in the presence or absence of P08514 /IIIa inhibition was determined . RESULTS : Although incubation with P08514 /IIIa inhibitors completely abolished platelet aggregation , stimulation dependent NO release was significantly enhanced . Superoxide is known to alter the bioavailability of NO , and its contribution to the P08514 /IIIa dependent increase in NO release was determined . In the presence of P08514 /IIIa inhibitors , platelet superoxide release was significantly decreased . Preincubation with P08514 /IIIa inhibitors also modified aggregation induced membrane translocation of the platelet proteins , endothelial NO synthase ( P29474 ) and NADPH oxidase ( p67phox and p47phox ) , known to contribute to the generation of NO and superoxide , respectively . In the presence of leukocytes , abciximab incubation led to enhanced NO release and attenuated superoxide generation . CONCLUSION : These observations suggest that the pharmacological effects of P08514 /IIIa antagonists on platelet function , apart from inhibition of aggregation , may contribute to their efficacy . Consequences of immunogenicity to the therapeutic monoclonal antibodies ReoPro and Remicade . The clinical consequences of immune antibodies generated to abciximab ( ReoPro ) and infliximab ( Remicade ) are described . DB00054 , a chimaeric Fab fragment that binds to the beta3 integrin of the P08514 /IIIa and alphavbeta3 receptors on human platelets , is approved in the US and Europe for use in percutaneous coronary intervention ( P05154 ) to prevent cardiac ischaemic complications . The effects of induced antibodies upon the safety and efficacy of repeat administration of abciximab have been evaluated in the ReoPro Re-administration Registry Study , in which 5.7 % of patients were HACA positive before re-treatment . An interim evaluation of 1000 patients has indicated that re-administration of abciximab can be accomplished in the setting of P05154 with an acceptable safety and efficacy profile . DB00065 is a chimaeric IgG1 antibody specific for human TNFalpha , and is approved in the US and Europe for the acute treatment of the signs and symptoms of Crohn 's disease and for the chronic treatment of rheumatoid arthritis ( RA ) . The incidence of antibodies to infliximab is reported to be approximately 10 % ; however , an inverse dose-immunogenicity relationship was observed , indicating that higher doses of infliximab ( > or = 3 to 10 mg/kg ) could reduce the incidence of immune antibodies . The induction of immune antibodies could also be reduced by concomitant administration of low-dose methotrexate and other immunosuppressant agents . Although antibodies to infliximab appeared to be associated with lower serum infliximab concentrations and a slightly higher incidence of infusion reactions , these immune antibodies were generally not associated with a reduction in clinical efficacy . In addition , the antibodies induced to infliximab are specific for infliximab , and do not cross-react with other currently available therapeutic antibodies . Genetic aspects of ischemic stroke : coagulation , homocysteine , and lipoprotein metabolism as potential risk factors . Stroke is one of the most common causes of death and long term disability throughout the world . It may be the outcome of a number of monogenic disorders or , more commonly , a polygenic multifactorial disease . Numerous studies have investigated the role of genetics in the pathogenesis of ischemic stroke , with varied and often contradictory results . The candidate ' stroke risk ' genes affecting haemostasis ( P12259 , F2 , P02671 / P02675 , P08709 , P00488 , P04275 , P00748 , P05121 , P05106 / P08514 , P17301 , P07359 , TPA , Q96IY4 , P07204 , PZ , P08758 ) , homocysteine metabolism ( P42898 , P35520 , Q99707 ) , and lipid metabolism ( apo E , P06858 , P11597 , O95477 , apo AI , apo CIII , apo AIV , apo AV , apo B , apo H , apo(a) , P27169 /2/3 , P01130 / P78380 ) are evaluated in this review . By examining meta-analyses and case-control studies , we made a classification of gene/gene polymorphisms according to the degree of association with ischemic stroke risk . The data assembled could be very useful for further meta-analysis and for future clinical applications . A new epigenetic challenge : systemic lupus erythematosus . In recent years , compelling evidence has been gathered that supports a role for epigenetic alterations in the pathogenesis of systemic lupus erythematosus ( SLE ) . Different blood cell populations of SLE patients are characterized by a global loss of DNA methylation . This process is associated with defects in P29323 pathway signalling and consequent P26358 1 downregulation . Hypomethylation of gene promoters has been described , which permits transcriptional activation and therefore functional changes in the cells and also hypomethylation of the ribosomal RNA gene cluster . Among the identified targets undergoing demethylation are genes involved in autoreactivity ( P20701 ) , osmotic lysis and apoptosis ( P14222 , P50281 and P80188 ) , antigen presentation ( Q99062 ) , inflammation ( MMP 14 ) , B- T-cell interaction ( P32970 and P29965 ) and cytokine pathways ( Q99062 , P05112 , P05231 and P38484 ) . DNA methylation inhibitors are also known to induce autoreactivity in vitro and cause a lupus-like disease in vivo . Further , altered patterns of histone modifications have been described in SLE . P01730 + lymphocytes undergo global histone H3 and H4 deacetylation and consequent skewed gene expression . Although multiple lines of evidence highlight the contribution of epigenetic alterations to the pathogenesis of lupus in genetically predisposed individuals , many questions remain to be answered . Attaining a deeper understanding of these matters will create opportunities in the promising area of epigenetic treatments . A bispecific antifibrin-antiplatelet urokinase conjugate ( BAAUC ) induces enhanced clot lysis and inhibits platelet aggregation . Thrombolysis is well established in the treatment of acute myocardial infarction . However , clinical application of thrombolytic agents has limitations with respect to efficacy and specificity . To achieve highly effective and at the same time clot-selective plasminogen activation urokinase was coupled to a bispecific antibody consisting of the monovalent Fab ' from the antifibrin monoclonal antibody 59D8 and the monovalent Fab ' from the anti-glycoprotein P08514 /IIIa monoclonal antibody DB00054 . The bispecific antifibrin-antiplatelet urokinase conjugate ( BAAUC ) was synthesized and characterized . Assays with either immobilized platelets , P08514 /IIIa or fibrin showed an increase in