Flow Cytometric Quantification of Human Platelet Membrane Glycoproteins in Citrated Whole Blood

2019 ◽  
Author(s):  
C. Berens ◽  
H. Rühl ◽  
J. Müller ◽  
J. Oldenburg ◽  
B. Pötzsch
Keyword(s):  
Whole Blood ◽  
Human Platelet ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Flow Cytometric

scholarly journals Analysis of human platelet glycoproteins IIb-IIIa and Glanzmann's thrombasthenia in whole blood by flow cytometry

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 173-179
Author(s):  
LK Jennings ◽  
RA Ashmun ◽  
WC Wang ◽  
ME Dockter
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Platelet Membrane ◽  
Parameter Analysis ◽  
Light Scatter ◽  
Membrane Glycoproteins ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

Antibodies that bind to human platelet membrane glycoproteins IIb and IIIa were used to develop methods for analyzing platelet membrane components by flow cytometry. Platelets were tentatively identified by their low-intensity light scatter profiles in whole blood or platelet- rich plasma preparations. Identification of this cell population as platelets was verified by using platelet-specific antibodies and fluorescein-conjugated antiimmunoglobulin. Two-parameter analysis of light scatter versus fluorescence intensity identified greater than 98% of the cells in the “platelet” light scatter profile as platelets due to their acquired fluorescence. Both platelet-rich plasma and whole blood were used to study platelet membrane glycoproteins IIb and IIIa on a single cell basis in an unwashed system. Prostacycline was included in these preparations as a precautionary step to inhibit platelet aggregation during analysis. Flow cytometry is a successful technique for rapid detection of platelet membrane defects such as Glanzmann's thrombasthenia. Platelets from Glanzmann's thrombasthenic individuals were readily distinguished from platelets with normal levels of glycoprotein IIb and IIIa and from platelets with glycoprotein levels characteristic of heterozygote carriers of this disorder. This technique provides a sensitive tool for investigating platelet functional defects due to altered expression or deficiency of platelet surface proteins.

scholarly journals Analysis of human platelet glycoproteins IIb-IIIa and Glanzmann's thrombasthenia in whole blood by flow cytometry

Blood ◽  
1986 ◽  
Vol 68 (1) ◽  
pp. 173-179 ◽  
Author(s):  
LK Jennings ◽  
RA Ashmun ◽  
WC Wang ◽  
ME Dockter
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Platelet Membrane ◽  
Parameter Analysis ◽  
Light Scatter ◽  
Membrane Glycoproteins ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

Abstract Antibodies that bind to human platelet membrane glycoproteins IIb and IIIa were used to develop methods for analyzing platelet membrane components by flow cytometry. Platelets were tentatively identified by their low-intensity light scatter profiles in whole blood or platelet- rich plasma preparations. Identification of this cell population as platelets was verified by using platelet-specific antibodies and fluorescein-conjugated antiimmunoglobulin. Two-parameter analysis of light scatter versus fluorescence intensity identified greater than 98% of the cells in the “platelet” light scatter profile as platelets due to their acquired fluorescence. Both platelet-rich plasma and whole blood were used to study platelet membrane glycoproteins IIb and IIIa on a single cell basis in an unwashed system. Prostacycline was included in these preparations as a precautionary step to inhibit platelet aggregation during analysis. Flow cytometry is a successful technique for rapid detection of platelet membrane defects such as Glanzmann's thrombasthenia. Platelets from Glanzmann's thrombasthenic individuals were readily distinguished from platelets with normal levels of glycoprotein IIb and IIIa and from platelets with glycoprotein levels characteristic of heterozygote carriers of this disorder. This technique provides a sensitive tool for investigating platelet functional defects due to altered expression or deficiency of platelet surface proteins.

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.

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.

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.

Wheat germ agglutinin affinity chromatography of human platelet membrane glycoproteins

1978 ◽  
Vol 12 (1) ◽  
pp. 91-104 ◽  
Author(s):  
Ralph L. Nachman ◽  
Ethan Tarasov ◽  
Babette B. Weksler ◽  
Barbara Ferris
Keyword(s):  
Affinity Chromatography ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Human Platelet ◽  
Platelet Membrane ◽  
Membrane Glycoproteins

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.

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