scholarly journals Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometry

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1401-1403 ◽  
Author(s):  
GI Johnston ◽  
EB Pickett ◽  
RP McEver ◽  
JN George
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Response ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Granule Membrane ◽  
Membrane Changes ◽  
Granule Secretion

Abstract Platelet membrane changes that accompany in vivo activation may be difficult to detect if only a small fraction of circulating platelets has undergone secretion. This study describes an approach to that problem by using a method to measure the number of molecules of fluorescein-labeled antibody bound to individual platelets by flow cytometry. The platelet response to different concentrations of thrombin was determined by measuring the binding of a monoclonal antibody (S12) to GMP-140, an alpha-granule membrane protein that becomes exposed on the platelet surface during alpha-granule secretion. Unstimulated platelets bound a mean of 1,120 molecules of S12 per cell, and 93% of platelets bound less than 2,000 molecules. Platelet stimulation by 0.25 U/mL thrombin caused maximum S12 binding with a mean of 7,529 molecules per cell. Even at low concentrations of thrombin (0.025 U/mL), 5% of platelets were maximally activated, binding over 7,000 molecules of S12 per cell. Conversely, at 0.25 U/mL thrombin, 13% of platelets continued to bind less than 2,000 molecules of S12 per cell. A mixture of as little as 5% thrombin-activated platelets with unstimulated platelets could be detected by this method. Therefore flow cytometry offers an important tool for investigating patients who may have circulating activated platelets as part of a disorder predisposing to thrombosis or hemorrhage.

scholarly journals Heterogeneity of platelet secretion in response to thrombin demonstrated by fluorescence flow cytometry

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1401-1403
Author(s):  
GI Johnston ◽  
EB Pickett ◽  
RP McEver ◽  
JN George
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Response ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Low Concentrations ◽  
Granule Membrane ◽  
Membrane Changes ◽  
Granule Secretion

Platelet membrane changes that accompany in vivo activation may be difficult to detect if only a small fraction of circulating platelets has undergone secretion. This study describes an approach to that problem by using a method to measure the number of molecules of fluorescein-labeled antibody bound to individual platelets by flow cytometry. The platelet response to different concentrations of thrombin was determined by measuring the binding of a monoclonal antibody (S12) to GMP-140, an alpha-granule membrane protein that becomes exposed on the platelet surface during alpha-granule secretion. Unstimulated platelets bound a mean of 1,120 molecules of S12 per cell, and 93% of platelets bound less than 2,000 molecules. Platelet stimulation by 0.25 U/mL thrombin caused maximum S12 binding with a mean of 7,529 molecules per cell. Even at low concentrations of thrombin (0.025 U/mL), 5% of platelets were maximally activated, binding over 7,000 molecules of S12 per cell. Conversely, at 0.25 U/mL thrombin, 13% of platelets continued to bind less than 2,000 molecules of S12 per cell. A mixture of as little as 5% thrombin-activated platelets with unstimulated platelets could be detected by this method. Therefore flow cytometry offers an important tool for investigating patients who may have circulating activated platelets as part of a disorder predisposing to thrombosis or hemorrhage.

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

1994 ◽  
Vol 72 (05) ◽  
pp. 745-749 ◽  
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.

scholarly journals Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1006-1016 ◽  
Author(s):  
AD Cox ◽  
DV Devine
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
A Chain ◽  
Functional Receptor

Abstract Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

scholarly journals Factor XIIIa binding to activated platelets is mediated through activation of glycoprotein IIb-IIIa

Blood ◽  
1994 ◽  
Vol 83 (4) ◽  
pp. 1006-1016 ◽  
Author(s):  
AD Cox ◽  
DV Devine
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Activation ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
A Chain ◽  
Functional Receptor

Stabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin- induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti- FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.

scholarly journals Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 307-315 ◽  
Author(s):  
SJ Shattil ◽  
M Cunningham ◽  
JA Hoxie
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Fluorescein Isothiocyanate ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Activated Platelets ◽  
Clinical Disorders ◽  
Granule Membrane

