scholarly journals Direct detection of activated platelets and platelet-derived microparticles in humans

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 128-138 ◽  
Author(s):  
CS Abrams ◽  
N Ellison ◽  
AZ Budzynski ◽  
SJ Shattil
Keyword(s):  
Flow Cytometry ◽  
Cardiopulmonary Bypass ◽  
Platelet Activation ◽  
Bleeding Time ◽  
Relative Proportion ◽  
Open Heart Surgery ◽  
Normal Range ◽  
In Vivo Models ◽  
Platelet Surface ◽  
Activated Platelets

Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha- granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet- derived microparticles in the circulation of humans.

scholarly journals Direct detection of activated platelets and platelet-derived microparticles in humans

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 128-138 ◽  
Author(s):  
CS Abrams ◽  
N Ellison ◽  
AZ Budzynski ◽  
SJ Shattil
Keyword(s):  
Flow Cytometry ◽  
Cardiopulmonary Bypass ◽  
Platelet Activation ◽  
Bleeding Time ◽  
Relative Proportion ◽  
Open Heart Surgery ◽  
Normal Range ◽  
In Vivo Models ◽  
Platelet Surface ◽  
Activated Platelets

Abstract Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha- granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet- derived microparticles in the circulation of humans.

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.

DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY

1987 ◽  
Author(s):  
S J Shattil ◽  
J A Hoxie ◽  
M Cunningham ◽  
C S Abrahms ◽  
J O’Brien ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Platelet Activation ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Membrane Surface ◽  
White Blood Cells ◽  
Color Flow ◽  
Platelet Surface ◽  
Activated Platelets

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. We have developed a direct test for activated platelets in whole blood that utilizes dual-color flow cytometry and requires no washing steps. Platelets were distinguished from erythrocytes and white blood cells in the flow cytometer by labeling the platelets with biotin-AP1, an antibody specific for membrane glycoprotein lb, and analyzing the cells for phycoerythrin-streptavidin fluorescence. Membrane surface changes resulting from platelet activation were detected with three different FITC-labeled monoclonal antibodies: 1) PAC1, an antibody specific for the fibrinogen receptor on activated platelets; 2) 9F9, which binds to the D-domain of fibrinogen and detects platelet-bound fibrinogen; and 3) S12, which binds to an alpha-granule membrane protein that associates with the platelet surface during secretion. Unstimulated platelets demonstrated no PAC1, 9F9, or S12-specific fluorescence, indicating that they did not bind these antibodies. Upon stimulation with agonists, however, the platelets demonstrated a dose-dependent increase in FITC-fluorescence. The binding of 9F9 to activated platelets required fibrinogen. Low concentrations of ADP and epinephrine, which induce fibrinogen receptors but little secretion, stimulated near-maximal PAC1 or 9F9 binding but little S12 binding. On the other hand, a concentration of phorbol myristate acetate that evokes full platelet aggregation and secretion induced maximal binding of all three antibodies. When blood samples containing activated and non-activated platelets were mixed, as few as 0.8% activated platelets could be detected by this technique. There was a direct correlation between ADP-induced FITC-PAC1 binding and binding determined in a conventional 125I-PAC1 binding assay (r = 0.99; p < 0.001). These studies demonstrate that activated platelets can be reliably detected in whole blood using activation-dependent monoclonal antibodies and flow cytometry. This method may be useful to assess the degree of platelet activation and the efficacy platelet inhibitor therapy in thrombotic disorders.

A Study of Platelet Activation during Human Cardiopulmonary Bypass and the Effect of S-Nitrosoglutathione

1997 ◽  
Vol 78 (06) ◽  
pp. 1516-1519 ◽  
Author(s):  
Edward J Langford ◽  
Andrew Parfitt ◽  
Adam J de Beider ◽  
Michael T Marrinan ◽  
John F Martin
Keyword(s):  
Nitric Oxide ◽  
Flow Cytometry ◽  
Cardiac Surgery ◽  
Cardiopulmonary Bypass ◽  
Platelet Activation ◽  
Surface Expression ◽  
Platelet Inhibition ◽  
Nitric Oxide Donor ◽  
Platelet Dysfunction ◽  
Platelet Surface

SummaryCardiac surgery is complicated by the occurrence of post-operative bleeding due to platelet dysfunction. This is largely caused by platelet activation and consumption during cardiopulmonary bypass. Patients undergoing cardiac surgery requiring cardiopulmonary bypass were studied to determine whether early platelet changes due to bypass could be inhibited using the platelet-selective nitric oxide donor S-nitroso-glutathione (GSNO). Flow cytometry was used to measure platelet surface expression of P-selectin (an α-granule protein) and glycoproteins (GP) IIb/IIIa and Ib (mediators of aggregation and adhesion) before and 5 and 10 min after commencing cardiopulmonary bypass, in 6 controls and 6 patients receiving GSNO 50 μg/min. Platelet P-selectin expression increased during bypass both in controls and patients receiving GSNO. Glycoproteins IIb/IIIa and Ib fell during bypass in control and GSNO-treated patients. There was no difference between control and GSNO-treated groups. Thus no significant platelet inhibition by S-nitrosoglutathione was demonstrated under these conditions.

