MH_dev_169

c7E3 Fab inhibits human tumor angiogenesis in a SCID mouse human skin xenograft model . The alphavbeta 3 integrin plays an important role in tumor growth and angiogenesis . Inhibition of this receptor by intact bivalent antibodies has been shown to inhibit angiogenesis and tumor growth . In this study we tested the chimeric Fab of DB00054 SUB ( c7E3 Fab ) , an antibody reactive with human platelet P08514 REA / IIIa and alphavbeta 3 to determine if it would inhibit in vivo angiogenesis and tumor growth in a SCID mouse / human skin tumor growth and angiogenesis model . c7E3 Fab inhibited human tumor angiogenesis and tumor growth . These data suggest monovalent antibody fragments devoid of antibody effector function can have efficacy in preclinical models of angiogenesis .

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 REA - 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 SUB ( 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 REA / 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 REA - 1 polymorphism on the inter-individual variability in the platelet inhibitory effects of the three P08514 REA / IIIa inhibitors approved for clinical use .

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 TXA 2 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 TXA 2 in this process , TXA 2 biosynthesis , determined a excretion of its enzymatic metabolite , 2,3- dinor-TXB 2 , was markedly increased during coronary thrombolysis . Furthermore , inhibition of TXA 2 by aspirin , given alone or in combination with a TXA 2 / prostaglandin endoperoxide receptor antagonist , accelerated reperfusion and partly inhibited cyclic flow variations during reperfusion . The delay in reperfusion and reocclusion induced by TXA 2 appeared to be mediated by platelet aggregation since the F ( ab ' ) 2 fragment of DB00054 SUB , a monoclonal antibody to the platelet P08514 REA / IIIa , also accelerated reperfusion and prevented reocclusion without altering TXA 2 biosynthesis . These finding suggest that platelet aggregation limits the response to coronary thrombolysis and that platelet activation in this setting is partly TXA 2 - dependent .

P08514 REA / IIIa antagonists and other anti-integrins . Platelet aggregation involves the binding of adhesive proteins ( fibrinogen , P04275 REA ) to the alphaIIbbeta 3 integrin , which assumes a high-affinity state for adhesive proteins during platelet activation . The occupied integrin sends signals back into the platelet , and the bound adhesive protein forms the bridges linking platelets together . Anti-integrin therapy is designed to inhibit this process in arterial thrombosis . DB00054 SUB , mouse-human chimeric Fab fragments , blocks platelet aggregation and provides proven clinical benefit in acute situations such as in patients with unstable angina undergoing angioplasty or stenting . DB00063 and tirofiban are small molecular mass inhibitors also in current use . In contrast , oral inhibitors of alphaIIbbeta 3 have proved disappointing , provoking increased mortality without assuring an adequate blockade of alphaIIbbeta 3 . The problems of using anti-integrin therapy are discussed in this article as are ways of improving its efficacity . Final thoughts provide ideas for a new generation of inhibitors .

Genetic markers in the EET metabolic pathway are associated with outcomes in patients with aneurysmal subarachnoid hemorrhage . Preclinical studies show that epoxyeicosatrienoic acids ( EETs ) regulate cerebrovascular tone and protect against cerebral ischemia . We investigated the relationship between polymorphic genes involved in EET biosynthesis / metabolism , cytochrome P450 ( CYP ) eicosanoid levels , and outcomes in 363 patients with aneurysmal subarachnoid hemorrhage ( aSAH ) . Epoxyeicosatrienoic acids and dihydroxyeicosatetraenoic acid ( DHET ) cerebrospinal fluid ( P04141 REA ) levels , as well as acute outcomes defined by delayed cerebral ischemia ( P42126 REA ) or clinical neurologic deterioration ( CND ) , were assessed over 14 days . Long-term outcomes were defined by Modified Rankin Scale ( P59665 REA ) at 3 and 12 months . P10632 REA * 4 allele carriers had 44 % and 36 % lower mean EET and DHET P04141 REA levels ( P= 0.003 and P= 0.007 ) and were 2.2- and 2.5- fold more likely to develop P42126 REA and CND ( P= 0.039 and P= 0.041 ) , respectively . P34913 REA 55Arg , P51589 REA * 7 , P10632 REA * 1B , and P10632 REA g . 36785A allele carriers had lower EET and DHET P04141 REA levels . P10632 REA g . 25369T and P10632 REA g . 36755A allele carriers had higher EET levels . Patients with P10632 REA * 2C and P34913 REA 404del variants had worse long-term outcomes while those with P34913 REA 287Gln , P51589 REA * 7 , and P11712 REA g . 816G variants had favorable outcomes . Epoxyeicosatrienoic acid levels were associated with Fisher grade and unfavorable 3 - month outcomes . Dihydroxyeicosatetraenoic acids were not associated with outcomes . No associations passed Bonferroni multiple testing correction . These are the first clinical data demonstrating the association between the EET biosynthesis / metabolic pathway and the pathophysiology of aSAH .

[ DB00054 SUB ( ReoPro ) in the treatment of acute coronary syndromes ] . Platelet activation plays a major role in the pathophysiology of acute coronary syndromes ( ACS ) . Inhibition of platelet function is the basic pharmacological treatment of ACS . P08514 REA / IIIa inhibitors , a new class of potent antiplatelet agents , have been used in the treatment of ACS and in the prevention of complications after percutaneous coronary interventions ( P05154 REA ) . Several large clinical trials have demonstrated the effectiveness of this class of agents . The first of these agents to show beneficial effects after coronary interventions was the mouse / human chimeric Fab fragment antibody c7E3 abciximab ( ReoPro ) . The purpose of this article is to describe the pharmacology of abciximab and to review the results of the clinical trials carried out with the drug in patients with ACS , treated either with or without acute / elective P05154 REA .

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 REA ] , 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 REA - P05106 REA , 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 REA - P05106 REA receptor complex of two human Fab autoantibody fragments , with no platelet activation . Dose-dependent fibrinogen binding to P08514 REA - P05106 REA and platelet aggregation were completely inhibited . One Fab fragment was competitively inhibited by abciximab and its murine analog monoclonal antibody ( mAb ) DB00054 SUB , 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 REA - P05106 REA 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 .

[ P08514 REA - IIIa inhibitors ] . Therapy involving the use of anti - P08514 REA - 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 SUB 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 REA - 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 REA - IIIa complexes .

