Measuring circulating cell-derived microparticles
J.-M. Freyssinet, Institut d'Hématologie & Immunologie, Faculté de Médecine, 4, rue Kirschleger – 67085 Strasbourg cedex, France.
Tel.: +33 390 243985; fax: +33 390 244016; e-mail: firstname.lastname@example.org
Solid-phase capture assay for the determination of cellular microparticles and associated deleterious potentials
Microparticles (MPs) are detectable in the peripheral blood of normal individuals, and are elevated in clinical situations where the atherothrombotic risk is increased [1,2]. Owing to platelet responsiveness under most pathophysiologic conditions, platelet-derived MPs appear to be the main procoagulant circulating species, leukocytic and endothelial origins being less represented, which does not, however, rule out a particular significance when also elevated.
Because they are pathogenic markers [1,2], MPs have to be assessed at two levels: first, for proportion or amount, and, second, for associated activities, procoagulant and others possibly indicated by, or related to, the presence of particular antigens or effectors .
Preparation of platelet-free plasma samples
Blood samples, 5 mL, are collected by vein puncture in 0.138 mol L−1 tri-sodium citrate at a final volume ratio of 9 : 1. Plasma is immediately obtained by centrifugation at 1500 × g for 15 min at room temperature. Platelet-free plasma is prepared by a second centrifugation at 13 000 × g for 2 min at room temperature, and can be frozen at −80 °C.
Just before addition to wells of the microtitration plate, d-phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) and 1,5-dansyl-glutamyl-glycyl-arginyl chloromethyl ketone (Dns-GGACK), two potent irreversible inhibitors of thrombin and factor Xa, respectively, are added to samples at a final concentration of 50 µm each. Samples are then recalcified by addition of 50 mm CaCl2 and added to the microtitration plate previously coated with biotinylated annexin V (annexin VBi) or antibodies.
Microparticle capture onto immobilized annexin V
Annexin VBi is insolubilized onto streptavidin-coated microtitration plates by contact of 100 µL/well of 350 ng mL−1 annexin VBi diluted in TBS-Ca2+ (50 mm Tris buffer, pH 7.5, containing 120 mm NaCl, 2.7 mm KCl, 1 mm CaCl2) supplemented with 45 µm human serum albumin (HSA). Incubation is allowed to proceed during 30 min at 37 °C, and plates are washed three times with TBS-Ca2+.
One hundred microliters per well of platelet-free plasma is then added and allowed to be in contact with insolubilized annexin VBi for 30 min at 37 °C. Four washing steps are then performed with Tris buffer containing 1 mm CaCl2 and 0.05% Tween 20, each with a 5-min incubation at 20 °C, the last one without Tween. Anionic phospholipid content can then be determined by prothrombinase assay as described below.
Microparticle capture onto immobilized antibodies
Biotinylated antibodies are insolubilized onto streptavidin-coated microtitration plates by adding 100 µL/well of the antibody solution (10 µg/mL in TBS-Ca2+, 45 µm HSA) during 30 min at 37 °C. Plates are washed three times with TBS-Ca2+. One hundred microliters per well of platelet-free plasma are then added, incubated and washed just as described above with annexin VBi. MP detection is achieved by prothrombinase assay as described below. Background values obtained with irrelevant IgGs are subtracted from those measured with specific antibodies.
Prothrombinase assay for the determination of the amount of captured microparticles
The phosphatidylserine content of MPs is measured through its ability to promote the activation of prothrombin to thrombin. The blood clotting factor concentrations have been determined to ensure that this phospholipid is the rate-limiting parameter of the reaction. Immobilized MPs are incubated with factor Xa (50 pmol L−1), factor Va (360 pmol L−1), prothrombin (1.3 µmol L−1), and 2.3 mmol L−1 CaCl2 in TBS-Ca2+ containing 45 µm HSA. After a 15-min incubation at 37 °C, Chromozym TH, a chromogenic substrate for thrombin, is added at a final concentration of 380 µmol L−1. Linear absorbance changes are recorded at 405 nm using a microtitration plate reader equipped with a kinetics software. Results are expressed as nanomolar phosphatidylserine equivalent by reference to a standard curve constructed by using liposomes of known concentration, containing 33% phosphatidylserine and 67% phosphatidylcholine (mol/mol).
Currently used biotin-conjugated monoclonal antibodies (mAbs) and annexin V for the determination of the following: MP-borne phosphatidylserine, annexin V; T lymphocyte-derived MPs, mAbs to CD3 and/or CD4; leukocyte-derived MPs, mAb to CD11a; platelet-derived MPs, mAb to human platelet glycoprotein Ibα (GPIbα); activated platelet-derived MPs, mAb to P-selectin; apoptotic endothelial cell-derived MPs, mAb to CD31; activated endothelial cell-derived MPs, mAb to E-selectin; and MP-borne tissue factor, mAb to tissue factor (TF).
Recombinant annexin V was produced and biotinylated in our laboratory. mAbs to CD3, CD4, CD11a as well as isotype-matched IgGs, all conjugated with biotin were from Leinco Technologies (Ballwin, MO, USA). The mAb to GPIbα was a kind gift from Dr F. Lanza (Unité 311 INSERM, Strasbourg, France); the mAb to CD31 was from Caltag Laboratoties (Burlingame, CA, USA). mAbs to P-selectin and E-selectin were from Ancell Corp. (Bayport, MN, USA), mAb to TF was from American Diagnostica (Stamford, CT, USA). High binding capacity streptavidin-coated microtitration plates and chromozym-TH were from Roche Diagnostics GmbH (Mannheim, Germany).
Advantages of the solid-phase capture assay
The system allows the capture of most of MPs, even when the PS proportion is as low as 1 : 50 (molar ratio with respect to other phospholipids), at [Ca2+]∼ 10 mm, or even lower if [Ca2+] 10 mm.
Expression of the results in ‘nmol L−1 phosphatidylserine equivalents’ makes sense regarding the procoagulant potential of circulating MPs in pathologies with an increased thrombotic risk, which is the case in most of cardiovascular disorders, or others with frequent thrombotic complications .
Possibility of automation, allowing easy, rapid, low-cost and reproducible measurements in large cohorts
Tissue factor (TF) content can be estimated, either directly when in rather large proportions, such as in the atherosclerotic plaque, through measurement of MP-associated TF activity by replacing the prothrombinase complex by the TF-dependent factor X activating complex , or indirectly by capture of TF-containing MPs onto insolubilized antibody to TF and determination by prothrombinase assay (see above).
Drawbacks of the solid-phase capture assay
Each method has its inherent drawbacks, which often depend on the quality of reagents, usually antibodies. In addition, the possible interference of soluble antigens may lead to underestimation of MP levels by antigenic capture, but when observed relative variations are generally significant.