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B S Coller

Publications and source records attributed to B S Coller.

At least 55 records · Page 3Linked to original sources
PubMed1997

In vitro effects of the platelet glycoprotein IIb/IIIa receptor antagonist c7E3 Fab on the activated clotting time.

BACKGROUND: In the evaluation of 7E3 for the Prevention of Ischemic Complications study (EPIC), the activated coagulation (clotting) times (ACTs) were longer in heparinized patients treated with c7E3 Fab than in those treated with placebo. The present study was designed to further investigate this observation by assessing whether the in vitro addition of c7E3 Fab to blood would affect the ACT. METHODS AND RESULTS: Native or heparinized blood obtained from normal volunteers was preincubated with antibodies c7E3 Fab (anti-GPIIb/IIIa and anti-alpha v beta 3), 10E5 (anti-GPIIb/IIIa), or LM609 (anti-alpha v beta 3). The ACTs of the heparinized, but not native samples were significantly prolonged by the addition of c7E3 Fab and 10E5 but not LM609, indicating that the prolongation was due to GPIIb/IIIa blockade. The addition of c7E3 Fab also significantly prolonged the ACTs of blood anticoagulated with the direct thrombin inhibitors hirudin and D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone, indicating that the effect of c7E3 Fab was not exclusively related to decreased release of PF4, a heparin-neutralizing factor, from platelets. CONCLUSIONS: These data support the conclusion that the prolongation of the ACT in patients in EPIC was due to c7E3 Fab blockade of GPIIb/IIIa receptors. This raises the possibility that in vivo c7E3 Fab functions not only as an antiplatelet agent but also as an anticoagulant; direct in vivo data will, however, be required for assessment of this possibility.

Abciximab↗
PubMed1997

GPIIb/IIIa antagonists: pathophysiologic and therapeutic insights from studies of c7E3 Fab.

Platelet GPIIb/IIIa receptor antagonists have been shown to be more potent than aspirin in inhibiting platelet function in vitro and decreasing thrombotic vascular events in animal models in vivo. One of these agents (c7E3 Fab, abciximab, ReoPro) significantly decreased the ischemic complications of percutaneous coronary interventions when given in conjunction with aspirin and heparin to patients at high risk of such complications, and other agents (Integrelin and tirofiban) have shown favorable trends. This review focuses on what we have learned from studies of patients treated with c7E3 Fab, including its pharmacology, its potential mechanism(s) of action, the ability to reverse its effects with platelet transfusion, and the risks of hemorrhage, thrombocytopenia, reinjection, and performing coronary artery bypass surgery. The future of GPIIb/IIIa antagonists therapy is considered, including the potential of orally active agents.

Antibodies, Monoclonal↗
PubMed1996

A Cys374Tyr homozygous mutation of platelet glycoprotein IIIa (beta 3) in a Chinese patient with Glanzmann's thrombasthenia.

A 20-year-old woman from a consanguineous family in the Hunan Province of the People's Republic of China was diagnosed as having Glanzmann's thrombasthenia based on (1) nearly a lifelong history of epistaxis, gum bleeding, petechiae, and purpura; (2) severe menorrhagia resulting in anemia and need for whole-blood transfusion; (3) normal coagulation assays; (4) prolonged bleeding time; (5) absent clot retraction; (6) decreased glass bead retention; (7) absent platelet aggregation in response to adenine diphosphate, epinephrine, and collagen; and (8) normal initial slope of platelet aggregation in response to ristocetin, but with a diminished maximal extent. The patient's platelets had a decreased level of platelet fibrinogen, but the deficiency was not as severe as in other Glanzmann's thrombasthenia patients. As judged by monoclonal antibody binding studies, surface glycoprotein (GP) IIb/IIIa (alpha IIb beta 3) expression was less than 15% of normal and alpha v beta 3 vitronectin receptor expression was 15% to 19% of normal, suggesting that the defect was in GPIIIa (beta 3). Immunoblotting of platelet lysates demonstrated decreased levels of GPIIb (approximately 30% to 35% of normal) and GPIIIa (approximately 10% of normal), and the GPIIb had undergone normal maturational processing into GPIIb heavy and light chains. Sequence analysis of the patient's GPIIIa RNA identified a G to A mutation at nucleotide 1219, predicting a Cys to Tyr substitution at residue 374. The patient's parents, who are first cousins, are asymptomatic and have only minor reductions in platelet aggregation. Direct sequencing of polymerase chain reaction-amplified cDNA and GPIIIa exon VIII indicated that the patient is homozygous and her parents are heterozygous for the mutation. Transient transfection studies in Chinese hamster ovary cells indicated that the mutation results in an 85% to 90% reduction in GPIIb/IIIa surface expression, but these cells retain the ability to mediate adhesion to immobilized fibrinogen. The relative preservation of platelet fibrinogen despite the very low level of platelet surface GPIIb/IIIa expression in this patient raises some interesting questions regarding the mechanism of fibrinogen uptake and the pathophysiology of Glanzmann's thrombasthenia.

