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Biomedical subjects

F C Chao

Publications and source records attributed to F C Chao.

At least 19 recordsLinked to original sources
PubMed1996

Infusible platelet membrane microvesicles: a potential transfusion substitute for platelets.

BACKGROUND: Several substitutes for intact, viable platelets have been used for transfusion, both to people and in animal models, with varied success. Infusible platelet membrane (IPM) is prepared from human platelets. IPM retains the glycoprotein (GP)lb receptor and has platelet factor 3 activity (procoagulant activity). However, factor V, serotonin, a cytoplasmic marker enzyme (purine nucleotide phosphorylase), GPIIb/IIIa complex, and HLA class I and II antigens are all absent in IPM. STUDY DESIGN AND METHODS: IPM is prepared from outdated platelets. The platelets were disrupted by freezing and thawing; they were washed and heated to inactivate possible viral contaminants, and then the sonicated membrane microvesicle fraction was separated and lyophilized. The hemostatic activity of IPM was measured by its ability to reduce the prolonged bleeding time in thrombocytopenic rabbits. RESULTS: Administration of IPM at a dose of 2 mg per kg results in a substantial reduction in the bleeding time. In a series of 23 experiments, a median preinjection bleeding time of 15 minutes was reduced to 6 minutes within 4 hours after IPM administration. Administration of IPM did show a mild enhancement in the thrombogenicity index, as measured in the Wessler rabbit model. This enhancement is, however, not significant, as a thrombogenicity index value of up to 0.6 is clinically acceptable. CONCLUSION: IPM may have clinical potential as a substitute for platelets in the treatment of bleeding due to thrombocytopenia.

Animals↗
PubMed1990

Human blood cells at microgravity: the NASA Initial Blood Storage Experiment.

The Initial Blood Storage Experiment (IBSE) probed the behavior of human red cells, white cells, and platelets during exposure to microgravity for 6 days and 2 hours on a National Aeronautics and Space Administration (NASA) shuttle mission, named STS 61-C, which was launched on January 12, 1986. IBSE involved carefully controlled comparisons between two identical sets of blood cells, one exposed to microgravity and the other held on the ground. Specially designed and fabricated, electrically powered environmental chambers provided appropriate environmental temperatures and air flow to support cell metabolism throughout the experiment. To circumvent the need for constant agitation of platelets during storage, a new thin-layer compression method for platelet preservation was developed. Blood cell samples were allocated to the two arms of the experiment, microgravity and earth gravity, by blind assignment. Moreover, to ensure unbiased assessment of the experiment's findings, postexperiment samples for measurement were identified by code. To optimize the chances of detecting possible gravitational effects, a wide array of measurements of cellular function, morphology, metabolism, and immunology were made. Analysis of variance was used in analyzing the data. The most striking finding was that platelets displayed markedly superior structural and functional integrity at microgravity. Granulocytes held on the ground were preserved slightly better than those that orbited in the shuttle, whereas red cells displayed few effects that were attributable to the gravitational variable. Polyvinylchloride-di-(2-ethylhexyl)phthalate (PVC-DEHP) was the plastic of choice for storage of red cells, while PVC-trioctyltrimellitate (TOTM) was superior to PVC-DEHP and polyolefin (PO) for platelets.

Aerospace Medicine↗
PubMed1987

Platelet antithrombin defect in malignancy: platelet protein alterations.

Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)-electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti-human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.

Antithrombins↗
PubMed1986

Thrombospondin in essential thrombocythemia.

