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Biomedical subjects

I F Charo

Publications and source records attributed to I F Charo.

64 records · Page 4Linked to original sources
PubMed1987

The platelet membrane glycoprotein IIb/IIIa complex. Structure, function, and relationship to adhesive protein receptors in nucleated cells.

The GPIIb/IIIa complex functions as the aggregation site on the platelet membrane surface. This complex has been purified, characterized biochemically and morphologically, and reconstituted into phospholipid vesicles. Fibrinogen and fibronectin bind to reconstituted GPIIb/IIIa with many of the properties that characterize their binding to intact platelets. The GPIIb/IIIa complex appears to be a member of a widely distributed family of cell-surface glycoproteins that mediate cellular interactions. The terms cytoadhesins30 and integrins39 have been suggested for the members of this family of two-subunit molecules. The aminotermini of the alpha subunits of these molecules have been sequenced and appear to be homologous. Three beta subunits have been identified for this family of receptors, indicating that many alpha subunits have a common beta subunit. The three beta subunits have been sequenced, and there is about a 40 to 50% identity among their amino acid sequences. It thus appears that the receptors mediating cellular interactions have evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1986

Chemotactic peptides modulate adherence of human polymorphonuclear leukocytes to monolayers of cultured endothelial cells.

We have used a new centrifugation assay to examine the effects of highly purified human C5a and C5a des Arg, as well as effects of N-formyl-methionyl-leucyl-phenylalanine (FMLP), on both the extent and strength of human polymorphonuclear leukocyte (PMN) adherence to monolayers of cultured human umbilical vein endothelial cells. At concentrations that were chemotactic for PMN, C5a (0.1 nM), C5a des Arg (5.0 nM), and FMLP (1.0 nM) significantly reduced the percentage of PMN that adhered to endothelial monolayers. Adherence also was reduced by C5a des Arg that was generated by incubating (37 degrees C, 30 min) fresh human serum with either zymosan or purified C5a. High concentrations of C5a (greater than 1.0 nM) and FMLP (greater than 50 nM) that diminished PMN chemotaxis significantly enhanced the percentage of PMN that adhered tightly to endothelial cells (adherent cells resisted a dislodgment force of 1200 X G). Tight adherence of PMN to endothelial cells also was increased by high concentrations of C5a that were added to human serum in which carboxypeptidase N activity was destroyed by heating (56 degrees C, 30 min), and by C5a that was generated by incubating (37 degrees C, 30 min) fresh human serum with zymosan in the presence of the carboxypeptidase N inhibitor, epsilon-aminocaproic acid. High concentrations of C5a des Arg (up to 80 nM) neither enhanced adherence of PMN to endothelial cells nor decreased PMN migration. Thus, a reciprocal relation exists between PMN migration and PMN adherence to endothelial cells in response to chemotactic factors. At concentrations that are chemotactic for human PMN, C5-derived peptides and FMLP reduce the adherence of PMN to endothelial monolayers. Only at concentrations that decrease PMN migration do C5a and FMLP augment PMN adherence.

Adult↗
PubMed1986

Platelet glycoproteins IIb and IIIa: evidence for a family of immunologically and structurally related glycoproteins in mammalian cells.

Human and bovine cultured cell lines and circulating leukocytes were examined for the presence of surface proteins similar to platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human endothelial cells, smooth muscle cells, and MG-63 fibroblast-like cells were found to have surface proteins that cross-reacted with platelet GPIIb and GPIIIa antibodies, existed as complexes, and had molecular weights similar to those of the corresponding platelet glycoproteins. Bovine endothelial cells and smooth muscle cells also expressed GPIIb- and GPIIIa-like surface proteins. Metabolic labeling studies with [35S]methionine demonstrated that the cultured cells synthesized these glycoproteins. The GPIIIa-like protein in human endothelial and smooth muscle cells had the same isoelectric point as platelet GPIIIa, whereas their GPIIb alpha-like protein was slightly more acidic than platelet GPIIb alpha (pI = 5.2-5.3 versus 5.5). Platelet and endothelial cell GPIIb alpha (but not GPIIIa) showed an increased electrophoretic mobility in Ca2+ -containing versus EDTA-containing gels, implying a Ca2+ -GPIIb alpha interaction. The amino acid sequence of the amino termini of platelet GPIIb alpha and GPIIb beta and of the alpha chains of the leukocyte LFA-1 and Mac-1 glycoprotein complexes had significant sequence homology. These data indicate that glycoproteins that have either immunological cross-reactivity or amino-terminal sequence homology with the platelet GPIIb-IIIa complex are widely distributed in human and non-human adherent cells and circulating leukocytes and suggest that these proteins may be the products of a large gene family whose expression is cell specific.

Amino Acid Sequence↗
PubMed1985

Human and bovine endothelial cells synthesize membrane proteins similar to human platelet glycoproteins IIb and IIIa.

Human umbilical vein endothelial (HUVE) and bovine aortic endothelial (BAE) cells in culture were examined to determine whether membrane proteins similar to human platelet glycoproteins (GP) IIb and IIIa were present. The HUVE and BAE cells were either 125I-surface labeled or metabolically labeled. Triton X-100 lysates of labeled cells were immunoprecipitated with polyclonal antibodies prepared against purified human platelet GP IIb-IIIa complex. Two membrane proteins were detected on both HUVE (Mr = 130,000 and 110,000) and BAE (Mr = 135,000 and 105,000) cells, which were similar to human platelet GP IIb (Mr = 125,000) and GP IIIa (Mr = 108,000). The two membrane proteins from HUVE cells and the two from BAE cells cosedimented in sucrose gradients, indicating that they exist as a complex. Unlike the human platelet GP IIb-IIIa complex, the HUVE and BAE membrane protein complexes were not dissociated by chelation of Ca2+. Platelet GP IIb and GP IIIa and the related membrane proteins on both HUVE and BAE cells showed similar changes in electrophoretic mobility upon disulfide reduction. These data demonstrate that human and bovine endothelial cells synthesize membrane proteins that have properties similar to the platelet membrane GP IIb-IIIa complex.

