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

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.

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

Blood ◽

10.1182/blood.v83.4.1006.1006 ◽

1994 ◽

Vol 83 (4) ◽

pp. 1006-1016 ◽

Cited By ~ 29

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.

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Factor VIII’s C Domains Bind to Platelets.

Blood ◽

10.1182/blood.v108.11.1715.1715 ◽

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

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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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DETECTION OF ACTIVATED PLATELETS IN WHOLE BLOOD BY FLOW CYTOMETRY

10.1055/s-0038-1643830 ◽

1987 ◽

Cited By ~ 4

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.

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

Blood ◽

10.1182/blood.v75.1.128.bloodjournal751128 ◽

1990 ◽

Vol 75 (1) ◽

pp. 128-138 ◽

Cited By ~ 3

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.

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Platelet Hyporeactivity in Very Low Birth Weight Neonates

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656093 ◽

1997 ◽

Vol 77 (05) ◽

pp. 1002-1007 ◽

Cited By ~ 84

Author(s):

Damodara Rajasekhar ◽

Marc R Barnard ◽

Francis J Bednarek ◽

Alan D Michelson

Keyword(s):

Flow Cytometry ◽

Binding Site ◽

Whole Blood ◽

Very Low Birth Weight ◽

Thromboxane A2 ◽

Platelet Surface ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Platelet Binding ◽

Thromboxane A2 Analogue

SummaryVery few studies have examined platelet function in very low birth weight (VLBW) preterm neonates, because of the relatively large volumes of blood required. In this study, platelet function in clinically stable VLBW neonates was examined by whole blood flow cytometry, which requires only 5 |jl1 of whole blood per assay. The following monoclonal antibodies were used: S12 (P-selectin-specific, reflecting a granule secretion), PAC1 (directed against the fibrinogen binding site exposed on the GPIIb-IIIa complex of activated platelets), F26 (directed against a conformational change in fibrinogen bound to the GPIIb-IIIa complex), and 6D1 (directed against the von Willebrand factor binding site on the GPIb-IX-V complex). VLBW neonates, like normal adults, did not have circulating activated platelets, as determined by the lack of binding of SI2, PAC1, and F26 in the absence of an added agonist. VLBW neonatal platelets were markedly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619 (a stable thromboxane A2 analogue), as determined by the extent of increase in the platelet binding of SI2, PAC1, and F26, and the extent of decrease in the platelet binding of 6D1. In summary, compared to adults, the platelets of VLBW neonates are markedly hyporeactive to thrombin, ADP/epinephrine and a thromboxane A2 analogue in the physiologic milieu of whole blood, as determined by: 1) the increase in platelet surface P-selectin; 2) the exposure of the fibrinogen binding site on the GPIIb-IIIa complex; 3) fibrinogen binding; and 4) the decrease in platelet surface GPIb. This platelet hyporeactivity may be a factor in the propensity of VLBW neonates to intraventricular hemorrhage. In addition to its previously defined use as a test of platelet hyperreactivity, the present study suggests that whole blood flow cytometry may be useful in the clinical assessment of platelet hyporeactivity.

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Studies on the Binding of 3H-SR121566, an Inhibitor of Gp IIb-IIIa Activation

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615656 ◽

2001 ◽

Vol 85 (04) ◽

pp. 702-709 ◽

Cited By ~ 3

Author(s):

P. Savi ◽

G. Zamboni ◽

O. Rescanières ◽

J. M. Herbert

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Platelet Aggregation ◽

Binding Sites ◽

Human Platelets ◽

Gamma Chain ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Direct Competition ◽

