Haematologica 2004;89: supplement no. 8 - Supplements ...

98PostersPO-008INTEGRIN α2β1 AND GLYCOPROTEIN VI DEPENDENTCALCIUM SIGNALS IN PLATELETS INTERACTING WITHACID-SOLUBLE TYPE I COLLAGEN UNDER FLOWMazzucato M, Cozzi MR, Battiston M, De Marco LCentro di Riferimento Oncologico, IRCCS, Aviano,ItalyWe studied the role of platelet integrin α2β1 andglycoprotein GPVI in stabilizing adhesion and modulatingsignal transmission of Fluo-3 AM plateletsperfused onto a surface of acid-soluble collagen typeI at wall shear rate of 250 s-1. We analyzed concurrentlythe instantaneous velocity and [Ca ++ ]i in singleplatelets interacting with collagen using a videoimagingmethod characterized by high-speed imageacquisition (25 frames/s) and high performance software(M. Mazzucato et al. Blood, 2002;100:2793-800). Perfusion of washed platelets resulted in singleplatelet adhesion that displayed a series of transient[Cav]i elevations. Two distinct peaks could bedefined on the basis of [Ca ++ ]i levels, duration, relationto motion on the surface and sensitivity to EGTA.Type α/β like Ca ++ oscillations were characterized bya rapid increase of 0.2 to 2 µM, lasting for 0.5-2.5seconds and EGTA insensitive. The discriminationbetween type α/β (typical of platelets translocatingover a surface of VWF at high shear rate) and α/β likewas obtained on the basis of dynamic behavior:platelets showing α/β like peaks were firmly adherentto collagen. Type γ like [Ca ++ ]i peaks were characterizedby a maximum level >2-3 µM lasting forseveral seconds (3-60) and stable adhesion to collagensurface and abolished by EGTA. MoAb againstintegrin α2β1 prevented platelet adhesion to collagensurface and the appearance of both type α/βlike and γ like peaks. Whereas only type γ like peak,which involve a transmembrane Ca ++ flux, was inhibitedby an anti-GPVI MoAb. Inhibitors of PI3-kinase(wortmannin and LY294002) and PLC (U73122) preventedthe appearance of both Ca ++ peaks, reducedthe arrest time from ~14 to ~3 seconds and preventedplatelet stable adhesion. Our results demonstratethat α2β1 partecipates in the initial stages ofplatelet adhesion and activation which is subsequentlyreinforced by activation through GPVI.PO-009COLLAGEN-INDUCED PLATELET ACTIVATION: NEW INSIGHTSRiondino S, Lotti L, Bronzi C, Pulcinelli FMDipartimento di Medicina Sperimentale e Patologia,Università degli Studi di Roma "La Sapienza”, ItalyShape change is platelets’ earliest response to stimuli;it is mainly dependent upon Ca 2+ /calmodulininteraction, subsequent to Ca2 + mobilization, and ismediated by MLCK. It has been recently hypothesizedthat collagen itself is not able to elicit platelet shapechange in the absence of ADP and thromboxane A2(TxA2) co-stimulation, while it is capable of inducingMLCK activation. Since we hypothesize that the morphologicalchanges of the few platelets that adhereto collagen might not be revealed by turbidimetry,aim of this study was to use electron microscopy (EM)as a tool to asses platelet shape change, in theabsence of the amplificatory feedback pathways ofADP and TxA2. For this purpose, platelets were treatedwith aspirin, AR-C66096 and MRS 2179 (P2Y12and P2Y1 ADP receptor antagonists, respectively),stimulated for 30 sec with collagen (25 µg/mL) in anaggregometer and then fixed for EM studies. Ourresults demonstrated that only those platelets thatwere more close to the insoluble collagen fibresunderwent a typical change in shape, while all otherplatelets did not modify their quiescent morphology.Turbidimetry failed to reveal such phenomenon.Moreover, since cAMP is known to inhibit plateletactivation by reducing Ca2 + mobilization in responseto all platelet agonists, except for collagen, in the presentstudy we sought to investigate whether cAMP isinvolved in the inhibition of collagen-induced plateletshape change. Platelets were thus treated with iloprost(28 nM) prior to stimulation. EM studies demonstratedthat iloprost did not modify collagen-inducedshape change, while immunoblotting studies showeda marked inhibition of collagen-induced MLC phosphorylationin the presence of enhanced cAMP levels.Since cAMP, contrariwise to what happens for allknown platelet agonists, enhances Ca2 + mobilizationin response to collagen, we can hypothesize thatcAMP exerts an inhibitory action on MLCK activation.PO-010GP91PHOX-DEPENDENT EXPRESSION OF PLATELETCD40 LIGANDPignatelli P,*° Sanguigni V,** Lenti L,* Ferro D,°Finocchi A,*** Rossi P,*** Violi F*°Divisione IV Clinica Medica°, Dipartimento diMedicina Sperimentale e Patologia*, Università diRoma "La Sapienza", Dipartimento di MedicinaInterna** e Dipartimento di Medicina Sperimentale,***Università di Roma Tor Vergata, ItalyBackground: CD40L expression on platelets ismediated by agonists but the underlying mechanismis still unclear. Methods: CD40L expression was measuredboth in platelets from healthy subjects - withand without the addition of antioxidants or a phospholipaseA2 (PLA2) inhibitor - and in platelets fromtwo patients with an inherited deficiency ofhaematologica vol. 89(suppl. n. 8):september 2004