Do ADP-activated platelets in vitro aggregate as discocytes ?

Abstract

E rnst F. Lüscher had posed somei nteresting questions in 2000 about the timeflowofthe first platelet reactionsafter activation: shape change (SC) or aggregation (1). Regarding in-vivo studies-c ommencing on the observations of Bizzozeroi n1 882 (2) -L üscherd educed fromt he literaturea nd from owninvestigations (3, 4) thatshape change whichprecedes aggregation would be an in-vitro artefact. Indeed,aplethoraof ultra-structural studies dealt with the examination of aggregated platelets starting with the investigations of Bornetal. 1980(5), and Borna nd manyo thers used transmission electronm icroscopy (TEM)tosupportthe viewthat the initial decrease in light transmission in plateleta ggregometryw as caused by platelet shape change.Ye t, recent morphological findings could attribute aggregatesofdiscoid platelets (designateddiscocytes) to the initialc hange in the aggregometer curve (6). More recently,a shear-dependenti n-vitro aggregation of discoid platelets was demonstrated using differential interferencec ontrast microscopyorscanning electronmicroscopy(7-9). These initiallyunstable aggregatesarise under the formation of membrane tethers between plateletsand an adhesive surface. Tetherformation involves the adhesivef unction of glycoprotein Ib/V/IX complex on vonWillebrand factor.The conversion of aggregatesfrom discoidplatelets into stableaggregatesconsisting of platelets showing shape change requiresthe release of ADP.Apartfrom astudy investigating aggregation in agenetic defective mouse(10), until nownoin-vitro aggregate formation between discoid mammalianp latelets could be observedi nt he TEM. Inspiredb yt he aforementioned implications (1) we investigated whether it is possible to demonstrateaggregation of discoid platelets in vitro . To induce aggregation we chose ADP, an agonist of platelets released fromdamaged cells, particularlyerythrocytes, at sites of vasculari njury( 11). Lüscheri nitiatedt hese investigations and inspiringlya ccompaniedt hem up to his sudden death in April 2002.Results presentedinthis reportindicate that already 1.5 seconds (s) after ADP-stimulation of discoid platelets in-vitro fibrinogen moleculesi nitiatedt he first contactsb etween platelets still retaining their discoid shape.The contacts were seen at any site of the plateletsurface. One second later,shape change took place, and focal contacts were formedo nt he plateletb ody associated with the constricting contractileg el.The existenceo f bridging fibrinogen moleculesduring the aggregation wasconfirmed by immunolabelling with anti-fibrinogen antibody.Thus, plateletaggregation appears to start withdiscoid platelet-fibrinogen contacts under the chosenin-vitro conditions, and the twostage process leads to formation of stable aggregatesofplatelets with achangedshape. Materials andmethodsAntibody Rabbit-antih uman-fibrinogen IgG Dakopatts (Denmark) was used. Platelet preparationPlatelet-rich plasma( PRP) waso btained by centrifugation (16 minutes[ min],2 30 g) from ACDN IH (formula A) anticoagulated venous blood of healthyv olun...