A new role for the P40763 REA inhibitor , Q9Y6X2 REA : a repressor of microphthalmia transcription factor . In vitro and in vivo evidence suggest that microphthalmia transcription factor ( O75030 REA ) plays a key regulatory role in tissue-specific gene regulation in several cell types , including melanocytes , osteoclasts , and mast cells . A yeast two-hybrid search , using a portion of a nonmutated O75030 REA gene as the bait in the screening of a mast cell library , resulted in the isolation of the P40763 REA inhibitor , Q9Y6X2 REA . Q9Y6X2 REA is a transcriptional inhibitor that acts by specifically inhibiting P40763 REA ' s DNA binding activity . We found that it can directly associate with O75030 REA using an in vitro pull-down assay . Immunoprecipitation of O75030 REA from rat basophilic leukemic cells or mouse melanocytes resulted in the specific co-immunoprecipitation of Q9Y6X2 REA . Co-transfection of O75030 REA with Q9Y6X2 REA in NIH 3T3 fibroblasts containing an mMCP - 6 promoter-luciferase reporter demonstrated up to 94 % inhibition of O75030 REA - mediated transcriptional activation . Using a gel-shift assay , it was shown that Q9Y6X2 REA can block DNA binding activity . It was also found that P40763 REA does not interfere , either in vitro or in vivo , with the interaction between Q9Y6X2 REA and O75030 REA . These data suggest that Q9Y6X2 REA functions in vivo as a key molecule in supressing the transcriptional activity of O75030 REA , a role of considerable importance in mast cell and melanocyte development .

A chimeric murine / human antibody Fab fragment directed against the platelet P08514 REA / 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 SUB ( 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 REA / 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 )

Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa . Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase , factor XIIIa ( FXIIIa ) . In addition to fibrin stabilization , FXIIIa acts on a number of platelet-reactive proteins , including fibronectin and vitronectin , as well as the platelet proteins , glycoprotein ( GP ) IIb-IIIa , myosin , and actin . However , conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified . The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin-induced activation events ; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin ( ogen ) binding . Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa , the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa . Immunoprecipitation of radiolabeled platelets using polyclonal anti-FXIII A-chain antibody identified two proteins corresponding to P08514 REA and P05106 REA . Preincubation of intact platelets with DB00054 SUB , a monoclonal antibody that blocks the fibrinogen binding site , or GRGDSP peptide inhibited FXIIIa binding by about 95 % when measured by flow cytometry ; FXIIIa binding to purified P08514 REA - IIIa was also inhibited by DB00054 SUB . The binding of FXIIIa to purified P08514 REA - IIIa was enhanced by the addition of fibrinogen , but not by that of fibronectin or thrombospondin , suggesting that FXIIIa also binds to fibrinogen associated with the complex . These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events .

Inhibition of angiogenesis and tumor growth by murine DB00054 SUB , the parent antibody of c7E3 Fab ( abciximab ; ReoPro ) . Angiogenesis plays an essential role in the growth and dissemination of solid tumor cancers . The expression of endothelial cell integrin alpha ( v ) beta 3 has been shown to increase during vascular proliferation associated with human tumors . Selective antagonists of alpha ( v ) beta 3 can block angiogenesis and tumor growth by inducing programmed cell death in proliferating endothelial cells . Monoclonal antibody DB00054 SUB , an antagonist of the human , but not murine , integrins alpha ( v ) beta 3 and alphaIIbbeta 3 ( P08514 REA / IIIa ) , inhibits platelet aggregation . It is the parent antibody of a mouse / human chimeric antibody fragment approved for adjunctive therapy of patients undergoing percutaneous coronary interventions to prevent ischemic complications ( c7E3Fab ; abciximab ; ReoPro ) . To evaluate the potential of 7E3 to inhibit human angiogenesis and tumor growth independent of its antiplatelet effects , we established integrin alpha ( v ) beta 3 - negative human melanoma tumors in full-thickness human skin grafted onto SCID mice . The resulting tumors induce a human angiogenic response as assessed by the immunoreactivity of vascular cells with monoclonal antibodies specific for human CD31 . Administration of 7E3 prevented or significantly inhibited the growth of tumors , and this effect correlated with a significant reduction in the number of blood vessels supplying the tumors . These results support the previous findings that blockade of integrin alpha ( v ) beta 3 inhibits angiogenesis and tumor growth and indicates that dual inhibitors of alpha ( v ) beta 3 and alphaIIbbeta 3 are effective in blocking tumor growth and angiogenesis .

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 SUB [ 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 SUB . 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 REA / IIIa inhibition in an experimental model for an extended time , demonstrating the duration of antiplatelet therapy required to prevent rethrombosis after thrombolysis .

A new algorithm for weekly phenprocoumon dose variation in a southern Brazilian population : role for P11712 REA , P08684 REA / 5 and Q9BQB6 genes polymorphisms . DB00946 MENMAX DB00946 MEN is widely used in prophylaxis and treatment of thromboembolic disorders . However , its pharmacokinetics and pharmacodynamics vary according to several genetic and non-genetic factors . DB00946 MEN metabolism is mediated by P11712 REA and CYP 3A enzymes . Moreover , Q9BQB6 is phenprocoumon target of action . Therefore , the aim of this study was to evaluate the association of single nucleotide polymorphisms ( SNPs ) in Q9BQB6 , P11712 REA , P08684 REA and P20815 REA genes with the variance of weekly phenprocoumon dose as well as to develop an algorithm for dose prediction based on genetic and environmental factors . A total of 198 patients with stable phenprocoumon dose , 81 % of European ancestry , were investigated . Genotypes were determined by allelic discrimination with TaqMan assays . Polymorphisms - 1639G > A and 1173C > T in Q9BQB6 and the presence of P11712 REA * 2 and / or P11712 REA * 3 are associated with lower doses . On the other hand , 3730G > A in Q9BQB6 gene is associated with higher doses . No association was found between P08684 REA * 1B , P20815 REA * 3 and P20815 REA * 6 polymorphisms . Among non-genetic factors , gender , height , age and use of captopril , omeprazole , simvastatin and β-blockers are associated with dose . Two algorithms were derived : one for the whole sample explained 42 % of dose variation and one for patients of European ancestry only which explained 46 % of phenprocoumon dose . The mean absolute difference between observed and predicted dose was low in both models ( 3.92 mg / week and 3.54 mg / week , for models 1 and 2 , respectively ) . However , more studies with other genes and environmental factors are needed to test and to improve the algorithm .

Enhancement of fibrinolysis by gel-filtered platelets and its quenching by cytochalasin B and P08514 REA / IIIa antagonists . The effects of gel-filtered platelets on euglobulin clot lysis time ( ECLT ) were analyzed to elucidate the possible role of platelets in thrombolysis . Gel-filtered platelet-supplemented ECLT ( plt-ECLT ) was significantly shorter than ECLT without platelets ( regular ECLT ) . DB00054 SUB , anti-glycoprotein IIb / IIIa ( P08514 REA / IIIa ) antibody , and cytochalasin B nullified the enhancement of ECLT by platelets , and increased plt-ECLT beyond regular ECLT . When gel-filtered platelets were used after disruption , ECLT was not shortened but rather became longer than regular ECLT , probably due to natural fibrinolysis inhibitors released from platelets . Therefore , for platelets to enhance fibrinolysis , intact cell structure and cytoskeletal reorganization after thrombin stimulation is required . Various P08514 REA / IIIa antagonists prolonged plt-ECLT . The concentrations of P08514 REA / IIIa antagonists required to prolong plt-ECLT , were varied . Interestingly , the effects of these antagonists were independent of their ability to inhibit thrombin-induced platelet aggregation , but dependent on their ability to induce clot retraction . T - 250 , a P08514 REA / IIIa antagonist , had the smallest effect on plt-ECLT . These drugs do not affect regular ECLT or tissue plasminogen activator ( tPA ) - catalyzed DB00142 - plasminogen activation in the presence of thrombin-activated platelets . Although their overall effect on thrombolysis is inhibitory , platelets could promote fibrinolysis through a P08514 REA / IIIa-dependent mechanism .