Stimulation Of

SummarySR121566 is a new synthetic agent which inhibits the binding of fibrinogen to activated platelets, and platelet aggregation. 3H-SR121566 bound with nanomolar affinity (KD ranging from 45 to 72 nM) to Gp IIb-IIIa expressing cells only. On activated human platelets, this ligand allowed the detection of a maximal number of 100-140,000 binding sites. The binding of SR121566 to platelets, was displaced by several agents including RGD-containing peptides and synthetic RGD mimetics, but not by ReoPro®, a humanised monoclonal antibody which inhibits the binding of fibrinogen to the Gp IIb-IIIa complex. Neither the fibrinogen dodecapeptide nor fibrinogen itself were able to compete with SR121566 whether platelets were activated or not.Flow cytometry studies indicated that SR121566 which did not activate Gp IIb-IIIa by itself, dose-dependently prevented the detection of activation-induced binding sites on TRAP-stimulated platelets in the presence or absence of exogenous fibrinogen, indicating a direct effect on the activation state of the Gp IIb-IIIa complex. Moreover, SR121566 was able to reverse the activation of Gp IIb-IIIa and to displace the binding of fibrinogen when added up to 5 min after TRAP stimulation of platelets. When added at later times (15 to 30 min), SR121566 failed to displace fibrinogen binding, even if SR121566 binding sites were still accessible and the Gp IIb-IIIa complex not activated.In conclusion, our study is in accordance with the finding that fibrinogen is recognised by the activated Gp IIb-IIIa complex through the dodecapeptide sequence present on its gamma chain, and that this interaction is inhibited by SR121566 by preventing and reversing the activated conformation of Gp IIb-IIIa and not by direct competition with fibrinogen.

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Sialic Acid Deficiency Impairs GPVI-Mediated Platelet Activation in ST3Gal-IV−/− Mice.

Blood ◽

10.1182/blood.v114.22.3005.3005 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3005-3005

Author(s):

Viktoria Rumjantseva ◽

Anne Louise Sørensen ◽

Karin M Hoffmeister ◽

Hervé Falet

Keyword(s):

Flow Cytometry ◽

Sialic Acid ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Receptor Expression ◽

Platelet Surface ◽

Lectin Domain ◽

Terminal Sialic Acid ◽

Fibrinogen Binding ◽

Surface Receptor

Abstract Abstract 3005 Poster Board II-972 Lack of terminal sialic acid residues on platelet surface glycoproteins results in rapid platelet clearance. Using null mice for the ST3Gal-IV sialyltransferase gene (ST3Gal-IV−/− mice), we have recently identified galactose residues on the N-terminus of the platelet Von Willebrand Factor receptor GPIbαa as a major counter receptor for the lectin domain of the asialoglycoprotein receptor on both hepatocytes and liver Kupffer cells (Sørensen et al., Blood 2009). ST3Gal-IV−/− mice have increased tail bleeding time. However, the role of terminal sialic acid residues on platelet activation is unclear. We investigated here whether loss of sialylation affects platelet activation mediated through the collagen receptor GPVI or by thrombin. Platelets were isolated from ST3Gal-IV−/− and ST3Gal-IV+/+ mouse littermates, stimulated with collagen-related peptide (CRP), convulxin (CVX), or thrombin, and platelet activation was evaluated by flow cytometry using P-selectin expression, as a marker for αa-granule secretion, and fibrinogen binding, as a marker for integrin αaIIbβ3 activation. Stimulation of ST3Gal-IV−/− platelets with CRP and CVX revealed a profound activation defect, compared to ST3Gal-IV+/+ platelets. The defect was not due to loss of surface receptor expression since ST3Gal-IV−/− and ST3Gal-IV+/+ platelets had comparable GPVI expression, as evidenced by flow cytometry. By contrast, activation of ST3Gal-IV−/− platelets with thrombin was normal. The data show that terminal sialic acid residues on GPVI are required for maximal platelet activation by CRP and CVX. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v69.5.1401.bloodjournal6951401 ◽

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.

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

Blood ◽

10.1182/blood.v75.1.128.128 ◽

1990 ◽

Vol 75 (1) ◽

pp. 128-138 ◽

Cited By ~ 301

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.

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