Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography

Clemetson, Kenneth J.; Pfueller, Sharron L.; Lüscher, E. F.; Jenkins, C. S. P.

doi:10.1016/0005-2736(77)90025-6

Cited by 139 publications

(79 citation statements)

References 28 publications

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“…Tritiated borohydride labeling of normal platelets after galactose oxidation or periodate treatment has also revealed a band in this same molecular weight region (16,17). Band 2 polypeptide, Mr 150,000, as isolated in the wheat germ lectin system, probably corresponds to what others have referred to as GPIb (4,5,8). In the Phillips two-dimensional system, unreduced GPIb has an apparent Mr of 170,000 that falls to 143,000 on reduction due to disulfidelinked nonidentical subunits (6).…”

Section: Resultssupporting

confidence: 54%

“…The glycoprotein I (GPI) complex on the surface of the human platelet may act as a von Willebrand factor receptor (4) and thus directly influence the adhesion of platelets to the subendothelium. Depending on the method of membrane solubilization and the gel system utilized for analysis, the platelet GPI complex has been found to consist of at least two distinct glycoproteins (GPIa, GPIb) (5,6). GPIc, which is part of the GPI system, does not stain with periodic acid/Schiff reagent and is probably not a glycoprotein (6).…”

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confidence: 99%

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Structural analysis of human platelet membrane glycoprotein I complex.

Nachman¹,

Kinoshita²,

Ferris³

1979

Proc. Natl. Acad. Sci. U.S.A.

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The glycoprotein I complex, consisting of two polypeptides of Mr 210,000 and 150,000, was isolated from human platelet membranes by wheat germ lectin affinity chromatography. Glycocalicin, a soluble loosely bound membrane glycoprotein of M, 150,000 related to the glycoprotein I system, was also purified. The isolated poly eptides were radioiodinated in sodium dodecyl sulfate/! Iyac lamide gels and digested with trypsin, and the labeled Reptide digest was analyzed by two-dimensional high-voltage electrophoresis and thin-layer chromatography. The two polypeptides of Mr 210,000 and 150,000 in the glycoprotein I complex had essentially identical radioactive peptide maps. Glycocalicin had a completely different t ptic peptide map. These studies shed light on the molecular relationships of some of the components of the platelet membrane glycoprotein I system. The possibility is raised that the receptorlike function of the intrinsic platelet membrane glycoproteins may be related to the polymeric subunit associations of the constituent polypeptides. The external surface glycoproteins of mammalian cells mediate important cellular reactions, including aggregation, adhesion, and surface contact interactions (1). It is therefore not surprising that platelet membrane glycoproteins have been implicated in von Willebrand factor-dependent platelet agglutination induced by ristocetin (2, 3). The glycoprotein I (GPI) complex on the surface of the human platelet may act as a von Willebrand factor receptor (4) and thus directly influence the adhesion of platelets to the subendothelium. Depending on the method of membrane solubilization and the gel system utilized for analysis, the platelet GPI complex has been found to consist of at least two distinct glycoproteins (GPIa, GPIb) (5, 6). GPIc, which is part of the GPI system, does not stain with periodic acid/Schiff reagent and is probably not a glycoprotein (6). Another constituent of the GPI complex, glycocalicin or GPIs, is easily removed from the platelet membrane by homogenization and is ordinarily detected in the soluble subcellular fractions of homogenized preparations (7). The platelets of patients with the Bernard-Soulier syndrome are deficient in GPI as well as in glycocalicin (2,8). These platelets fail to agglutinate in the presence of von Willebrand factor and ristocetin and also do not bind thrombin normally (9). The possibility has been raised that glycocalicin may act as a single receptor for ristocetin-induced and thrombin-induced platelet aggregation (10). We have previously isolated two proteins of the GPI complex from human platelet membranes by using wheat germ lectin affinity columns and demonstrated that these proteins mediate von Willebrand factor-dependent platelet agglutination induced by ristocetin (11). At present, it is not clear what the structural relationships are among the isolated separate constituents of the GPI complex. This study presents a comparison of the radioactive tryptic peptide maps of the isolated polypeptides of the platelet-GPI...

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Section: Resultssupporting

confidence: 54%

mentioning

confidence: 99%

Structural analysis of human platelet membrane glycoprotein I complex.

Nachman¹,

Kinoshita²,

Ferris³

1979

Proc. Natl. Acad. Sci. U.S.A.