Adult↗
PubMed1996

Inhibition of platelet-mediated, tissue factor-induced thrombin generation by the mouse/human chimeric 7E3 antibody. Potential implications for the effect of c7E3 Fab treatment on acute thrombosis and "clinical restenosis".

The murine/human chimeric monoclonal antibody fragment (c7E3 Fab) blocks GPIIb/IIIa and alpha v beta 3 receptors, inhibits platelet aggregation, and decreases the frequency of ischemic events after coronary artery angioplasty in patients at high risk of suffering such events. Although inhibition of platelet aggregation is likely to be the major mechanism of c7E3 Fab's effects, since activated platelets facilitate thrombin generation, it is possible that c7E3 Fab also decreases thrombin generation. To test this hypothesis, the effects of c7E3 Fab and other antiplatelet agents were tested in a thrombin generation assay triggered by tissue factor. c7E3 Fab produced dose-dependent inhibition of thrombin generation, reaching a plateau of 45-50% inhibition at concentrations > or = 15 micrograms/ml. It also inhibited thrombin-antithrombin complex formation, prothrombin fragment F1-2 generation, platelet-derived growth factor and platelet factor 4 release, incorporation of thrombin into clots, and microparticle formation. Antibody 6D1, which blocks platelet GPIb binding of von Willebrand factor, had no effect on thrombin generation, whereas antibody 10E5, which blocks GPIIb/IIIa but not alpha v beta 3 receptors decreased thrombin generation by approximately 25%. Combining antibody LM609, which blocks alpha v beta 3 receptors, with 10E5 increased the inhibition of thrombin generation to approximately 32-41%. The platelets from three patients with Glanzmann thrombasthenia, who lacked GPIIb/IIIa receptors but had normal or increased alpha v beta 3 receptors, supported approximately 21% less thrombin generation than normal platelets. We conclude that thrombin generation initiated by tissue factor in the presence of platelets is significantly inhibited by c7E3 Fab, most likely in part through both GPIIb/IIIa and alpha v beta 3 blockade, and that this effect may contribute to its antithrombotic properties.

Acute Disease↗
PubMed1996

Analysis of GPIIb/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 GPIIb/IIIa receptors using specific monoclonal antibodies (MoAbs) or fibrinogen binding have, however, yielded varying estimates of receptor number. To refine the quantitative estimation of GPIIb/IIIa receptors on resting platelets, we have used the MoAb 7E3, which has high affinity for GPIIb/IIIa. Quantitative binding studies were performed using radiolabeled conjugates of 7E3 IgG, as well as fragments and derivatives of 7E3. 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 GPIIb/IIIa number and that the number of GPIIb/IIIa receptors is approximately 80,000 per platelet.

Antibodies, Bispecific↗
PubMed1996

The effects of thrombocytopenia on the activated coagulation time.