Essential thrombocythemia is a myeloproliferative disorder characterized by frequent bleeding and thrombotic complications. On a molecular level, two abnormalities of platelet thrombospondin have been identified: abnormal glycosylation of the intact 185,000-dalton chain has been detected and a shortened form of the thrombospondin chain is present. We have used two monoclonal antibodies and Lens culinaris lectin to probe the structure of thrombospondin in the platelets from three patients with essential thrombocythemia; one patient with polycythemia vera and two patients with secondary thrombocytosis. The presence of abnormal thrombospondin fragments with molecular weights of 160,000 and 30,000 was detected in the intact platelets and in the supernatant from thrombin-treated platelets, in all of the individuals except one of the secondary thrombocytosis patients. Monoclonal antibody binding studies indicate that both fragments are produced by proteolysis at a single site, which results in the removal of a 30,000-dalton fragment from the NH2-terminal. Lens culinaris lectin-binding studies revealed that some of the carbohydrate moieties of thrombospondin are near this cleavage site. The results are consistent with the hypothesis that the abnormal thrombospondin fragments observed under conditions of increased platelet production are due to increased susceptibility to proteolysis which, in turn, may be due to defective glycosylation.

Antibodies, Monoclonal↗
PubMed1985

The structure of human platelet thrombospondin.

Two distinct murine monoclonal antibodies, designated MA-I and MA-II, and limited proteolysis with thrombin and trypsin have been used to probe the structure of human platelet thrombospondin. The results indicate that each of the constituent chains of thrombospondin comprise four distinct polypeptide segments. The production of these segments is influenced by the presence of calcium, the enzyme employed, the temperature of digestion, and the enzyme-to-substrate ratio. Thrombin digestion in the presence of calcium results in the release of a 30,000-dalton fragment, designated segment I, which contains the epitope for MA-II and the heparin-binding site. Prior EDTA treatment results in the concomitant cleavage of a 25,000-dalton fragment, designated segment IV, from the other terminus. Limited tryptic digestion in the absence of calcium produces a 47,000-dalton fragment (segment III) which is adjacent to segment IV. Segment III contains the epitope for MA-I. Segment II is an 85,000-dalton fragment which contains the interchain disulfide bonds. Calcium inhibits proteolysis at cleavage sites between segments II and III and between segments III and IV. In the presence of calcium, an 85,000-dalton fragment is produced, which is derived from portions of segments II, III, and possibly IV. Electron microscopy of platinum replicas produced by low angle rotary shadowing reveals that thrombospondin is composed of four well-defined globular regions connected by thin flexible regions. Three of the globular regions, designated globular region C, appear to be at the ends of the three thin connecting regions. The fourth globular region, designated globular region N, appears to be close to the site where the chains are cross-linked. Globular region N can be resolved into three separate smaller globular structures which are 70 +/- 7.1 A in diameter. This region is selectively removed by thrombin digestion in the presence of calcium and binds a monoclonal antibody directed against the heparin-binding peptides. These data indicate that globular region N comprises the three NH2-terminal portions (segment I) from each of the three chains of thrombospondin. Globular region C is located at the ends of each of the three thin connecting regions which are each approximately 291 +/- 46 A long. The removal of calcium results in a decrease in the size of globular region C from 118 +/- 18.6 A to 80 +/- 7.4 A and an increase in the length of the adjacent thin connecting region to 383 +/- 30 A.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1985

Diadenosine 5',5'''-p1,p4-tetraphosphate deficiency in blood platelets of the Chédiak-Higashi syndrome.

Diadenosine tetraphosphate (AP4A) is an unusual nucleotide found in a variety of cells, including platelets. It has been suggested that platelet AP4A is stored in the dense granules and is metabolically inactive. We have studied the AP4A content of blood platelets in two patients and three cattle with Chédiak-Higashi syndrome (CHS), a hereditary platelet defect with dense granule deficiency. Acid-soluble extractions of whole blood and platelets were neutralized. The adenosine triphosphate (ATP) level was measured by luminescence technique. To measure the AP4A content, the neutralized extract was treated with phosphomonoesterase for removal of ATP. The AP4A content was then measured by coupling the phosphodiesterase and luciferase reaction. The AP4A content was 0.43 nmol/mg protein for normal human platelets and 0.004 nmol/mg protein for CHS platelets. The ATP/AP4A ratio was 67 for normal and 3,023 for CHS platelets. The whole blood AP4A was reduced by 89% in CHS patients who had only a slight decrease in ATP level (26% reduction). Similarly, bovine platelets with CHS showed a marked decrease of AP4A content and a moderate reduction of the ATP level. The platelet ATP/AP4A ratio was 351 and 3,133 for normal and CHS cattle, respectively. Results demonstrate a marked reduction of AP4A in CHS platelets and suggest that AP4A may be a useful marker for the measurement of dense granule content in platelets.

