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R McMillan

Publications and source records attributed to R McMillan.

At least 19 recordsLinked to original sources
PubMed1992

Different specificities of platelet-associated and plasma autoantibodies to platelet GPIIb-IIIa in patients with chronic immune thrombocytopenic purpura.

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to antiplatelet autoantibodies, many of which are directed against platelet membrane glycoprotein (GP) IIb-IIIa or GPIb-IX. In a recent study, we described plasma autoantibodies from 13 selected ITP patients, which required the presence of the putative GPIIIa cytoplasmic region for antibody binding. Since this region may not be available for antibody binding under physiologic conditions, we evaluated the frequency of binding to this or other regions of GPIIb-IIIa by platelet-associated and plasma autoantibody from a group of chronic ITP patients. We studied platelet-associated autoantibodies in 27 patients and plasma antibodies in 21 patients; in 15 patients, both were studied. To determine if autoantibodies were directed to the cytoplasmic portion of GPIIIa or to another portion of the GPIIb-IIIa molecule, antibody eluted from patient platelets or plasma antibody was tested in an antigen capture assay for binding to GPIIb-IIIa obtained from Chinese hamster ovary (CHO) cells transfected with GPIIb and either intact GPIIIa or GPIIIa lacking the carboxy terminal 35 residues. Of the 21 plasma autoantibodies tested, 13 bound primarily to the carboxy terminus of GPIIIa and eight to other epitopes. Conversely, all 26 platelet-associated autoantibodies, including eight of the 13 with anti-carboxy terminus antibodies, bound to epitopes in other regions of GPIIb-IIIa. Comparison of the degree of antibody adsorption by intact or lysed platelets indicated that epitopes on the c-terminal region of GPIIIa are relatively inaccessible on the surface of intact washed platelets when compared with other epitopes. We conclude that the importance of plasma autoantibodies in chronic ITP patients should be interpreted cautiously, since their specificity may differ from that of antibodies bound to the platelet. Whether antibodies against the c-terminus of GPIIIa are of pathogenetic importance remains to be determined, although patients with these antibodies have particularly severe disease.

Animals↗
PubMed1992

Localization of a PlA1 epitope to the amino terminal 66 residues of platelet glycoprotein IIIa.

A platelet glycoprotein (GP) IIIa epitope library was constructed by insertion of randomly cleaved GPIIIa cDNA fragments in the prokaryotic expression vector lambda gt22 and screened with purified anti-PlA1 antibodies for clones expressing a PlA1 epitope. Five independent clones were isolated and characterized by nucleotide sequencing. The smallest anti-PlA1 reactive clone obtained encoded the amino terminal 66 residues of mature GPIIIa. Substitution of leucine33 (PlA1) with a proline33 (PlA2) by in vitro mutagenesis resulted in the loss of anti-PlA1 reactivity; however, this clone still reacted with anti-GPIIIa polyclonal antibodies. These data indicate that a PlA1 alloantigenic epitope is located within a small, unglycosylated fragment of GPIIIa containing the polymorphism responsible for the PIA phenotype. Furthermore, these results prove that small recombinant mimics of a PlA1 epitope may be synthesized and used for detection of these alloantibodies.

Alleles↗
PubMed1992

Molecular cloning and preliminary characterization of a novel cytoplasmic antigen recognized by myasthenia gravis sera.

A cDNA clone was isolated by screening of a lambda gt11 endothelial expression library with serum from a patient with myasthenia gravis (MG). Rabbit antisera raised against the recombinant protein and human MG sera reactive with the clone immunoblotted an M(r) integral of 250,000 polypeptide (gravin) present in endothelial cells and several adherent cells. Gravin was not detected in platelets, leukocytes, U937, or human erythroleukemic (HEL) cell lines, but was expressed in HEL cells after induction with phorbol myristate acetate. Northern blot analysis showed two transcripts of approximately 6.7 and 8.4 kb in endothelial cells but not U937 or HEL cells. Indirect immunofluorescence of permeabilized cells revealed a trabecular network of gravin staining with a distinct linear component. Antibodies to gravin, were present in sera from 22:72 (31%) of MG patients. In contrast 0:50 normal sera and 1:72 sera from patients with other autoimmune diseases contained antigravin antibodies. Gravin is not likely to be a nonerythroid spectrin, talin, myosin, or actin-binding protein based on the lack of reactivity of antigravin with these polypeptides in immunoblots. The nucleotide sequence of the immunoreactive clone indicated that it encodes a highly acidic polypeptide fragment that contains the carboxyl terminus of the protein. Neither amino acid nor nucleotide sequences were present in Genbank, EMBL, or Swissprot databases as of March, 1992. These data indicate that gravin is an inducible, cell type-specific cytoplasmic protein and that auto-antibodies to gravin may be highly specific for MG.