The activated coagulation (clotting) time (ACT) is widely used to monitor heparin therapy during cardiopulmonary bypass (CPB). Since thrombocytopenia occurs during and for some time after CPB, we considered the possibility that thrombocytopenia may affect the ACT. Blood samples were obtained from 12 healthy volunteers. Thrombocytopenic samples were created by a differential centrifugation technique. Blood samples from five thrombocytopenic patients were also studied. The platelet counts of the control citrated whole blood samples were 254,000 +/- 35,000/microL (mean +/- SD), whereas the platelet counts of the thrombocytopenic citrated samples were 49,000 +/- 8000/microL. There were no significant differences in the ACTs between these two groups (143 +/- 8 vs 145 +/- 9 s, respectively, P = 0.16). There were also no significant differences in the ACTs of the heparinized control samples compared with the ACTs of the heparinized thrombocytopenic samples. The platelet counts of the five thrombocytopenic patients were 43,000, 18,000, 16,000, 8000, and 7000/microL, and the ACTs of the unanticoagulated samples were 162, 240, 191, 210, and 215s, respectively. We conclude that the ACT is not affected by moderate thrombocytopenia and therefore this test may be used in patients who are moderately thrombocytopenic.

Anticoagulants↗
PubMed1996

The anti-GPIIb-IIIa agents: fundamental and clinical aspects.

The platelet GPIIb/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 7E3 (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 GPIIb/IIIa receptors. This new class of agents thus holds promise for improving the therapy of angioplasty as well as perhaps other thrombotic phenomena.

Abciximab↗
PubMed1995

Glanzmann's thrombasthenia associated with deletion-insertion and alternative splicing in the glycoprotein IIb gene.

Glanzmann's thrombasthenia is a bleeding disorder characterized by a decrease or absence of the functional platelet membrane glycoprotein (GP) complex, GPIIb/IIIa (alpha IIb beta 3). We describe a new deletion-insertion mutation in the GPIIb gene causing type I Glanzmann's thrombasthenia in two siblings of a consanguineous Iranian-Jewish family. The proband's platelets bound more antibodies against the vitronectin receptor-alpha V beta 3 than normal platelets, suggesting a normal GPIIIa (beta 3) gene and a defect in the GPIIb gene. Sequencing of amplified cDNA and genomic DNA fragments showed a 6-bp deletion and 31-bp insertion in exon 25 of the GPIIb gene. The predominant platelet GPIIb mRNA of the proband was a product of the splicing of exon 24 to a cryptic AG acceptor site in the insertion and encoded for deletion of amino acids Leu817-Asn826 and insertion of eight different amino acids. Cotransfection of COS-7 cells with expression vectors containing wild-type GPIIIa cDNA and the mutated GPIIb cDNA failed to produce detectable amounts of GPIIb/IIIa on the surface of the cells. Allele-specific restriction analysis of genomic DNA of family members showed homozygosity for the mutation in the affected siblings, heterozygosity in the parents, and homozygosity for the normal allele in an unaffected sibling. The observed mutation is in a region that is conserved from rodents to humans and has been suggested to be involved in the interaction between GPIIb and GPIIIa when these GPs are complexed in solution.

Adult↗
PubMed1995

The immunogenicity of the 7E3 murine monoclonal Fab antibody fragment variable region is dramatically reduced in humans by substitution of human for murine constant regions.

A murine monoclonal antibody (7E3) directed against the platelet glycoprotein IIb/IIIa was engineered to reduce immunogenicity by substituting human for murine constant regions. The chimeric antibody is functionally identical to the murine antibody in vitro. Results from clinical trials with 7E3 Fab antibody fragments, however, show that the 7E3 variable region, which elicits the vast majority of the immune response to murine 7E3 Fab, is rendered dramatically less immunogenic (incidence reduced from 17% to 1%) when the identical variable region is linked to human rather than murine constant regions. Neither murine nor human constant regions were highly immunogenic themselves. We conclude that the constant regions of the Fab fragments are critical in modulating the immune response elicited by the linked 7E3 variable region. Because naturally occurring anti-human Fab fragment antibodies are prevalent both in the normal human population and in the patient population studied here, murine 7E3 Fab and chimeric 7E3 Fab may be fundamentally different in their interactions with the human immune system. This difference may be related to the dramatic difference in immunogenicity observed between murine 7E3 Fab and chimeric 7E3 Fab.

