Thesis (Ph.D.) -- University of Adelaide, Dept. of Physiology, 2000. / Copies of author's previously published articles inserted. Bibliography: leaves 267-303.

Thesis (M.S.)--Catholic University of America. / Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisors: Ulhas P. Naik, Dept. of Biological Sciences and Brian J. Bahnson, Dept. of Chemistry and Biochemistry. Includes bibliographical references.

When platelets come into contact with collagen, they adhere to it then release the contents of their dense granules and granules and aggregate together. This interaction is dependent on the conformation and degree of multimerisation of the collagen as well as the experimental conditions. The fibrillar state of collagen varies with the pH and ionic strength of the solution so collagen was immobilised on to an insoluble support in order that its fibrillar state should remain unchanged during exposure to platelets and during modification. The first support used was polyamide sheet, but although the resulting collagen-strips induced measurable platelet adhesion and aggregation, there was insufficient collagen present to induce adequate aggregation or serotonin release to be of use in investigating these interactions. Sepharose 6B was then used as a support for collagen and this proved satisfactory, inducing approximately 20% serotonin release from platelets. The release reaction is nearer to the initial interaction between platelets and collagen than aggregation, which is the final step, and it therefore allows a better insight into the conditions necessary for the interaction to take place. The amino groups of collagen-Sepharose were modified in three ways, using succinic anhydride, trinitrobenzene sulphonic acid and dinitrofluorobenzene. Succinylation almost abolished serotonin release while di-and trinitrophenylation enhanced this reaction. amino groups of collagen are therefore not in themselves important in the recognition of collagen by platelets and the subsequent reactions, since they were blocked by the modifications. The suggestion is made that succinylation disrupted the fibrils because of the change in charge whereas di- and trinitrophenylation merely neutralised the charge and permitted realignment of any previously misaligned areas of the molecule. These results reinforce theories that the correct quaternary structure of collagen must be present in order that it will be recognised by platelets.

Platelets are small and anucleate blood cells that are well known for their role in hemostasis. Other less recognized platelet functions include contributions to cancer vascularization, growth, and metastasis. Although the participation of platelets in cancer biology has been broadly studied in mouse models, there is no information in the literature regarding the crosstalk of platelets and cancer cells in dogs. The objective of this dissertation was to explore the interaction of canine platelets and tumor cells in vitro. The main hypothesis was that canine platelets were similar to human and murine platelets, and would interact with tumor cells, resulting in a change in the behavior of these cells. Using confocal immunofluorescence, we show that fibrinogen and von Willebrand factor have little colocalization within platelets, providing evidence that canine platelets have selective packaging and different alpha-granule subtypes, as shown in mice and humans. Then, we demonstrate canine platelet activation by osteosarcoma and mammary carcinoma cells, utilizing platelet aggregometry. Next, we show that intact platelets, platelet lysate, and thrombin-activated platelet releasate have an inhibitory effect on the migration of canine osteosarcoma and mammary carcinoma cell lines, contrary to what is described in humans and mice. We also demonstrate that releasate from canine platelets activated by collagen induces cancer cell migration, the opposite of the effect of releasates derived from thrombin or adenosine diphosphate activation. Lastly, we show that platelets can downregulate epithelial-to-mesenchymal-related transcription factors in canine cancer cells, suggesting that platelets may play an important role regulating this process in canine cancer progression. In conclusion, the results of this study have revealed important interactions between canine cancer cell lines and platelets in vitro. Our findings suggest that platelets most likely have a significant role in cancer growth in dogs and that cancer cells most likely affect platelet function in cancer patients.

Immune-mediated hemolytic anemia (IMHA) is one of the most common causes of anemia in dogs. Despite aggressive therapy, there is a 50% mortality rate in IMHA patients, and the most common cause of death is thromboembolic disease, particularly pulmonary thromboembolism. With the high thromboembolism rate in dogs with IMHA, anti-platelet therapy with aspirin can be a life-saving preventative therapy. Along with anti-platelet therapy, immunosuppressive therapy is needed to decrease erythrocyte destruction. Cyclosporine has become a popular medication for immunosuppression in IMHA patients. Unfortunately, recent human reports have suggested that cyclosporine could activate platelets and contribute to a hypercoagulable state. With the goal of improving therapy, these studies investigated the role aspirin plays in inhibiting platelet function and cyclooxygenase expression, an enzyme that enhances platelet reactivity. The effect of cyclosporine on platelet reactivity and hypercoagulablity was investigated to determine if this medication would create activated platelets and a prothrombotic state.

