scholarly journals Further studies on the interaction between human platelet membrane glycoproteins IIb and IIIa in triton X-100

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 894-904 ◽  
Author(s):  
D Pidard ◽  
JP Rosa ◽  
TJ Kunicki ◽  
AT Nurden
Keyword(s):  
Membrane Proteins ◽  
Divalent Cation ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Indirect Evidence ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Nonionic Detergent ◽  
Triton X

Analysis of human platelet membrane proteins by crossed immunoelectrophoresis (CIE) in the presence of Triton X-100 (TX-100) has previously shown that glycoproteins (GP) IIb and IIIa are located in a single immunoprecipitate, band 16.2 To investigate whether IIb and IIIa are associated in a complex, we have analyzed TX-100-solubilized 125I-labeled membrane proteins by density gradient ultracentrifugation using 10%-40% sucrose gradients containing the nonionic detergent. studies were performed using soluble proteins derived from membranes isolated in the presence or absence of EDTA. Analysis of gradient fractions by SDS-polyacrylamide gel electrophoresis showed that in the absence of divalent cation chelation, GP IIb and IIIa penetrated well into the gradient (fractions 15–17). Analysis of fractions 15–17 by CIE revealed the presence of band 16. In contrast, when the membrane proteins were incubated with EDTA prior to or after TX-100 solubilization, IIb and IIIa remained near the top of the gradient (fractions 8–11) and gave separate immunoprecipitates during CIE. Incubation of washed platelet lysates with leupeptin, an inhibitor of the Ca2+-dependent protease of human platelets, had no effect on the shape of the band 16 immunoprecipitate. Thus, for the first time, direct evidence has been obtained that GP IIb and IIIa may form a divalent cation-mediated complex. Calibration of the sedimentation profiles using proteins of known molecular weight suggests that the complex is of limited size. Indirect evidence suggests that the complex is a heterodimer.

scholarly journals Further studies on the interaction between human platelet membrane glycoproteins IIb and IIIa in triton X-100

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 894-904 ◽  
Author(s):  
D Pidard ◽  
JP Rosa ◽  
TJ Kunicki ◽  
AT Nurden
Keyword(s):  
Membrane Proteins ◽  
Divalent Cation ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Indirect Evidence ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Nonionic Detergent ◽  
Triton X

Abstract Analysis of human platelet membrane proteins by crossed immunoelectrophoresis (CIE) in the presence of Triton X-100 (TX-100) has previously shown that glycoproteins (GP) IIb and IIIa are located in a single immunoprecipitate, band 16.2 To investigate whether IIb and IIIa are associated in a complex, we have analyzed TX-100-solubilized 125I-labeled membrane proteins by density gradient ultracentrifugation using 10%-40% sucrose gradients containing the nonionic detergent. studies were performed using soluble proteins derived from membranes isolated in the presence or absence of EDTA. Analysis of gradient fractions by SDS-polyacrylamide gel electrophoresis showed that in the absence of divalent cation chelation, GP IIb and IIIa penetrated well into the gradient (fractions 15–17). Analysis of fractions 15–17 by CIE revealed the presence of band 16. In contrast, when the membrane proteins were incubated with EDTA prior to or after TX-100 solubilization, IIb and IIIa remained near the top of the gradient (fractions 8–11) and gave separate immunoprecipitates during CIE. Incubation of washed platelet lysates with leupeptin, an inhibitor of the Ca2+-dependent protease of human platelets, had no effect on the shape of the band 16 immunoprecipitate. Thus, for the first time, direct evidence has been obtained that GP IIb and IIIa may form a divalent cation-mediated complex. Calibration of the sedimentation profiles using proteins of known molecular weight suggests that the complex is of limited size. Indirect evidence suggests that the complex is a heterodimer.

