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S Karpatkin

Publications and source records attributed to S Karpatkin.

At least 19 recordsLinked to original sources
PubMed1992

Serum platelet-reactive IgG of autoimmune thrombocytopenic purpura patients is not F(ab')2 mediated and a function of storage.

Serum platelet-reactive and glycoprotein (GP) IIb-GPIIIa-reactive IgG and F(ab')2 was examined in 39 patients with classic autoimmune thrombocytopenic purpura (ATP), two patients with anti-PLA1 antibody and 25 control subjects in an enzyme-linked immunosorbent assay. IgG was purified by diethyl aminoethyl chromatography and centrifuged at 100,000g before testing of the supernatant. Significant IgG binding (threefold to fourfold control IgG binding) was noted with 8 of 17 ATP patients' IgG, 2 anti-PLA1 IgGs, and 2 ATP patients with multiple platelet transfusions. However, F(ab')2 fragments of nine of nine positive ATP IgGs were nonreactive; F(ab')2 from the two anti-PLA1 and two multiply transfused ATP IgGs were as reactive as their intact IgG. Antiplatelet or anti-GPIIb-GPIIIa reactivity of ATP IgG could be adsorbed to fixed platelets or solid-phase GPIIb-GPIIIa and eluted with 0.1 mol/L glycine, pH 2.5. However, binding of IgG to GPIIb-GPIIIa could not be inhibited with F(ab')2 of ATP IgG or Fc fragments of control subjects. When platelet- or GPIIb-GPIIIa-reactive ATP IgG was applied to a Sephacryl 300 gel filtration column, no reactivity was noted in the 7S region, whereas anti-PLA1 localized to this region. Antiplatelet or anti-GPIIb-GPIIIa reactivity was noted in the void volume and accompanied by a high molecular weight protein region. An immunoblot of the void volume fraction with goat antihuman IgG (gamma chain) antibody showed high molecular weight bands greater than 250 Kd, which after reduction converted to a 55-Kd heavy-chain band. Fresh samples of ATP and control IgG processed within 1 to 2 days of blood withdrawal had no reactivity for GPIIb-GPIIIa. After storage at -20 degrees C for greater than 3 months, 5 of 19 ATP IgG became reactive, whereas 16 of 16 controls were nonreactive. Thus, platelet-reactive IgG of ATP sera appears to be caused by the development of IgG aggregates held together by disulfide bonds that develop on storage, and is not F(ab')2 mediated.

Binding Sites, Antibody↗
PubMed1992

Effect of thrombin treatment of tumor cells on adhesion of tumor cells to platelets in vitro and tumor metastasis in vivo.

Seven different tumor cell lines (human melanoma SK MEL 28; hamster melanoma HM29; murine melanomas B16F10 and amelanotic melanoma B16a; human colon carcinoma HCT8; murine colon carcinoma CT26; and murine Lewis lung carcinoma) were treated with thrombin at 0.5-1 unit/ml and examined for their ability to bind to adherent platelets; HM29 was studied for its ability to bind to fibronectin and von Willebrand factor; CT26, B16F1, B16F10, and B16a were studied for their ability to form pulmonary metastasis after i.v. injection of thrombin-treated tumor cells; CT26 was studied for its ability to grow s.c. Five of 7 thrombin-treated tumor cell lines increased their adhesion to adherent platelets 2-to 3-fold. HM29 increased its adherence to fibronectin and von Willebrand factor 2- to 3-fold. CT26, B16F1, B16F10, and B16a increased experimental pulmonary metastasis 10- to 156-fold. Thrombin-treated CT26 cells demonstrated 2-fold greater growth in vivo after s.c. injection. The mechanism of enhanced adhesion of thrombin-treated tumor cells to platelets required the platelet integrin GPIIb-GPIIIa since it could be inhibited by agents known to block adhesion of ligands to GPIIb-GPIIIa (monoclonal antibody 10E5, tetrapeptide RGDS, disintegrin Albolabrin); as well as a "GPIIb-GPIIIa-like" structure on tumor cells since it could be inhibited by treatment of thrombin-treated tumor cells with 10E5 and RGDS. The thrombin effect on tumor cells was optimum at 1 h of incubation with thrombin, did not require active thrombin on the tumor cell surface, and did not require protein synthesis (not inhibited by cycloheximide). Thus, thrombin-treated tumor cells markedly enhance pulmonary metastasis. It is suggested that this may be secondary to thrombin-induced enhanced adhesion as well as growth of tumor cells.