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Platelets are involved in many processes ranging from fighting microbial infections and triggering inflammation to promoting tumor angiogenesis and metastasis. Nevertheless, the primary physiological function of platelets is to act as essential mediators in maintaining homeostasis of the circulatory system by forming hemostatic thrombi that prevent blood loss and maintain vascular integrity. The p21-activated kinases (PAKs) are a family of serine/threonine kinases known to be the downstream effectors of GTPases, Cdc42 and Rac1. PAKs are the key regulators of actin polymerization and have been shown to play an important role in platelet spreading and aggregation in thrombin-stimulated platelets. Whereas several signaling cascades downstream of heterotrimeric G proteins that regulate platelet functions have been characterized, little attention is paid towards the signaling cascades that involve small G proteins effectors such as PAK. A few studies have characterized the role of PAK, downstream of the Rho family of small G proteins, in outside-in signaling, but its role in the regulation of platelet functional responses by inside-out signaling events have not been elucidated. PAK is reported to interact with numerous proteins including Akt, PDK1 and PI3-kinase in different cell lines. PAK’s function as a scaffolding protein expands the role of this protein in cellular functions. Although PAK is known to have non-catalytic scaffolding functions and is shown to associate and translocate Akt in other cell systems, the catalytic and possible non-catalytic scaffolding role in platelet functions are not clearly defined. In this dissertation we propose to elucidate the scaffolding function of PAK and also its role platelet functional responses using molecular genetics approach. Akt is an important signaling molecule regulating platelet aggregation. Akt is phosphorylated upon translocation to the membrane through Gi signaling pathways by a PIP3-dependent mechanism. However, Akt is more robustly phosphorylated by thrombin compared to ADP in platelets. In this study, we investigated the mechanisms of Akt translocation as a possible explanation for this difference. Stimulation of washed human platelets with protease-activated receptor (PAR) agonists caused rapid translocation of Akt to the membrane, whereas Akt phosphorylation occurred later. The translocation of Akt was abolished in the presence of a Gq-selective inhibitor or in Gq-deficient murine platelets, indicating that Akt translocation is regulated downstream of Gq signaling pathways. Interestingly, PI3-kinase inhibitors or P2Y12 antagonist abolished Akt phosphorylation without affecting Akt translocation to the membrane, suggesting that Akt translocation occurs through a PI3-kinase/PIP3/ Gi-independent mechanism. An Akt scaffolding protein, PAK, translocates to the membrane upon stimulation with PAR agonists in a Gq-dependent manner with the kinetics of translocation similar to that of Akt. Co-immunoprecipitation studies showed constitutive association of PAK and Akt, suggesting a role of PAK in Akt translocation. These results show for the first time an important role of the Gq signaling pathway in mediating Akt translocation to the membrane in a novel Gi/PI3-kinase/PIP3-independent mechanism. PAK contains an autoinhibitory domain that suppresses the catalytic activity of its kinase domain. This autoregulatory domain found within PAK kinase provides a unique target for chemical inhibitors. IPA3, a small molecule allosteric inhibitor of PAK activation, binds covalently to the PAK regulatory domain and prevents binding to its upstream activators. IPA3 has been used in various cells, including platelets, to evaluate the role of PAK in signaling. Herein, we investigated the specificity and selectivity of IPA3 as a PAK inhibitor in the human platelets. Stimulation of platelets pretreated with IPA3 using a PAR-4 or GPV1 agonist resulted in a concentration dependent inhibition of aggregation, as was suggested by earlier studies. Interestingly, we found that incubation of washed human platelets with IPA3 leads to a non-specific increase in phosphorylation of several proteins in absence of any agonist. However, this phosphorylation is not sufficient for aggregation of platelets by IPA3. In summary, we demonstrate that IPA3 by itself can phosphorylate several proteins in human platelets and thus its use is not an appropriate strategy for investigating PAK function in platelets. PAKs are classified into two groups based on their structural differences. Human platelets have been shown to express both group I (PAK1, PAK2, and PAK3) and group II PAKs (PAK4). Previous studies showing the role of PAK were performed with nonspecific inhibitors of PAK, such as IPA3, that do not distinguish isoforms. Thus, we propose to evaluate the function of specific PAK isoforms in platelets using knockout murine platelets, which are more selective tools to study the role of individual isoforms of PAK. We observed that Pak2 null mice showed enhanced secretion responses upon stimulation with 2MeS ADP and collagen, and delayed clot retraction. Interestingly, Pak1 null murine platelets did not have any functional defects, suggesting redundancy with other PAK isoforms. The studies proposed in my thesis will provide further insights into the molecular mechanisms of platelet activation and hence provide a basis for development of PAK as novel antithrombotic therapeutic targets. Furthermore, PAK inhibitors are currently being developed by pharmaceutical companies to treat malignancies, although this enzyme is ubiquitously expressed in the body. A thorough understanding of the role of PAK in platelets can predict the effect of these drugs on hemostatic functions, which helps during clinical trials. In the future, targeted inhibition of signaling molecules in platelets could be developed and that would solely target platelet signaling pathways. / Organ Systems & Translational Medicine