plasminogen activation compared to uncoupled urokinase by 10-fold , 58-fold and 13-fold , respectivley ( p < 0.0001 each ) . In vitro clot lysis was performed on platelet-rich and fibrin-rich clots and revealed an up to 5-fold higher potency of BAAUC compared to uncoupled urokinase ( p < 0.0001 ) . In vitro platelet aggregation was effectively inhibited by the hybrid molecule , whereas urokinase had no effect . BAAUC and two monospecific urokinase-conjugates , UK-59D8-IgG and UK-7E3-(Fab')2 were compared with each other with regard to similar tests . In vitro clot assays with platelet-rich and platelet-poor clots were performed . BAAUC achieved a significantly higher plasminogen activation compared to each of the monospecific conjugates ( p < 0.05 , respectively ) . We conclude that BAAUC , a bispecific plasminogen activator with antifibrin and antiplatelet properties has the potency to lyse both fibrin-rich and platelet-rich thrombi with high efficacy and to effectively inhibit platelet aggregation . Effect of glycoprotein IIb-IIIa receptor antagonism on platelet membrane glycoproteins after coronary stent placement . Platelet membrane glycoproteins play a crucial role in ischemic complications after coronary stenting . Glycoprotein IIb-IIIa blockade reduces adverse clinical events after angioplasty but is associated with rare but profound thrombocytopenia that might increase hemorrhagic complications . Changes in platelet membrane glycoproteins of patients with angina who underwent coronary stenting and were treated with the P08514 -IIIa antagonist abciximab ( n=20 ) or with heparin ( n=23 ) were studied . GPIb-IIIa receptor blockade and membrane glycoproteins were evaluated with immunological markers in venous blood samples taken before . 10 , 24 , 48 , 72 , and 96 h after initial treatment with either abciximab or heparin . Patients receiving abciximab therapy showed a rapid inhibition of binding of fluorochrome-conjugated mAb CD41 and c7E3 concomitant with a reduction in platelet aggregation which was restored in part in the days after termination of abciximab infusion . Induction of ligand-induced binding sites on P08514 -IIIa was increased in patients receiving abciximab . The expression of ligand-induced binding sites correlated inversely with platelet count . No significant change in platelet membrane markers were found in the heparin group . In vitro studies showed that abciximab induces ligand-induced binding sites on isolated platelets and on nuclear cells bearing recombinant P08514 -IIIa . DB00054 rapidly achieves P08514 -IIIa receptor blockade after coronary stent placement that might be beneficial in high-risk settings to bridge the delayed action of ticlopidine . Significant alterations of platelet membrane glycoproteins during P08514 -IIIa antagonism might contribute to development of acute profound thrombocytopenia . The role of matrix metalloproteinase in the intimal sarcoma-like cells derived from endarterectomized tissues from a chronic thromboembolic pulmonary hypertension patient . Sarcoma-like cells ( SCLs ) were derived from endarterectomized tissue of a single chronic thromboembolic pulmonary hypertension ( CTEPH ) patient during incubation of those thrombi at second passage as described at our previous report . These cells had malignant potential , with an increased expression of matrix metalloproteinase-14 ( P50281 ) , leading to tumor emboli within pulmonary arteries in in vivo studies . The purpose of this study was to perform a more detailed evaluation of the characteristics of SCLs , and to elucidate the role of the increased expression of P50281 expression in the growth and death of these cells . In order to elucidate the characteristics of SCLs and to confirm the protein expression of P50281 , three-dimentional culture , invasion assays , a Western blot analysis and immunohistochemical studies were performed . To examine the role of P50281 in tumorigenesis , the metalloproteinase inhibitor , batimastat , was administered to SCID mice which were subcutaneously injected with SCLs . Those mice were sacrificed on day 14 and the tumor volume was evaluated . A Western blot analysis showed the increased expression of P50281 in comparison to the expression in lung adenocarcinoma cells ( A549 ) . Immunohistochemistry showed that SCLs were positive for vimentin , P50281 , P08253 and P16070 . However , endothelial markers , such as CD31 and P04275 ( P04275 ) , were negative . The in vivo studies demonstrated that batimastat could suppress the growth of the subcutaneous tumors formed by the SCLs . This study suggested that MMPs had critical roles on the pathological activities of SCLs and that batimastat might have anti-proliferative and anti-invasive effects on these cells . DB00171 -sensitive potassium channels ( K( DB00171 ) ) in retina : a key role for delayed ischemic tolerance . The objectives of the present study were to determine the localization of K( DB00171 ) channels in normal retina and to evaluate their potential roles in ischemic preconditioning ( IPC ) in a rat model of ischemia induced by increased intraocular pressure ( IOP ) . Brown Norway rats were subjected to sublethal 3- , lethal 20- and 40-min ischemia and the functional recovery was evaluated using electroretinography . The time interval between ischemic insults ranged from 1 to 72 h . The effects of K( DB00171 ) channel blockade on IPC protection were studied by treatment with 0.01 % glipizide . IPC was mimicked by injection of K( DB00171 ) channel openers of 0.01 % (-)cromakalim or 0.01 % P1060 72 h before 20-min ischemia . Co-expression of K( DB00171 ) channel subunits Kir6.2/ Q09428 was observed in the retinal pigment epithelium , inner segments of photoreceptors , outer plexiform and ganglion cell layers and at the border of the inner nuclear layer . In contrast to a 20- or 40-min ischemia , a 3-min ischemia induced no alteration of the electroretinogram ( ERG ) and constituted the preconditioning stimulus . An ischemic challenge of 40 min in preconditioned rats induced impairment of retinal function . However , animals preconditioned 24 , 48 and 72 h before 20-min ischemia had a significant improvement of the ERG . (-)Cromakalim and P1060 mimicked the effect of IPC . DB01067 significantly suppressed the protective effects of preconditioning . In conclusion , activation of K( DB00171 ) channels plays an important role in the mechanism of preconditioning by enhancing the resistance of the retina against a severe ischemic insult . The effects of P08514 -IIIa antagonists and a combination of three other antiplatelet agents on platelet-leukocyte interactions . The effects of the P08514 -IIIa antagonists abciximab and MK-852 on platelet-leukocyte interactions in vitro were studied and the results compared with those obtained with a combination of aspirin , dipyridamole and AR-C69931 ( DB00128 /Dip/AR-C ) . Platelet-monocyte ( P/M ) and platelet-neutrophil ( P/N ) conjugate formation increased when blood was stirred or a platelet agonist was added . Leukocyte activation also occurred as judged by expression of surface tissue factor antigen and CD11b . DB00054 and MK-852 potentiated P/M , especially when collagen was used . They also increased the amount of tissue factor on the monocytes , but not CD11b . The DB00128 /Dip/AR-C did not enhance P/M or tissue factor exposure . Augmented tissue factor expression on monocytes in the presence of a P08514 -IIIa antagonist may be relevant to the increased mortality associated with trials of such antagonists when given orally in patients with vascular disease . The DB00128 /Dip/AR-C was superior to abciximab and MK-852 in inhibiting platelet and leukocyte function . Suppressed Ca2+/ P62158 /CaMKII-dependent K( DB00171 ) channel activity in primary afferent neurons mediates hyperalgesia after axotomy . Painful axotomy decreases K( DB00171 ) channel current ( IK( DB00171 ) ) in primary afferent neurons . Because cytosolic Ca(2+) signaling is depressed in injured dorsal root ganglia ( Q86YR7 ) neurons , we investigated whether Ca(2+)-calmodulin ( P62158 ) -Ca(2+)/ P62158 -dependent kinase II ( CaMKII ) regulates IK( DB00171 ) in large Q86YR7 neurons . Immunohistochemistry identified the presence of K( DB00171 ) channel subunits Q09428 , SUR2 , and Kir6.2 but not Kir6.1 , and pCaMKII in neurofilament 200-positive Q86YR7 somata . Single-channel recordings from cell-attached patches revealed that basal and evoked IK( DB00171 ) by ionomycin , a Ca(2+) ionophore , is activated by CaMKII . In axotomized neurons from rats made hyperalgesic by spinal nerve ligation ( Q16658 ) , basal K( DB00171 ) channel activity was decreased , and sensitivity to ionomycin was abolished . Basal and Ca(2+)-evoked K( DB00171 ) channel activity correlated inversely with the degree of hyperalgesia induced by Q16658 in the rats from which the neurons were isolated . Inhibition of IK( DB00171 ) by glybenclamide , a selective K( DB00171 ) channel inhibitor , depolarized resting membrane potential ( O94763 ) recorded in perforated whole-cell patches and enhanced neurotransmitter release measured by amperometry . The selective K( DB00171 ) channel opener diazoxide hyperpolarized the O94763 and attenuated neurotransmitter release . Axotomized neurons from rats made hyperalgesic by Q16658 lost sensitivity to the myristoylated form of autocamtide-2-related inhibitory peptide ( AIPm ) , a pseudosubstrate blocker of CaMKII , whereas axotomized neurons from Q16658 animals that failed to develop hyperalgesia showed normal IK( DB00171 ) inhibition by AIPm . AIPm also depolarized O94763 in control neurons via K( DB00171 ) channel inhibition . Unitary current conductance and sensitivity of K( DB00171 ) channels to cytosolic DB00171 and ligands were preserved even after painful nerve injury , thus providing opportunities for selective therapeutic targeting against neuropathic pain . New anticoagulant strategies . The limitations of standard heparin have prompted the development of a variety of newer antithrombotic agents . In fact , a LMWH preparation has recently been approved for clinical use in North America . Of these novel preparations , LMWH , the direct thrombin inhibitors , and inhibitors of P08514 -IIIa have been used clinically and are in advanced stages of evaluation . Not only is LMWH effective in the prevention of venous thromboembolic disease in high-risk patients , but its more predictable dose response makes it an ideal candidate for the treatment of venous thrombosis . Further studies are needed to determine whether LMWH is superior to standard heparin as adjunctive therapy in patients undergoing coronary thrombolysis or angioplasty . Particularly promising in the setting of arterial thrombosis are hirudin , hirulog , and DB00054 . With the encouraging results reported to date , it is likely that these agents will soon find their way into the treatment armamentarium of arterial thrombosis . No evidence for an influence of the human platelet antigen-1 polymorphism on the antiplatelet effects of glycoprotein IIb/IIIa inhibitors . This study investigated the hypothesis that the human platelet antigen-1 ( Q9Y251 -1 ) polymorphism may influence the antiplatelet effects of glycoprotein (GP)IIb/IIIa inhibitors . DB00640 diphosphate ( 30 micro mol ) -induced fibrinogen binding was measured by flow cytometry . DB00054 ( 0.03-3 micro g/ml ) , tirofiban ( 0.3-30 nmol/l ) or eptifibatide ( 0.01-1 micro g/ml ) were incubated for 15 min with the samples prior to stimulation . IC(50) values for the inhibition of fibrinogen binding were determined from each experiment . All subjects were genotyped by GALIOS and automated fluorescence correlation spectroscopy . Although a marked variability in the inhibitory effects of all three P08514 /IIIa inhibitors was confirmed , there were no significant differences between the genotypes with respect to the inhibition of fibrinogen binding . Thus , the present study does not provide evidence for an effect of Q9Y251 -1 polymorphism on the inter-individual variability in the platelet inhibitory effects of the three P08514 /IIIa inhibitors approved for clinical use . Progress in the field of P08514 /IIIa antagonists . Platelet aggregation plays an important role in pathological situations such as myocardial infarction , unstable angina , peripheral artery disease , and stroke . Thus , pharmacological agents that specifically inhibit platelet aggregation are of great interest in the treatment and prevention of these cardiovascular diseases . Since binding of activated glycoprotein IIb/IIIa complex , a platelet surface integrin , to fibrinogen is the final step leading to platelet aggregation regardless of the initial stimulus , many researches have focused on the development of drugs that could antagonize this integrin . Three intravenous glycoprotein IIb/IIIa antagonists are currently marketed for the prevention of myocardial infarction in patients undergoing percutaneous intervention : DB00054 , DB00063 and Tirofiban . To further test the clinical efficacy of these agents , oral glycoprotein IIb/IIIa antagonists have been developed but only led to disappointing clinical results . Nevertheless , due to recognized usefulness of oral agents for the prevention and treatment of cardiovascular diseases , a great number of new orally