Advancements in Flow Cytometry Fluidics: A Hypothesis

SummaryThe anti-platelet monoclonal antibody P256 is currently undergoing development for in vivo detection of thrombus. We have examined the actions of P256 and two fragments on human platelet function. P256, and its divalent fragment, caused aggregation at concentrations of 10−9−3 × 10−8 M. A monovalent fragment of P256 did not cause aggregation at concentrations up to 10−7 M. P256–induced platelet aggregation was dependent upon extracellular calcium ions as assessed by quin2 fluorescence. Indomethacin partially inhibited platelet aggregation and completely inhibited intracellular calcium mobilisation. Apyrase caused partial inhibition of aggregation. Aggregation induced by the divalent fragment was dependent upon fibrinogen and was inhibited by prostacyclin. Aggregation induced by the whole antibody was only partially dependent upon fibrinogen, but was also inhibited by prostacyclin. P256 whole antibody was shown, by flow cytometry, to induce fibrinogen binding to indomethacin treated platelets. Monovalent P256 was shown to be a specific antagonist for aggregation induced by the divalent forms. In–111–labelled monovalent fragment bound to gel-filtered platelets in a saturable and displaceable manner. Monovalent P256 represents a safer form for in vivo applications

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A P-Selectin/CD62P Monoclonal Antibody (LYP-20), in Tandem with Flow Cytometry, Detects in vivo Activated Circulating Rat Platelets in Severe Vascular Trauma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648952 ◽

1994 ◽

Vol 72 (05) ◽

pp. 745-749 ◽

Cited By ~ 7

Author(s):

Elza Chignier ◽

Maud Parise ◽

Lilian McGregor ◽

Caroline Delabre ◽

Sylvie Faucompret ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Activation ◽

Peripheral Blood ◽

Vascular Trauma ◽

Activated Platelets ◽

Group I ◽

Group Ii ◽

Rat Platelets

SummaryP-selectin, also known as CD62P, GMP140 or PADGEM, is present in platelet a-granules and endothelial cell Weibel-Palade bodies and is very rapidly expressed on the surface of these cells on activation. In this study, an anti P-selectin monoclonal antibody (LYP20) was used, in tandem with flow cytometry, to identify activated platelets at the site of induced vascular trauma or in peripheral blood. Moreover, electron microscopy was performed to characterize sites of vascular trauma and quantify the number of adhering platelets. The same induced vascular trauma was observed to result into animals responding in 2 different ways (Group I, Group II) following the degree of platelet activation. Five rats, out of 14 with induced vascular trauma, had more than half of their circulating platelets expressing P-selectin when drawn at the site of the trauma (67.4% ± 3.44) or in peripheral blood (78.5% ± 2.5) (Group I). In the remaining 9 animals a much smaller proportion of circulating platelets expressed P-selectin when assayed from trauma sites (18% ± 3.34) or in peripheral blood (18.0% ± 4.30) (Group II). Enhanced P-selectin expression by circulating platelets in Group I, compared to Group II, appears to be linked to the degree of activated platelets adhering at sites of trauma (171 ± 15 × 103 platelets versus 48 ± 31 × 103 platelets per mm2). In the 5 control animals, that were not operated on, platelets expressing P-selectin when drawn at the site of a mock trauma (7.0% ± 1.84) or in the peripheral blood (11.2% ± 3.30) showed little activation. In addition, no platelet adhesion was seen on the vascular bed of these animals. Results from this study show that analysis of P-selectin (CD62P) expression, in circulating platelets, is a valuable and rapid marker of platelet activation following severe vascular trauma induced in rats. However, activated platelets were not detected to the same extent in the peripheral blood of all animals having undergone vascular trauma. It is conceivable that platelets, depending on the degree of activation, may be actively sequestered in organs and prevented from circulating. Alternatively, P-selectin may be rapidly endocytosed, or not expressed, by activated circulating platelets depending on the type of agonists implicated in vivo activation.

