Linköping University Medical Dissertations No. 569

Intracellular Signalling and Cellular Interaction

Magnus Grenegård

Akademisk avhandling
som för avläggande av medicine doktorsexamen kommer att offentligt försvaras i Berzeliussalen, Hälsouniversitetet, Linköping, fredagen den 9 oktober 1998, kl 13.00

Docent Hans Gyllenhammar, Enheten for Hematologi, Huddinge Sjukhus, Huddinge


Activation of platelets is essential to prevent excessive blood loss at the site of vascular injury. These highly reactive cell fragments are also involved in several pathological conditions, in particular arterial thrombosis, which, in the coronary vessels, can lead to reduced blood flow, vessel occlusion and myocardial infarction.

The present research was focused on the anti-platelet properties, mechanisms of actions, and interactions of GEA 3175 and adenosine. The former a novel nitric oxide-containing and cyclic GMP-elevating compound, the latter a cyclic AMP-elevating purine nucleoside. Separately, the two substances significantly reduced the cytosolic Ca2+ responses, but only marginally suppressed subsequent functional responses in thrombin-stimulated human platelets. However, coadministration of the compounds synergistically inhibited platelet aggregation and abolished dense granule secretion and exposure of P-selectin, and also markedly reduced binding of fibrinogen to the surface of platelets. The results imply that the stronger inhibition of platelet functions was due to potent blocking of the initial Ca2+ transient and abrogation of mechanisms involved in the influx of extracellular Ca2+. Together, the findings clearly manifest the importance of an interaction between different inhibitory intracellular pathways in order to completely suppress the activity of thrombin-activated platelets.

Cell- and drug-comparative studies revealed that GEA 3175 provoked a remarkable long-acting inhibition of contractile responses and a concomitant sustained increase in cyclic GMP levels in airway smooth muscle tissue. The mechanisms of relaxation involved iberiotoxin-sensitive K+ channels and okadaic acid-sensitive phosphatases. Separately, GEA 3175 and adenosine markedly inhibited the respiratory burst in chemoattractant-activated neutrophil granulocytes. Opposite to the situation in platelets, the two compounds did not interact synergistically and coadministration had only a negligible effect on cytosolic Ca2+ signals in neutrophils. These data clearly demonstrate cell type-specific responses to cyclic GMP- and cyclic AMP-elevating agents.

This thesis also delineates the regulatory effects of platelets on neutrophil cellular and intracellular responses. Simple mixture of suspensions of human platelets and neutrophils caused prominent changes in the neutrophil in regard to cytoskeletal arrangement, cytosolic Ca2+ responsiveness, and capacity to generate reactive oxygen species. Platelet-derived factor(s) induced a marked suppression of the respiratory burst in activated neutrophils, an effect attributed to peripheral accumulation of filamentous actin and enhanced release of endogenous adenosine from the neutrophil. The chemotactic-peptide-induced rise in cytosolic Ca2+ in neutrophils was dramatically amplified in the presence of platelets, and ATP released from the platelets may play a role in this priming phenomenon, However, additional experiments showed that the amplified Ca2+ response was virtually independent of the state of activation of the platelet, required extracellular Ca2+ ions, and was completely insensitive to the NO-donor GEA 3175. Further analysis strongly indicated that platelets affected the cyclic AMP-sensitive phase of the Ca2+ response in neutrophils. In conclusion, multiple mediators and mechanisms participate in the platelet-mediated modulation of neutrophil responses, and greater understanding of the complex mechanisms and consequences of the interactions between these blood cells may provide useful information for the design of pharmacological tools and methods to control the inflammatory reaction.

Division of Pharmacology, Department of Medicine and Care, Faculty of Health Sciences, Linköpings Universitet, S-581 85, Linköping, Sweden

Linköping 1998

ISBN 91-7219-055-8     ISSN 0345-0082