Amino Acid Sequence↗
PubMed1992

Histological comparison of autogenous canine fascia lata, Gore-Tex, lyophilized human fascia lata, and autogenous canine vein for vascular patch graft material in a canine arteriotomy model.

Autogenous fascia lata has found little clinical use as a vascular patch graft material. Previous experience, however, suggests that it possesses attributes that might make it useful in this regard. To assess its efficacy as a vascular patch graft, nine adult mongrel dogs each underwent four arteriotomies with placements of patch grafts. The four sites included both carotid arteries and both femoral arteries. In each animal, one of four patch graft materials (autogenous canine fascia lata, Gore-Tex, lyophilized human fascia lata, and autogenous canine vein) were placed as patch material at the arteriotomy site utilizing 7-0 running sutures and loop magnification. The site for placement of each graft material was rotated serially in the animals so that each site would have equal numbers of all four graft materials applied. The animals were killed at either 6 to 8 weeks or 11 to 12 weeks after angiography of all four vessels. The specimens were then evaluated histologically. No difference was observed among any of the patch graft materials with regard to myofibroblast plaque formation. Inflammatory responses were noted to be substantially less in the canine fascia lata group than in the other three groups. Granuloma formation, however, appeared to be most significant in the autogenous canine vein group. Only one vessel was occluded. Aneurysm or pseudoaneurysm formation was not noted in any specimen. It appears from the above results that autogenous fascia lata may be an appropriate alternative to currently utilized arterial patch graft materials and that it should be evaluated further for this purpose.

Animals↗
PubMed1991

Autoantibodies to the presumptive cytoplasmic domain of platelet glycoprotein IIIa in patients with chronic immune thrombocytopenic purpura.

Chronic immune thrombocytopenic purpura (ITP) is an autoimmune disorder due to autoantibodies against platelets that result in their destruction. In some patients, these autoantibodies bind to platelet glycoprotein (GP) IIIa. With the aim of better defining the antigenic epitopes, plasma from 13 selected patients with chronic ITP known to have anti-GPIIb/IIIa autoantibodies was tested for reactivity with nine synthetic peptides corresponding to different regions of the GP IIIa molecule. Of these plasmas, five bound significantly (P less than .001) with either peptide 8 (amino acids 721-744) or peptide 9 (amino acids 742-762), which together form most of the carboxyterminal region presumed to be the cytoplasmic domain. Three of these positive plasmas, were tested further. In two of these positive plasmas, the anti-peptide antibodies represented greater than 80% of the detectable circulating autoantibody. To further evaluate the importance of the carboxyterminal region as an antigenic site, the chronic ITP plasmas were tested against Chinese hamster ovary cells transfected with GPIIb and either whole GPIIIa or GPIIIa lacking amino acids 728 to 762. Ten of the 13 plasmas required the presence of this region for significant autoantibody binding. We conclude that the carboxyterminal region is an important area for stimulating antiplatelet autoantibody formation in some patients with chronic ITP. It is not known whether these autoantibodies to the presumed cytoplasmic domain play an important role in the pathogenesis of the disease or occur as a secondary phenomenon during the course of platelet destruction.

Amino Acid Sequence↗
PubMed1990

Analysis of platelet aggregation disorders based on flow cytometric analysis of membrane glycoprotein IIb-IIIa with conformation-specific monoclonal antibodies.