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PAICA: A Method for Characterizing Platelet-Associated Antibodies - Its Application to the Study of Idiopathic Thrombocytopenic Purpura and to the Detection of Platelet-bound c7E3

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650702 ◽

1996 ◽

Vol 76 (06) ◽

pp. 1020-1029 ◽

Cited By ~ 26

Author(s):

Laurent Macchi ◽

Gisèle Clofent-Sanchez ◽

Gérald Marit ◽

Claude Bihour ◽

Catherine Durrieu-Jais ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Idiopathic Thrombocytopenic Purpura ◽

Antithrombotic Therapy ◽

Membrane Glycoproteins ◽

Serum Antibodies ◽

Platelet Destruction ◽

Thrombocytopenic Purpura ◽

Capture Assay ◽

Specific Membrane

SummaryIn idiopathic thrombocytopenic purpura (ITP), autoantibodies reacting with antigens on the platelet membrane bring about accelerated platelet destruction. We now report PAICA (“Platelet-Associated IgG Characterization Assay”), a method for detecting autoantibodies bound to specific membrane glycoproteins in total platelet lysates. This monoclonal antibody (MAb) capture assay takes into account the fact that antibodies on circulating platelets may be translocated to internal pools as well as being on the surface. A total of twenty ITP patients were examined by PAICA, and the results compared with those obtained by measuring (i) serum antibodies bound to paraformaldehyde-fixed control platelets by ELISA, (ii) IgG bound to the surface of the patient’s own platelets by flow cytometry (PSIgG), (iii) total platelet-associated IgG (PAIgG) by ELISA and (iv) serum antibodies reacting with control platelets by MAIPA (“Monoclonal Antibody-specific Immobilization of Platelet Antigens”). Of twelve patients with elevated PAIgG, nine had increased PSIgG yet eleven reacted positively in PAICA. Of these, eight possessed antibodies directed against GP Ilb-IIIa, two against GP Ib-IX and one patient possessed antibodies directed against GP Ilb-IIIa and GP Ia-IIa respectively. Only seven of the patients possessed serum antibodies detectable by MAIPA. PAICA was also able to detect platelet-associated c7E3 (the chimeric form of Fab fragments of the MAb 7E3) following its infusion during antithrombotic therapy, when it proved more sensitive over a seven-day period than a MAIPA assay adapted for assessing surface-bound antibody. We propose that PAICA provides added sensitivity to the detection of platelet-associated antibodies in immune thrombocytopenias or following therapy with humanized MAbs.

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Internalization of an Anti-Glycoprotein IIb/IIIa Antibody by HEL Cells

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653767 ◽

1995 ◽

Vol 73 (02) ◽

pp. 291-296 ◽

Cited By ~ 1

Author(s):

Kenjiro Hamamoto ◽

Shosaku Nomura ◽

Masahiko Suzuki ◽

Shigetoshi Ohga ◽

Shirou Fukuhara

Keyword(s):

Flow Cytometry ◽

Monoclonal Antibodies ◽

Immunoelectron Microscopy ◽

Surface Membrane ◽

Intracellular Pool ◽

Adhesion Proteins ◽

Hel Cells

SummaryPlatelets are known to internalize monoclonal antibodies directed against the glycoprotein (GP) IIb/IIIa complex. We investigated whether an antibody directed against this complex (NNKY 2-11) was transported from the surface membrane to the intracellular pool in HEL cells. Flow cytometry showed that the percent binding of NNKY 2-11 to the surface membrane of HEL cells was decreased after incubation for 24 h compared with 1 h, while the binding of an anti-GPIb antibody (NNKY 5-5) did not change. It did not seem likely that the GP Ilb/IIIa complex antibody was shed from the surface membrane of the HEL cells during incubation, because the medium conditioned by incubation with these cells for 24 h showed almost no binding to washed platelets. In addition, immunoelectron microscopy demonstrated that GP IIb/IIIa complex antibodies were incorporated into the intracellular pool of HEL cells and were associated with alpha granules. These findings indicated that an anti-GP IIb/IIIa antibody could be internalized by megakaryocytes, as has been previously shown with platelets, suggesting that megakaryocyte GP IIb/IIIa may act as a carrier for various adhesion proteins.

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