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 Monocytes and platelets share the glycoproteins IIb and IIIa that are absent from both cells in Glanzmann's thrombasthenia type I

1983 ◽  
Vol 214 (2) ◽  
pp. 331-337 ◽  
Author(s):  
G Gogstad ◽  
Ø Hetland ◽  
N O Solum ◽  
H Prydz
Keyword(s):  
Platelet Membrane ◽  
Type I ◽  
Membrane Glycoproteins ◽  
Edta Treatment ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia

By means of an antiserum specific to the complex of the platelet membrane glycoproteins IIb and IIIa we demonstrate here that monocytes and purified monocyte membranes share these glycoproteins with platelets. The monocyte glycoprotein IIb-IIIa complex showed complete immunological identity with the platelet counterpart and, furthermore, dissociated after EDTA treatment exactly as did the platelet complex. In Glanzmann's thrombasthenia type I, monocytes as well as platelets lack this antigen completely.

Crossed Immunoelectrophoresis In The Identification Of Platelet Membrane Glycoproteins And Complexes

1981 ◽  
Author(s):  
S Karpatkin ◽  
S Shulman ◽  
L Howard ◽  
S Sadanandan
Keyword(s):  
Concanavalin A ◽  
Surface Antigens ◽  
Platelet Membrane ◽  
Surface Proteins ◽  
Membrane Glycoproteins ◽  
Glanzmann’S Thrombasthenia ◽  
Crossed Immunoelectrophoresis ◽  
Platelet Membranes ◽  
Glanzmann's Thrombasthenia ◽  
Major Antigen

Human platelet membranes were solubilized in 1% Triton X-100 and subjected to crossed immunoelectrophoresis, employing a rabbit anti-piatelet membrane antibody. Ten different antigens were observed fairly consistently; one could be identified as albumin, the other as fibrinogen. Surface antigens were determined by antibody adsorbtion experiments, and cell surface labeling with 125I-lactoperoxidase. Four surface antigens reacted with concanavalin A, when this was employed as an intermediate spacer gel. A major surface antigen, 10, was present on all preparations and was inversely related to antigens 13 and 18, which moved more cathodally. Membranes from preparations with full 10 antigen peaks had absent or diminished 13 and 18 antigen peaks, whereas preparations with absent to incomplete cathodal curves had increased 13 and 18 antigen peaks. Digestion of intact washed platelets with α chymotrypsin resulted in a decrease in the 10 antigen peak and an increase in 13 and 18, suggesting a structural relationship. Extraction of platelet membranes in EDTA or EGTA resulted in the disappearance of 10 and appearance of 13 and 18 (which react with concanavalin A) and 15 and 16 (which do not). Splitting of the major antigen into 13, 18, 15 and 16 could be prevented by addition of excess Ca++ relative to EGTA (but not by excess Mg++). Similar results were obtained in the absence of chelating agents following dialysis at 60-200 hrs against ‘Ca++-free’ buffer. Five patients with Glanzmann’s thrombasthenia have been studied. All k components of the major membrane antigen are missing. We conclude that the major antigen, 10, is composed of 2 glycoproteins, 13 and 18, and 2 other surface proteins, 15 and 16, which are held together by Ca++. It is conceivable that patients with Glanzmann’s thrombasthenia are lacking a membrane receptor for Ca++ or the platelet membrane (glyco)protein which anchors these A components.

scholarly journals Platelet aggregation in whole blood from patients with Glanzmann's thrombasthenia

Blood ◽  
1987 ◽  
Vol 69 (1) ◽  
pp. 38-42 ◽  
Author(s):  
ME Burgess-Wilson ◽  
SR Cockbill ◽  
GI Johnston ◽  
S Heptinstall
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Normal Subjects ◽  
Normal Response ◽  
Glanzmann’S Thrombasthenia ◽  
Low Concentrations ◽  
Glanzmann's Thrombasthenia ◽  
Per Se ◽  
Light Absorbance

Abstract We examined platelet aggregation in platelet-rich plasma (PRP) and in whole blood from two patients with Glanzmann's thrombasthenia. In PRP, aggregation was measured by monitoring the changes in light absorbance that occurred in response to aggregating agents; to measure platelet aggregation in whole blood, we used a platelet counting technique. In PRP, the patients' platelets showed defective aggregation in response to ADP, adrenaline, arachidonic acid (AA), and collagen, but normal agglutination occurred in response to ristocetin. In whole blood, however, platelet aggregation in response to the aggregating agents appeared to be either very similar to that which occurred in blood from normal subjects or only slightly reduced. There was a reduced response to all concentrations of ADP and to low concentrations of collagen but a normal response to all concentrations of adrenaline, AA, and higher concentrations of collagen. Conversely, there seemed to be an increased agglutination response to ristocetin. The abnormality in our two patients with Glanzmann's thrombasthenia probably lies in the inability of their platelets to form large, macroscopic aggregates rather than in platelet aggregation per se.

scholarly journals Residual amounts of glycoproteins IIb and IIIa may be present in the platelets of most patients with Glanzmann's thrombasthenia