Animals↗
PubMed1995

Differential inhibition of platelet aggregation induced by adenosine diphosphate or a thrombin receptor-activating peptide in patients treated with bolus chimeric 7E3 Fab: implications for inhibition of the internal pool of GPIIb/IIIa receptors.

OBJECTIVES: This study sought to describe in detail the pharmacokinetics and pharmacodynamics of chimeric monoclonal 7E3 Fab (c7E3 Fab) and to compare platelet responses to adenosine diphosphate (ADP) and the 11-amino acid thrombin receptor-activating peptide (TRAP [SFLLRNPNDKY-NH2]) in patients undergoing elective coronary angioplasty. BACKGROUND: Inhibition of platelet aggregation with monoclonal antibody c7E3 Fab directed against glycoprotein (GP) IIb/IIIa has been shown to reduce ischemic complications after angioplasty and is being considered for treatment of other acute ischemic syndromes. METHODS: Patients undergoing elective coronary angioplasty received aspirin (325 mg orally), heparin (12,000 U intravenously) and a bolus of c7E3 Fab (0.25 mg/kg body weight). Surface GPIIb/IIIa receptor blockade and aggregation in response to 20 mumol/liter ADP, 5 micrograms/ml collagen and 7.5 and 15 mumol/liter TRAP were assessed. RESULTS: Surface GPIIb/IIIa receptor blockade by c7E3 Fab was 80% 2 h after injection and decreased to 50% at 24 h. Platelet aggregation in response to 20 mumol/liter ADP was inhibited by 73% at 2 h, and this inhibition decreased to 27% at 24 h. Platelet aggregation in response to 7.5 mumol/liter TRAP was inhibited by 53% at 2 h and 30% at 24 h. In contrast, aggregation in response to 15 mumol/liter TRAP was inhibited only 37% at 2 h and 10% at 24 h (p < 0.001 and p = 0.006, respectively vs. 20 mumol/liter ADP). Addition of exogenous c7E3 Fab to platelet-rich plasma led to more complete inhibition of 7.5 mumol/liter TRAP-induced aggregation. CONCLUSIONS: After c7E3 Fab treatment, inhibition of platelet aggregation depends on the agonist and can be overcome by increased thrombin activity but is restored if additional c7E3 Fab is added to block additional GPIIb/IIIa receptors. This phenomenon may be related to an internal pool of GPIIb/IIIa receptors joining the surface membrane and has implications concerning the duration of therapy with c7E3 Fab for patients with unstable angina or acute myocardial infarction.

Abciximab↗
PubMed1995

The role of platelets in arterial thrombosis and the rationale for blockade of platelet GPIIb/IIIa receptors as antithrombotic therapy.

Platelet aggregation plays a crucial role in ischaemic arterial thrombosis. Recent biochemical data indicate that the platelet glycoprotein (GP) IIb/IIIa receptor mediates platelet aggregation by binding fibrinogen, von Willebrand factor, or other ligands, that can span between platelets. New antiplatelet agents that block the binding sites on GPIIb/IIIa are efficacious in arterial thrombosis animal models and are now being evaluated in human disease. A mouse/human chimeric monoclonal antibody fragment (c7E3 Fab) and agents modelled after the arginine-glycine-aspartic acid (RGD) cell binding motif are in development. c7E3 Fab showed significant efficacy in reducing ischaemic complications after angioplasty in patients at high risk of such complications in the EPIC study, and thus has been approved for use in the U.S. and several European and Scandinavian countries. These new agents also hold considerable promise in the treatment of other thrombotic disorders, including unstable angina and myocardial infarction.

Animals↗
PubMed1995

New antiplatelet agents: platelet GPIIb/IIIa antagonists.

