PubMed Health⌕ Search

Biomedical subjects

G P Tuszynski

Publications and source records attributed to G P Tuszynski.

At least 91 records · Page 5Linked to original sources
PubMed1984

Identification of human platelet membrane fibrinogen receptors by immunochemical techniques.

The fibrinogen receptors of platelets were investigated with the use of three types of anti-platelet membrane antibodies and three types of platelets. We found that antisera raised in rabbits against membranes prepared from human intact, chymotrypsin- or pronase-treated platelets inhibited the fibrinogen-induced aggregations of ADP-stimulated intact platelets, chymotrypsin-treated platelets and pronase-treated platelets. These antisera also blocked the binding of 125I-fibrinogen to ADP-stimulated intact, chymotrypsin-treated, and pronase-treated platelets. These results suggest that all three antisera blocked the interaction of fibrinogen with its receptor on the surface of the three types of platelets studied. Fibrin clot retraction by intact platelets was also inhibited by these three antibodies indicating an important role of platelet membrane proteins in clot retraction. As demonstrated by techniques using 125I-surface labeling, Staphylococcus aureus immunoprecipitation, SDS-polyacrylamide gel electrophoresis and autoradiography, anti-intact platelet membrane antibody immunoprecipitated the membrane glycoproteins GPIIb, GPIII and a protein with an apparent molecular weight of 66 000 from detergent solubilized surface 125I-iodinated chymotrypsin-treated platelets. Anti-chymotrypsin and anti-pronase-treated platelet membrane antisera immunoprecipitated mostly GPIII and the 66 000 molecular weight protein from detergent solubilized, surface 125I-iodinated chymotrypsin-treated platelets. The 66 000 Mr protein was not found on the surface of intact (unstimulated) platelets which do not bind 125I-fibrinogen and are not aggregated by fibrinogen without the prior addition of ADP. The ability of anti-platelet membrane antibodies to block fibrinogen-induced platelet aggregation and fibrinogen binding to platelets correlated with their ability to immunoprecipitate a 66 000 Mr protein from the platelet surface. It is proposed that the 66 000 Mr protein may be the fibrinogen binding domain of GPIII which becomes permanently exposed on the surface of chymotrypsin and pronase-treated platelets following proteolysis and which becomes exposed upon stimulation of intact platelets by agents such as ADP.

Autoradiography↗
PubMed1984

Factor V: a platelet cytoskeletal associated protein.

Platelet cytoskeletons prepared from thrombin activated platelets contain specifically associated Factor Va. We postulate that Factor Va associates with the cytoskeleton through receptors present on the platelet surface. The following evidence supports this hypothesis. (a) Prior secretion of Factor Va is necessary for the association of Factor Va with the cytoskeleton. (b) Reagents that inactivate Factor Va on the surface of the platelet such as EDTA and proteolytic enzymes also inactivate the Factor Va on the cytoskeleton. (c) The platelet Factor Xa binding sites (i.e. Factor Va) are quantitatively retained on the platelet cytoskeleton. (d) Platelet cytoskeletons prepared from platelets that are thought to be deficient in Factor Va binding sites contain 25 percent of Factor Va activity of normal platelet cytoskeletons and platelet cytoskeletons prepared from platelets deficient in Factor Va contain no Factor Va activity but regain control levels of Factor Va only when Factor Va is added to the platelets prior to the preparation of cytoskeletons.

Antigens, Surface↗
PubMed1984

The possible role of platelets in bypassing the contact phase of blood coagulation.

Data presented herein and previously support an active role for platelets in promoting the interaction and activation of the coagulation proteins of the contact phase of intrinsic coagulation. The platelet membrane, activated by ADP collagen or thrombin, can promote the proteolytic activation of factor XII to factor XIIa in the presence of kallikrein and high molecular weight kininogen. The zymogen factor XI associates with high molecular weight kininogen in plasma and becomes bound to a site on the membrane of thrombin or collagen activated platelets. Thereafter, platelet bound factor XI can be proteolytically activated to factor XIa either in the presence of factor XIIa or in the presence of kallikrein. These observations could explain the absence of bleeding complications in patients with factor XII deficiency. In addition, platelets contain a molecule which has a higher molecular weight than plasma factor XI and possibly consists of a tetramer of four identical subunits of 52000 daltons each of which is functionally and immunologically similar to plasma factor XI. Since this molecule is present in the platelets of patients with severe plasma factor XI deficiency and no evidence of bleeding, we postulate that platelet factor XI can substitute for plasma factor XI in hemostasis and possibly account for the considerable variability in clinical severity observed in patients with factor XI deficiency.

