Ras-like small GTPases in platelet biology

Abstract

In a human body, the behaviour of each cell is controlled by signals like hormones or growth factors. A cell receives such messages by means of specific receptors. These transmit the messages to other proteins in the cell and so on. Such cascades control for example the pattern of active genes or metabolic responses. In this way, proliferation, differentiation, growth and cell death are tightly regulated. The complex of biochemical reactions that occurs in the cell upon signals is called signal transduction. Members of the Ras-like small GTPases protein family function as molecular switches in signalling pathways. Activating signals induce the GTP-bound form of Ras proteins. In this conformation these proteins pass the signals on to other proteins. Hydrolysis of GTP returns them into the inactive, GDP-bound version and signalling is terminated. Certain mutations in the prototype family member Ras are known which render the protein continuously active. This contributes to oncogenic transformation. The function of the family members Rap1 and Ral is largely unknown. However, they are abundant and rapidly activated upon a variety of signals in platelets. This suggests these GTPases participate in signalling that controls platelet functions. Upon activation, platelets change their shape and concomitantly start sticking to each other (adhesion) by activation of adhesion molecules (integrins). This results in the formation of a blood clot which stops bleeding. Activation under inproper conditions may result in thrombosis, heart or brain infarct. Knowledge and insight with respect to signal transduction is thus required to understand platelet behaviour (and cells in general) under normal and pathological circumstances. In this thesis, the focus has been on how Ras-like small GTPases are involved in signalling pathways as they occur in platelets. In chapter 1 the Ras proteins and their behaviour and function in blood platelets are discussed. In chapter 2 the calcium-induced Ras-independent Ral activation mechanism in platelets is studied. This research describes the identification and characterization of the Ral-specific guanine nucleotide exchange factor RalGEF2. In chapter 3, new insight is provided on the role of Rap1 in integrin-mediated cell adhesion. Rap1 is required for ?1 and ?2 integrin-mediated adhesion induced by Mn2+- or integrin activating antibodies. Treatment with Mn2+ or the activating antibodies did not induce Rap1 activation. Rap1 may fulfil a facilitating function. In chapter 4 the proposed Rap1 function in cAMP-induced PKA-independent elevation of the intracellular calcium concentration is investigated in megakaryocytes. Also in platelets Rap1 has been connected with the regulation of intracellular calcium concentration. However, no evidence was found for the involvement of Rap1 in this process. The experiments described in chapter 5 show Rap1 activation in a diversity of human blood cell lines representing megakaryocytes, monocytes, B and T lymphocytes as the consequence of shear stress. Moreover, Rap1 activity was required for ?IIb?3-mediated adhesion of human megakaryocytes. These findings suggest a connection between shear stress, Rap1 and integrin regulation, but also link Rap1 with the most important platelet integrin. Proper control of this integrin is inevitable for the correct functioning of blood platelets. This connects Rap1 with one of the most elementary processes in platelet activation