Animals↗
PubMed1985

Adherence of human polymorphonuclear leukocytes to endothelial monolayers: effects of temperature, divalent cations, and chemotactic factors on the strength of adherence measured with a new centrifugation assay.

Polymorphonuclear leukocytes (PMNs) adhere to endothelial cells at sites of acute inflammation. To examine this phenomenon in vitro, we have developed a new assay to measure adherence of PMNs to cultured endothelial cells. Human PMNs were labeled with 111indium-oxine and incubated in microtiter wells with monolayers of either human umbilical vein or bovine aortic endothelial cells. Following incubation, the wells were sealed, inverted, and centrifuged at varying speeds. Results are expressed as the percentage of PMNs added initially that remained attached to the monolayers after being subjected to dislodgment forces (ie, relative centrifugal forces) ranging from 1 to 1,200 g. Adherence of PMNs to endothelial monolayers was temperature dependent, dependent on the concentration of extracellular Mg2+ (but not Ca2+), and enhanced significantly by the chemotactic peptides, N-formyl-methionyl-leucyl-phenylalanine (fMLP) and human C5a. It was found that fMLP and C5a not only increased the number of PMNs that adhered to endothelial cells, but also increased the strength of adherence.

Animals↗
PubMed1984

Prostaglandin I2 is not a major metabolite of arachidonic acid in cultured endothelial cells from human foreskin microvessels.

Prostaglandin I2 (PGI2), a potent vasodilator and inhibitor of platelet aggregation, is a major product of arachidonic acid metabolism in endothelial cells that are derived from large blood vessels (e.g., umbilical veins). We have examined whether PGI2 is also a major product of arachidonic acid metabolism in cultured endothelial cells that are derived from dermal microvessels in human newborn foreskin. Supernatants from confluent monolayers of endothelial cells that had been incubated for 20 min with [3H]arachidonic acid and the calcium ionophore A23187 (10 microM) were assayed for prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 (PGE2), and 6-keto-prostaglandin F1 alpha (PGF1 alpha) (the stable metabolite of PGI2) by using authentic standards and high performance liquid chromatography. Whereas supernates from stimulated umbilical vein endothelial cells contained 6-keto-PGF 1 alpha much greater than PGF 2 alpha much greater than PGE2, supernates from stimulated foreskin microvessel endothelial cells contained PGF 2 alpha congruent to PGE2 much greater than 6-keto-PGF 1 alpha. Similar results were obtained when supernates from stimulated, unlabeled endothelial cells were analyzed by radioimmunoassay. These data indicate that PGI2 is not a major metabolite of arachidonic acid in cultured endothelial cells from human foreskin microvessels.

Arachidonic Acid↗
PubMed1978

Evidence that calcium regulates platelet function.

It is generally believed that calcium ions play a key role in regulation of platelet function. This is based on 3 types of evidence. 1. Analogies with other cells. Calcium ions are known to trigger secretion and contraction in many cells, possibly reflecting a general role for calcium in all secretion and contraction. 2. Indirect evidence. Platelet aggregation and secretion are induced by divalent cation ionophores. The response to the ionophore A23187 is identical to that induced by other potent stimuli. 3. Direct evidence. Platelet activation can be blocked by drugs (e. g. certain local anesthetics) that block release of calcium ions from sarcoplasmic reticulum; the inhibition can be overcome by addition of extracellular calcium in the presence of a calcium ionophore. While this does not constitute definitive proof, the central role for calcium ions remains an attractive hypothesis that justifies attempts to further define calcium pools and fluxes in platelets.

Blood Platelets↗
PubMed1977

Interrelations of platelet aggregation and secretion.

The mechanism of stimulus-response coupling in human platelets was investigated with a new instrument that simultaneously monitors aggregation and secretion in the same sample of plateletrich plasma. When platelets were stimulated by high concentrations of ADP, secretion began only after aggregation was almost complete. With lower concentrations of ADP or with epinephrine, biphasic aggregation was observed, and secretion began simultaneously with, or slightly after, the second phase of aggregation. When platelets were stimulated with high concentrations of gamma-thrombin or A23187, secretion and aggregation began essentially together. With very low concentrations of gamma-thrombin or A23187, biphasic aggregation was observed with secretion paralleling the second phase. At every concentration of collagen, secretion and aggregation appeared to be parallel events. Under every condition where the beginning of secretion lagged behind aggregation, secretion was dependent upon aggregation and was inhibited by indomethacin; this is referred to as aggregation-mediated platelet activation. When secretion began at the same time as aggregation, it also occurred in the absence of aggregation and was not blocked by indomethacin; this is referred to as directly induced platelet activation. These observations are consistent with a simple model of platelet stimulus-response coupling that includes two mechanisms for activation; aggregation-mediated activation is inhibited by indomethacin, while direct activation does not depend upon aggregation and is not inhibited by indomethacin. Secretion and second wave aggregation appear to be parallel events, with little evidence for second wave aggregation being a consequence of secretion as usually described.

Adenosine Diphosphate↗