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an antibody specific for an alpha-granule membrane protein that associates with the platelet surface during secretion. Platelet-bound antibodies were detected with streptavidin conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Platelets were differentiated from the larger erythrocytes and WBCs by their light- scatter profile. Alternatively, platelets could be identified with FITC- AP1, an antibody specific for platelet membrane glycoprotein Ib, and analyzed further for PAC1 or S12 binding with PE-streptavidin. No centrifugation or washing steps were required. With gel-filtered platelets, there was a direct correlation between ADP-induced biotin- PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = .99; P less than .001). Furthermore, as few as 0.8% activated platelets could be detected by flow cytometry when activated platelets were mixed with unstimulated platelets. In whole blood, unstimulated platelets demonstrated no PAC1- or S12-specific fluorescence, indicating that they did not bind these antibodies. On stimulation with agonists, however, the platelets demonstrated a dose- dependent increase in fluorescence similar to that observed for platelets in plasma or buffer. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate (TPA) that evokes full platelet aggregation and secretion induced maximal PAC1 and S12 binding. Activated platelets could also be analyzed in whole blood samples that had been fixed with paraformaldehyde. These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This technique may be useful to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders.

Factor VIII’s C Domains Bind to Platelets.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1715-1715
Author(s):  
Ting-Chang Hsu ◽  
Kathleen P. Pratt ◽  
Arthur R. Thompson
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Factor Viii ◽  
Platelet Activation ◽  
Von Willebrand Factor ◽  
C2 Domain ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract The C domains of factor VIII contain the primary binding site for the cofactor, activated factor VIII, to interact with the phospholipid membranes, including those on the platelet surface. Isolated C2 domain has been shown to bind to phosphotidyl-L-serine-rich lipids and platelets; under flow cytometry, binding to activated platelets was confirmed. For comparison, C1C2, expressed in E.coli, was prepared with up to mg quantities isolated. Fresh, gel-filtered platelets were then studied in a flow cytometer either with or without activation by the thrombin receptor peptide, SFLLRN-amide. Depending upon the conditions, up to 80% of the platelets could be stained with a monoclonal antibody to C2 (ESH8) that is known not to compete with lipid or von Willebrand factor binding. The results were confirmed using a S2296C mutant C1C2 where the free suflhydryl group was either biotinylated and detected by fluorescein labeled streptavidin or directly labeled with fluorescein. As shown in the figure, essentially all platelets bound directly fluorescein labeled C1C2. Using standardized, labeled microbeads, it was estimated that there are 7000–10,000 binding sites per platelet. After platelet activation, the number of platelets binding C1C2 increased with all three detecting systems but only by 15–30%. In contrast, binding of isolated C2, as determined either by ESH8 or as a C2296 biotinylated species, was much lower when the same molar amounts were added, and was primarily detectable following platelet activation. C1C2 binding appeared independent of von Willebrand factor as platelets from two unrelated subjects with severe, type 3 von Willebrand disease gave the same patterns on flow cytometry as seen in platelets from normal subjects. ESH4, a monoclonal antibody known to inhibit binding of C2 to lipid membranes effectively competed C1C2 binding to platelets. Although an indirect alteration the C2 domain conformation cannot be excluded, results support a direct role of C1 in enhancing platelet binding. Binding of direct florescein-labeled C1C2 to SFLLRN-amide-activated platelets Binding of direct florescein-labeled C1C2 to SFLLRN-amide-activated platelets

scholarly journals Detection of activated platelets in whole blood using activation- dependent monoclonal antibodies and flow cytometry

Blood ◽  
1987 ◽  
Vol 70 (1) ◽  
pp. 307-315 ◽  
Author(s):  
SJ Shattil ◽  
M Cunningham ◽  
JA Hoxie
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Fluorescein Isothiocyanate ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Activated Platelets ◽  
Clinical Disorders ◽  
Granule Membrane