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“…CD36 is an 88,000-MW integral membrane protein expressed on platelets (1,2), professional phagocytes (3,4), microvascular endothelium (4), adipocytes (5), muscle cells (6), and certain specialized epithelium (6)(7)(8). It is a multifunctional receptor with independent capacity to bind at least 3 major classes of ligands: modified phospholipids, long-chain fatty acids, and thrombospondins.…”

Section: Introductionmentioning

confidence: 99%

“…Although CD36 was first isolated and structurally characterized from platelets (1), where it was termed glycoprotein IV or IIIb, its functional role on platelets has been obscure. CD36 -mice have normal tail vein bleeding times and over many years of observation do not exhibit a bleeding diathesis.…”

Section: Introductionmentioning

confidence: 99%

Platelet CD36 mediates interactions with endothelial cell–derived microparticles and contributes to thrombosis in mice

Li²,

et al. 2008

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CD36 is a scavenger receptor that binds multiple ligands, including phosphatidyl serine (PS). Although CD36 -mice do not have a bleeding diathesis, we show here that they do have significantly prolonged thrombotic occlusion times in response to FeCl 3 -induced vascular injury. Because cell-derived microparticles (MPs) are generated in response to vascular injury and circulate in patients with prothrombotic diseases, we hypothesized that PS exposed on their surfaces could be an endogenous CD36 ligand that transmits an activating signal to platelets. We found that MPs prepared from human ECs, monocytes, or platelets or isolated from blood of normal subjects bound to platelets. Binding was not observed with platelets from CD36 -donors and was inhibited by an anti-CD36 antibody or by blockade of exposed PS by annexin V or anti-PS IgM. Preincubation of platelets with MPs led to CD36-dependent augmentation of platelet activation in response to low doses of ADP, as assessed by measuring α 2b β 3 activation, P-selectin expression, and aggregation. Immunofluorescence confocal microscopy of murine carotid thrombi from CD36 -mice showed a significant decrement in endothelial antigen accumulation, which suggests that CD36 plays a role in MP recruitment into thrombi. These results provide what we believe to be a novel role for CD36 in thrombosis.

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“…However, this has been achieved using a new batch of anti-platelet antibodies (unpublished results). Clemetson and coworkers [27] have utilized the different lectin binding properties of the platelet glycoproteins for preparative purposes. Thus, by affinity chromatography of deoxycholate-solubilized platelets using lectins from Lens culinaris, wheat germ and Ahrus precatorius, they were able to prepare glycoprotein G P I in a pure state and greatly purify two other glycoproteins.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Human Platelet Proteins Solubilized with Triton X‐100 and Examined by Crossed Immunoelectrophoresis

Hagen

Bjerrum

Solum

1979

European Journal of Biochemistry

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Whole human platelets and platelet membranes have been solubilized in 1 % Triton X-100, and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies raised against either whole platelets or isolated membranes. 90 % of the platelet proteins were solubilized by this extraction. About twenty immunoprecipitates were observed using the extracts obtained from whole platelets, whereas normally eight immunoprecipitates were seen with extracts from isolated membranes. Albumin, factor VIII and fibrinogen were identified with monospecific antibodies.Correlation of the patterns obtained for platelets or membranes was obtained by addition experiments, by crossed-line immunoelectrophoresis and by crossed immunoelectrophoresis of a mixture of extracts from unlabeled whole platelets and membranes isolated from platelets labeled by lactoperoxidase-catalyzed "' I iodination.Four sialoglycoproteins were identified by their reduced electrophoretic migration after neuraminidase treatment, and six proteins interacted with various lectins, indicating them to be glycosylated.Seven amphiphilic proteins were identified by charge-shift crossed immunoelectrophoresis, and nine by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose.The topographical arrangement of the membrane proteins was examined with lactoperoxidasecatalyzed "'I-labeled platelets as antigens, and by antibodies absorbed with a suspension of whole platelets. Four and six radioactively labeled precipitates could be identified using the platelet and membrane extracts, respectively, indicating them to be exposed at the outer platelet surface. This was confirmed by the use of antibodies absorbed with intact platelets.Blood platelets play a crucial role in the arrest of bleeding. Adhesion to subendothelial tissue, secretion of intracellularly stored material as well as aggregate formation are essential steps in the sequence of reactions which platelets undergo during the hemostatic process [I]. There is evidence that platelet glycoproteins play important roles in these reactions, and it is thus of interest to characterize the platelet protein composition and surface structure. Lactoperoxidasecatalyzed 1251 iodination of whole platelets followed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis has demonstrated more than twenty polypeptides, and at least seven of these were radioactively labeled indicating a surface location in the intact cell [2]. However, functional studies on proteins solubilized by sodium dodecyl sulphate are difficult since this detergent tends to denature proteins [3]. A -.~ Enzyme. Neuraminidase (EC 3.2.1.18). high degree of solubilization has been achieved by extraction of membrane proteins with non-ionic detergents [3,4]. Since the antigenicity of the proteins is retained, such extracts are suitable for immunochemical examination, e.g. quantitative immunoelectrophoresis [4]. Erythrocyte membrane proteins solubilized in a non-ionicdetergent have been examined in crossed immunoelectropho...

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Isolation of the membrane glycoproteins of human blood platelets by lectin affinity chromatography

Cited by 139 publications

References 28 publications

Structural analysis of human platelet membrane glycoprotein I complex.

Structural analysis of human platelet membrane glycoprotein I complex.

Platelet CD36 mediates interactions with endothelial cell–derived microparticles and contributes to thrombosis in mice

Characterization of Human Platelet Proteins Solubilized with Triton X‐100 and Examined by Crossed Immunoelectrophoresis

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