==================================BSR33================================== 33. Characterization of platelet membrane proteins (in particular, glycoproteins) in primates (non-human) and small mammals (especially, dog, sheep, rabbit). Ristocetin induced aggregation of platelets in primates (non-human) and small mammals. 1 UI - 87041455 AU - Charo IF ; Fitzgerald LA ; Steiner B ; Rall SC Jr ; Bekeart LS ; Phillips DR TI - Platelet glycoproteins IIb and IIIa: evidence for a family of immunologically and structurally related glycoproteins in mammalian cells. AB - 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. MH - Amino Acid Sequence ; Animal ; Antibody Specificity ; Antigens, Surface/ ANALYSIS ; Blood Platelets/*ANALYSIS ; Calcium/METABOLISM ; Cattle ; Cells, Cultured ; Electrophoresis, Polyacrylamide Gel ; Human ; Platelet Membrane Glycoproteins/*BLOOD/IMMUNOLOGY ; Support, U.S. Gov't, P.H.S. SO - Proc Natl Acad Sci USA 1986 Nov;83(21):8351-5 2 UI - 87026584 AU - Kunicki TJ ; Nugent DJ ; Piotrowicz RS ; Lai CS TI - Covalent attachment of sulfhydryl-specific, electron spin resonance spin-labels to Fab' fragments of murine monoclonal antibodies that recognize human platelet membrane glycoproteins. Development of membrane protein specific spin probes. AB - A general method for the production of high-affinity, nitroxide-labeled, protein-specific spin probes is described in this paper. Fab' fragments are generated from protein-specific, murine monoclonal antibodies by pepsin digestion and mild reduction with cysteine. The free sulfhydryl group located in the carboxy-terminal region of these molecules and produced de novo by this manipulation is then alkylated by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), thereby generating spin-labeled Fab' fragments of these monoclonal antibodies. Two prototypic monoclonal antibodies were tested, each specific for a different integral membrane glycoprotein of human blood platelets. The results indicate that Fab' spin probes generated by this method retain the ability to bind to these glycoproteins within the membrane of intact platelets. These reagents thus represent probes that can be generally used to monitor integral membrane protein mobility on the surface of the intact cell. MH - Animal ; *Antibodies, Monoclonal/ISOLATION & PURIFICATION ; Antigen-Antibody Complex ; Blood Platelets/*ANALYSIS ; Cell Membrane/ ANALYSIS ; Cyclic N-Oxides ; Electron Spin Resonance ; Glycoproteins/ *BLOOD ; Human ; IgG/ISOLATION & PURIFICATION ; *Immunoglobulins, Fab/ ISOLATION & PURIFICATION ; Membrane Proteins/*BLOOD ; Mice ; Spin Labels ; Sulfhydryl Compounds/ANALYSIS ; Support, U.S. Gov't, P.H.S. SO - Biochemistry 1986 Sep 9;25(18):4979-83 3 UI - 87024615 AU - Aiken M ; Ciaglowski RE ; Walz DA TI - Isolation and identification of a 23,000-dalton heparin binding fragment from the amino terminus of bovine thrombospondin. AB - Bovine freeze-thaw lysed platelets were fractionated by dextran sulfate affinity chromatography and a purified protein of 23,000 Da was subsequently obtained by G-75 gel filtration of the 0.5 M NaCl fraction. This protein had an amino terminal sequence of Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-Asp-Ile-Phe-Glu-Leu- Thr-Gly-Ala-Ala-Trp-Lys-, a sequence identical to that reported for human thrombospondin. Thrombin-released platelets, fractionated in an identical manner, yielded a protein of 30,000 Da. Immunoblotting of purified bovine platelet thrombospondin and the 150,000- and 30,000-Da plasmin-generated thrombospondin fragments indicated that polyclonal antisera raised against the 23,000-Da protein cross-reacted with intact thrombospondin and the 30,000-Da fragment but not the 150,000-Da fragment. The 23,000-Da protein possessed weak heparin neutralization activity. MH - Amino Acid Sequence ; Animal ; Blood Platelets/*METABOLISM ; Cattle ; Chromatography, Affinity ; Chromatography, Gel ; Glycoproteins/*BLOOD ; Heparin/*BLOOD ; Immunochemistry ; Molecular Weight ; Peptide Fragments/ BLOOD/ISOLATION & PURIFICATION ; Receptors, Endogenous Substances/ *ISOLATION & PURIFICATION ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. SO - Arch Biochem Biophys 1986 Oct;250(1):257-62 4 UI - 86078059 AU - Levene RB ; Rabellino EM TI - Platelet glycoproteins IIb and IIIa associated with blood monocytes are derived from platelets. AB - Platelet glycoprotein IIb/IIIa (GP IIb/IIIa), the receptor complex for fibrinogen, has been regarded as a megakaryocyte/platelet lineage-restricted antigen. Recently, however, it has been reported that GP IIb/IIIa is expressed in blood monocytes. Studies were performed to establish the origin and immunological characteristics of monocyte-associated glycoproteins IIb and IIIa (GPs IIb and IIIa). Preparations of blood monocytes containing varying platelet-monocyte ratios were metabolically labeled with [35S]methionine with the expectation that any newly synthesized GPs IIb and IIIa would be monocyte-derived, since platelets have only rudimentary protein synthetic apparatuses. Analyses of sodium dodecyl sulfate (SDS) gels of homogenates of cell preparations containing from 200 to 5:1 platelet-monocyte ratios revealed that unlabeled GPs IIb and IIIa were readily immunoisolated using protein A-Sepharose immunobeads. However, fluorographic analyses of the same cell preparations pulse-labeled with [35S]methionine failed to demonstrate synthesis of GP IIb or IIIa. Additionally, no GP IIb or IIIa was detected when immunoisolation was carried out in pure preparations of monocytes containing less than 1:100 platelet-monocyte ratios and SDS acrylamide gels were stained by the sensitive silver stain method. Furthermore, heterologous polyspecific antisera and two monoclonal antibody preparations against GPs IIb and IIIa, which bound to platelets, failed to bind to monocyte membranes. Thus, evidence was presented that indicated that monocytes do not synthesize platelet GPs IIb and IIIa and that detection of these molecules in blood monocyte preparations reflects platelet contamination. MH - Animal ; Antigens/ANALYSIS ; Blood Platelets/*ANALYSIS ; Glycoproteins/ BIOSYNTHESIS/*BLOOD/ISOLATION & PURIFICATION ; Human ; Membrane Proteins/ BIOSYNTHESIS/*BLOOD/ISOLATION & PURIFICATION ; Monocytes/*ANALYSIS/ METABOLISM ; Rabbits ; Receptors, Endogenous Substances/BLOOD ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. SO - Blood 1986 Jan;67(1):207-13 5 UI - 86104698 AU - Moon DG ; Kaplan JE ; Mazurkewicz JE TI - The inhibitory effect of plasma fibronectin on collagen-induced platelet aggregation. AB - Plasma fibronectin (Fn) has been proposed to have an antithrombotic effect, protecting against platelet and fibrinogen consumption after injury. The current study was designed to determine the effect of plasma fibronectin on collagen-induced platelet aggregation. In vitro aggregometry using an isolated homologous rat system, demonstrated a significant (P less than .05) inhibitory effect of 120 micrograms/mL Fn on platelet aggregation as induced by 60 micrograms/mL fibrillar collagen (type I). The inhibition was evidenced by a threefold increase in lag time and a significant decrease in the rate and extent of aggregation. The hypothesis was also tested using an in vivo model of collagen-induced platelet aggregation. The model used was intravenous injection of 2 mg/kg of homologous type I collagen into anesthetized Sprague-Dawley rats. Injection of collagen preincubated with 4 mg/kg Fn resulted in significantly less thrombocytopenia and fibrinogen consumption as compared with injection of collagen alone. The results of both the in vitro and in vivo studies are consistent with the proposed antithrombotic effect of plasma fibronectin. MH - Animal ; Blood Platelets/*DRUG EFFECTS ; Collagen/*PHARMACODYNAMICS ; Comparative Study ; Diphosphonates/PHARMACODYNAMICS ; Fibronectins/BLOOD/ *PHARMACODYNAMICS ; Human ; In Vitro ; Male ; Platelet Aggregation/*DRUG EFFECTS ; Rats ; Solubility ; Support, Non-U.S. Gov't ; Support, U.S. Gov't, P.H.S. ; Thrombin/PHARMACODYNAMICS SO - Blood 1986 Feb;67(2):450-7 r