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 REA / 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 REA / IIIa-specific monoclonal antibody DB00054 SUB , 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 REA / 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 .

Dynamics of P08514 REA / 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 SUB ( anti - P08514 REA / IIIa ( alphaIIbbeta 3 ) + alphaVbeta 3 ) or tirofiban ( anti - P08514 REA / 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 REA / IIIa ) and platelets from beta 3 - 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 beta 3 - null platelets demonstrated a more uniform deposition than predicted . Thus , in this model system , absence or blockade of P08514 REA / IIIa receptors interferes with thrombus formation and alters the pattern of platelet deposition .

DB01109 potentiation of collagen-induced platelet aggregation is related to the P08514 REA / P05106 REA 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 SUB ( MAb DB00054 SUB ) , 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 SUB , 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 SUB . 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 REA 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 SUB , 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 .

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 REA / IIIa antagonists . Several P08514 REA / 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 REA / 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 SUB , the latter demonstrating that compounds must enter cells to induce apoptosis through caspase activation . DB00063 had no effect . Binding studies with ( 3 ) H-SC 52012B , a P08514 REA / IIIa antagonist analogue of orbofiban , showed no specific binding to cardiomyocytes , but the radioligand accumulated intracellularly over 72 h . ( 3 ) H-SC 52012B also bound directly to human recombinant caspase - 3 ( K ( d ) , 59 + / - 2 nm ) , and this was prevented by orbofiban , xemilofiban , and the monoclonal DB00054 SUB 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 .

Repetitive profound thrombocytopenia after treatment with tirofiban : a case report . The P08514 REA / 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 SUB 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 .

7E3 F ( ab ' ) 2 , a monoclonal antibody to the platelet P08514 REA / IIIa receptor , protects against microangiopathic hemolytic anemia and microvascular thrombotic renal failure in baboons treated with C4b binding protein and a sublethal infusion of Escherichia coli . We have used our previously described baboon model of infusion of both a sublethal dose of Escherichia coli and C4b binding protein to assess the impact of inhibiting platelet function with the F ( ab ' ) 2 fragment of the monoclonal antibody DB00054 SUB , directed against the platelet glycoprotein ( GP ) IIb / IIIa receptor , on the characteristic microvascular changes . At a dose of 0.25 to 0.35 mg / kg bolus plus an infusion of 0.25 to 0.35 mg / kg over 6 hours , c7E3 F ( ab ' ) 2 had only a minimal impact on fibrinogen consumption and delayed but did not prevent , the development of thrombocytopenia . Treatment with 7E3 F ( ab ' ) 2 , however , produced significant protection from the development of microangiopathic hemolysis and renal insufficiency . Histologic examination supported these observations , with treated animals having fewer schistocytes on blood smear and less evidence of ischemic renal changes . Treated animals also had more rapid recovery of peripheral white blood counts , suggesting a possible protective effect of treatment on ischemic damage to the bone marrow . These data indicate that potent inhibition of platelet function via P08514 REA / IIIa receptor blockade can decrease ischemic organ damage in this animal model that has features similar to those found in diffuse intravascular coagulation , hemolytic uremic syndrome , and thrombotic thrombocytopenic purpura .

Using ImageJ for the quantitative analysis of flow-based adhesion assays in real-time under physiologic flow conditions . This article intends to close the gap between the abundance of regular articles focusing on adhesive mechanisms of cells in a flow field and purely technical reports confined to the description of newly developed algorithms , not yet ready to be used by users without programming skills . A simple and robust method is presented for analysing raw videomicroscopic data of flow-based adhesion assays using the freely available public domain software ImageJ . We describe in detail the image processing routines used to rapidly and reliably evaluate the number of adherent and translocating platelets in videomicroscopic recordings . The depicted procedures were exemplified by analysing platelet interaction with immobilized P04275 REA and fibrinogen in flowing blood under physiological wall shear rates . Neutralizing GPIbalpha function reduced shear-dependent platelet translocation on P04275 REA and abolished firm platelet adhesion . DB00054 SUB , Tirofiban and DB00063 completely inhibited P08514 REA / IIIa-dependent stable platelet deposition on fibrinogen . The presented method to analyse videomicroscopic recordings from flow-based adhesion assays offers the advantage of providing a simple and reliable way to quantify flow-based adhesion assays , which is completely based on ImageJ and can easily be applied to study adhesion mechanisms of cells in non-fluorescent modes without the need to deviate from the presented protocol .

Regulation of megakaryocytopoiesis by thrombopoietin and stromal cells . P40225 REA ( Tpo ) is a cytokine which stimulates megakaryocyte maturation . We found that Tpo is constitutively and ubiquitously expressed in all tissues examined , including bone marrow stromal cells , even in thrombocytopenia , thrombosis and steady-state condition in mice . Thus , platelet level in circulation is not regulated by Tpo gene expression . Furthermore , when the purified megakaryocytes were cocultured with the stromal cells , most of the megakaryocytes adhered to the stromal cells and remained unchanged , while free megakaryocytes induced proplatelet formation . Thus the stromal cells in bone marrow secrete Tpo and stimulate megakaryocytopoiesis , but the interaction of megakaryocytes with the stromal cells may suppress platelet formation . Study on signal transduction through Q9UHA4 revealed that Tpo induces activation of O60674 REA and Tyk 2 , which in turn activate P42224 REA , P40763 REA and P42229 REA . Further , Tpo stimulates transcription factors P15976 REA and NF-E 2 , which induce differentiation markers , P08514 REA / IIIa and Pm - 1 . In addition , Shc , Vav , Ras , P04049 REA , MAPKK , MAPK and Pim - 1 are also activated . Thus , Tpo activates a lineage-specific cascade as well as a specific JAK - P35610 REA cascade and a common signaling cascade .

Delayed immunologic thrombocytopenia induced by abciximab . DB00054 SUB is an anti - P08514 REA - IIIa drug widely used to prevent thrombotic complications during percutaneous coronary intervention . We now report on the immunologic origin of thrombocytopenia developing between 7 and 12 days after the onset of abciximab infusion . Antibodies directed against abciximabcoated platelets were located in 5 patients with delayed thrombocytopenia , just as they were present in a patient whose platelet count fell within a few hours after receiving the drug . DB00054 SUB - dependent IgG antibody was revealed in serum using control platelets in the monoclonal antibody immobilization of platelet antigens assay ( MAIPA ) performed with SZ22 , a MoAb to P08514 REA . The presence of IgG antibodies specific for platelets sensitized with abciximab was confirmed by flow cytometry . They were not located in 13 patients receiving abciximab but whose platelet counts remained stable . For three patients , antibodies were transient and their presence related to the extent of the thrombocytopenia . Surprisingly , antibodycontaining plasma from three patients induced abciximabdependent activation and aggregation of normal platelets , a finding confirmed by electron microscopy . Immunogold labeling revealed that abciximab was associated with platelets in the aggregate , suggesting that its inhibitory effect was overcome by the platelet stimulation . In summary , these results show that abciximab-dependent thrombocytopenia can be delayed and potentially prothrombotic .