Abstract Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We developed a direct test for activated platelets in whole blood using flow cytometry. Whole blood was incubated with either biotin-PAC1, a monoclonal antibody specific for the fibrinogen receptor on activated platelets, or biotin-S12, an antibody specific for an alpha-granule membrane protein that associates with the platelet surface during secretion. Platelet-bound antibodies were detected with streptavidin conjugated with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). Platelets were differentiated from the larger erythrocytes and WBCs by their light- scatter profile. Alternatively, platelets could be identified with FITC- AP1, an antibody specific for platelet membrane glycoprotein Ib, and analyzed further for PAC1 or S12 binding with PE-streptavidin. No centrifugation or washing steps were required. With gel-filtered platelets, there was a direct correlation between ADP-induced biotin- PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = .99; P less than .001). Furthermore, as few as 0.8% activated platelets could be detected by flow cytometry when activated platelets were mixed with unstimulated platelets. In whole blood, unstimulated platelets demonstrated no PAC1- or S12-specific fluorescence, indicating that they did not bind these antibodies. On stimulation with agonists, however, the platelets demonstrated a dose- dependent increase in fluorescence similar to that observed for platelets in plasma or buffer. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate (TPA) that evokes full platelet aggregation and secretion induced maximal PAC1 and S12 binding. Activated platelets could also be analyzed in whole blood samples that had been fixed with paraformaldehyde. These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This technique may be useful to assess the degree of platelet activation and the efficacy of antiplatelet therapy in clinical disorders.

Effects of a Monoclonal Antibody to Glycoprotein IIb/IIIa (P256) and of Enzymically Derived Fragments of P256 on Human Platelets

1991 ◽  
Vol 65 (04) ◽  
pp. 432-437 ◽  
Author(s):  
A W J Stuttle ◽  
M J Powling ◽  
J M Ritter ◽  
R M Hardisty
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Calcium Ions ◽  
Human Platelet ◽  
Human Platelets ◽  
In Vivo Detection ◽  
Fibrinogen Binding ◽  
Specific Antagonist

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

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

PSGL-1 regulates platelet P-selectin-mediated endothelial activation and shedding of P-selectin from activated platelets

2007 ◽  
Vol 98 (10) ◽  
pp. 806-812 ◽  
Author(s):  
Vandana Dole ◽  
Wolfgang Bergmeier ◽  
Ian Patten ◽  
Junichi Hirahashi ◽  
Tanya Mayadas ◽  
...  
Keyword(s):  
Endothelial Activation ◽  
Leukocyte Rolling ◽  
Wild Type ◽  
Platelet Surface ◽  
Activated Platelets ◽  
And Function ◽  
Body Secretion

SummaryWe have previously shown that activated platelets in circulation stimulate release of endothelial Weibel-Palade bodies thus increasing leukocyte rolling in venules. P-selectin on the activated platelets mediates adhesion to leukocytes via PSGL-1 and is rapidly shed into plasma. We were interested in studying the role of PSGL-1 in regulating expression and function of platelet P-selectin. We show here that PSGL-1 is critical for the activation of endothelial cells in venules of mice infused with activated platelets. The interaction of platelet P-selectin with PSGL-1 is also required for P-selectin shedding, as P-selectin was retained significantly longer on the surface of activated platelets infused into PSGL-1-/- compared to wild-type mice. The leukocyte integrin αMβ2 (Mac-1) was not required for P-selectin shedding. In addition to shedding, P-selectin can be downregulated from the platelet surface through internalization and this is the predominant mechanism in the absence of PSGL-1. We demonstrate that leukocyte- neutrophil elastase,known to cleave P-selectin in vitro, is not the major sheddase for P-selectin in vivo. In conclusion, interaction of platelet P-selectin with PSGL-1 is crucial for activation of the endothelium andWeibel-Palade body secretion. The interaction with PSGL-1 also results in rapid shedding of P-selectin thus downregulating the inflammatory potential of the platelet.

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