active compounds are under clinical or preclinical evaluation . The aim of this review is to describe the chemical , pharmacological and clinical properties of existing and forthcoming glycoprotein IIb/IIIa antagonists . Antiaggregatory and proangiogenic effects of a novel recombinant human dual specificity anti-integrin antibody . BACKGROUND : beta(3)-Integrins are involved in platelet aggregation via alpha(IIb)beta(3) [ glycoprotein (GP)IIb- P05106 ] , and in angiogenesis via endothelial alpha(V)beta(3) . Cross-reactive ligands with antiaggregatory and proangiogenic effects , both desirable in peripheral vasculopathies , have not yet been described . OBJECTIVES : In vitro and in vivo characterization of antiaggregatory and proangiogenic effects of two recombinant human Fab fragments , with emphasis on beta(3)-integrins . METHODS : Recombinant Fab fragments were obtained by phage display technology . Specificity , affinity and IC(50) were determined by immunodot assays , enzyme-linked immunosorbent assay ( ELISA ) , and Scatchard plot analysis , and by means of human umbilical vein endothelial cells ( HUVECs ) . Functional analyses included ELISA for interaction with fibrinogen binding to P08514 - P05106 , flow cytometry for measurement of activation parameters and competitive inhibition experiments , human platelet aggregometry , and proliferation , tube formation and the chorioallantoic membrane ( P62158 ) assay for measurement of angiogenic effects . RESULTS : We observed specific and high-affinity binding to an intact P08514 - P05106 receptor complex of two human Fab autoantibody fragments , with no platelet activation . Dose-dependent fibrinogen binding to P08514 - P05106 and platelet aggregation were completely inhibited . One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody ( mAb ) DB00054 , whereas the other Fab fragment bound to cultured HUVECs , suggesting cross-reactivity with alpha(V)beta(3) , and also demonstrated proangiogenic effects in tube formation and P62158 assays . CONCLUSIONS : These Fab fragments are the first entirely human anti- P08514 - P05106 Fab fragments with full antiaggregatory properties ; furthermore , they do not activate platelets . The unique dual-specificity anti-beta(3)-integrin Fab fragment may represent a new tool for the study and management of peripheral arterial vasculopathies . Ca2+-calmodulin and janus kinase 2 are required for activation of sodium-proton exchange by the Gi-coupled 5-hydroxytryptamine 1a receptor . The type 1 sodium-proton exchanger ( P19634 ) is expressed ubiquitously and regulates key cellular functions , including mitogenesis , cell volume , and intracellular pH . Despite its importance , the signaling pathways that regulate P19634 remain incompletely defined . In this work , we present evidence that stimulation of the 5-hydroxytryptamine 1A ( P08908 ) receptor results in the formation of a signaling complex that includes activated O60674 ( Jak2 ) , Ca2+/calmodulin ( P62158 ) , and P19634 , and which involves tyrosine phosphorylation of P62158 . The signaling pathway also involves rapid agonist-induced association of P62158 and P19634 as assessed by coimmunoprecipitation studies and by bioluminescence resonance energy transfer studies in living cells . We propose that P19634 is activated through this pathway : P08908 receptor --> G(i2)alpha and/or G(i3)alpha --> Jak2 activation --> tyrosine phosphorylation of P62158 --> increased binding of P62158 to P19634 --> induction of a conformational change in P19634 that unmasks an obscured proton-sensing and/or proton-transporting region of P19634 --> activation of P19634 . The G(i/o)-coupled P08908 receptor now joins a handful of Gq-coupled receptors and hypertonic shock as upstream activators of this emerging pathway . In the course of this work , we have presented clear evidence that P62158 can be activated through tyrosine phosphorylation in the absence of a significant role for elevated intracellular Ca2+ . We have also shown for the first time that the association of P62158 with P19634 in living cells is a dynamic process . Catalog of 178 variations in the Japanese population among eight human genes encoding G protein-coupled receptors ( GPCRs ) . We screened DNAs from 48 Japanese individuals for single-nucleotide polymorphisms ( SNPs ) in eight genes encoding G protein-coupled receptors ( GPCRs ) by directly sequencing the entire relevant genomic regions except for repetitive-sequence elements . This approach identified 147 SNPs and 31 insertion/deletion polymorphisms among the eight GPCR genes . On average , we identified one SNP in every 584 nucleotides . Of the 147 SNPs , 69 were identified in P30556 , 12 in P50052 , nine in P35414 , 20 in P37288 , nine in P30518 , 16 in P21728 , six in P08514 , and six in P43119 . Twenty-one SNPs were located in 5' flanking regions , 76 in introns , 32 in exons , and 18 in 3' flanking regions . These variants should contribute to investigations of possible correlations between genotypes and phenotypes as regards susceptibility to disease or responsiveness to drug therapy . Opposed effects of lithium on the MEK- P29323 pathway in neural cells : inhibition in astrocytes and stimulation in neurons by GSK3 independent mechanisms . DB01356 is widely used in the treatment of bipolar disorder , but despite its proven therapeutic efficacy , the molecular mechanisms of action are not fully understood . The present study was undertaken to explore lithium effects of the MEK/ P29323 cascade of protein kinases in astrocytes and neurons . In asynchronously proliferating rat cortical astrocytes , lithium decreased time- and dose-dependently the phosphorylation of MEK and P29323 , with 1 mM concentrations achieving 60 and 50 % inhibition of P29323 and MEK , respectively , after a 7-day exposure . DB01356 also inhibited [3H]thymidine incorporation into DNA and induced a G2/M cell cycle arrest . In serum-deprived , quiescent astrocytes , pre-exposure to lithium resulted in the inhibition of cell cycle re-entry as stimulated by the mitogen endothelin-1 : under this experimental setting , lithium did not affect the rapid , peak phosphorylation of MEK taking place after 3-5 min , but was effective in inhibiting the long-term , sustained phosphorylation of MEK . DB01356 inhibition of the astrocyte MEK/ P29323 pathway was independent of inositol depletion . Further , compound SB216763 inhibited Tau phosphorylation at Ser396 and stabilized cytosolic beta-catenin , consistent with the inhibition of glycogen synthase kinase-3 beta ( P49841 ) , but failed to reproduce lithium effects on MEK and P29323 phosphorylation and cell cycle arrest . In cerebellar