Hematopoietic differentiation of embryonic stem cells : an in vitro model to study gene regulation during megakaryocytopoiesis . We are interested in the regulation of the tissue specificity of the megakaryocyte-specific platelet glycoprotein IIb gene . The murine embryonic stem ( ES ) cells are able to differentiate into erythroid , mast and granulomonocytic cells in appropriate culture conditions . Our goal is to optimize the production of myeloid cells including megakaryocytes ( MKs ) by ES cells . We have found that coculture with MS - 5 stromal cells and the presence of a cocktail of hematopoietic growth factors ( HGFs ) [ stem cell factor , interleukin 3 ( P08700 REA ) , P05231 REA , IL - 11 , G - P04141 REA and erythropoietin ] had a high synergistic activity on differentiation of ES cells into pure and MK-containing myeloid colonies from day 12 embryoid bodies . P40225 REA increased the number of MKs only when added to the P14210 REA cocktail in the presence of MS - 5 cells . Interestingly , many MKs exhibited a " hairy " appearance evocative of pseudopodial proplatelet formation . Expression of genes specific for the megakaryocytic lineage , P08514 REA , P02776 REA , mpl and P05106 REA , was detected by reverse transcriptase-polymerase chain reaction ( RT-PCR ) during differentiation of ES cells , and their relative time course was evaluated . This demonstrates that optimized culture conditions for the differentiation of ES cells into the MK lineage provide a useful tool for the study of the regulation of expression of genes during megakaryocytopoiesis .

Concentration-dependent effect of abciximab on platelets and neutrophils in a model of cardiopulmonary bypass . DB00054 SUB is a P08514 REA / IIIa antagonist used in percutaneous coronary interventions to avoid platelet activation , thrombosis and inflammation . We investigated whether abciximab influenced platelet activation and platelet interaction with neutrophils and polyvinyl chloride ( PVC ) in a cardiopulmonary bypass ( P15086 REA ) model . Isolated platelets , preincubated with and without 0.1- 20 microg / mL of abciximab , were resuspended with neutrophils in plasma and recirculated by a roller pump . Platelet , but not neutrophil adhesion to PVC was inhibited by abciximab . Only high doses of abciximab reduced platelet aggregation size , but simultaneously increased platelet-neutrophil aggregation . DB00054 SUB had no effect on platelet CD62P expression or degranulation , but platelet activation on platelet-neutrophil aggregates increased with high doses . Only low doses inhibited neutrophil degranulation . The concentration-dependent effect of abciximab on platelet-neutrophil interaction reduces its usefulness and stresses the dependency on experimental design in the evaluation of abciximab . Our study does not support the use of abciximab alone in P15086 REA . However , incorporation of surface-coating the biomaterial with abciximab may be an interesting option .

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 REA - IIIa ( A2A9 , DB00054 SUB , PAC 1 ) 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 REA - IIIa and is fully supported by the intracellular pool of P08514 REA - 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 REA - 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 REA - IIIa to include platelet vesiculation and suggests that one or all of its binding ligands play a role .

The retrograde ventriculo-sinus shunt ( El Shafei RVS shunt ) . Rationale , evolution , surgical technique and long-term results . Since 1990 , 110 retrograde ventriculo-sinus ( RVS ) shunts were implanted ; 98 patients ( 89.1 % ) benefited - 1 of them ( 0.9 % ) after shunt revision . The manifestations of high intra cranial pressure ( ICP ) disappeared , there were no problems related to improper cerebrospinal fluid ( P04141 REA ) drainage , and the transcranial Doppler ( P24386 REA ) resistive index ( RI ) measurements decreased to within normal ranges . Radiologically , the ventriculomegaly showed no regression in patients with open craniums and variable degrees of mild regression in patients with rigid craniums . Complications that needed shunt removal or revision occurred in 13 patients ( 11.8 % ) ; 1 patient ( 0.9 % ) died before shunt revision ; they were all due to technical errors committed during the stages of evolution of the surgical technique for shunt implantation . The follow-up ranged between 4 months and 11 years ( mean 3.42 years ) . CONCLUSION : the RVS shunt is a simple , minimally invasive , physiological procedure for treatment of hydrocephalus and is suitable for all ages .

Arterial reocclusion and persistent distal occlusion after thrombus aspiration . BACKGROUND AND PURPOSE : Early reocclusion of intracranial arteries can lead to poor clinical outcome . We report reocclusion detection after endovascular clot aspiration , followed by administration of P08514 REA - IIIa antagonist under continuous ultrasound monitoring . CASE DESCRIPTION : A 73 - year-old man developed the right middle cerebral artery ( MCA ) occlusion with NIHSS 17 points , 6 days after aortic valve replacement . Recanalization was achieved with Penumbra ™ system and reocclusion was detected with transcranial Doppler ( P24386 REA ) 30 minutes postcompletion of intra-arterial procedure . Proximal recanalization was achieved with the second thrombus aspiration while M2 MCA occlusion persisted beyond the reach of the device . Intravenous abciximab was administered under continuous P24386 REA monitoring . Recanalization with Thrombolysis in Brain Ischemia ( TIBI ) flow grade 4 was observed at 60 minutes postintervention accompanied with clinical recovery to NIHSS 3 points . DB00054 SUB was given for 12 hours with no hemorrhagic transformation on repeat CT scan . Patient was discharged home with mild left pronator drift and facial droop , and his modified ranking score was 1 at 6 - week follow-up visit . CONCLUSIONS : Early arterial reocclusion can occur after successful thrombus aspiration while P08514 REA - IIIa antagonist administration may lead to subsequent recanalization of persisting distal occlusions not amenable to mechanical removal .

Potential future clinical applications for the P08514 REA / IIIa antagonist , abciximab in thrombosis , vascular and oncological indications . DB00054 SUB ( ReoPro ) is a mouse-human chimeric monoclonal antibody Fab fragment of the parent murine monoclonal antibody DB00054 SUB , 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 REA ) . DB00054 SUB binds with high avidity to both the non-activated and activated form of the P08514 REA / 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 SUB 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 REA / 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 REA / 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 .

Characterization of the response of human bone marrow endothelial cells to in vitro irradiation . Endothelial cell dysfunction is a classic consequence of radiation damage . Bone marrow endothelial cells ( BMEC ) are a critical component of the stroma in the regulation of haemopoiesis . In animal models , radiation-induced injury of BMEC has been described and a role for BMEC in haemopoietic regeneration after irradiation has been suggested . However , functions of BMEC involved in the haemopoietic regeneration have not been assessed . Therefore we studied the functional response of human BMEC to irradiation using the transformed human BMEC line ( TrHBMEC ) irradiated with 2 . 5 or 10Gy . Our results showed a time - and a dose-dependent increase in damage to irradiated TrHBMEC measured by a decreased number of adherent cells which correlated with increased apoptosis and augmented release of soluble P05362 REA and P04275 REA . 2 Gy irradiated TrHBMEC expressed more P05362 REA on their surface than non-irradiated cells , whereas no change in P19320 REA , P16581 REA and P16284 REA expression was observed . An increased production of DB00099 , GM - P04141 REA , P10145 REA , P05231 REA , IL - 1alpha , IL - 11 , MIP - 1alpha and P21583 REA and no production of P15018 REA , P01375 REA , P07202 REA and P08700 REA by 2 Gy irradiated TrHBMEC was observed . The haemopoietic supportive function of TrHBMEC was not altered after a 2 Gy exposure . These results suggest that although radiation induces endothelial cell damage , irradiated cells still support the proliferation and the differentiation of P28906 REA + haemopoietic cells .

