Integrin αIIbβ3 regulation in platelets

Abstract

In summary, the studies described in this thesis show that discrete parts of the platelet integrin αIIbβ3 are involved in discrete ligand binding sub processes. A tripeptide sequence in the A3 domain of vWF is involved in preactivation of αIIbβ3, leading to an increase of both adhesion to immobilized fibrinogen as well as outside-in signalin. Dependent on whether the fibrinogen is in suspension or immobilized, two membrane distal parts of the b3-cytoplasmic tail are involved in: 1) maintaining the ligand bound state with regard to platelets and ligand in suspension (E-N) 2) attenuating adhesion to immobilized fibrinogen under flow (E-N) 3) maintaining a binding to the cytoskeleton upon ligand binding (T-T) 4) controlling of platelet spreading under flow (T-T). The basis for the different and opposite roles of the investigated regions may be due to the fact that under flow the conformational changes of immobilized fibrinogen enable platelet adhesion without prior activation, whereas, in suspension platelets have to be activated before ligand binding occurs. In the future, further analysis of the signal transduction routes affected by the presence of the peptide in platelets under flow, may give more insight in the difference in the involvement of the β3-cytoplasmic tail regions compared to platelets in suspension. Of special interest are rac, cdc42 and foremost rho, being involved in the formation of stress fibres and their upstream effector SYK. It would be interesting to elucidate the molecular basis of the interaction of region T-T and the cytoskeleton. For this, studies in which the behaviour upon adhesion to fibrinogen of individual platelets could give insights in the spreading process. It would also be interesting to study the activational state of αIIbβ3, which is located on top of the adhered platelets, in the presence of peptides E-N and T-T. The integrins may be affected by the presence of the peptides similar to integrins on platelets in suspension. Thr of Tyr- phosphorylated peptides could aid in the investigation of signalin and ligand binding processes dependent on phosphorylated residues. The extracellular parts of αIIbβ3 can be influenced by parts of ligands, for instance VPW in vWF. In vitro, the VPW sequence may generate a preactivation or micro clustering of αIIbβ3. VPW increases αIIbβ3-mediated adhesion to immobilized fibrinogen, and increases the tyrosine phosphorylation of the clustering dependent kinase SYK. The cooperativity of the binding of RGD, fibrinogen and VPW is necessary for optimal αIIbβ3 adhesion to fibrinogen under flow. In vivo vWF could contribute to platelet adhesion on the site of injury by increasing fibrinogen-αIIbβ3 interaction. One of the questions that remain to be answered is the location of the VPW-binding site on αIIbβ3. Mapping studies using the fibrin specific β316-322 sequence or antibodies directed against this part of fibrinogen could provide more insight. It would be interesting to further study the involvement of VPW in micro clustering, by determining if VPW is able to induce dimers or small multimers of purified αIIbβ3. Perfusion studies at high shear rate may lead to better understanding of the effect of VPW on platelets layering (aggregation). Whether or not VPW preactivates αIIbβ3, it will be difficult to investigate, however, electron microscopy of VPW-αIIbβ3 complexes may show the prevalence of this complex for an intermediate state αIIbβ3 closer resembling the fully activated conformation.