granule neurons , millimolar concentrations of lithium enhanced MEK and P29323 phosphorylation in a concentration-dependent manner , again through an inositol and P49841 independent mechanism . These opposing effects in astrocytes and neurons make lithium treatment a promising strategy to favour neural repair and reduce reactive gliosis after traumatic injury . Increased thromboxane biosynthesis during coronary thrombolysis . Evidence that platelet activation and thromboxane A2 modulate the response to tissue-type plasminogen activator in vivo . Platelet activation is markedly increased during coronary thrombolysis and limits the response to thrombolytic therapy . A possible mediator of platelet activation in this setting is thromboxane ( TX ) A2 , a potent platelet agonist formed in greatly increased amounts during coronary thrombolysis in man . To address this hypothesis , we examined the role of TXA2 in modulating the response to intravenous tissue-type plasminogen activator ( t-PA ) in a chronic canine model of coronary thrombosis . Reperfusion occurred in 60 +/- 5 minutes and was complicated by spontaneous reocclusion . The times to reperfusion and reocclusion were platelet-dependent . Consistent with a role for TXA2 in this process , TXA2 biosynthesis , determined a excretion of its enzymatic metabolite , 2,3-dinor-TXB2 , was markedly increased during coronary thrombolysis . Furthermore , inhibition of TXA2 by aspirin , given alone or in combination with a TXA2/prostaglandin endoperoxide receptor antagonist , accelerated reperfusion and partly inhibited cyclic flow variations during reperfusion . The delay in reperfusion and reocclusion induced by TXA2 appeared to be mediated by platelet aggregation since the F(ab')2 fragment of DB00054 , a monoclonal antibody to the platelet P08514 /IIIa , also accelerated reperfusion and prevented reocclusion without altering TXA2 biosynthesis . These finding suggest that platelet aggregation limits the response to coronary thrombolysis and that platelet activation in this setting is partly TXA2-dependent . Analysis of human platelet glycoprotein IIb-IIIa by fluorescein isothiocyanate-conjugated disintegrins with flow cytometry . Disintegrins are a group of snake venom peptides which inhibit human platelet aggregation by acting as glycoprotein IIb-IIIa ( P08514 -IIIa ) antagonists . They are cysteine-rich , DB00125 - DB00145 - DB00128 ( RGD ) -containing peptides , and bind to P08514 -IIIa complex on platelet membrane with a very high affinity ( Kd , 10(-7)-10(-8) M ) . In this study , we analyzed P08514 -IIIa complex on platelet membrane by flow cytometry using fluorescein isothiocyanate ( FITC ) -conjugated disintegrins as probes . Of these FITC-conjugated disintegrins , FITC-Rhodostomin is the most sensitive probe because Rhodostomin was conjugated with more FITC molecules than Trigramin and Halysin were . The binding fluorescence intensity of FITC-Trigramin ( FITC-Tg ) , FITC-Halysin ( FITC-Hy ) and FITC-Rhodostomin ( FITC-Rn ) was measured in both resting and ADP-activated platelets of diluted human platelet-rich plasma . The binding fluorescence of FITC-disintegrins was abolished by DB00974 and DB00054 , a monoclonal antibody against P08514 -IIIa . ADP markedly increased the fluorescence intensity of FITC-Tg and FITC-Hy bound on platelets especially when lower doses of these probes were used , whereas it had little effect on that of FITC-Rn . Therefore , FITC-Tg and FITC-Hy can be used for the detection of the activated platelets as noted by a higher ratio of fluorescence intensity ( approx. 2-4 ) between ADP-activated and resting platelets as compared with that ( approx. 1-1.3 ) in the case of FITC-Rn as the probe . The platelets from three patients with Glanzmann 's thrombasthenia were probed with FITC-disintegrins. ( ABSTRACT TRUNCATED AT 250 WORDS ) The anti- P08514 -IIIa agents : fundamental and clinical aspects . The platelet P08514 /IIIa receptor mediates platelet aggregation induced by all physiologic agonists . Blockade of the receptor , either by monoclonal antibodies or small molecules patterned after the arginine glycine-aspartic acid ( RGD ) cell recognition domain , prevents arterial thrombosis in animal models much better than does aspirin . c7E3 Fab , the Fab fragment of the mouse/human chimeric antibody DB00054 ( abciximab : ReoPro ) , was shown to reduce ischemic events after angioplasty when given in conjunction with heparin and aspirin to patients at high risk in the EPIC study , but its was associated with an increase in bleeding . Preliminary data from the subsequent EPILOG study , in which a lower dose of heparin was used , demonstrated efficacy in low risk as well as high risk patients and no significant increase in major bleeding . Preliminary data from the CAPTURE study support the use of c7E3 Fab in patients with unstable angina who are candidates for PTCA within 24 hours . Positive trends toward decreased thrombotic events have also been observed in patients treated with small molecule inhibitors of P08514 /IIIa receptors . This new class of agents thus holds promise for improving the therapy of angioplasty as well as perhaps other thrombotic phenomena . DB01356 inhibits glycogen synthase kinase-3 activity and mimics wingless signalling in intact cells . BACKGROUND : Exposing eukaryotic cells to lithium ions ( Li+ ) during development has marked effects on cell fate and organization . The phenotypic consequences of Li+ treatment on Xenopus embryos and sporulating Dictyostelium are similar to the effects of inhibition or disruption , respectively , of a highly conserved protein serine/threonine kinase , glycogen synthase kinase-3 ( GSK-3 ) . In Drosophila , the GSK-3 homologue is encoded by zw3sgg , a segment-polarity gene involved in embryogenesis that acts downstream of wg . In higher eukaryotes , GSK-3 has been implicated in signal transduction pathways downstream of phosphoinositide 3-kinase and mitogen-activated protein kinases . RESULTS : We investigated the effect of Li+ on the activity of the GSK-3 family . At physiological doses , Li+ inhibits the activity of human P49841 and Drosophila Zw3Sgg , but has no effect on other protein kinases . The effect of Li+ on GSK-3 is reversible in vitro . Treatment of cells with Li+ inhibits GSK-3-dependent phosphorylation of the microtubule-associated protein Tau . Li+ treatment of Drosophila S2 cells and rat PC12 cells induces accumulation of cytoplasmic Armadillo/beta-catenin , demonstrating that Li+ can mimic Wingless signalling in intact cells , consistent with its inhibition of GSK-3 . CONCLUSIONS : Li+ acts as a specific inhibitor of the GSK-3 family of