DB00054 SUB : cost-effective survival advantage in clinical trials and clinical practice . Adjunctive blockade of the platelet glycoprotein ( GP ) IIb / IIIa receptor during either percutaneous coronary intervention ( P05154 REA ) or for patients who present with non-ST segment elevation acute coronary syndromes has demonstrated efficacy in reducing platelet-mediated adverse cardiovascular ischemic events . The three currently available agents ( abciximab , eptifibatide , tirofiban ) differ markedly in pharmacodynamic and pharmacokinetic profiles , receptor affinity , and cost . Although pharmacoeconomic substudies are available from placebo-controlled randomized trials of platelet P08514 REA / IIIa blockade during P05154 REA , " real-world " cost-effectiveness data from high-volume practice are lacking . Therefore , in-hospital and late ( 6 - month ) clinical outcomes and cumulative cost / charge data were analyzed on 1472 consecutive P05154 REA procedures ( 70 % received abciximab ) performed by high-volume operators at a single institution . ( 1 ) Data were adjusted for lack of randomized treatment allocation with the use of a propensity scoring technique . Adjunctive abciximab therapy for P05154 REA was associated with a significant ( 3.4 % ) reduction in mortality to 6 months . Based on the economic cost-effectiveness concept of cost per life year gained relative to standard therapy , ( 2,3 ) abciximab provided a cost-effective survival advantage in high-volume interventional practice that compares very favorably with currently accepted standards . Clinical and procedural demographics associated with increased cost-effectiveness include multivessel coronary intervention , stent deployment , recent ( < 1 week ) myocardial infarction ( MI ) , and impaired left-ventricular ( LV ) function .

P15018 REA functions as a growth factor in pancreas carcinoma cells : Involvement of regulation of P15018 REA and its receptor expression . P15018 REA ( P15018 REA ) is a pleiotrophic cytokine , which plays an important role in inducing cancer cachexia . We have previously reported that P15018 REA promotes cell proliferation in some human carcinoma cells through c-fos , jun-B and cyclin-E expression . In the present study , we analyzed the regulation of P15018 REA and its receptor ( P42702 REA ) expression in pancreatic carcinoma cells . Seven pancreatic carcinoma cells expressed constitutively P15018 REA and its heterodimer receptor ( P42702 REA and P40189 REA ) mRNA in RPMI - 1640 medium without FBS . The amount of P15018 REA immunoreactive protein was 132.5+ / - 52 pg / 10 ( 6 ) cells in culture supernatants without FBS . Pro-inflammatory cytokines , such as P01375 REA , IL - 1beta , P05231 REA , P10145 REA , or P15018 REA , enhanced the expression of P15018 REA mRNA in Hs - 700T and Hs - 766T cells . Addition of P15018 REA significantly induced cell proliferation of Hs700T in 13 days P15018 REA dose-dependently . However , anti - P15018 REA IgG failed to suppress cell proliferation in Hs - 700T cells . P15018 REA acted as a paracrine growth factor in Hs - 700T cells , which expressed low amount of P15018 REA without stimuli . Cellular signal transductions by P15018 REA was down-regulated by inhibitors of protein kinase C ( PKC ) , protein tyrosine kinase ( PTK ) , and Ca / P62158 . P15018 REA induced phosphorylation of P40763 REA . Moreover , exogenous P15018 REA upregulated the expression of P42702 REA mRNA . Antisense P42702 REA oligonucleotide significantly suppressed cell growth in the presence of P15018 REA in Hs - 700T cells . These results suggest that cytokine network might alter the expression and responsiveness to P15018 REA in tumor microenvironment .

Therapeutic Implications of a Specific Murine Monoclonal Antibody ( DB00054 SUB ) to the Platelet Receptor P08514 REA / 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 .

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 SUB ( a monoclonal antibody to P08514 REA / IIIa that inhibits platelet aggregation ) both contortrostatin and multisquamatin demonstrated P08514 REA / IIIa specific binding to human and canine platelets . The IC50 for contortrostatin displacement of 7E3 binding to human and canine P08514 REA - / 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 REA / IIIa .

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 SUB , 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 REA / IIIa overlapping with that of DB00054 SUB , leading to the blockade of fibrinogen binding to its receptor .

Contribution of platelets and the vessel wall to the antithrombotic effects of a single bolus injection of Fab fragments of the antiplatelet P08514 REA / 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 SUB ( 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 )

The anti - P08514 REA - IIIa agents : fundamental and clinical aspects . The platelet P08514 REA / 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 SUB ( 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 REA / IIIa receptors . This new class of agents thus holds promise for improving the therapy of angioplasty as well as perhaps other thrombotic phenomena .

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 REA / IIIa monoclonal antibody DB00054 SUB . The bispecific antifibrin-antiplatelet urokinase conjugate ( BAAUC ) was synthesized and characterized . Assays with either immobilized platelets , P08514 REA / 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 .

Time course of the effects of a single bolus injection of F ( ab ' ) 2 fragments of the antiplatelet P08514 REA / 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 SUB [ 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 )

Cytokine signaling in the human brain capillary endothelial cell line hCMEC / D3 . Brain microvascular endothelial cells are part of the blood-brain barrier and participate actively in immunological processes including cytokine-mediated inflammatory reactions . Using the human brain capillary endothelial cell line hCMEC / D3 , activation of JAK / P35610 REA signaling pathways were studied in response to stimulation by cytokines . The phenotype of hCMEC / D3 cells was confirmed by flow cytometry analysis of cell adhesion factors ( cluster of differentiation molecules CD31 and P28906 REA ) and the P04275 REA endothelial marker was detected by immunofluorescence . Strong P42224 REA , P42226 REA and P40763 REA activation was observed in response to interferon-gamma ( P01579 REA ) , interleukin 4 ( P05112 REA ) and interleukin 6 ( P05231 REA ) , respectively . Nuclear translocation of phosphorylated P35610 REA proteins was visualized by confocal microscopy . Treatment of hCMEC / D3 cells with P01579 REA resulted in interferon-induced upregulation of major histocompatibility complex ( MHC ) class I within 48h . Interferon-alpha ( IFN-alpha ) did not activate P42224 REA or P40763 REA nor did it induce MHC class I upregulation . Therefore , hCMEC / D3 cells were judged to be non-responsive to IFN-alpha . We also observed that hCMEC / D3 cells exhibit functional expression of alternative cytokine signal transduction pathways ( i . e . P01375 REA mediated activation of NF-kappaB ) . Together these results indicate that human blood-brain barrier hCMEC / D3 cells are responsive towards stimulation with various cytokines . We conclude that this unique cell line can be used to explore in vitro human blood-brain barrier functionality under proinflammatory conditions .