Blood Coagulation↗
PubMed1984

Differential cytotoxic effect of gossypol on human melanoma, colon carcinoma, and other tissue culture cell lines.

Gossypol, a yellow pigment from the cotton plant (Gossypium) was found to have anti-tumor cell effects against several tumor cell lines grown in tissue culture. Most sensitive to the drug were melanoma and colon carcinoma cells. After 24 hr of treatment with 10 microM gossypol, over 90% of these cells were killed. A rough correlation existed between very rapidly growing cells and their sensitivity to the drug. For example, slow growing cells, such as normal embryonic lung fibroblasts and mammary adenocarcinoma, were least sensitive, requiring over 30 microM gossypol in order to kill 90% of cells after 48 hr of treatment. These results indicate that gossypol has differential cytotoxic effects against certain tumor cell types, such as melanoma and colon carcinoma, and suggest that it might be of potential therapeutic value. The drug appears to act as a metabolic poison rather than as an agent interfering with DNA synthesis at the concentrations used in the study. Cells sensitive to the drug contain the more cathodic forms of lactate dehydrogenase, lactate dehydrogenases I to III.

Breast Neoplasms↗
PubMed1983

Inhibition of fibrinogen receptor-mediated platelet aggregation by heterologous anti-human platelet membrane antibody. Significance of an Mr = 66,000 protein derived from glycoprotein IIIa.

Heterologous anti-human platelet membrane antisera were raised in rabbits against membranes prepared from human intact or chymotrypsin- or pronase-treated platelets. Anti-intact, anti-chymotrypsin, and anti-pronase-treated platelet membrane antibodies (IgG and Fab fragments) inhibited the fibrinogen-induced aggregation of ADP-stimulated platelets and of chymotrypsin-treated platelets. The specific binding of 125I-fibrinogen to these platelets was also inhibited. As revealed by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 125I-surface-radiolabeled platelet proteins and by the electrophoresis of the immunoprecipitates prepared from these labeled proteins, predominant components on the surface of intact platelets were glycoproteins IIb and IIIa. These proteins were immunoprecipitated by all three antibodies. In addition, chymotrypsin-treated platelets contained an Mr = 66,000 protein on their surface that was also immunoprecipitated by the three types of anti-platelet membrane antibodies. The appearance of this Mr = 66,000 protein on the surface of chymotrypsin-treated platelets correlated with the exposure of fibrinogen receptors on the platelet surface as evidenced by the increased platelet aggregation and the enhanced 125I-fibrinogen binding shown by chymotrypsin-treated platelets. The origin of the Mr = 66,000 protein labeled on chymotrypsin-treated platelets was studied by first labeling intact platelets with 125I and then treating these platelets with chymotrypsin. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total labeled proteins present after chymotrypsin treatment and those immunoprecipitated by anti-pronase-treated platelet membrane antibody from detergent extracts of chymotrypsin-treated platelets suggested that the Mr = 66,000 protein was a proteolytic cleavage product of glycoprotein IIIa. Analysis on sodium dodecyl sulfate gels of the major chymotryptic cleavage products of partially purified glycoprotein IIIa and analysis of the peptide of glycoprotein IIIa immunoprecipitated by anti-pronase-treated platelet membrane antibody revealed that the Mr = 66,000 protein produced by chymotrypsin on the platelet surface was the major chymotryptic cleavage product of glycoprotein IIIa. We propose a hypothesis that the Mr = 66,000 is a part of glycoprotein IIIa present on the surface of proteolytically treated platelets and it may function in fibrinogen binding and fibrinogen-induced platelet aggregation and that the 66,000-dalton region of glycoprotein IIIa in intact platelets may represent the fibrinogen-binding domain of glycoprotein IIIa.

Adenosine Diphosphate↗
PubMed1983

Fibrinogen interaction with platelet receptors.

In summary: Incubation of platelets with ADP or proteolytic enzymes (chymotrypsin or pronase) results in an exposure of two classes of specific binding sites on platelet surface: low and high affinity fibrinogen receptors. Fibrinogen interaction with these receptors results in platelet aggregation. High affinity fibrinogen receptors are not exposed on thrombasthenic platelets stimulated by ADP but are rendered available on chymotrypsin-treated thrombasthenic platelets; low affinity receptors cannot be exposed by ADP or chymotrypsin on these platelets. Availability of high affinity fibrinogen receptors on thrombasthenic platelets may depend on the residual glycoprotein IIIa. Fibrinogen receptors appear to be associated with glycoproteins IIb, IIIa and a 66,000 Mr platelet membrane component that is exposed during proteolysis of platelet membranes. Some of the platelet-binding sites on the fibrinogen molecule appear to be associated with the COOH-terminal portion of the gamma chain (gamma 374-411). Additional binding sites may also be located in the COOH-terminal portion of the A alpha chain. The conformation of the fibrinogen molecule may be important in its interaction with platelets. Platelet aggregation may result from bridging platelets by fibrinogen molecule in the presence of bivalent cations. In conclusion, platelet interaction with fibrinogen is a complex process involving different binding sites of the fibrinogen molecule. Our own data and review of literature suggest that platelet-interaction with fibrinogen is of major significance in hemostasis.