protein kinases in vitro and in intact cells , and mimics Wingless signalling . This reveals a possible molecular mechanism of Li+ action on development and differentiation . Q13547 ( Q13547 ) participates in the down-regulation of corticotropin releasing hormone gene ( crh ) expression . The paraventricular nucleus of the hypothalamus ( PVH ) plays a central role in regulating the hypothalamic-pituitary-adrenal ( Q9Y251 ) axis . Medial parvocellular neurons of the PVH ( mpPVH ) integrate sensory and humoral inputs to maintain homeostasis . Humoral inputs include glucocorticoids secreted by the adrenals , which down-regulate Q9Y251 activation . A primary glucocorticoid target is the population of mpPVH neurons that synthesize and secrete corticotropin-releasing factors , the most potent of which is corticotropin-releasing hormone ( P06850 ) . Although P06850 gene ( crh ) expression is known to be down-regulated by glucocorticoids , the mechanisms by which this process occurs are still poorly understood . To begin this study we postulated that glucocorticoid repression of crh involves HDAC recruitment to the region of the crh proximal promoter . To evaluate this hypothesis , we treated hypothalamic cells that express P06850 with the HDAC inhibitor trichostatin A ( P32119 ) . As predicted , treatment with P32119 led to increased P06850 mRNA levels and crh promoter activity . Although co-treatment with DB00514 ( 10(-7)M ) reduced the P32119 effect on mRNA levels , it failed to reduce promoter activity ; however co-transfection of Q13547 but not 3 restored DB00514 inhibition . A distinction between Q13547 and 3 was also apparent with respect to crh promoter occupancy . DB00514 led to increased Q13547 but not O15379 occupancy . In vivo studies revealed that P06850 -immunoreactive ( -ir ) neurons contained Q13547 - and O15379 -ir . Collectively , these data point to a role for Q13547 in the physiologic regulation of crh . Flow cytometric analysis of platelet activation throughout normal gestation . OBJECTIVE : To measure platelet activation in normal pregnancy , before and after stimulation with agonists , with a whole blood flow cytometric technique . METHODS : In a cross-sectional study , 5 mL of whole blood was collected from healthy volunteers ( nine in the first trimester , ten in the second trimester , 35 in the third trimester , and 32 nonpregnant controls ) . Platelets were treated with an agonist ( thrombin or U-46619 , a thromboxane A2 analogue ) or buffer and were exposed to saturating concentrations of monoclonal antibodies directed against platelet membrane glycoproteins ( GPs ) : DB00054 ( fibrinogen receptor P08514 /IIIa ) , P28222 ( alpha granule marker P16109 ) , and 6D1 ( P04275 receptor GPIb ) . Mean fluorescence intensity was determined for 5000 platelets per sample by using a flow cytometer . RESULTS : In the absence of agonist , no significant difference between groups was found in antibody binding . At no stage of pregnancy were circulating activated platelets detected . Platelets from third-trimester subjects bound significantly less 7E3 than platelets of controls or of first- or second-trimester subjects after stimulation with high-dose thrombin ( P < .05 for all comparisons ) . Down-regulation of 6D1 on platelets after stimulation with high-dose U-46619 was significantly greater in third-trimester gravidas than in controls or first-trimester subjects ( P < .05 ) . CONCLUSION : Pregnancy does not increase the percentage of activated platelets in the circulation . Platelet reactivity is altered in the third trimester , as evidenced by decreased antibody binding to fibrinogen receptor epitope and enhanced down-regulation of a P04275 receptor epitope . Anti-thrombotic and anticoagulant treatment in interventional cardiology . Efforts to improve Percutaneous Transluminal Coronary Angioplasty ( PTCA ) have resulted in the usage of new antiplatelets , and antithrombotic agents . These new agents may increase bleeding complications . However , EPIC , EPILOG and CAPTURE trials showed benefits of DB00054 , a P08514 /IIIa platelets receptor blocker , in high risk PTCA patients . On the other hand , direct thrombin inhibitors , Hirudin and DB02351 , did not clearly show any benefit when compared to heparin in patients with unstable angina undergoing PTCA . Combination of oral antiplatelets , ticlopidine and aspirin , is widely utilized following stent implantation . However , its benefit over aspirin alone has not been demonstrated . This article aims to review mechanisms and benefits of these new agents in cardiovascular field . Therapeutic Implications of a Specific Murine Monoclonal Antibody ( DB00054 ) to the Platelet Receptor P08514 /IIIa . The murine monoclonal antibody 7E3 blocks the platelet glycoprotein IIb/IIIa receptor , and is a potent inhibitor of platelet function in both animals and man . Animal models of the acute coronary syndromes suggest that 7E3 abolishes the in vivo formation of platelet thrombi , accelerates thrombolysis with tissue plasminogen activator , and prevents subsequent reocclusion . Human studies with 7E3 suggest that complete inhibition of platelet function may be safely undertaken for periods of up to 36 h , and preliminary studies indicate effectiveness in the therapy of clinical unstable angina . Potential problems with its use include immunogenicity and thrombocytopenia . The outcome of the acute coronary thrombotic syndromes , which include unstable angina , acute myocardial infarction and abrupt closure following coronary angioplasty , may be significantly improved with 7E3 therapy . A new short chain RGD-containing disintegrin , accutin , inhibits the common pathway of human platelet aggregation . A new short-chain disintegrin , accutin , was purified from the Formosan Agkistrodon acutus venom by using of gel filtration , ion exchanger and reverse phase HPLC . The homogeneous protein is a 47-residue polypeptide with a molecular mass of 5241 Da containing an DB00125 - DB00145 - DB00128 sequence and seven cysteinyl residues at positions highly homologous to other disintegrins . Accutin dose-dependently inhibited human platelet aggregation stimulated by ADP , collagen , thrombin or the thromboxane analogue U46619 in platelet suspension with IC50 values of 66-267 nM . It was also active in inhibiting platelet aggregation of platelet-rich plasma . However , accutin apparently did not affect the shape change caused by these agonists . Accutin also inhibited fibrinogen-induced aggregation of human elastase-treated platelets in a dose-dependent manner . Furthermore , accutin dose-dependently inhibited the binding reaction of fluorescein