Further Observations On The Possible Association Of Human Platelet Membrane Glycoproteins IIb And IIIa

1981 ◽  
Author(s):  
J-P Rosa ◽  
D Pidard ◽  
T Kunicki ◽  
A T Nurden
Keyword(s):  
Platelet Aggregation ◽  
Membrane Proteins ◽  
Divalent Cation ◽  
Human Platelet ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Normal Human ◽  
Triton X

Studies are described which represent a continuation of our investigation into the role of membrane glycoproteins (GP’s) IIb and IIIa during human platelet aggregation. The surface proteins of washed platelets were labelled with 125I by the lactoperoxidase-catalysed method prior to membrane isolation by the glycerol lysis procedure. Solubilisation of the membrane proteins by triton X-100 was followed by their analysis by crossed immunoelectrophoresis (CIE) using a rabbit antibody prepared against normal human platelets. In the absence of divalent cation chelation GP IIb and Ilia were contained within a single 125I-labelled immunoprecipitate. When the isolated membranes were solubilised by triton X-100 in the presence of 5mM EDTA, GP IIb and IIIa formed distinctand separate immunoprecipitates during CIE. In order to further investigate this finding 125I-labelled membrane proteins solubilised by triton X-100 in the presence or absence of EDTA were subjected to centrifugation for 18 h at 100,000 g over a 10-40% sucrose gradient containing the nonionic detergent. The results confirmed that in the presence of divalent cations lib and Ilia were associated in a complex, and that this complex is dissociated by EDTA. The IgG..L is an alloantibody isolated from a polytransfused thrombasthenic patient that has been shown in previous studies to inhibit ADP-induced platelet aggregation and the binding of 125I-fibrinogen to normal human platelets in the presence of ADP. When the IgG..L was incorporated in an intermediate gel during CIE it was shown to precipitate the complex containing IIb/IIIa but under identical conditions it did not precipitate the individual glycoproteins dissociated by EDTA. Divalent cation-mediated changes in the orientation of lib and Ilia in the platelet membrane should be considered in assessing the role of these GP’s in platelet function.

scholarly journals The human platelet membrane glycoprotein complex GP IIb-IIIa expresses antigenic sites not exposed on the dissociated glycoproteins

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1246-1253
Author(s):  
JP Rosa ◽  
N Kieffer ◽  
D Didry ◽  
D Pidard ◽  
TJ Kunicki ◽  
...  
Keyword(s):  
Human Platelet ◽  
Divalent Cations ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Glycoprotein Complex ◽  
Weak Binding ◽  
The Individual ◽  
Triton X ◽  
Murine Monoclonal Antibodies ◽  
Partial Release

A number of recent reports have described murine monoclonal antibodies that react specifically with the complex formed by human platelet membrane glycoproteins (GP) IIb and IIIa. We show that the IgG L, a previously described human alloantibody isolated from a polytransfused thrombasthenia patient, has similar properties. When used in non- precipitating amounts in crossed immunoelectrophoresis (CIE), 125I-IgG L bound strongly to the IIb-IIIa complex. However, after dissociation of the complex with EDTA, only a weak binding to GP IIb and no binding to GP IIIa was detected. In further studies, increased amounts of IgG L were interacted with 125I-labeled membrane glycoproteins in (a) CIE and (b) classical indirect immunoprecipitation experiments. Although the antibody was able to quantitatively precipitate the IIb-IIIa complex from Triton X-100-soluble extracts of platelet membranes, no precipitation of GP IIb or GP IIIa was observed after divalent cation chelation. Addition of EDTA to immunoprecipitates containing GP IIb- IIIa resulted in dissociation and partial release of both glycoproteins. The interaction of the IgG L with electrophoretically separated GP IIb and GP IIIa was studied using a Western blot procedure in the presence of Ca2+, Mg2+, or EDTA. The presence of divalent cations did not increase the reactivity of the antibody with the individual glycoproteins. Overall, our results show that acquired antibodies to IIb-IIIa, such as the IgG L, may predominantly react with complex-dependent determinants.

