Calcium involvement in aminophospholipid exposure and microparticle formation during platelet activation: A study using Ca2+-ATPase inhibitors

Abstract

The development of procoagulant activity and microparticle formation during platelet activation is known to depend on an increase in cytosolic Ca2+ levels. We have studied the mechanisms leading to these events using FITC-labeled recombinant annexin V, a protein which binds with a high affinity to aminophospholipids, in flow cytometry. In particular, we show that the Ca(2+)-ATPase inhibitors thapsigargin and cyclopiazonic acid are as potent inducers of aminophospholipid exposure and microparticle formation as the ionophore A23187. In contrast, 2,5-di-tert-butyl-1, 4-benzohydroquinone induced negligible microparticle formation, although platelets abundantly bound annexin V-FITC. That platelet activation had occurred was confirmed by binding studies with VH10, a monoclonal antibody specific for the alpha-granule membrane glycoprotein GMP-140, and by prothrombinase activity measurements. These results demonstrate that microvesiculation is not an automatic response to aminophospholipid exposure. The Ca(2+)-ATPase inhibitors induced different intracellular Ca2+ levels as measured using fluo-3 as a calcium dye. These were 10 +/- 4 microM (n = 11) for thapsigargin (3 microM), 19.6 +/- 2.2 microM (n = 8) for cyclopiazonic acid (100 microM), and 0.619 +/- 0.137 microM (n = 8) for 2,5-di-tert-butyl-1,4-benzohydroquinone (100 microM). Calpain activity, as assessed in platelets by analyzing the degradation of cytoskeletal proteins, was only observed with agents that stimulated microparticle formation. Phospholipid transbilayer movement was studied by measuring annexin V binding during platelet activation. Results showed that aminophospholipid exposure induced by ionophore A23187 (t1/2 = 133 +/- 14 s) was more rapid than that induced by TG (t1/2 = 280 +/- 30 s), although the rate-limiting step in the assay was the binding of annexin V to activated platelets (t1/2 = 70-80 s). Interestingly, the presence of annexin V itself during the activation inhibited microparticle formation, although degradation of platelet proteins by calpain continued to occur. Our results clearly show (i) that aminophospholipid exposure and platelet microvesiculation are independent but closely regulated events and (ii) that while both processes are associated with an increase in intracellular Ca2+, microvesiculation additionally requires Ca(2+)-induced calpain activation and a fusion process inhibited by annexin V.

Systemic Inflammatory Priming in Normal Pregnancy and Preeclampsia: The Role of Circulating Syncytiotrophoblast Microparticles

“…One study showed a marked diminishment in Foxp3 mRNA up-regulation after TGF-␤ induction of peripheral T cells stimulated in the presence of anti-CD28 Abs (7). In contrast, a second study found that CD28 costimulation was important for Foxp3 expression in TGF-␤-treated CD4 ϩ CD25 Ϫ thymocytes, primarily by enhancing cell survival (42). In multiple experiments using highly purified Ab-stimulated T lymphocytes, we failed to observe either a beneficial or deleterious role for CD28 costimulation in Foxp3 up-regulation.…”

“…Circulating microparticles may be derived from leukocytes (40), endothelial cells (41), platelets (42), or RBC (43). The particles carry bioactive molecules that deliver intercellular signals (42). They are thought to have important proinflammatory and procoagulatory roles (44,45), including promoting interaction between endothelial cells and monocytes (40), platelet activation (46), and apoptosis (47).…”

Increased soluble and platelet-associated CD40 ligand in essential thrombocythemia and reactive thrombocytosis

“…In experiments performed to control the extent of platelet activation, whole blood was also collected in the presence of inhibitors of platelet activation (theophylline, adenosine, and dipyridamole [CTAD] tubes, Becton Dickinson). To assess platelet activation, flow cytometry was performed on whole blood with the following monoclonal antibodies: VH10, 8 recognizing P selectin; PAC-1 (Immunotech, Marseille, France); and AP6 (gift from Dr T.S. Kunicki), 9 recognizing the active conformation of glycoprotein IIbIIIa.…”

Involvement of extracellular calcium in phosphatidylserine exposure during apoptosis

“…PS can also be exposed by red ~lood cells and is involved in their clearance from circulation 11]. Platelet activation causes rapid increase in intracellular 2a 2+, and pharmacological modulation with Ca 2+ ionophores ~r Ca2+-ATPase inhibitors can stimulate the exposure of large lmounts of PS by platelets within minutes [12]. Ca 2+ inacti- vates the aminophospholipid translocase which normally constrains PS to the inner leaflet [13], but this alone is insufficient for exposure.…”