Animals↗
PubMed1992

B-cell subsets and platelet counts in HIV-1 seropositive subjects.

A subset of B lymphocytes positive for the CD5 antigen have been implicated in several autoimmune disorders. To investigate their role in human immunodeficiency virus type 1 (HIV-1) infection, we studied peripheral-blood B and T lymphocytes from HIV-1-positive patients with (n = 13) and without (n = 18) thrombocytopenia, 8 patients with classic autoimmune thrombocytopenia, and 16 healthy controls. The proportion of CD5-positive B cells was significantly higher in the HIV-1-positive thrombocytopenic patients than in the healthy controls, as a result of both higher numbers of CD5-positive B cells and lower numbers of CD5-negative B cells. Platelet count was positively correlated with CD5-negative B-cell count (r = 0.6, p less than 0.001) and negatively correlated with proportion of B cells that were CD5 positive (r = -0.5, p less than 0.01) among the HIV-1-positive patients. The high concentrations of IgM-containing immune complexes in HIV-1-positive patients with autoimmune disorders may be due to changes in the CD5-positive B-cell subset.

Adult↗
PubMed1992

A monoclonal antibody (LK-4) which differentiates PLA1 from PLA2 platelet extracts but not intact platelets.

A unique murine monoclonal antibody (LK-4) is described which differentiates PLA1/PLA1 platelet extracts from PLA2/PLA2 and PLA1/PLA2 platelet extracts on solid phase ELISA and immunoblot at the 100kD GPIIIa location, but not on intact platelets. LK-4 reacts equally with intact PLA1/PLA2 and PLA2/PLA2 platelets. Adsorbtion of LK-4 with PLA1/PLA1 platelets results in loss of reactivity for intact platelets as well as platelet extracts on ELISA or immunoblot. LK-4 inhibits platelet aggregation induced by ADP, epinephrine, collagen and thrombin, suggesting reactivity at or near the fibrinogen binding site on GPIIIa. It is suggested the LK-4 reacts with a conformation-induced common epitope for PLA1 and PLA2 on GPIIIa, with loss of this conformation for PLA2 GPIIIa following solubilization with Triton X-100.

Adult↗
PubMed1992

Internal-image anti-idiotype HIV-1gp120 antibody in human immunodeficiency virus 1 (HIV-1)-seropositive individuals with thrombocytopenia.

Anti-CD4 antibody was found in 30% of human immunodeficiency virus (HIV-1)-seropositive thrombocytopenic patients compared with 5% of nonthrombocytopenic seropositive patients (chi 2 = 21.7, P less than 0.001) and was shown by the following observations to contain internal-image anti-idiotype antibody (Ab2) directed against the antibody (Ab1) to gp120, the HIV-1 envelope glycoprotein that binds to CD4: (i) affinity-purified anti-CD4 (Ab2) bound to affinity-purified anti-HIV-1gp120 (Ab1) on solid-phase radioimmunoassay, and binding could be blocked by recombinant CD4 (rCD4) as well as recombinant gp120 (rgp120); (ii) F(ab')2 fragments of Ab1 inhibited the binding of Ab2 to rCD4; (iii) Ab2 inhibited the binding of Ab1 to HIV-1 beads; (iv) Ab2 inhibited the binding of Ab1 to gp120 on immunoblot; (v) Ab2 bound to the CD4 receptor on a CD4-bearing T-cell line, H9; (vi) Ab3 (anti-rgp120) could be produced in vivo by immunizing mice with Ab2, and binding of Ab3 to rgp120 could be blocked with rCD4; and (vii) three different Ab2 preparations bound to two different homologous Ab1 preparations. Ab1 or Ab2 alone did not bind to platelets, whereas the idiotype-anti-idiotype complex did bind to platelets in a concentration-dependent manner. Binding of the internal-image complex was 10-fold greater than that of a non-internal-image Ab1-Ab2 complex composed of anti-HIV-1gp120 and anti-anti-HIV-1gp120. Thus, patients with HIV-1 thrombocytopenia contain internal-image idiotype-anti-idiotype complexes that could be affecting CD4 cell number or function, inhibiting HIV-1 binding to CD4 cells or contributing to HIV-1 thrombocytopenia.

Animals↗
PubMed1992

Development of a monoclonal antibody capable of differentiating platelet PLA1/PLA1, PLA1/PLA2 and PLA2/PLA2 genotypes.