Effects of Thrombin, Chymotrypsin and Aggregated Gamma-Globulins on the Proteins of the Human Platelet Membrane

1977 ◽  
Vol 37 (03) ◽  
pp. 396-406 ◽  
Author(s):  
B Podolsak
Keyword(s):  
Molecular Weight ◽  
Platelet Activation ◽  
Gel Electrophoresis ◽  
Human Platelet ◽  
Polyacrylamide Gel Electrophoresis ◽  
Platelet Membrane ◽  
Low Molecular Weight ◽  
Membrane Glycoproteins ◽  
Membrane Component ◽  
Before And After

SummaryAnalysis of platelet membrane proteins and glycoproteins by SDS Polyacrylamide gel electrophoresis was carried out before and after treatment with thrombin. Extended incubation with thrombin (in the presence of EDTA or adenosine, which inhibit aggregation) produced extensive changes in the bands observed. With incubation times of a few minutes however, the changes were restricted to a glycopeptide, GP IV (approx. 90,000 Daltons) and one or two polypeptides of low molecular weight, in particular polypeptide 16 (approx. 23,000 Daltons). At 0–3° C only polypeptide 16 was still hydrolyzed.Chymotrypsin, which does not activate platelets, attacked glycopeptides I, II, III but no changes were apparent in GP IV and polypeptide 16. When chymotrypsin-treated platelets were further incubated with thrombin, only GP IV and one to two low molecular weight polypeptides, especially polypeptide 16, were affected. As polypeptide 16 appears to be an integral membrane component it is possible that it, either by itself or in combination with GP IV, represents the primary thrombin substrate involved in platelet activation.Aggregated IgG, which also activates platelets, does not modify the membrane glycoproteins but does change the low molecular weight region in particular band 16.

Absence of Myosin on Platelet Membranes Supported by Membrane Iodination, Immuno-Fluorescence and Functional Studies

1979 ◽  
Author(s):  
J.C. Mattson ◽  
W.J. Esselman ◽  
M.L. Schwarz ◽  
C.A. Zuiches
Keyword(s):  
Membrane Proteins ◽  
Protein Complex ◽  
Human Platelet ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Clot Retraction ◽  
Platelet Surface ◽  
Functional Studies ◽  
Sds Page ◽  
Formalin Fixed

Immunofluorescence and functional studies were performed utilizing the immunoglobulin fraction of antisera raised against chromatographically purified human platelet myosin. When non-permeable, formalin fixed platelets were used, no FITC staining of the platelet membrane occurred indicating the myosin is not present on the platelet surface. When similar studies were performed on formalin fixed platelets using antibodies raised against the contractile protein complex, thrombosthenin, membrane fluorescence occurred. Autoradiographs of SDS-PAGE gels of the immune precipitate produced by reacting 125I labeled human platelet thrombosthenin with antithrombosthenin demonstrated that anti-thrombosthenin antisera was capable of reacting with at least three iodinated proteins In addition to myosin. 125I lactoperoxidase catalyzed labeling of the external membrane proteins of resting and ADP stimulated platelets indicated no external labeling of myosin although actin appeared to be labeled. In functional studies, incubation of human platelets with antimyosin did not produce aggregation nor did it Inhibit subsequent aggregation by ADP, collagen or epinephrine. Similarly, treatment of intact platelets with antimyosin did not cause Inhibition of clot retraction. These studies support the thesis that myosin is not localized on the external platelet membrane.

scholarly journals The formation of Ca++-dependent complexes of platelet membrane glycoproteins IIb and IIIa in solution as determined by crossed immunoelectrophoresis

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 268-278 ◽  
Author(s):  
TJ Kunicki ◽  
D Pidard ◽  
JP Rosa ◽  
AT Nurden
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Tetraacetic Acid ◽  
Rabbit Antibody ◽  
Membrane Glycoproteins ◽  
Normal Platelet ◽  
Crossed Immunoelectrophoresis ◽  
Glycoprotein Antigen ◽  
Glycoprotein Iiia ◽  
Triton X