Blood ◽  
1985 ◽  
Vol 65 (4) ◽  
pp. 1021-1024 ◽  
Author(s):  
AT Nurden ◽  
D Didry ◽  
N Kieffer ◽  
RP McEver
Keyword(s):  
Bleeding Disorder ◽  
Platelet Membrane ◽  
Type I ◽  
Membrane Glycoproteins ◽  
Glanzmann’S Thrombasthenia ◽  
Polyclonal Rabbit ◽  
Glanzmann's Thrombasthenia ◽  
Genes Encoding ◽  
Or Genes

Abstract Glanzmann's thrombasthenia is an inherited bleeding disorder characterized by abnormalities of platelet membrane glycoproteins (GP) IIb and IIIa. Most patients, usually designated as type I, have been reported to have undetectable levels of GP IIb and GP IIIa with the assay used. We have used polyclonal rabbit antibodies against GP IIb and GP IIIa in a sensitive immunoblot procedure capable of revealing trace amounts of these glycoproteins. Platelets from nine thrombasthenic patients, including seven with type I disease, were studied. GP IIIa, although decreased, was clearly detectable in platelets of eight patients and GP IIb was identified in five. Our findings suggest that residual quantities of GP IIb and GP IIIa are present in most patients with thrombasthenia and therefore that major deletions in the gene or genes encoding these proteins are uncommon.

Platelet Membrane Glycoproteins and their Possible Relationship to the ADP Mechanism of Platelet Aggregation

1975 ◽  
Author(s):  
D. R. Phillips ◽  
C. S. P. Jenkins ◽  
D. Meyer ◽  
M.-J. Larrieu ◽  
E. F. Luscher
Keyword(s):  
Platelet Aggregation ◽  
Surface Structure ◽  
Marked Reduction ◽  
Membrane Surface ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Altered Expression ◽  
Glanzmann’S Thrombasthenia ◽  
Glanzmann's Thrombasthenia ◽  
Induced Aggregation

Platelets isolated from patients with Glanzmann’s Thrombasthenia release in the presence of thrombin and other stimuli but fail to respond to ADP. Since the iutia.l interaction, between the platelet and ADP is at the membrane surface, it would appear that this surface lacks the necessary receptor for ADP. The surface structure of normal and thrombasthenia platelets was compared using the lactoperoxidase iodination technique. Iodination of normal platelets results in the labelling of four glycoproteins, I, IIa IIb and III, with relative ratios of 1 : 1 : 1 : 3 plus other non-characterised polypeptides. Thrombasthenic platelets similarly treated revealed a drastically altered expression of the glycoproteins on the membrane. The relative ratios (1.5: 1:0.4:0.5) revealed the decrease of glycoprotein IIb and the marked reduction of glycoprotein III. Arguments and data will be presented which point to the possibility that glycoprotein IIb is involved in ADP-induced aggregation.

Analysis of Human Platelet Glycoprotein llb-llla by Fluorescein Isothiocyanate-Conjugated Disintegrins with Flow Cytometry

1994 ◽  
Vol 72 (06) ◽  
pp. 919-925 ◽  
Author(s):  
Chao-Zong Liu ◽  
Yi-Wen R Wang ◽  
Ming-Ching Shen ◽  
Tur-Fu Huang
Keyword(s):  
Flow Cytometry ◽  
Fluorescence Intensity ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Fluorescein Isothiocyanate ◽  
Platelet Membrane ◽  
Platelet Glycoprotein ◽  
Type I ◽  
Activated Platelets ◽  
Very High

SummaryDisintegrins are a group of snake venom peptides which inhibit human platelet aggregation by acting as glycoprotein Ilb-IIIa (GPIIb-Ilia) antagonists. They are cysteine-rich, Arg-Gly-Asp (RGD)-containing peptides, and bind to GPIIb-Ilia complex on platelet membrane with a very high affinity (Kd, 10−7 ∼ 10−8 M). In this study, we analyzed GPIIb-Ilia complex on platelet membrane by flow cytometry using fluorescein isothiocyanate (FITC)-conjugated disintegrins as probes. Of these FITC-conjugated disintegrins, FITC-Rhodostomin is the most sensitive probe because Rhodostomin was conjugated with more FITC molecules than Trigramin and Halysin were. The binding fluorescence intensity of FITC-Trigramin (FITC-Tg), FITC-Halysin (FITC-Hy) and FITC-Rhodostomin (FITC-Rn) was measured in both resting and ADP-activated platelets of diluted human platelet-rich plasma. The binding fluorescence of FITC-disintegrins was abolished by EDTA and 7E3, a monoclonal antibody against GPIIb-Ilia. ADP markedly increased the fluorescence intensity of FITC-Tg and FITC-Hy bound on platelets especially when lower doses of these probes were used, whereas it had little effect on that of FITC-Rn. Therefore, FITC-Tg and FITC-Hy can be used for the detection of the activated platelets as noted by a higher ratio of fluorescence intensity (approx. 2-4) between ADP-activated and resting platelets as compared with that (approx. 1-1.3) in the case of FITC-Rn as the probe. The platelets from three patients with Glanzmann’s thrombasthenia were probed with FITC-disintegrins. As a result these three patients could be classified as type I thrombasthenia based on the extremely low level of GPIIb-Ilia detected by this method (<5% of normal value).

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