Antibody-mediated neuronal cell signaling in behavior and movement disorders . Behavioral and movement disorders may have antibody responses where mimicry and signal transduction may lead to neuropsychiatric abnormalities . In our study , antibodies in pediatric autoimmune neuropsychiatric disorders associated with streptococci ( PANDAS ) reacted with the neuronal cell surface and caudate-putamen and induced calcium-calmodulin dependent protein ( P62158 ) kinase II activity in neuronal cells . Depletion of serum IgG abrogated P62158 kinase II cell signaling and reactivity of P04141 REA was blocked by streptococcal antigen N-acetyl-beta-d-glucosamine ( GlcNAc ) . Antibodies against GlcNAc in PANDAS sera were inhibited by lysoganglioside G ( M1 ) . Results suggest that antibodies from an infection may signal neuronal cells in some behavioral and movement disorders .

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 SUB ( P08514 REA - IIIa complex-specific ) . As determined by P28222 REA ( a P16109 REA - 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 REA and the P08514 REA - IIIa complex , and the extent of decrease in the platelet surface expression of the GPIb-IX complex . ( ABSTRACT TRUNCATED AT 250 WORDS )

Covariates of corticotropin-releasing hormone ( P06850 REA ) concentrations in cerebrospinal fluid ( P04141 REA ) from healthy humans . BACKGROUND : Define covariates of cerebrospinal corticotropin-releasing hormone ( P06850 REA ) levels in normal humans . CRHCSF was measured in 9 normal subjects as part of an intensive study of physiological responses stressors in chronic pain and fatigue states . CRHCSF was first correlated with demographic , vital sign , Q9Y251 REA axis , validated questionnaire domains , baseline and maximal responses to pain , exercise and other stressors . Significant factors were used for linear regression modeling . RESULTS : Highly significant correlations were found despite the small number of subjects . Three models were defined : ( a ) CRHCSF with blood glucose and sodium ( explained variance = 0.979 , adjusted R2 = 0.958 , p = 0.02 by 2 - tailed testing ) ; ( b ) CRHCSF with resting respiratory and heart rates ( R2 = 0.963 , adjusted R2 = 0.939 , p = 0.007 ) ; and ( c ) CRHCSF with SF - 36 Vitality and Multidimensional Fatigue Inventory Physical Fatigue domains ( R2 = 0.859 , adjusted R2 = 0.789 , p = 0.02 ) . CONCLUSIONS : Low CRHCSF was predicted by lower glucose , respiratory and heart rates , and higher sodium and psychometric constructs of well being . Responses at peak exercise and to other acute stressors were not correlated . CRHCSF may have reflected an overall , or chronic , set-point for physiological responses , but did not predict the reserves available to respond to immediate stressors .

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 REA - 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 REA - 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 REA - IIIa . DB00054 SUB rapidly achieves P08514 REA - 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 REA - IIIa antagonism might contribute to development of acute profound thrombocytopenia .

DB00338 MEN , a gastric proton pump inhibitor , inhibits melanogenesis by blocking Q04656 REA trafficking . DB00338 MEN is a proton pump inhibitor used in the treatment of peptic ulcer disease and gastrosophageal reflux disease and acts by irreversibly blocking P20648 REA , a P-type H + / K + ATPase in gastric parietal cells . We found that omeprazole and its closely related congeners inhibited melanogenesis at micromolar concentrations in B16 mouse melanoma cells , normal human epidermal melanocytes , and in a reconstructed human skin model . DB00338 MEN topically applied to the skin of UV-irradiated human subjects significantly reduced pigment levels after 3 weeks compared with untreated controls . DB00338 MEN had no significant inhibitory effect on the activities of purified human tyrosinase or on the mRNA levels of tyrosinase , dopachrome tautomerase , Pmel 17 , or O75030 REA mRNA levels . Although melanocytes do not express P20648 REA , they do express Q04656 REA , a copper transporting P-type ATPase in the trans-Golgi network that is required for copper acquisition by tyrosinase . Q04656 REA relocalization from the trans-Golgi network to the plasma membrane in response to elevated copper concentrations in melanocytes was inhibited by omeprazole . DB00338 MEN treatment increased the proportion of EndoH sensitive tyrosinase , indicating that tyrosinase maturation was impaired . In addition , omeprazole reduced tyrosinase protein abundance in the presence of cycloheximide , suggestive of increased degradation . Our findings are consistent with the hypothesis that omeprazole reduces melanogenesis by inhibiting Q04656 REA and by enhancing degradation of tyrosinase .

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 REA / 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 REA / IIIa monoclonal antibody DB00054 SUB . Both agents completely inhibited platelet aggregation caused by all agonists tested except ristocetin . Further , both inhibited human platelet adhesion to P04275 REA , 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 REA / IIIa was precipitated from the solubilized fraction of platelets by both agents . In contrast , integrin alphavbeta 3 was precipitated from the solubilized fraction of human umbilical vein endothelial cells by abciximab but not by YM337 . DB09222 binding to purified P08514 REA / IIIa was dose-dependently inhibited by both agents . In contrast , vitronectin binding to purified integrin alphavbeta 3 was dose-dependently inhibited by abciximab but not by YM337 , supporting the idea that abciximab reacts to integrin alphavbeta 3 . Therefore , YM337 was suggested to bind to a different epitope of P08514 REA / IIIa from abciximab . These results suggest that YM337 specifically acts on platelet P08514 REA / IIIa receptors and has similar inhibitory properties on platelet aggregation and platelet adhesion to abciximab .

Progress in the field of P08514 REA / 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 SUB , 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 .

DB00054 SUB : 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 REA ) . 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 SUB has been investigated in the clinical field far more extensively than the other P08514 REA / IIIa inhibitors . DB00054 SUB 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 SUB , 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 REA 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 .

The high molecular mass , glycoprotein Ib-binding protein flavocetin-A induces only small platelet aggregates in vitro . The direct effects of snake venom glycoprotein ( GP ) Ib-binding proteins on platelet receptors during the formation of platelet aggregates were determined by a particle counting method using light scattering . Flavocetin-A induces small platelet aggregates , but not medium or large ones . However , neither jararaca GPIb-BP nor tokaracetin induce platelet aggregation . The flavocetin-A dose-response curve for formation of small aggregates is bell-shaped , with maximal effect at 1 to 2 microg / mL . The formation of small aggregates was not observed when fixed human platelets were used . Jararaca GPIb-BP , the anti-GPIb monoclonal antibody GUR 83-35 , prostaglandin I2 , and ethylene diamine-N , N-dimethylformamide all inhibited flavocetin-A-induced small aggregate formation , but acetylsalicylic acid did not . Furthermore , anti - P08514 REA / IIIa monoclonal antibodies , DB00054 SUB , and YM337 significantly but partially inhibited aggregate formation , but the anti - P04275 REA monoclonal antibody NMC - 4 had no effect . The formation of small aggregates required extracellular calcium , but flavocetin-A did not elevate cytosolic calcium . These results suggest that flavocetin-A binds to intact platelets , initiating platelet responses and inducing platelet aggregate formation by cross-linking platelets . Consequently , flavocetin-A may be a useful tool to study the mechanism of GPIb-mediated platelet activation and the structure-function relationships of GPIb .