Triton X-100 soluble proteins from 125I-labeled human platelets were studied by crossed immunoelectrophoresis employing a multispecific rabbit antibody raised against whole normal platelets. Emphasis was placed upon an analysis of immunoprecipitates containing 125I-labeled major membrane glycoproteins, and in particular, a prominent immunoprecipitate containing a glycoprotein antigen (s) previously designated as protein 16. SDS-polyacrylamide gel electrophoresis of protein 16 precipitated by a monospecific alloantibody. IgG L . . . , confirmed the presence of both glycoproteins IIb and IIIa. 125I-IgG L . . . , at concentration below that capable of precipitating protein 16 by itself, bound specifically to the precipitate containing protein 16 produced by the multispecific rabbit antibody. No other precipitates formed by the rabbit antibody contained either glycoprotein IIb or IIIa. When platelet proteins, incubated with optimum concentrations of ethylenediamine tetraacetic acid (EDTA) or ethyleneglycol bis (B- aminoethylether) NN1-tetraacetic acid (EGTA), were electrophoresed against the rabbit antibody, previously unobserved immunoprecipitates that contained either free glycoprotein IIb or free glycoprotein IIIa were detected. Upon readdition of excess Ca++, but not Mg++, to the same protein samples, a single immunoprecipitate containing both glycoproteins was once again observed. It is thus demonstrated that glycoproteins IIb and IIIa can form Ca++-dependent complexes (protein 16) in Triton X-100 extracts of normal platelets. The potential significance of the reversible association of these glycoproteins to normal platelet function is discussed.

scholarly journals The formation of Ca++-dependent complexes of platelet membrane glycoproteins IIb and IIIa in solution as determined by crossed immunoelectrophoresis

Blood ◽  
1981 ◽  
Vol 58 (2) ◽  
pp. 268-278 ◽  
Author(s):  
TJ Kunicki ◽  
D Pidard ◽  
JP Rosa ◽  
AT Nurden
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Tetraacetic Acid ◽  
Rabbit Antibody ◽  
Membrane Glycoproteins ◽  
Normal Platelet ◽  
Crossed Immunoelectrophoresis ◽  
Glycoprotein Antigen ◽  
Glycoprotein Iiia ◽  
Triton X

Abstract Triton X-100 soluble proteins from 125I-labeled human platelets were studied by crossed immunoelectrophoresis employing a multispecific rabbit antibody raised against whole normal platelets. Emphasis was placed upon an analysis of immunoprecipitates containing 125I-labeled major membrane glycoproteins, and in particular, a prominent immunoprecipitate containing a glycoprotein antigen (s) previously designated as protein 16. SDS-polyacrylamide gel electrophoresis of protein 16 precipitated by a monospecific alloantibody. IgG L . . . , confirmed the presence of both glycoproteins IIb and IIIa. 125I-IgG L . . . , at concentration below that capable of precipitating protein 16 by itself, bound specifically to the precipitate containing protein 16 produced by the multispecific rabbit antibody. No other precipitates formed by the rabbit antibody contained either glycoprotein IIb or IIIa. When platelet proteins, incubated with optimum concentrations of ethylenediamine tetraacetic acid (EDTA) or ethyleneglycol bis (B- aminoethylether) NN1-tetraacetic acid (EGTA), were electrophoresed against the rabbit antibody, previously unobserved immunoprecipitates that contained either free glycoprotein IIb or free glycoprotein IIIa were detected. Upon readdition of excess Ca++, but not Mg++, to the same protein samples, a single immunoprecipitate containing both glycoproteins was once again observed. It is thus demonstrated that glycoproteins IIb and IIIa can form Ca++-dependent complexes (protein 16) in Triton X-100 extracts of normal platelets. The potential significance of the reversible association of these glycoproteins to normal platelet function is discussed.

scholarly journals Monoclonal antibodies against porcine platelet membrane glycoproteins Ib and IIb/IIIa

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1740-1747 ◽  
Author(s):  
H Takami ◽  
WL Nichols ◽  
SE Kaese ◽  
RS Miller ◽  
JA Katzmann ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Platelet Membrane ◽  
In Vivo Studies ◽  
Membrane Glycoproteins ◽  
Fab Fragments

Abstract We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3–4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3–4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3–4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3–3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3–3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3–3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3–3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3–4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3–3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3–4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3–3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.

scholarly journals The human platelet membrane glycoprotein complex GP IIb-IIIa expresses antigenic sites not exposed on the dissociated glycoproteins