Normal primary platelet aggregation requires agonist-mediated activation of membrane GPIIb-IIIa, binding of fibrinogen to GPIIb-IIIa, and cellular events after ligand binding. PAC1 monoclonal antibody distinguishes between resting and activated states of GPIIb-IIIa, and other antibodies preferentially recognize GPIIb (PMI-1) or IIIa (anti-LIBS1) after the binding of fibrinogen or fibrinogen-mimetic peptides, such as GRGDSP. Using these antibodies and platelet flow cytometry, we studied two distinct persistent platelet aggregation abnormalities. Platelets from a thrombasthenic variant, which contained near-normal amounts of GPIIb-IIIa, failed to aggregate or bind PAC1 in response to agonists. In addition, GRGDSP, which binds to normal GPIIb-IIIa without prior cell activation, failed to increase the binding of PMI-1 or anti-LIBS1 to the thrombasthenic platelets, suggesting a primary defect in ligand binding. Chromatography of detergent-solubilized platelets on a KYGRGDS affinity column confirmed that the patient's GPIIb-IIIa lacked the fibrinogen binding site. In another patient with myelofibrosis and defective aggregation, PAC1 failed to bind to adenosine diphosphate-stimulated platelets, but did bind when protein kinase C was directly activated with phorbol myristate acetate. Furthermore, the binding of PMI-1 and anti-LIBS1 increased in response to GRGDSP, confirming a defect in agonist-mediated fibrinogen receptor activation rather than in fibrinogen binding or events distal to binding. These studies indicate that this immunochemical approach is useful in classification of clinical abnormalities of platelet aggregation as defects in either (a) fibrinogen receptor activation, (b) fibrinogen binding, or (c) postoccupancy events.

Antibodies, Monoclonal↗
PubMed1990

Autologous bone marrow transplantation for acute myeloid leukemia using monoclonal antibody-purged bone marrow.

We report our experience from a clinical trial of autologous bone marrow transplantation (ABMT) in the treatment of 30 patients with acute myeloid leukemia (AML) using monoclonal antibody (MoAb) and complement-treated bone marrow. All patients were in complete remission (CR) at the time of transplant: 6 patients were in first CR, 18 in second CR, and 6 in third CR. The median age of all patients was 42 years (range 11 to 57 years). For marrow ablation, 28 patients were treated with cyclophosphamide and total body irradiation. One patient was treated with busulfan and cyclophosphamide and one was treated with busulfan and VP-16. Each patient was then transfused with autologous bone marrow that had been harvested previously and treated with two MoAbs, PM-81 and AML-2-23, and rabbit complement. Median time to recovery of neutrophils (500/microL) was 30 days, and platelets (20,000/microL) was 45 days. Median time for initial erythrocyte engraftment, assessed by a flow cytometric reticulocyte assay, was 13 days. Median overall and relapse-free survival of first CR patients was at least 17.4 months post-ABMT and the 2- and 3-year actuarial overall and relapse-free survival was 67% (+/- 19%). Median survival for the 24 patients in second or third CR was 6.8 months post-ABMT and 9.3 months since CR; however, six patients survived disease-free from 16 to 61 months post-ABMT. For the second and third CR group it was observed that six patients (5 of the 6 survivors) showed "inversions," when their post-ABMT remission lasted longer than any previous one. Actuarial 2- and 3-year disease-free and overall survival of patients in second and third CR was 25% (+/- 9%) and 18% (+/- 9%), and 29% (+/- 9%) and 23% (+/- 9%), respectively. ABMT avoids the problems of graft-versus-host disease and of finding suitable donors for allogeneic marrow transplantation.

Adolescent↗
PubMed1990

Nonsense mutation in the glycoprotein Ib alpha coding sequence associated with Bernard-Soulier syndrome.

Three distinct gene products, the alpha and beta chains of glycoprotein (GP) Ib and GP IX, constitute the platelet membrane GP Ib-IX complex, a receptor for von Willebrand factor and thrombin involved in platelet adhesion and aggregation. Defective function of the GP Ib-IX complex is the hallmark of a rare congenital bleeding disorder of still undefined pathogenesis, the Bernard-Soulier syndrome. We have analyzed the molecular basis of this disease in one patient in whom immunoblotting of solubilized platelets demonstrated absence of normal GP Ib alpha but presence of a smaller immunoreactive species. The truncated polypeptide was also present, along with normal protein, in platelets from the patient's mother and two of his four children. Genetic characterization identified a nucleotide transition changing the Trp-343 codon (TGG) to a nonsense codon (TGA). Such a mutation explains the origin of the smaller GP Ib alpha, which by lacking half of the sequence on the carboxyl-terminal side, including the trans-membrane domain, cannot be properly inserted in the platelet membrane. Both normal and mutant codons were found in the patient, suggesting that he is a compound heterozygote with a still unidentified defect in the other GP Ib alpha allele. Nonsense mutation and truncated GP Ib alpha polypeptide were found to cosegregate in four individuals through three generations and were associated with either Bernard-Soulier syndrome or carrier state phenotype. The molecular abnormality demonstrated in this family provides evidence that defective synthesis of GP Ib alpha alters the membrane expression of the GP Ib-IX complex and may be responsible for Bernard-Soulier syndrome.