Adenosine Diphosphate↗
PubMed1983

Labeling of platelet surface proteins with 125I by the iodogen method.

A procedure for the 125I-iodination of platelet suspensions is described. The procedure utilizes Iodogen, a solid-phase oxidizing agent similar to chloramine-T. Platelets were labeled under a variety of conditions, including in the presence of 0.1% albumin, and showed between 7 and 28% incorporation of 125I. Best labeling results were obtained at low platelet concentrations (3-5 x 10(8) platelets/ml), short reaction times (15 min), and with 2-ml glass vials coated with 100 micrograms of Iodogen. Analysis of the labeled platelet proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography revealed that the same major protein bands were labeled by this procedure as were labeled by the lactoperoxidase procedure. At low platelet concentrations, the Iodogen procedure gives twice the amount of iodine incorporation.

Blood Platelets↗
PubMed1983

High-molecular weight kininogen. A secreted platelet protein.

Human platelets were studied immunochemically to determine if they contain high-molecular weight kininogen. On crossed immunoelectrophoresis with total kininogen antisera (antisera that recognizes both high- and low-molecular weight kininogen) extracts of platelets contained total kininogen antigen. Platelet total kininogen antigen showed complete antigenic identity with plasma total kininogen and displayed the same electrophoretic migration as plasma total kininogen. Using antisera monospecific to high molecular weight kininogen, a competitive enzyme-linked immunosorbent assay (CELISA) was developed to directly measure platelet high-molecular weight kininogen. By CELISA, 27-101 ng of high molecular weight kininogen antigen per 10(8) platelets was quantitated in detergent-soluble lysates of washed human platelets from nine normal donors with a mean level of 60 ng +/- 24/10(8) platelets. Plasma high-molecular weight kininogen, either in the platelet suspending medium or on the surface of the platelets, could only account for 5% of antigen measured in the solubilized platelets. On the CELISA, platelet high-molecular weight kininogen was immunochemically identical to plasma and purified high-molecular weight kininogen. Platelet high-molecular weight kininogen was secreted from platelets after exposure to ionophore A23187 (3-15 microM), collagen (5-150 micrograms/ml), and thrombin (1.6 U/ml). Secreted platelet high-molecular weight kininogen did not become a part of the platelet Triton-insoluble cytoskeleton. On cross immunoelectrophoresis secreted platelet total kininogen antigen had a similar electrophoretic migration to plasma total kininogen. Thus, human platelets contain high-molecular weight kininogen that can be secreted from platelets and that may participate in plasma coagulation reactions.

Adult↗
PubMed1983

Human platelet cytoskeletons: specific content of glycolipids and phospholipids.

The lipid composition of platelet cytoskeletons was analyzed. Triton X-100 (0.5%) was used to prepare cytoskeletons from thrombin-treated platelets. The lipid/protein ratio of platelet cytoskeletons was 0.260 and the phospholipid/protein ratio was 0.177, which were comparable to the ratios present in platelets. However, there was a selective enrichment of platelet lipids in platelet cytoskeletons. Only 2 of the 5 major platelet phospholipids were detected. About 14% platelet sphingomyelin and 2% platelet phosphatidylcholine were present in platelet cytoskeletons. Only 1 of the 4 platelet neutral glycolipids, trihexosyl ceramide, was detected and was about 7% of that in intact platelets. Two percent of platelet hematoside, the predominant ganglioside in platelets, was found in cytoskeletons. Six percent of platelet cholesterol was present in platelet cytoskeletons, while no other neutral lipid could be detected. The study demonstrates that the lipid/protein ratio of platelet cytoskeletons is similar to that in platelets, but the composition of cytoskeleton lipids is specific and distinctly different from that in platelets. The selective glycolipid and phospholipid composition of cytoskeletons may be important for cytoskeleton and platelet function.

Blood Platelets↗
PubMed1982

Factor XI antigen and activity in human platelets.

