Annexin V as a probe of aminophospholipid exposure and platelet membrane vesiculation: a flow cytometry study showing a role for free sulfhydryl groups

Abstract

Annexin V, a protein with a high affinity and a strict specificity for aminophospholipids at physiologic calcium concentrations, was used to probe platelet activation and the development of procoagulant activity. Platelet secretion was studied in parallel using VH10, a murine monoclonal antibody specific for GMP-140, an alpha-granule membrane glycoprotein. Both proteins were labeled with fluorescein isothiocyanate and platelet activation was assessed by flow cytometry. Microparticles, which are shed from the platelet surface and also support procoagulant activity, were distinguished from platelets according to their associated light scattering signal. The relative ability of different inducers to trigger exposure of the procoagulant surface and microparticle formation was: ionophore A23187 = thrombin plus collagen = collagen = thrombin. The density of aminophospholipid on microparticles was higher than on remnant platelets. Platelet activation by these agonists was accompanied by GMP-140 exposure, both on platelets and microparticles. Here, thrombin was the most efficient agonist. The mechanisms responsible for the above processes were investigated using E-64-d, a specific membrane-permeable inhibitor of Ca(2+)-activated protease (calpain); tetracaine, an activator of calpain; and N-ethylmaleimide and diamide, two sulfhydryl-reactive agents. These agents were added to platelets alone or before stimulation by agonists. Calpain activity was assessed by the hydrolysis of cytoskeletal proteins as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Results showed that calpain activity is not essential for aminophospholipid translocation or for secretion. In contrast, although sulfhydryl-reactive agents alone can trigger procoagulant activity, they inhibit microvesicle formation and platelet secretion induced by the above agonists, suggesting that different mechanisms account for these phenomena. The use of annexin V in flow cytometry is a rapid method to assess procoagulant activity in platelets and the loss of phospholipid asymmetry in cell membranes.

Phosphatidylserine surface expression and integrin αIIbβ3 activity on thrombin/convulxin stimulated platelets/particles of different sizes

“…In our preliminary experiments (Fig 1 and data not shown) we established that low concentrations of Thr/Cvx (0AE005 U/ml/5 ng/ml) are sufficient to induce platelet apoptosis-like events during short stimulation (2 min). Several reports described that activated platelets are not homogeneous (Dachary-Prigent et al, 1993;Pasquet et al, 1998;Tonon et al, 2002;Dale et al, 2005;Remenyi et al, 2005;Leytin et al, 2006;Keuren et al, 2007). Three main platelet populations appeared after Thr/Cvx stimulation.…”

“…Not all activated platelets demonstrate PS expression (Dachary-Prigent et al, 1993;Pasquet et al, 1998;Tonon et al, 2002;Dale et al, 2005;Remenyi et al, 2005;Leytin et al, 2006). After adhesion to fibrinogen and activation by thrombin, approximately 3% of platelets show PS expression, while adhesion to collagen induces PS exposure in 57% of platelets.…”

Pathological events in platelets of Wiskott‐Aldrich syndrome patients

“…Also, the duration of the Ca 2 transient may be equally or more important. For example, thrombin plus collagen, but not either agent alone, induces substantial PS exposure and microparticle release (Bevers et al, 1983;Dachary-Prigent et al, 1993). Whereas the Ca 2 transient produced by either agent alone is of brief duration, that produced by thrombin plus collagen is long-lived (Weiss & Lages, 1997).…”

“…This ®nding strongly suggests that WAS platelets are abnormally susceptible to PS exposure. Pretreatment of normal platelets with calpeptin, an inhibitor of the protease calpain, is known to potentiate agonist-induced PS exposure (Dachary-Prigent et al, 1993). When pretreated with calpeptin, WAS platelets, like normal platelets, showed potentiation of A23187-dependent PS exposure ( Fig 5B), indicating that the enhanced susceptibility of WAS platelets to PS exposure is not due to blockage of calpain-mediated events.…”

“…In physiological conditions, normal platelets release microparticles upon activation with certain agonists (Bevers et al, 1983;Dachary-Prigent et al, 1993). We studied isolated platelets for microparticle release using CD41 staining and small size to identify microparticles.…”