Adenine Nucleotides↗
PubMed1982

Alteration in plasma proteins and platelet functions with aging and cigarette smoking in healthy men.

Blood samples were obtained on four different occasions from 18 cigarette smoking and 34 non-smoking healthy men (age 40-69) and analyzed to assess age- and smoking-associated changes in plasma proteins, blood coagulation and platelet functions. Collagen-induced platelet aggregation was significantly increased with aging in non-smokers. Significant changes in chronic smokers were increases in platelet count and fibrinogen in plasma; elevation of platelet factor-3 (PF-3) activity in platelet-poor plasma (PPP); increase in serum levels of alpha 1-antitrypsin, orosomucoid, haptoglobin and properdin factor B; and shortening of the lag period of collagen-induced platelet aggregation. Filtration of PPP through Millipore filters removed PF-3 membranes. The differences in PF-3 activities in filtered plasma were no longer significant between smokers and non-smokers. Results suggest that chronic smokers have higher levels of acute phase proteins reflecting underlying inflammatory processes, and higher levels of PF-3 activity in plasma due to liberation of PF-3 membranes from platelets.

Adult↗
PubMed1981

Thrombin inhibition by malignant and normal cells: a cell-bound antithrombin effect.

Analysis of fresh surgical specimens of normal tissue and tumor tissue show a cellular antithrombin activity to be present in certain organs. In normal tissues it was noted chiefly in normal colon, testes, breast, and uterus. In malignant tissues it was prominent in adenocarcinomas of the colon, breast, and lung. No epidermoid tumors showed evidence of thrombin binding. The thrombin- binding activity required the presence of intact cells and was distinct from the soluble antithrombins normally present in plasma and serum. There is growing evidence to suggest an interrelationship between clotting and the growth and dissemination of cancer. The implications of cellular antithrombins are reviewed in this context.

Adenocarcinoma↗
PubMed1980

Ionophore-induced disassembly of blood platelet microtubules: effect of cyclic AMP and indomethacin.

Cytoplasmic calcium levels are believed to be important in blood platelet activation. Upon activation, the discrete marginal microtubule band, which maintains the discoid shape of non-activated platelets, becomes disrupted. Present studies demonstrate that the extent of assembly of the marginal microtubule band is related to cytoplasmic calcium levels. The divalent cationophore, A23187, causes platelet aggregation, secretion, and contraction by promoting calcium transport from intraplatelet storage sites into the cytoplasm. A23187 caused disassembly of platelet microtubules. Quantitation of electron micrographs revealed that numbers of microtubules were reduced by approximately 80% after A23187 treatment. Secondly, assembled microtubules in homogenates of platelets, in which microtubules were stabilized prior to homogenization, were decreased in favor of free tubulin in A23187-treated platelets. Thirdly, A23187 increased 14C-colchicine binding by intact platelets; this also indicated a shift in the microtubule subunit equilibrium to favor free, colchicine-binding tubulin subunits. In control experiments, A23187 did not affect the stability of platelet tubulin, the colchicine binding reaction, or the total tubulin content of platelets. Stimulation of colchicine binding depended on A23187 concentration (0.05-0.5 microM) and did not require extracellular calcium. A23187-stimulation of colchicine binding was blocked by dibutyryl cyclic AMP (0.80 mM) and/or 3-isobutyl-1-methylxanthine (50 microM) and by indomethacin (10 microM). Cyclic AMP or indomethacin also interferes with A23187-induced platelet activation, but indomethacin is not likely to completely inhibit the perturbation of intraplatelet calcium gradients by A23187. It is suggested that A23187-induced microtubule disassembly may be an indirect effect of calcium on microtubules.

Anti-Bacterial Agents↗
PubMed1980

Localizaton of fibrin converted by thrombin from released platelet fibrinogen.

