The interplay between platelets and coagulation

Abstract

Platelet activation and blood coagulation are two processes often studied separately, but which cannot be seen independently from each other. Platelets play a pivotal role in coagulation, not only by providing negatively charged phospholipids, but also in localizing the coagulation process from a diffuse plasma environment to an anionic surface, most likely via specific receptors on the platelets. On the other hand, the main product of coagulation, thrombin, is a potent platelet activator, thereby contributing to the exposure of negatively charged phospholipids on the activated platelet surface. In this thesis, the interaction of coagulation factors with platelet surfaces will be discussed. The main focus will be on the role glycoprotein Ib-alpha (GPIb-alpha) plays in this process. The consequences of ligand binding to GPIb-alpha on the function of these ligands and the role of GPIb-alpha as an adhesive receptor will be described. The first part of the thesis deals with the consequences of interaction of coagulation factors with the activated platelet on the process of coagulation itself. In chapter 3, the role of GPIb-alpha in the mechanism of action of rFVIIa is studied. The molecular interaction between rFVIIa and GPIb-alpha and the effect of the interaction with GPIb-alpha on thrombin generation mediated by rFVIIa has been investigated. In chapter 4, platelet adhesion to FIX(a) has been examined under static conditions as well as under conditions of flow. Furthermore, the effect of GPIb-alpha on FIXa-mediated FXa-generation has been investigated. In the second part of the thesis, the consequences of ligand binding to GPIb-alpha on platelet adhesion will be discussed. In chapter 5, the interaction between thrombin and GPIb-alpha has been investigated, by studying platelet adhesion to immobilized thrombin. The contribution of the different exosites of thrombin has been examined by using several thrombin mutants. Furthermore, platelet adhesion to thrombin bound to fibrin has been investigated. Chapter 6 examines the interaction between FXII and von Willebrand factor and describes a new role for FXII in thrombus formation, which has been studied using an in vitro flow model. Chapter 7 deals with the platelet activating properties of SSL5, a small protein secreted by S. aureus. More specific, the effect of SSL5 on platelets has been investigated using platelet aggregometry and platelet adhesion studies to immobilized SSL5. Furthermore, platelet receptors that are involved in this process have been studied. In chapter 8, the role of GPIb-alpha and the activated platelet in coagulation will be discussed in a broader perspective.