Washed platelets, contaminated with less than 0.20% plasma factor XI, were examined for the presence of factor XI antigen and activity. These platelets contained a factor-XI-like coagulant activity (0.67 +/- 0.11 U/10(11) platelets) that remained constant after successive washes. By means of indirect immunofluorescence, a monospecific antibody to factor XI showed specific staining of both normal platelets and platelets from patients deficient in plasma factor XI. Radiolabeled Triton extracts of washed platelets and labeled purified factor XI solutions were analyzed for factor XI antigen by Staph A immunoprecipitation analysis using antibody to purified plasma factor XI followed by SDS gel electrophoresis. On unreduced gels, the platelet material ran as a single band having an apparent molecular weight of 220,000 daltons, whereas purified plasma factor XI gave a single band at 160,000 daltons. On reduced gels, the platelet material analyzed as a single band at 52,000 daltons, whereas purified factor XI gave a single band of 80,000 daltons. Analysis of a partially purified factor XI preparation from platelets by immunoelectrophoresis revealed that the platelet preparation displayed a slightly lower cathodal electrophoretic mobility at pH 8.6 than did plasma factor XI and yet appeared to possess complete antigenic identity with plasma factor XI. These results indicate that platelets possess a form of factor XI that exists as a disulfide-linked 52,000-dalton tetramer in contrast to the plasma form that circulates as a 80,000-dalton disulfide-linked dimer.

Antigens↗
PubMed1979

The detection of smooth muscle desmin-like protein in BHK21/C13 fibroblasts.

Three distinct proteins, actin (42,000 daltons), the principal form of fibroblast 10 nm filament protein (55,000 daltons), and a protein with a molecular weight of 52,000 and a pI of 5.8 were detected in nonionic detergent-insoluble cytoskeletal and 10 nm filament preparations of control BHK21/C13 and line 9 hamster fibroblasts. Cytoskeletal preparations of other hamster fibroblast cell types, such as NIL8 and primary embryo fibroblasts, contained the 55,000-dalton component but lacked the 52,000-dalton protein. A Rous sarcoma virus transformant of the BHK21/C13 line and an adenovirus transformant of line 9, resembled the NIL8 and other fibroblast types in that they contained only the 55,000- and 42,000-dalton polypeptides. The identity of the 52,000-dalton protein in BHK21/C13 cells was studied. This protein co-electrophoreses on both one- and two-dimensional polyacrylamide gels with the predominant muscle form of 10 nm filament protein. Further, one-dimensional peptide maps of the hamster smooth muscle 10 nm filament protein and the hamster fibroblast 52,000-dalton protein are identical to one another and distinct from the peptide maps of both the 42,000- and the 55,000-dalton components of the fibroblast cytoskeletal preparations. We conclude that BHK21/C13 cells contain both the fibroblast and the muscle form of 10 nm filament protein.

Animals↗
PubMed1979

Alterations of membrane glycoproteins in malignant cells.

Comparisons have been made of the carbohydrates bound to membrane proteins of control and malignant cells in culture. It has been found that most individual glycoproteins of control and malignant cells differ in their carbohydrate components. Differences between control and malignant cells have been foundin the glycoproteins of the various membrane systems (surface, nucleus, mitochondria, endoplasmic reticulum). It is suggested that the extensive changes in bound carbohydrates of the malignant cell permit the cell to live and divide but isolate the cell functionally. The extensive structural changes may also be responsible for the cascading of involvement of various processes within the malignant cell.

Animals↗
PubMed1978

Glycopeptides derived from individual membrane glycoproteins from control and Rous sarcoma virus-transformed hamster fibroblasts.

The membrane glycoproteins from control (BHK21/C13) and Rous sarcoma virus-transformed (C13/B4) baby hamster kidney cells grown in medium containing [14C]- or D-[3H]glucosamine have been separated into two distinct classes: a phenol-soluble fraction and an aqueous fraction. The membrane glycoproteins from both BHK21/C13 and C13/B4 partitioned similarly into these two fractions. The phenol and aquesous-soluble glycoproteins differed in their sodium dodecyl sulfate-polyacrylamide gel profiles, polyacrylamide isoelectric focusing profiles, and glycopeptide distribution on Sephadex G-50. A number of aqueous and phenol-soluble glycoproteins from BHK21/C13 and C13/B4 cells were purified to near homogeneity by means of polyacrylamide electrophoresis and gel electrofocusing. These glycoproteins range in molecular weight from 179,000 to 31,000 and have isoelectric points of 7.5 to 3.0. Our results show that the pronase glycopeptides of 20 out of 24 homologous membrane glycoproteins of equivalent molecular weight and isoelectric point from BHK21/C13 and C13/B4 cells are dissimilar as measured by Sephadex G-50 gel filtration.

Avian Sarcoma Viruses↗