A monoclonal antibody, LK-4, has been developed which distinguishes platelet PLA1/PLA1, PLA1/PLA2 and PLA2/PLA2 genotypes on platelet glycoprotein GPIIIa of Triton-solubilized platelet extracts. An ELISA assay has been developed which traps GPIIIa with Concanavalin A, enriching the platelet extract for the PLA antigens. A second monoclonal antibody, DEK-10, which reacts equally with GPIIIa of PLA1/PLA1 and PLA2/PLA2 platelet extracts is employed as an internal standard to correct for individual differences in GPIIIa content, GPIIIa extracted by Triton X-100 and GPIIIa trapped with Concanavalin A. This ELISA assay clearly differentiated 11 different PLA1/PLA1 subjects from eight PLA2/PLA2 women with a history of neonatal alloimmune thrombocytopenia as well as six unrelated obligate heterozygotes and should be useful in evaluating the PLA genotype of pregnant women and their families.

Alleles↗
PubMed1992

Autoimmune anti-HIV-1gp120 antibody with antiidiotype-like activity in sera and immune complexes of HIV-1-related immunologic thrombocytopenia.

Autoimmune antiidiotype-like antibody (Ab2) directed against anti-HIV-1gp120 (Ab1) was found in high titer in the sera of 10 consecutive homosexual and 11 narcotic addict HIV-1-related immunologic thrombocytopenia (HIV-1-ITP) patients, was barely detectable in 10 nonthrombocytopenic HIV-1 sero-positive individuals, and was not detectable in 5 normal subjects by use of a solid-phase RIA. Reactivity of autologous Ab2 for Ab1 was 4-120-fold greater than Ab2 for homologous Ab1. Affinity-purified Ab2 did not block the binding of affinity-purified Ab1 to its HIV-1gp120 epitopes on immunoblot, indicating the absence of "internal image" antiidiotype. Both Ab1 and Ab2 are precipitable from sera with polyethylene glycol (PEG) and present in a macromolecular complex that is excluded by gel filtration on G200 and contains IgG, IgM, C3, and the anti-F(ab')2 antiidiotype-like complex. PEG-precipitable complexes bind to platelets in a saturation-dependent manner. Neither affinity-purified Ab1 nor Ab2 binds to platelets. However, the combination of Ab1 and Ab2 (preincubated for 2 h at 22 degrees C) binds to platelets in a saturation-dependent manner at an optimum ratio range of 10-20:1. Ab2 reactivity correlates with serum PEG-precipitable immune complex level (r = 0.91; P less than 0.001) and with thrombocytopenia (r = 0.89; P less than 0.001). We suggest that the anti-HIV-1gp120 antiidiotype-like complex contributes to the markedly elevated platelet Ig and C3 level of HIV-1-ITP patients and propose that this may contribute to their thrombocytopenia.

Acquired Immunodeficiency Syndrome↗
PubMed1991

A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope.

We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105-129 and 452-428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67-Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.

Amino Acid Sequence↗
PubMed1991

Thrombin stimulates tumor-platelet adhesion in vitro and metastasis in vivo.

Recent studies have revealed a role for platelets and the platelet-adhesive proteins, fibronectin and von Willebrand factor (vWF) in platelet-tumor cell interaction in vitro and metastasis in vivo. The present report documents the effect of thrombin treatment of platelets on this interaction in vitro and in vivo. In vitro, thrombin at 100-1,000 mU/ml maximally stimulated the adhesion of six different tumor cell lines from three different species two- to fivefold. As little as 1-10 mU/ml was effective. The effect of thrombin was specific (inhibitable by hirudin, dansyl-arginine N-(3-ethyl-1,5 pentanediyl) amide and unreactive with the inactive thrombin analogue N-P-tosyl-L-phenylchloromethylketone-thrombin and D-phenylalanyl-L-propyl-L-arginine chloromethylketone-thrombin (PPACK-thrombin), and required high-affinity thrombin receptors (competition with PPACK-thrombin but not with N-P-tosyl-L-lysine-chloromethyl-ketone-thrombin). Functionally active thrombin was required on the platelet surface. Binding of tumor cells to thrombin-activated platelets was inhibitable by agents known to interfere with the platelet GPIIb-GPIIIa integrin: monoclonal antibody 10E5, tetrapeptide RGDS and gamma chain fibrinogen decapeptide LGGAKQAGDV, as well as polyclonal antibodies against the platelet adhesive ligands, fibronectin and vWF. In vivo, thrombin at 250-500 mU per animal increased murine pulmonary metastases fourfold with CT26 colon carcinoma cells and 68-413-fold with B16 amelanotic melanoma cells. Thus, thrombin amplifies tumor-platelet adhesion in vitro two- to fivefold via occupancy of high-affinity platelet thrombin receptors, and modulation of GPIIb-GPIIIa adhesion via an RGD-dependent mechanism. In vivo, thrombin enhances tumor metastases 4-413-fold with two different tumor cell lines.