isothiocyanate ( FITC ) -conjugated arietin , a member of the disintegrin family , to human platelets . In addition , the binding of FITC-conjugated accutin to platelets was almost completely blocked by a monoclonal antibody , DB00054 , raised against the platelet glycoprotein IIb/IIIa complex . On the other hand , accutin as well as other disintegrins , rhodostomin and arietin , exhibited an inhibitory effect on 7E3 binding toward platelets and endothelial cells in a dose-dependent manner . It is concluded that accutin , a new platelet aggregation inhibitor belonging to the short-chain disintegrin family , acts specifically on a binding epitope of P08514 /IIIa overlapping with that of DB00054 , leading to the blockade of fibrinogen binding to its receptor . Comparison of the novel antipsychotic ziprasidone with clozapine and olanzapine : inhibition of dorsal raphe cell firing and the role of P08908 receptor activation . Ziprasidone is a novel antipsychotic agent which binds with high affinity to P08908 receptors ( Ki = 3.4 nM ) , in addition to P28221 , 5-HT2 , and D2 sites . While it is an antagonist at these latter receptors , ziprasidone behaves as a P08908 agonist in vitro in adenylate cyclase measurements . The goal of the present study was to examine the P08908 properties of ziprasidone in vivo using as a marker of central P08908 activity the inhibition of firing of serotonin-containing neurons in the dorsal raphe nucleus . In anesthetized rats , ziprasidone dose-dependently slowed raphe unit activity ( ED50 = 300 micrograms/kg i.v. ) as did the atypical antipsychotics clozapine ( ED50 = 250 micrograms/kg i.v. ) and olanzapine ( ED50 = 1000 micrograms/kg i.v. ) . Pretreatment with the P08908 antagonist WAY-100,635 ( 10 micrograms/kg i.v. ) prevented the ziprasidone-induced inhibition ; the same dose of WAY-100,635 had little effect on the inhibition produced by clozapine and olanzapine . Because all three agents also bind to alpha 1 receptors , antagonists of which inhibit serotonin neuronal firing , this aspect of their pharmacology was assessed with desipramine ( DB01151 ) , a NE re-uptake blocker previously shown to reverse the effects of alpha 1 antagonists on raphe unit activity . DB01151 ( 5 mg/kg i.v. ) failed to reverse the inhibitory effect of ziprasidone but produced nearly complete reversal of that of clozapine and olanzapine . These profiles suggest a mechanism of action for each agent , P08908 agonism for ziprasidone and alpha 1 antagonism for clozapine and olanzapine . The P08908 agonist activity reported here clearly distinguishes ziprasidone from currently available antipsychotic agents and suggests that this property may play a significant role in its pharmacologic actions . DB00054 : a reappraisal of its use in coronary care . Platelet reactivity plays a pivotal role in the pathogenesis of ischemic adverse events during and after acute coronary syndromes ( ACS ) , and percutaneous coronary intervention ( P05154 ) . Glycoprotein ( GP ) IIb/IIIa inhibitors are the strongest antiplatelet agents currently available on the market and three different compounds , namely abciximab , tirofiban , and eptifibatide , have been approved for clinical use . DB00054 has been investigated in the clinical field far more extensively than the other P08514 /IIIa inhibitors . DB00054 is an anti-integrin Fab fragment of a human - mouse chimeric monoclonal antibody with high affinity and a slow dissociation rate from the GP IIb/IIIa platelet receptor . DB00054 , given shortly before the coronary intervention , is superior to placebo in reducing the acute risk of ischemic complications ( EPIC , EPISTENT , EPILOG trials ) ; moreover , in the ISAR-REACT 2 study abciximab has been shown to reduce the risk of adverse events in patients with non ST-segment elevation ACS who are undergoing P05154 even after optimal pre-treatment with 600 mg of clopidogrel . Finally , abciximab has been also used in abciximab-coated stent , with only bolus administration regimen and for direct intracoronary use with promising results that may extend and/or modify its current use in clinical practice in future . Repetitive profound thrombocytopenia after treatment with tirofiban : a case report . The P08514 /IIIa inhibitors are used in the acute coronary syndromes and interventional cardiology as antiplatelet agents . These drugs induce thrombocytopenia in approximately 1-5 % of patients . Thrombocytopenia is rapid in onset and antibody mediated . DB00054 is associated with higher incidence of thrombocytopenia than eptifibatide and tirofiban . Profound thrombocytopenia has reportedly been an issue with abciximab , but not with tirofiban . We reported a case of acute profound thrombocytopenia due to on tirofiban treatment in the same patient at two different times . Novel cinnamyl hydroxyamides and 2-aminoanilides as histone deacetylase inhibitors : apoptotic induction and cytodifferentiation activity . Four novel series of cinnamyl-containing histone deacetylase ( HDAC ) inhibitors 1-4 are described , containing hydroxamate ( 1 and 3 ) or 2-aminoanilide ( 2 and 4 ) derivatives . When screened against class I ( maize HD1-B and human Q13547 ) and class II ( maize HD1-A and human P56524 ) HDACs , most hydroxamates and 2-aminoanilides displayed potent and selective inhibition toward class I enzymes . Immunoblotting analyses performed in U937 leukemia cells generally revealed high acetyl-H3 and low acetyl-α-tubulin levels . Exceptions are compounds 3 f-i , 3 m-o , and 4 k , which showed higher tubulin acetylation than DB02546 . In U937 cells , cell-cycle blockade in either the G₂/M or G₁/S phase was observed with 1-4 . Five hydroxamates ( compounds 1 h-l ) effected a two- to greater than threefold greater percent apoptosis than DB02546 , and in the CD11c cytodifferentiation test some 2-aminoanilides belonging to both series 2 and 4 were more active than MS-275 . The highest-scoring derivatives in terms of apoptosis ( 1 k , 1 l ) or cytodifferentiation ( 2 c , 4 n ) also showed antiproliferative activity in U937 cells , thus representing valuable tools for study in other cancer contexts . 1-(3-Trifluoromethylphenyl) piperazine ( TFMPP ) in the ventral tegmental area reduces the effect of desipramine in the forced swimming test in rats : possible role of serotonin receptors . 