Treatment of platelets (10(9) cells/ml) with thrombin (1 U/ml) resulted in rapid disappearance of fibrinogen from the system as measured by the tanned red cell hemagglutination inhibition immunoassay (TRCHII). Plasmin digestion of individual pellet and supernatant fractions that had been previously separated from thrombin-treated platelet suspensions by centrifugation resulted in recovery of TRCHII-detectable material in platelet pellets. To elucidate the specific association of fibrin to platelet membranes, control and thrombin-treated platelets were homogenized by a modified glycerol-loading and nitrogen decompression technique. Ultracentrifugation of homogenates through 27% sucrose cushions yielded three subcellular fractions: supernatant, small membrane vesicles, and a particulate fraction for controls; and supernatant membrane vesicles, and aggregated membrane "ghosts" for thrombin preparations. Ultrastructurally identifiable fibrin was noted only in the thrombin fraction containing membrane ghosts. Fibrinogen recovered from 3 thrombin fractions was markedly decreased (3% of the control). Plasmin digestion produced 23% and 46-fold increase in TRCHII-detectable material from 3 subcellular fractions of control and thrombin preparations, respectively. More than 97% of TRCHII material recovered from thrombin preparations was in the fraction containing aggregated membrane fractions. Results suggest that platelet plasma membranes function as surfaces for fibrin deposition.

Blood Platelets↗
PubMed1979

Platelet alpha granules contain a growth factor for fibroblasts.

Platelets contain a polypeptide growth factor that stimulates the replication of normal connective tissue cells; this platelet-derived growth factor (PDGF) is released during the clotting process. Human platelets from normal volunteers were disrupted by nitrogen cavitation, and the subcellular organelles were fractionated by ultracentrifugation through a 30%--60% sucrose gradient. Electron microscopy revealed that fraction 7 (density 1.23 g/liter) contained the largest number of alpha granules. The specific activity of platelet fibrinogen, an alpha-granule marker, was also highest in this fraction. The subcellular fractions were assay for the presence of PDGF and for beta-thromboglobulin. PDGF was assayed quantitatively by the stimulation of DNA synthesis in confluent growth-arrested BALB/c-3T3 cells, whereas the concentration of beta-thromboglobulin was determined by radioimmunoassay. The highest concentrations of both PDGF and beta-thromboglobulin were found in the alpha-granule fraction. In contrast, beta-glucuronidase, a lysosomal enzyme, was more diffusely distributed and had its highest specific activity in fractions of lower density than those for PDGS, beta-thromboglobulin, or fibrinogen. The data demonstrate that the alpha granules of platelets provide a unique delivery system for PDGF, a polypeptide hormone with growth-promoting activity for connective tissue cells.

Animals↗
PubMed1978

Plateletpheresis by discontinuous centrifugation: effect of collecting methods on the in vitro function of platelets.

The in vitro function of platelets collected by two different methods during centrifugal plateletpheresis was compared. The RBC method involves collecting platelets with red cells followed by a supplementary spin to remove them, whereas the no-RBC method requires collecting platelets only from the buffy coat without red cells. Platelet response to adenosine diphosphate (ADP), epinephrine and collagen was slightly reduced in platelet-rich plasma (PRP) prepared by no-RBC technique and was markedly decreased in samples obtained by the RBC technique when compared to prepheresis controls. The decrease in platelet response to ADP, epinephrine and collagen was apparent in three testing systems: aggregation, release of serotonin and reptilase clot retraction. Both plasma and platelets appeared to be affected by the pheresis procedure. Platelet preparations obtained by both RBC and no-RBC techniques showed an increase of platelet factor 3 activity and an enhancement of aggregation, release of serotonin and clot retraction induced by thrombin as compared to prepheresis controls. Postpheresis platelet-poor plasma contains platelet membrane fragments which exhibit a high platelet factor 3 activity. The results showed that the RBC method, although providing a higher platelet yield, caused more qualitative alterations in platelets than in those obtained by no-RBC method, and that both methods of collecting platelets activated the procoagulant activity of platelets.

Blood Platelets↗