Animals↗
PubMed1990

Regulation of autoimmune anti-platelet antibody-mediated adhesion of monocytes to platelet GPIIb/GPIIIa: effect of armed monocytes and the Mac-1 receptor.

Platelet autoantigen-autoantibody-monocyte interaction was studied by utilization of a specific monoclonal antibody (MoAb) 10E5 to trap and immobilize the GPIIb-GPIIIa complex on microtiter plates. Peripheral blood mononuclear cells (PBMC) or purified monocytes formed distinct morphologic clusters after incubation with immobilized antigen for 18 hours at 37 degrees C. PBMC of 18 and 19 patients with autoimmune thrombocytopenic purpura (ATP) formed 48 +/- 6.8 (SEM) clusters/well compared with 7.4 +/- 1.0 for control subjects, P less than .001. The number of clusters per well correlated inversely and exponentially with platelet count, r = -.8, n = 21, indicating that the GPIIb-GPIIIa autoantigen is pathophysiologically relevant. Binding of ATP PBMC to immobilized GPIIb-GPIIIa could be inhibited by F(ab')2 fragments of immunoglobulin (Ig) G of ATP patients, indicating that monocyte IgG bound to autoantigen by its F(ab')2 domain. Optimal cluster formation could be obtained with normal monocytes if preincubated with ATP IgG but not with F(ab')2 fragments of ATP IgG, indicating that ATP IgG binds to monocytes by its Fc domain. Armed monocytes (ie, normal monocytes preincubated with ATP IgG) bound to immobilized autoantigen 5.8-fold greater than normal monocytes incubated with immobilized autoantigen opsonized with ATP IgG. Armed monocyte adhesion could be inhibited 81% from 18.9 +/- 1.6 to 3.6 +/- 0.5 clusters/well by prior fixation with 0.1% formalin, whereas fixation of IgG before arming of monocytes was not inhibitory. MoAb MM41, directed against the alpha m-chain of the Mac-1 adhesive protein receptor of monocytes, inhibited cluster formation by 79%. Thus, (1) armed monocyte interaction with autoantigen is considerably more effective than monocyte interaction with opsonized autoantigen; (2) armed monocyte interaction requires specific F(ab')2-antigen recognition; and (3) monocyte-autoantigen interaction requires a secondary nonimmunologic adhesive event.

Antigens, CD↗
PubMed1990

Micromethod for demonstrating increased platelet surface immunoglobulin G: findings in acute, chronic, and human immunodeficiency virus-1-related immunologic thrombocytopenias.

A method has been developed for the demonstration of increased platelet surface IgG that uses 1 ml of blood regardless of the platelet count. Platelets are gel filtered to remove plasma and contaminating lymphocytes. They are then reacted with fluorescein-conjugated antihuman IgG and analysed by flow cytometry. Percent positive staining cells vary from 10% to 80% of total cells examined. A platelet antibody index is derived from the product of percent positive staining cells X mean fluorescence intensity of positive staining cells. All patients studied with chronic idiopathic thrombocytopenia purpura (ITP) or human immunodeficiency virus-1 (HIV-1)-related thrombocytopenia had increased platelet surface IgG. Twelve acute children and 11 chronic children had indices averaging 3.5- and 8.9-fold greater than 12 normal children, respectively. Five of 12 children with acute ITP had normal platelet IgG. There was no linear correlation between the platelet antibody index and platelet count. Platelets of patients with acute, chronic, or HIV-1-related ITP displayed autofluorescence. In chronic ITP, the percentage of platelets displaying autofluorescence had a significant negative correlation with the platelet count. This technique will be a valuable diagnostic tool in the pediatric population.

Acute Disease↗
PubMed1990

Expression of CD4 by human megakaryocytes.