1-(3-Trifluoromethylphenyl)piperazine ( TFMPP ) , a serotonin1 ( 5-HT1 ) receptor agonist , injected i.p. in doses of 0.1 and 0.6 mg/kg , did not modify the immobility time of rats in the forced swimming test but significantly antagonized the effect of a 7 days treatment with 10 mg/kg per day desipramine ( DB01151 ) . A similar effect was found on infusing 1 and 5 micrograms/microliters TFMPP bilaterally into the ventral tegmental area ( VTA ) . Infusion of 5 micrograms/microliters TFMPP into the nucleus accumbens or into the globus pallidus did not modify the effect of DB01151 . The effect of 5 micrograms TFMPP infused into the VTA was prevented by the i.p. administration of 5 mg/kg metergoline , a non-selective serotonin receptor antagonist . Infusion of 5 micrograms/microliters 8-hydroxy-2-(di-n-propylamino)tetralin , a specific P08908 receptor agonist , into the VTA did not modify the effect of DB01151 . Besides acting as a P28222 receptor agonist , TFMPP may also act on other 5-HT receptor types , but available evidence suggests that its former action is more important . It thus appears that 5-HT1 receptors in the VTA , presumably of the P28222 type , act by preventing the anti-immobility effect of DB01151 . The role of VTA dopamine and non-dopamine cells in the effect of TFMPP is discussed . Differences in transcript levels of ABC transporters between pancreatic adenocarcinoma and nonneoplastic tissues . OBJECTIVES : The aim of this study was to evaluate transcript levels of all 49 human DB00171 -binding cassette transporters ( ABCs ) in one of the most drug-resistant cancers , namely , the pancreatic ductal adenocarcinoma ( PDAC ) . Association of ABCs levels with clinical-pathologic characteristics and P01116 mutation status was followed as well . METHODS : Tumors and adjacent nonneoplastic tissues were obtained from 32 histologically verified PDAC patients . The transcript profile of ABCs was assessed using quantitative real-time polymerase chain reaction with a relative standard curve . P01116 mutations in exon 2 were assessed by high-resolution melting analysis and sequencing . RESULTS : Most ABCs were deregulated in PDAC and 10 ABCs were associated with clinical-pathologic characteristics . P01116 mutations did not change the global expression profile of ABCs . CONCLUSIONS : The expression of ABC transporters was significantly deregulated in PDAC tumors when compared to nonmalignant tissues . The observed up-regulation of P21439 , O95342 , P33527 , O15438 , O15440 , Q5T3U5 , and Q9UNQ0 in tumors may contribute to the generally poor treatment response of PDAC . The up-regulation of O95477 , Q8IZY2 , and P45844 implicates a serious impairment of cellular cholesterol homeostasis in PDAC . On the other hand , the observed down-regulation of Q99758 , O95255 , P13569 , and Q09428 suggests a possible role of stem cells in the development and progression of PDAC . DB01109 potentiation of collagen-induced platelet aggregation is related to the P08514 / P05106 receptor and not to the GPIb receptor , as tested by whole blood aggregometry . To determine whether heparin potentiation of platelet aggregation is related to platelet GP IIb/IIIa and GP Ib receptors , four series of experiments were performed on blood from normal volunteers . In the first experiment pretreatment with the monoclonal antibody DB00054 ( MAb DB00054 ) , which antagonizes at the GP IIb/IIIa receptor , potently inhibited the collagen-induced platelet aggregation ( p less than 0.001 ) . With heparin added to blood pretreated with MAb DB00054 , the aggregation increased ( p less than 0.005 ) to an extent similar to that when only saline was used for pretreatment . In the second experiment , monoclonal antibody 10E5 ( MAb 10E5 ) and peptide RGDS , substances which also antagonize at the GP IIb/IIIa receptor , decreased collagen-induced platelet aggregation to an extent similar to that after pretreatment with MAb DB00054 . Following pretreatment with RGDS , heparin increased platelet aggregation ( p less than 0.03 ) , while after pretreatment with antibody MAb 10E5 heparin did not enhance platelet aggregation . In the third experiment aurin , an inhibitor of P04275 and its interaction with the platelet GPIb receptor , decreased platelet aggregation dose-dependently . In the fourth experiment heparin enhanced platelet aggregation to a similar extent ( p less than 0.005 ) , regardless of pretreatment of the blood with saline , aurin or monoclonal antibody 6D1 ( MAb 6D1 ) , the latter an antagonist at the GP Ib receptor . In conclusion , the potentiation of collagen-induced platelet aggregation by heparin was not inhibited by MAb DB00054 , RGDS , aurin or MAb 6D1 , but was abolished by MAb 10E5 , implying that the heparin effect is related to activation of the platelet GP IIb/IIIa receptor complex . Histone deacetylase inhibitors increase microRNA-146a expression and enhance negative regulation of interleukin-1β signaling in osteoarthritis fibroblast-like synoviocytes . OBJECTIVE : MiR-146a exerts negative control on inflammatory responses by suppressing cytokine-induced expression of interleukin-1 receptor-associated kinase-1 ( P51617 ) and tumor necrosis factor receptor-associated factor 6 ( Q9Y4K3 ) by impairing NF-κB activity and inhibiting the expression of target genes . Recent study suggests that histone deacetylases ( HDACs ) are involved in the regulation of microRNA ( miRNA ) expression . Therefore , we determined whether HDAC inhibitors can increase miR-146a expression , thereby inhibiting interleukin-1β ( IL-1β ) -induced signaling in osteoarthritis fibroblast-like synoviocytes ( OA-FLS ) . METHOD : MiRNA expression was analyzed using real-time PCR . IL-1β-induced downstream signals and cytokine expression were evaluated using Western blotting and ELISA . Transcription factors regulating promoter activation were identified using chromatin immunoprecipitation assays . RESULTS : IL-1β treatment of OA-FLS induced a mild ( 1.7-fold ) increase in miR-146a expression that was unable to appropriately downregulate P51617 and Q9Y4K3 expression . HDAC inhibitors , DB02546 ( vorinostat ) , and LBH589 ( DB06603 ) significantly ( 6.1- and 5.4-fold ) elevated miR-146a expression by increasing the binding of the transcription factor NF-κB to the miR-146a promoter , and negatively regulated IL-1β-induced IKK/IκB/p65 phosphorylation signaling and P05231 secretion . The increase in miR-146a expression induced by the HDAC inhibitors was prevented by transfection of miR-146a inhibitor or Q13547 ( class I HDAC ) , P56524 ( class IIa HDAC ) , and Q9UBN7 ( class IIb HDAC ) overexpression , suggesting that they were due to inhibition of HDAC activity . CONCLUSIONS : Our study demonstrated that HDAC inhibitor treatment in OA-FLS significantly increased miR-146a expression and mediated markedly negative regulation to inhibit IL-1β-induced signaling and cytokine secretion . Our results indicate the potential rationale of anti-inflammatory effects for HDAC inhibitors .