Blood ◽  
1984 ◽  
Vol 64 (6) ◽  
pp. 1246-1253 ◽  
Author(s):  
JP Rosa ◽  
N Kieffer ◽  
D Didry ◽  
D Pidard ◽  
TJ Kunicki ◽  
...  
Keyword(s):  
Human Platelet ◽  
Divalent Cations ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Glycoprotein Complex ◽  
Weak Binding ◽  
The Individual ◽  
Triton X ◽  
Murine Monoclonal Antibodies ◽  
Partial Release

Abstract A number of recent reports have described murine monoclonal antibodies that react specifically with the complex formed by human platelet membrane glycoproteins (GP) IIb and IIIa. We show that the IgG L, a previously described human alloantibody isolated from a polytransfused thrombasthenia patient, has similar properties. When used in non- precipitating amounts in crossed immunoelectrophoresis (CIE), 125I-IgG L bound strongly to the IIb-IIIa complex. However, after dissociation of the complex with EDTA, only a weak binding to GP IIb and no binding to GP IIIa was detected. In further studies, increased amounts of IgG L were interacted with 125I-labeled membrane glycoproteins in (a) CIE and (b) classical indirect immunoprecipitation experiments. Although the antibody was able to quantitatively precipitate the IIb-IIIa complex from Triton X-100-soluble extracts of platelet membranes, no precipitation of GP IIb or GP IIIa was observed after divalent cation chelation. Addition of EDTA to immunoprecipitates containing GP IIb- IIIa resulted in dissociation and partial release of both glycoproteins. The interaction of the IgG L with electrophoretically separated GP IIb and GP IIIa was studied using a Western blot procedure in the presence of Ca2+, Mg2+, or EDTA. The presence of divalent cations did not increase the reactivity of the antibody with the individual glycoproteins. Overall, our results show that acquired antibodies to IIb-IIIa, such as the IgG L, may predominantly react with complex-dependent determinants.

scholarly journals Two-Dimensional Electrophoretic “Mapping” of Human Platelet Membrane Proteins

1977 ◽  
Author(s):  
W. L. Nichols ◽  
K. G. Mann
Keyword(s):  
Membrane Proteins ◽  
Human Platelet ◽  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Platelet Membrane ◽  
Total Protein Content ◽  
Two Dimensional ◽  
Analytical Technique ◽  
Gel Isoelectric Focusing ◽  
Triton X

Two-dimensional electrophoresis of solubilized human platelet membrane proteins utilizing Polyacrylamide gel isoelectric focusing followed by sodium dodecyl sulfate (SDS) slab gel electrophoresis with discontinuous buffer systems yields a high resolution “fingerprint” of platelet membrane proteins. Membranes were prepared from citrate-phosphate-dextrose (CPD) platelet concentrates by differential centrifugation, repeated washes and lysis by means of sonication or glycerol hypotonic lysis. Membrane vesicles were isolated on step gradients of 27%(w/v) or 30% (w/w) sucrose. Following further washing of the vesicles, membrane pellets were extracted by heating in SDS and β-mercaptoethanol. After pelleting the insoluble material, the resulting supernatant was found to contain most of the total protein content of the membrane, and to yield SDS gel patterns similar to platelet membranes totally solubilized with larger amounts of SDS. Extracts were dialyzed against 9M urea, 2% Triton X-100 at 37° for 12 hrs., then electrofocused in large-pore gels containing 9M urea, 1% Triton X-100 and 1.6% to 2.0% ampholyte (pH 3–10). Electrofocused samples were equilibrated with 1% SDS in half-strength stacking gel buffer (D. M. Neville, J. Biol. Chem. 246: 6328, 1971)and electrophoresed in the second dimension into slab gels.Coomassie Blue R-250 staining revealed more than 40 components, the majority of which electro-focused between pH 4.5 – 7. The major glycoproteins, as assessed by periodic acid-Schiff staining, electrofocus at acidic pH. The greater resolution obtained by this analytical technique will be useful in the further characterization of the membrane proteins of normal and abnormal platelets.

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