The CD4 antigen, which serves as the receptor for human immunodeficiency virus type 1 (HIV-1) on T cells, has been detected on human megakaryocytes. Recent evidence of impaired thrombopoiesis in HIV-1-related thrombocytopenia suggested that these cells could be directly infected by the virus and prompted a search for a receptor on megakaryocytes of normal subjects that could permit entry of HIV-1. Bone marrow specimens from uninfected normal control subjects were centrifuged over Ficoll-Hypaque (1.077 g/ml) and analyzed by three-color analysis with a flow cytometer utilizing monoclonal antibodies against CD4 and a glycoprotein present on the surface of megakaryocytes and platelets (GPIIb/IIIa; CD41), as well as 7-aminoactinomycin D, a stain for DNA. Cells presumed to be megakaryocytes were identified by having a DNA content greater than tetraploid and staining brightly with anti-CD41. Approximately 0.4% of the nucleated cells of the marrow met these criteria. Twenty-five percent of these megakaryocytes stained as brightly as CD4+ T cells. Several clones of antibody recognizing different epitopes of the CD4 molecule gave similar results. Platelets were CD4-. Staining of megakaryocytes with anti-CD4 was confirmed by direct microscopic examination of Percoll-gradient-enriched megakaryocytes employing two-color (CD4-phycoerythrin and CD41-fluorescein) immunofluorescence analysis and phase-contrast microscopy. The proportion of double-labeled cells among 112 phase-contrast-identifiable megakaryocytes from five bone marrow specimens varied between 20% and 26% with a mean and SD of 22% +/- 2.5%. Thus some human megakaryocytes express CD4 on their surface that should be capable of binding the HIV-1 gp120 envelope protein. This could serve as a portal of entry for HIV-1.

Antibodies, Monoclonal↗
PubMed1990

In vitro suppressor T lymphocyte dysfunction in autoimmune thrombocytopenic purpura associated with a complement-fixing antibody.

Ig secretion of peripheral blood mononuclear cells (PBMC) was measured in Epstein Barr virus (EBV) seropositive autoimmune thrombocytopenic purpura (ATP) patients and controls following in vitro infection with EBV. EBV infected PBMC from patients with ATP secreted Ig for a longer period of time than EBV infected control cells and had higher peak Ig production. Removal of T cells or CD8 cells from control PBMC increased the duration and level of Ig production to that achieved by ATP PBMC. Total T or CD8 cell depletion of ATP PBMC had no effect on the enhanced level or duration of Ig secretion. Depletion of control CD4 lymphocytes decreased Ig production. Treatment of EBV infected control PBMC with either ATP sera or purified IgG (plus complement) increased the duration and level of production of Ig to that observed in ATP PBMC, control PBMC depleted of all T cells or control PBMC depleted of CD8 cells. This antibody effect could be reversed by reconstitution with control T cells. Thus, patients with ATP produce an antibody which leads to suppressor cell dysfunction. Defective function of these cells may lead to a disorder of immune regulation in which autoantibodies are produced against platelets.

Adult↗
PubMed1990

Posttransfusion purpura: conversion of PLA1-negative platelets to the PLA1-positive phenotype by stored plasma is not due to the presence of soluble PLA1 antigen.

Three previous articles have reported a PLA1 antigen in the plasma of stored blood which is capable of binding to PLA1-negative platelets in the presence of divalent cations, rendering them PLA1 positive. Such a mechanism could explain the enigma of posttransfusion purpura (PTP), i.e., severe thrombocytopenia in a healthy subject with PLA1-negative platelets secondary to the infusion of blood containing PLA1-positive platelets. We find that the PLA1 antigen of stored blood is due to the presence of platelet fragments which can be removed by centrifugation and that divalent cation-chelating agents play no role in the apparent binding of these fragments to platelets. The apparent conversion of PLA1-negative platelets to the PLA1-positive phenotype by incubation with stored plasma from a PLA1-positive subject is due to the cosedimentation of platelet fragments with the platelets. No soluble PLA1 antigen was found in the plasma of five patients with acute posttransfusion purpura.

Antigens, Human Platelet↗
PubMed1990

HIV-1-related thrombocytopenia.

Immunologic thrombocytopenia has recently been described in HIV-1 seropositive patients: homosexuals, narcotic addicts, hemophiliacs, and heterosexual partners of HIV-1 infected patients. All patients have markedly elevated platelet-bound IgG and C3C4 as well as circulating immune complexes. The immune complexes of homosexuals and narcotic addicts contain anti-F(ab') against anti-HIV-1 antibody. These are present in the serum and on the platelets, and could be responsible for the thrombocytopenia.

Adrenal Cortex Hormones↗