sodium-dodecyl-sulfate and Thrombocytopenia

Integrin alpha2beta1, which is a membrane protein consisting of noncovalently bound alpha2 and beta1 chains, mediates cell binding to collagen and plays a role in platelet functions. DNAs encoding the chimeric proteins in which the extracellular domains of each alpha2 and beta1 chain was fused to hinge and Fc regions of human IgG(1)gamma chain were cotransfected into CHO cells. Soluble integrin alpha2beta1 (salpha2beta1) in which alpha2 and beta1 chains were covalently bound by disulfide bonds was recovered from the culture supernatant. salpha2beta1 maintained functional characteristics of cell surface alpha2beta1 as indicated by cation-dependent binding to collagen and conformational changes induced by cations or ligand. Intravenously administered salpha2beta1 in rats colocalized with collagen in inflamed microvessels. Moreover, salpha2beta1-conjugated liposome administered intravenously reduced bleeding time of the thrombocytopenic mice. These results indicated that salpha2beta1 has pharmaceutical utilities as an agent for detecting injured vessels and a component of platelet substitute.

Topics: Animals; Bleeding Time; Blood Platelets; CHO Cells; Cricetinae; Dimerization; Electrophoresis, Polyacrylamide Gel; Epitopes; Female; Humans; Immunoglobulin G; Integrins; Kinetics; Ligands; Lipopolysaccharides; Male; Mice; Precipitin Tests; Protein Binding; Protein Structure, Tertiary; Rats; Rats, Wistar; Receptors, Collagen; Sodium Dodecyl Sulfate; Thrombocytopenia; Time Factors

Patients with the heparin-induced thrombocytopenia syndrome (HIT) have heparin-associated antibodies (HAb+), which, in the presence of heparin, are responsible for platelet activation and aggregation. This study addressed the questions: (1) are the antibodies specific for heparin; and (2) how do the antibodies cause platelet aggregation?. Plasmas from 79 patients with HIT were divided into seven plasma samples: HAb+ plasma sample 1 (24 pooled plasmas); HAb+ plasma sample 2 (50 pooled plasmas); and HAb+ plasma samples 3 through 7 (individual plasmas). Normal patient plasmas were used as controls (HAb-).. All seven HAb+ plasma samples caused platelet aggregation (PLA) in the presence of heparin and formed a precipitation line with heparin in gel immunodiffusion plates (HAb- plasmas did neither). The HAb+ plasma samples reacted with heparin, as determined by immunoprecipitation in sodium dodecylsulfate-polyacrylamide gel, with the production of a band at 50 kd (no band with HAb- plasmas). The plasma samples 1 and 2 were passed over heparin sepharose beads three times; the unabsorbed plasmas produced 3+ PLA, the first effluent produced 2+ PLA, and the second and third effluents produced no PLA. The heparin sepharose beads stained 3+, 2+, and 1+, after the respective passages, with fluorescein-labeled goat sera containing anti-human immunoglobulin G antibody. HAb+ plasma samples were digested with pepsin to separate the F(ab')2 fragments from the Fc fragments. The F(ab')2 fragments reacted with heparin as determined by immunoprecipitation in sodium dodecylsulfate-polyacrylamide gel with the production of a band at 25 kd, but did not cause PLA in the presence of heparin.. Patients with HIT have heparin-specific antibodies that react with heparin in a classic F(ab')2 reaction and require the Fc fragment for platelet aggregation.

Topics: Absorption; Antibodies; Electrophoresis, Polyacrylamide Gel; Fluorescein-5-isothiocyanate; Fluorescent Antibody Technique; Heparin; Humans; Immunodiffusion; Immunoelectrophoresis; Immunoglobulin Fab Fragments; Immunoglobulin Fc Fragments; Immunoglobulin G; Platelet Activation; Platelet Aggregation; Precipitin Tests; Sepharose; Sodium Dodecyl Sulfate; Syndrome; Thrombocytopenia

Successive chromatographic procedures made it possible to isolate thrombocytopoietin from the serum of thrombocytopenic rats. The following steps were taken: DEAE-cellulose phosphate chromatography, Sephadex chromatography, exclusion chromatography on DEAE-Sephadex A-50 gel. The apparent molecular weight of thrombocytopoietin was about 48,000 daltons. Like erythropoietin, thrombocytopoietin is a glycoprotein, its molecular weight is similar. A single form is obtained only when the different purification steps last less than 10 days. Beyond that time, partial degradation may occur.

Topics: Animals; Blood Platelets; Chromatography, DEAE-Cellulose; Electrophoresis, Polyacrylamide Gel; Glycoproteins; Hematopoiesis; Radioisotopes; Rats; Rats, Inbred Strains; Selenium; Selenomethionine; Sodium Dodecyl Sulfate; Thrombocytopenia; Thrombopoietin

Spectrophotometric and gas-liquid chromatographic analyses on the carbohydrate moiety of tryptic erythrocyte glycopeptides from persons with Tn-syndrome reveal a selective lowering of the galactose and sialic acid content, the degree being dependent on the percentage of polyagglutinable cells. Alkaline borohydride specifically releases N-acetylgalactosaminitol, and the amount is correlated to the percentage of pathological acetylgalactosaminitol, and the amount is correlated to the percentage of pathological erythrocytes. It is concluded that the alkali-labile carbohydrate chains of Tn-polyagglutinable red cells solely consist of N-acetylgalactosamine linked to serine or threonine. Experiments with heterophile agglutinins whose specificity is known are in line with the above-mentioned results. As judged from SDS-polyacrylamide gel electrophoresis the three major membrane glycoproteins are affected to a different extent by the defect.

Topics: Anemia, Hemolytic; Antibodies, Heterophile; Blood Glucose; Blood Protein Electrophoresis; Cell Membrane; Chromatography, Gas; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Glycopeptides; Hemagglutination; Hemagglutination Tests; Hexoses; Humans; Leukopenia; Molecular Weight; Sialic Acids; Sodium Dodecyl Sulfate; Syndrome; Thrombocytopenia; Trypsin