title: Collagen-induced platelet shape change is not affected by positive feedback pathway inhibitors and cAMP-elevating agents.
abstract: Shape change is the earliest response of platelets to stimuli; it is mainly dependent upon Ca(2+)/calmodulin interaction subsequent to Ca(2+) mobilization and is mediated by myosin light chain kinase (MLCK) activation. It has been recently suggested that collagen itself is not able to elicit platelet shape change in the absence of ADP and thromboxane A(2) costimulation but is capable of inducing MLCK activation. Since we hypothesize that the morphological changes of the few platelets that adhere to collagen might not be revealed by turbidimetry, the aim of this study was to assess platelet shape change using transmission electron microscopy, in the absence of the amplificatory feedback pathways of ADP and thromboxane A(2). Our results demonstrated that only the platelets in contact with insoluble collagen fibers underwent a typical shape change, whereas those further away remained quiescent. Moreover, since cAMP enhances Ca(2+) mobilization in response to collagen, in the present study, we also investigated whether cAMP is involved in the inhibition of collagen-induced platelet shape change and MLC phosphorylation. Platelets were thus treated with iloprost (28 nm) prior to stimulation. Electron microscopy studies demonstrated that iloprost did not modify collagen-induced shape change, whereas immunoblotting studies showed a slight inhibition of MLC phosphorylation in the presence of enhanced cAMP levels. We can thus conclude that collagen is able to cause platelet shape change through activation of Ca(2+)/calmodulin-dependent MLCK, without the involvement of amplificatory pathways. Enhanced cytosolic cAMP levels do not inhibit collagen-induced platelet shape change but exert a weak inhibitory action on MLCK.
authors.affiliation: Department of Experimental Medicine and Pathology, University La Sapienza, Roma, Italy. silvia.riondino@uniroma1.it
authors.forename: Silvia
authors.initials: S
authors.lastname: Riondino
authors.forename: Lavinia V
authors.initials: LV
authors.lastname: Lotti
authors.forename: Lucia
authors.initials: L
authors.lastname: Cutini
authors.forename: Fabio M
authors.initials: FM
authors.lastname: Pulcinelli
mesh_terms: D001792:Blood Platelets / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D020013:Calcium Signaling; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology*; D000242:Cyclic AMP / Q000819:agonists / Q000502:physiology*; D025461:Feedback, Physiological; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D046529:Microscopy, Electron, Transmission; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects
chemical_list: D003094:Collagen; D000242:Cyclic AMP; D009219:Myosin-Light-Chain Kinase; D016285:Iloprost