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 Platelet activation leads to activation and propagation of the complement system

2005 ◽  
Vol 201 (6) ◽  
pp. 871-879 ◽  
Author(s):  
Ian del Conde ◽  
Miguel A. Crúz ◽  
Hui Zhang ◽  
José A. López ◽  
Vahid Afshar-Kharghan
Keyword(s):  
Flow Cytometry ◽  
Surface Plasmon Resonance ◽  
Platelet Activation ◽  
Vascular Injury ◽  
Complement System ◽  
Cell Activation ◽  
Additional Mechanism ◽  
Platelet Surface ◽  
Activated Platelets ◽  
The Complement System

Inflammation and thrombosis are two responses that are linked through a number of mechanisms, one of them being the complement system. Various proteins of the complement system interact specifically with platelets, which, in turn, activates them and promotes thrombosis. In this paper, we show that the converse is also true: activated platelets can activate the complement system. As assessed by flow cytometry and immunoblotting, C3 deposition increased on the platelet surface upon cell activation with different agonists. Activation of the complement system proceeded to its final stages, which was marked by the increased generation of the anaphylotoxin C3a and the C5b-9 complex. We identified P-selectin as a C3b-binding protein, and confirmed by surface plasmon resonance binding that these two proteins interact specifically with a dissociation constant of 1 μM. Using heterologous cells expressing P-selectin, we found that P-selectin alone is sufficient to activate the complement system, marked by increases in C3b deposition, C3a generation, and C5b-9 formation. In summary, we have found that platelets are capable of activating the complement system, and have identified P-selectin as a receptor for C3b capable of initiating complement activation. These findings point out an additional mechanism by which inflammation may localize to sites of vascular injury and thrombosis.

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

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.

Immunological Evidence for Prothrombin in Human Platelets

1986 ◽  
Vol 55 (02) ◽  
pp. 268-270
Author(s):  
R J Alexander
Keyword(s):  
Electrophoretic Mobility ◽  
Platelet Activation ◽  
Double Diffusion ◽  
Human Platelets ◽  
Secreted Proteins ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Similar Protein ◽  
Diffusion Assay ◽  
Supernatant Solution

SummaryAn attempt was made to isolate from plasma the platelet surface substrate for thrombin, glycoprotein V (GPV), because a GPV antigen was reported to be present in plasma (3). Plasma fractionation based on procedures for purification of GPV from platelets revealed a thrombin-sensitive protein with appropriate electrophoretic mobility. The protein was purified; an antiserum against it i) reacted with detergent-solubilized platelet proteins or secreted proteins in a double diffusion assay, ii) adsorbed a protein from the supernatant solution of activated platelets, and iii) inhibited thrombin-induced platelet activation, but the antiserum did not adsorb labeled GPV. The purified protein was immunochemically related to prothrombin rather than to GPV. Other antibodies against prothrombin were also able to adsorb a protein from platelets. It is concluded that 1) plasma does not contain appreciable amounts of GPV, and 2) platelets contain prothrombin or an immunochemically similar protein.

Platelet Activation and Cytokine Production during Hypothermic Cardiopulmonary Bypass – A Possible Correlation?

1998 ◽  
Vol 80 (07) ◽  
pp. 58-64 ◽  
Author(s):  
P. Ferroni ◽  
G. Speziale ◽  
G. Ruvolo ◽  
A. Giovannelli ◽  
F. M. Pulcinelli ◽  
...  
Keyword(s):  
Cardiac Surgery ◽  
Cardiopulmonary Bypass ◽  
Inflammatory Response ◽  
Platelet Activation ◽  
Cytokine Production ◽  
Inverse Correlation ◽  
Moderate Degree ◽  
Platelet Response ◽  
Activated Platelets

SummaryCardiopulmonary bypass (CPB) is associated with impaired platelet function and a systemic inflammatory response. The present study was designed to evaluate whether any correlation between platelet activation and inflammatory response during CPB exists. The results obtained from 8 patients undergoing hypothermic CPB for cardiac surgery showed the occurrence of a moderate degree of platelet activation during CPB, demonstrated by an increase of platelet CD62P expression in correlation with an increase of β-thromboglobulin levels, with a concomitant decrease of in vitro platelet response. Plasma IL-1β levels significantly increased during CPB, with a peak between 1 and 4 h after CPB. Similarly, IL-6 levels were elevated 30 min from CPB starting, peaked at 4 h, and remained elevated after 24 h. A direct correlation was found between plasma IL-1β and IL-6 levels. A significant correlation between plasma IL-1β and β-thromboglobulin levels was also found. In turn, plasma β-thromboglobulin levels correlated with CD62P expression on activated platelets. An inverse correlation was found between in vitro platelet aggregation and plasma IL-1β or IL-6 levels. From the present results it may be speculated that platelet activation during CPB may contribute, through the release of IL-1β, to activation of endothelial cells and subsequent release of other cytokines with chemotactic and pro-inflammatory properties, thus playing an important role in the inflammatory response associated with CPB.

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