Differential regulation of platelet aggregation and aminophospholipid exposure by calpain

Abstract

Summary Aggregation, exposure of procoagulant phospholipids and shedding of microparticles are platelet responses that depend on activating conditions. To determine how these different responses are interconnected, we simultaneously measured fibrinogen (Fg) binding and aminophospholipid exposure on activated platelets by means of flow cytometry. Low calcium ionophore (A23187) concentrations induced Fg binding but not annexin V binding. In contrast, high A23187 concentrations induced annexin V binding but not Fg binding. Collagen, both alone and in the presence of thrombin, induced both Fg and annexin V binding. Dual labelling was found on 38 ± 9% of platelets stimulated by thrombin plus collagen. The regulatory role of calpain in these platelet functions was investigated. When calpain was partially inhibited by 2 μg/ml calpeptin, Fg binding still occurred but aminophospholipid exposure was limited. By contrast, complete inhibition of calpain by 100 μg/ml calpeptin or E64d decreased Fg binding but enhanced aminophospholipid exposure. In these latter conditions, cytosolic calcium‐extruding systems were inhibited. The results suggest that (i) conditions that favour aminophospholipid exposure tend to decrease the aggregation process and (ii) calpain determines the switch to either aggregation or aminophospholipid exposure by controlling intracellular calcium.

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

“…In addition, we analysed the effect of these inhibitors on the distribution (proportions) of platelets/particles of different size upon Thr/Cvx stimulation. In general, our data confirm previous observations (Inomata et al , 1996; Schoenwaelder et al , 1997; Croce et al , 1999; Bachelot‐Loza et al , 2006) that calpain inhibition significantly reduced integrin α IIb β 3 activation (Fig 5B). However, more detailed analysis showed that this reduction was not equal in all platelets/particles, and was most prominent on the particles located in gate R3 (Fig 5B), which is due to a decreased expression of αIIbβ3 integrins (Fig 5C).…”

“…Simultaneous analysis of PS exposure and αIIbβ3 integrin activation in stimulated platelets by flow cytometry showed up to three platelet populations. The first exhibited exclusively activated αIIbβ3, the second group consisted of platelets exposing only PS, and the third population showed both PS expression and activated αIIbβ3 exposure (Bachelot‐Loza et al , 2006; Munnix et al , 2007). However, platelet groups that show distinct PS exposure or αIIbβ3 integrin activation were not defined in platelet populations of different size.…”

Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

“…In human platelets, the elevation in intracellular Ca 2+ concentration regulates various platelet functions, such as integrin activation, granule secretion, and rapid procoagulant phosphatidylserine (PS) exposure [26], [27]. One important initiator of Ca 2+ signaling is the activation of G q pathways, which induce the generation of diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP 3 ) to activate PKC and Ca 2+ store depletion, respectively [28].…”

Procoagulant properties of microparticles released from red blood cells in paroxysmal nocturnal haemoglobinuria

“…It has been reported that PKC is involved in human RBC Ca 2+ entry (Andrews et al, 2002) and subsequent PS exposure on RBC (de Jong et al, 2002). In the case of platelets, calpain (Bachelot-Loza et al, 2006) or caspase (Shcherbina & Remold-O'Donnell, 1999) is suggested to be involved in the mechanisms inducing PS exposure and/or MP release.…”