The GPIIb/IIIa (alpha IIb beta 3) receptor plays a crucial role in platelet aggregation and platelet thrombus formation. Inhibition of GPIIb/IIIa with the Fab fragment of the mouse/human chimeric monoclonal antibody 7E3, snake venom peptides containing the arginine-glycine-aspartic acid (RGD) sequence, or peptides or peptidomimetics based on the RGD sequence results in abolition of platelet aggregation and platelet thrombus formation. This results in profound inhibition of thrombotic occlusions in animal models. The Phase III EPIC study demonstrated that c7E3 Fab, given as bolus followed by a 12 h infusion, reduced the risk of acute ischemic complications after coronary angioplasty by approximately 35% in patients at high risk of suffering such complications. Treated patients had an approximately 2-fold increased risk of major bleeding, but no increase in cerebral hemorrhage or lethal bleeding. Treatment with c7E3 Fab may have had a beneficial effect on clinical restenosis at 6 months, but this needs to be confirmed. A possible anticoagulant effect of c7E3 Fab was also identified in EPIC, and in vitro studies support this possibility. With the approval of c7E3 Fab (abciximab; ReoPro) for patients undergoing high-risk angioplasty in the US and several European and Scandinavian countries, GPIIb/IIIa inhibition joins the armamentarium of antithrombotic agents.

Abciximab↗
PubMed1994

Identification of a novel thrombin receptor sequence required for activation-dependent responses.

Thrombin receptor (TR) activation by alpha-thrombin requires proteolytic cleavage, although synthetic peptides modeled after the new N-terminus directly effect receptor activation without cleavage, presumably by interacting with an unidentified region of the receptor. To further define critical residues responsible for receptor activation, we performed epitope mapping of anti-TR1-160, a previously described polyclonal antibody that inhibits peptide ligand-induced receptor activation in various cell types expressing a functional TR. An enzyme-linked immunosorbent assay (ELISA) using overlapping decapeptides derived from the TR extracellular domains identified four immunodominant peaks within the long N-terminal extension centered between amino acids 34-44, 48-67, 65-79, and 87-94. Soluble peptides derived from regions 83-94, but not those from other regions of the receptor, neutralized the ability of anti-TR1-160 to inhibit peptide ligand-induced platelet aggregation, suggesting that antibodies directed against this region of the TR are important in ligand-mediated activation. Thrombin receptor mutants lacking discrete regions of the TR were subsequently evaluated using microinjected Xenopus oocytes. Whereas a TR mutant lacking amino acid residues Thr67-Lys82 (TR delta 67-82) showed normal to exaggerated responses to either alpha-thrombin or synthetic peptide ligands, only TR mutants with limited deletions spanning the residues Gln83-Ser93 exhibited dysfunctional responses to either agonist (200 nmol/L alpha-thrombin or 200 mumol/L TR42-47). These data provide a model for receptor activation that implicates a discrete and previously uncharacterized sequence within the TR N-terminal extension that is necessary for initiation of signal transduction events independent of the initiating agonist.

Amino Acid Sequence↗
PubMed1994

Glanzmann thrombasthenia secondary to a Gly273-->Asp mutation adjacent to the first calcium-binding domain of platelet glycoprotein IIb.

We studied the defect responsible for Glanzmann thrombasthenia in a patient whose platelets expressed < 5% of the normal amount of GPIIb-IIIa. Genetic and biochemical evidence indicated that the patient's GPIIIa genes were normal. However, DNA analysis revealed the patient homozygous for a G818-->A substitution in her GPIIb genes, resulting in a Gly273-->Asp substitution adjacent to the first GPIIb calcium-binding domain. To determine how this mutation impaired GPIIb-IIIa expression, recombinant GPIIb containing the mutation was coexpressed with GPIIIa in COS-1 cells. The GPIIb mutant formed stable GPIIb-IIIa heterodimers that were not immunoprecipitated by either of two heterodimer-specific monoclonal antibodies, indicating that the mutation disrupted the epitopes for these antibodies. Moreover, the GPIIb in the heterodimers was not cleaved into heavy and light chains, indicating that the heterodimers were not transported from the endoplasmic reticulum to the Golgi complex where GPIIb cleavage occurs, nor were the mutant heterodimers expressed on the cell surface. These studies demonstrate that a Gly273-->Asp mutation in GPIIb does not prevent the assembly of GPIIb-IIIa heterodimers, but alters the conformation of these heterodimers sufficiently to impair their intracellular transport. The impaired GPIIb-IIIa transport is responsible for the thrombasthenia in this patient.

Adenosine Diphosphate↗