Exploring the effects of Cadherin-derived Peptides on Platelet and Endothelial Cell Function
The primary role of platelets is hemostasis, the prevention of excessive bleeding following vascular injury. However, platelets also play a role in the pathology of cardiovascular disease. Platelet mediated thrombosis on ruptured atherosclerotic plaques underlies the acute complications of cardiovascular disease including myocardial infarction and ischemic stroke. Arterial thrombus formation is a complex process involving various cell adhesion molecules and secondary mediators, such as ADP, secreted from activated platelets, which help to recruit platelets. There is an ongoing search for anti-platelet agents that can prevent thrombotic events whilst minimizing bleeding.
Previous work in our laboratory identified a palmitoylated peptide derived from Kidney Cadherin (K-Cadherin) as a significant inhibitor of platelet function. Based on this observation, the anti-platelet effects of other cadherin-derived peptides were investigated. Firstly, the effect of peptides derived from membrane-adjacent cytoplasmic regions of K-, Epithelial Cadherin (E-Cadherin) and Neural Cadherin (N-Cadherin) sequences were assessed in assays of platelet function. Although many of these peptides were shown to inhibit platelet secretion and aggregation responses, it emerged that the corresponding control peptide sequences showed a similar ability to inhibit platelet function. Thus it seemed that many of the observed effects were non-specific.
Secondly, I identified a novel junctional protein, Vascular Endothelial Cadherin (VE-Cadherin) and its associated protein, P120-catenin in human platelets and demonstrated, for the first time, that VE-Cadherin could form a heterophilic interaction with platelet integrin allb(33. This interaction was Arginine-Glycine-Aspartic acid (RGD) dependent. Peptides derived from juxtamembrane domain (JMD) of VE-Cadherin, such as those studied in the first results chapter, had only non-specific effects on platelet function.
Thirdly, the effect of the VE-Cadherin-derived peptides on endothelial cell function and angiogenesis were investigated. Non-specific activity was carefully monitored using matched control peptides and lower peptide doses than in previous studies. This study demonstrates that certain VECadherin peptides specifically inhibited endothelial cell functions, including angiogenesis, migration and proliferation. These results highlight an important role played by the JMD in VE-Cadherin and identified potential inhibitors of cadherin function that may have therapeutic relevance.
In the final chapter of this thesis, I investigated which parameters of cadherin-derived peptides, and their control peptides, were associated with the anti-platelet effect. It was found that water-soluble peptides with positively charged amino acids at their N-terminus are associated with anti-platelet activity. In addition, this prediction was also experimentally verified by using peptides with positively charged amino acids.
Taken together, this thesis explores the cellular effects of cell-permeable peptides derived from target proteins. It clearly identifies a need for appropriate control peptides in all such experiments to control for sequence, dose and toxicity. Within these constrains, novel peptides were identified that inhibit VE-Cadherin function in endothelial cells.