Rapid and sustained coronary artery recanalization with combined bolus injection of recombinant tissue-type plasminogen activator and monoclonal antiplatelet P08514 REA / 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 SUB ) against the human platelet P08514 REA / 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 )

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 SUB , a P08514 REA / 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 .

Glycoprotein IIb / IIIa blockade inhibits platelet-mediated force development and reduces gel elastic modulus . The effects of P08514 REA / IIIa blockade on clot retraction were studied utilizing an instrument which directly measures force produced by platelets . P08514 REA / IIIa disruption by calcium chelation , and P08514 REA / IIIa blockade by peptides and anti - P08514 REA / IIIa antibodies were investigated . One mM DB00974 suppressed ADP-induced platelet aggregation by 72 % and reduced force developed at 1200 s by 33 % . At 234 microM , the tetrapeptide DB00125 - DB00145 - DB00128 - DB00133 ( RGDS ) suppressed platelet aggregation by 74 % , reduced force at 1200 s by 45 % and reduced gel elastic modulus by 19 % . At 10 microM , the peptide D - DB00125 - DB00145 - DB00128 - L-Try ( D-RGDW ) completely suppressed platelet aggregation , reduced force development by 38 % and reduced gel elastic modulus by 29 % . At 0.133 microM , monoclonal anti - P05106 REA antibody ( AP - 3 ) reduced force development by 74 % and reduced gel modulus by 60 % . Murine antiGPIIb / IIIa antibodies 10E5 and 7E3 markedly suppressed force development . At 0.133 microM , 10E5 reduced force by 89 % and reduced gel modulus by 67 % . At 0.053 microM , 7E3 completely stopped force development and reduced gel modulus by 46 % . Platelet aggregation was blocked by 0.027 microM DB00054 SUB . Selective P08514 REA blockade by antibodies did not affect force development . None of the agents studied altered fibrin structure as monitored by effects of fibrin mass / length ratios . Suppression of platelet aggregation occurred at inhibitor concentrations substantially lower than those required to suppress force development . Complete suppression of platelet aggregation did not assure inhibition of clot retraction probably due to profound platelet activation by thrombin . ( ABSTRACT TRUNCATED AT 250 WORDS )

7E3 F ( ab ' ) 2 , an effective antagonist of rat alphaIIbbeta 3 and alphavbeta 3 , blocks in vivo thrombus formation and in vitro angiogenesis . DB00054 SUB ( c7E3 Fab , ReoPro ) blocks P08514 REA / IIIa and alphavbeta 3 and inhibits thrombotic and proliferative events only in humans and non-human primates . The bivalent F ( ab ' ) 2 fragment is an effective anti-thrombotic agent in canine models . In the present study , 7E3 F ( ab ' ) 2 was also found to bind to rat P08514 REA / IIIa ( KD = 27 + / - 4 microg / mL ) and alphavbeta 3 ( KD = 9 + / - 8 microg / mL ) , to block in vitro rat platelet aggregation ( IC50 = 16 + / - 6 microg / mL ) , and to inhibit alphavbeta 3 - mediated microvessel sprout formation in a rat aortic ring assay . Following administration of 7E3 F ( ab ' ) 2 ( 4 mg / kg ) to rats , platelet aggregation was completely blocked for up to 6 h and thrombus formation in response to a rat abdominal aorta double crush injury was prevented . Effective chronic dosing was achieved with 6 mg / kg daily I . P . injections . In vitro mixing experiments indicated that 7E3 F ( ab ' ) 2 redistributed to unlabeled platelets in 2 h . Ex vivo , 7E3 F ( ab ' ) 2 was detected on platelets for up to 4 days after a single 4 - mg / kg injection . These data suggest that 7E3 F ( ab ' ) 2 may be a useful agent to study the effects of P08514 REA / IIIa and alphavbeta 3 blockade in rat models of thrombosis and vascular disease .

P40189 REA - linked signal transduction promotes the differentiation and maturation of dendritic cells . In order to explore the role of P40189 REA - linked signal transduction in the differentiation and maturation of dendritic cells ( DC ) , the mAb , B - P28222 REA , an agonist of P40189 REA , was used for the activation of P40189 REA on DC . The effects of cytokines and of anti - P40189 REA mAb on the proliferation of DC , and their expression of IL - 12 and P33681 REA ( P33681 REA - 1 ) by DC were evaluated . DC differentiating from peripheral blood mononuclear cells did not express the P05231 REA receptor alpha chain , but expressed P40189 REA . Anti - P40189 REA mAb promoted the proliferation of DC , induced by P05112 REA and granulocyte macrophage colony stimulating factor ( GM - P04141 REA ) , by up-regulating the GM - P04141 REA receptor on DC . DC induced by P40189 REA mAb and cytokines expressed DC-derived CC chemokine , as measured by RT-PCR . Induced DC also stimulated strong proliferation of autologous T cells in mixed lymphocyte reaction since an up-regulated expression of IL - 12 and P33681 REA ( P33681 REA - 1 ) was observed in DC activated by anti - P40189 REA mAb . Thus , P40189 REA signal transduction is important for the differentiation and maturation of DC .

The effects of P08514 REA - IIIa antagonists and a combination of three other antiplatelet agents on platelet-leukocyte interactions . The effects of the P08514 REA - 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-C 69931 ( 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 SUB 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 REA - 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 .

DB00054 SUB pharmacodynamics are unaffected by antecedent therapy with other P08514 REA / 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 REA ) during infusion of these drugs , conversion to abciximab , which has long term proven clinical efficacy and cost-effectiveness , following P05154 REA 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 REA / 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 REA / 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 REA / IIIa receptor is not altered by immediate prior exposure of platelets to small molecule P08514 REA / 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 REA / IIIa antagonists .

DB00054 SUB 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 SUB , a human murine chimeric antibody to the P08514 REA / 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 REA ) . 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 REA / 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 SUB was a breakthrough drug in the management of high risk ACS patients undergoing P05154 REA . However , with newer available therapies and improvement in P05154 REA technology , dose and delivery of this drug have evolved as we try to extract maximum benefit while minimizing the adverse effects associated with it .

Characterization of canine platelet P16109 REA ( CD 62 ) and its utility in flow cytometry platelet studies . 1 . P16109 REA ( P16109 REA , GMP 140 , or CD62 ) a member of lectin-like adhesive proteins is expressed on the surface of activated degranulated canine platelets and is the calcium-dependent receptor for leukocyte adhesion . 2 . The electrophoretic mobility of P16109 REA , by Western blot analysis and immunoprecipitation from radiolabeled membranes of canine and human platelets , was similar or identical and immunocytochemical studies localized P16109 REA in internal vesicles similar to the alpha granule localization in human platelets . 3 . Two antibodies to human P16109 REA KC4 . 1 and Q99440 . 2 crossreacted with canine platelets whose surface binding , in response to agonists thrombin , calcium ionophore ( A23187 ) , phorbol esters and ADP , was similar . 4 . Anti - P16109 REA antibodies in conjunction with crossreacting anti - P08514 REA / IIIa antibodies ( A2A9 , DB00054 SUB , RUU-PL 7F12 ) enables the analysis of activated platelets , platelet-derived microparticles and platelet-leukocyte interactions in canine models by flow cytometry .