Adult↗
PubMed1990

Chronic ITP as a model for immunization by platelets.

In summary, ITP is an autoimmune disorder due to the development of autoantibodies which in the majority of cases are directed against normal platelet membrane glycoproteins. The autoantibody binds to both platelets and megakaryocytes. The binding to platelets can cause phagocytosis by the immune system via an Fc receptor mechanism and in addition can activate the complement pathway which could cause more efficient phagocytosis or in vivo cell lysis. It seems likely that antibody bound to megakaryocytes might influence platelet production as well. At the present time, the immunoregulatory defect which results in this autoimmune disorder is unknown.

Autoantibodies↗
PubMed1989

Therapy of chronic idiopathic thrombocytopenic purpura in adults.

Chronic ITP is a common hematologic illness. Approximately three fourths of the patients respond to corticosteroids or splenectomy and need no further treatment. Patients refractory to these two therapeutic approaches are relatively resistant to present forms of treatment and are at much greater risk for morbidity and mortality. Future clinical studies evaluating therapy in this refractory group would be best performed in a cooperative group setting in which large numbers of patients could be treated in a prospective randomized manner.

Adrenal Cortex Hormones↗
PubMed1989

Inhibition of autoantibody binding to platelet glycoprotein IIb/IIIa by anti-idiotypic antibodies in intravenous gammaglobulin.

Intravenous immunoglobulin (IVIgG) causes an acute rise in the platelet count in the majority of patients with chronic immune thrombocytopenic purpura (ITP) but the mechanism(s) of action is still unknown. We evaluated the ability of three different IVIgG preparations to inhibit the in vitro binding of autoantibody to platelet glycoprotein (GP) IIb/IIIa. ITP plasma, known to contain anti-GPIIb/IIIa antibodies, was incubated overnight with either IVIgG or bovine serum albumin (BSA) followed by measurement of the autoantibody titer. Binding of autoantibody from eight ITP patients was inhibited by IVIgG in proportion to the IVIgG concentration. Using 3.2% IVIgG, compatible with therapeutic concentrations expected in vivo, mean inhibition of autoantibody binding ranged from 20.2% to 41.3%. No inhibition by IVIgG of alloantibody binding to the same or different molecules was detected (five patients with anti-GPIIb/IIIa and two with anti-HLA alloantibodies). F(ab')2 fragments of IVIgG also inhibited the binding of both plasma autoantibodies and purified anti-GPIIb/IIIA autoantibodies prepared by elution from antigen affinity columns. A portion of the anti-idiotypic antibodies could be adsorbed from IVIgG using insolubilized, purified anti-GPIIb/IIIa autoantibody. These results show that IVIgG preparations from normal donors contain anti-idiotypic antibodies directed against idiotypes located on GPIIb/IIIa autoantibodies but do not have anti-idiotypes to platelet alloantibodies against the same or different molecules. The importance of these anti-idiotypic antibodies in the therapeutic response to IVIgG remains to be established.

Antibodies, Anti-Idiotypic↗
PubMed1987

Autoantibodies against platelet GPIIb/IIIa in chronic ITP react with different epitopes.

Some patients with chronic immune thrombocytopenic purpura have autoantibodies to the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) complex. To determine whether these autoantibodies are directed towards the same or different epitopes, we evaluated the ability of four murine monoclonal anti-GPIIb/IIIa antibodies specific for different epitopes to block autoantibody binding. We noted a variation in blocking patterns among autoantibodies from patients with chronic ITP. In addition, we were able to map the relative epitope locations of both the autoantibodies and the monoclonal antibodies. These data show that the anti-GPIIb/IIIa monoclonal autoantibodies in chronic ITP are directed towards different epitopes.

Autoantibodies↗
PubMed1986

Direct quantitation of activated C3 in human plasma with monoclonal anti-iC3b-C3d-neoantigen.

We developed a microtiter solid-phase radioimmunoassay for quantitating C3 breakdown products (iC3b, C3dg, C3d) in human plasma with a unique monoclonal antibody specific for a neoantigen present on iC3b and C3d (MoAb 130). This monoclonal antibody reacts with a neoantigen which appears when C3b is converted to iC3b. The neoantigen is also present on the C3dg and C3d fragments derived from iC3b. The concentration of the neoantigen is elevated in the plasma of most patients with rheumatoid arthritis and systemic lupus erythematosus as compared to normal volunteers. Some patients with glomerulonephritis also had elevated concentration of the neoantigen in their plasma.

Antibodies, Monoclonal↗