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 REA - IIIa ) antagonists . They are cysteine-rich , DB00125 - DB00145 - DB00128 ( RGD ) - containing peptides , and bind to P08514 REA - IIIa complex on platelet membrane with a very high affinity ( Kd , 10 ( - 7 ) - 10 (-8 ) M ) . In this study , we analyzed P08514 REA - 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 SUB , a monoclonal antibody against P08514 REA - 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 )

Antithrombotic therapy in the acute phase : new approaches . Both anticoagulants and antiplatelet agents have been advocated , used and studied for the treatment of acute ischemic stroke . Randomized trials of unfractionated heparin , low-molecular-weight heparin and heparinoids have failed to show an overall benefit to these agents largely because the benefits in reducing thromboembolic events are offset by the increased risk of bleeding complications . The International Stroke Trial , the Trial of ORG 10172 Acute Stroke Treatment and studies of fraxaripine all failed to show an overall benefit to anticoagulation in the patients studied . DB00945 has been shown to offer a modest benefit when studied in patients treated within 48 h of stroke onset . DB05099 is an antithrombotic agent that acts by reducing circulating fibrinogen levels . Patients treated within 3 h of stroke symptom onset had a better functional outcome at 90 days compared to placebo-treated patients with both the benefits and the risk of intracerebral bleeding related to the fibrinogen lowering achieved . DB00054 SUB is a blocker of the platelet P08514 REA / IIIa receptor . A dose finding safety study suggests that in doses up to that typically given in patients with acute coronary occlusion syndromes , there is no increased risk of symptomatic intracerebral bleeding and suggestions of potential benefits on neurological outcome .

Analysis of P08514 REA / IIIa receptor number by quantification of 7E3 binding to human platelets . A large number of glycoprotein ( GP ) IIb / IIIa receptors are present on the surface of platelets . Studies to define precisely the number of P08514 REA / IIIa receptors using specific monoclonal antibodies ( MoAbs ) or fibrinogen binding have , however , yielded varying estimates of receptor number . To refine the quantitative estimation of P08514 REA / IIIa receptors on resting platelets , we have used the MoAb DB00054 SUB , which has high affinity for P08514 REA / IIIa . Quantitative binding studies were performed using radiolabeled conjugates of 7E3 IgG , as well as fragments and derivatives of DB00054 SUB . For platelets obtained from any single individual , the numbers of 7E3 F ( ab ' ) 2 and IgG molecules bound per platelet were equivalent ( approximately 40,000 ) , whereas the number of Fab molecules bound per platelet was consistently approximately twofold higher ( approximately 80,000 ) . To investigate the basis of the quantitative disparity in binding of intact 7E3 and 7E3 F ( ab ' ) 2 versus 7E3 Fab , we studied the binding of a newly constructed , bispecific ( Fab ' ) 2 molecule containing only a single 7E3 combining site . Because this construct bound to the same extent as the Fab species , the larger size of the intact 7E3 and 7E3 F ( ab ' ) 2 molecules could not explain the reduced number of molecules that bound per platelet compared to the Fab fragment . Rather , it appears that the valency of the antibody is the critical factor determining the number of antibody molecules bound per platelet . Thus , we conclude that the binding of 7E3 Fab corresponds most closely with surface P08514 REA / IIIa number and that the number of P08514 REA / IIIa receptors is approximately 80,000 per platelet .

Exercise enhances insulin and leptin signaling in the cerebral cortex and hypothalamus during dexamethasone-induced stress in diabetic rats . Exercise and dexamethasone ( DEX ) are known to have opposite effects on peripheral insulin resistance . However , their effects and mechanism on brain glucose metabolism have been poorly defined . We investigated the modulation of the hypothalamo-pituitary-adrenal ( Q9Y251 REA ) axis and insulin / leptin signaling associated with glucose utilization in the brains of 90 % pancreatectomized diabetic rats , which had been administered two dosages of DEX and exercised for 8 weeks . The data revealed that the administration of a high dose ( 0.1 mg / kg body weight / day ) of DEX ( HDEX ) attenuated insulin signaling in the cerebral cortex and hypothalamus , whereas exercise potentiated their insulin signaling along with induction of Q9Y4H2 REA expression . In parallel with the modulated signaling , glucose utilization , such as glycogen storage and glycogen synthase activity , was suppressed by DEX in the cortex and hypothalamus , while exercise offset the DEX effects . Despite a decrease in epididymal fat mass , HDEX increased serum leptin levels , possibly due to an activated Q9Y251 REA axis , while exercise suppressed the increment . However , DEX reduced leptin-induced P40763 REA phosphorylation in the cortex and hypothalamus , and it increased AMP-activated protein kinase ( AMPK ) phosphorylation only in the hypothalamus . Exercise reversed the phosphorylation of P40763 REA and AMPK which had been modulated by DEX . In conclusion , exercise improves insulin and leptin signaling in the cerebral cortex and hypothalamus of diabetic rats exacerbated with HDEX , contributing to the regulation of body weight and glucose homeostasis .

Monoclonal antibody against the platelet glycoprotein ( GP ) IIb / IIIa receptor prevents coronary artery reocclusion after reperfusion with recombinant tissue-type plasminogen activator in dogs . Localized thrombosis was produced in the left anterior descending ( LAD ) coronary artery of open chest dogs by constricting a segment so as to produce greater than 90 % stenosis ( reducing blood flow to 40 + / - 10 % of baseline ) , and placing a thrombus in the segment immediately proximal to the stenosis by inducing endothelial cell injury and instilling a mixture of blood and thrombin . Intravenous infusion of recombinant tissue-type plasminogen activator ( rt-PA ) at a rate of 15-30 micrograms / kg per min for 30 or 60 min in eight dogs induced coronary artery reperfusion within 23 + / - 7 min ( mean + / - SD ) , but reocclusion occurred despite heparin anticoagulation in all but one of these dogs within 7 + / - 5 min . Intravenous injection of 0.8 mg / kg of the F ( ab ' ) 2 fragment of a monoclonal antibody ( DB00054 SUB ) directed against the platelet P08514 REA / IIIa receptor , prevented reocclusion in 10/10 dogs during an observation period of 2 h ( P less than 0.001 vs . rt-PA alone ) . The antibody abolished ADP-induced platelet aggregation and markedly prolonged the bleeding time . Intravenous aspirin or dipyridamole prevented reocclusion for 1 h or more in only 2/7 and 1/6 dogs , respectively . We conclude that the monoclonal antibody is very effective in preventing reocclusion after successful thrombolysis of occluded coronary arteries with rt-PA .