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Reason: Under embargo until Nov 2014. After this date a copy can be supplied under Section 51(2) of the Australian Copyright Act 1968 by submitting a document delivery request through your library

Investigation of signaling cross-talk between platelets and neutrophils

thesis
posted on 2017-01-09, 00:56 authored by Ghasemzadeh, Mehran
Summary Platelet activation is known to be associated with the release of a vast array of chemokines and proinflammatory lipids which induce pleiotropic effects on a wide variety of tissues and cells, including neutrophils. Protein ectodomain shedding and microparticle formation are also important processes occurring during platelet activation, although their physiological functions have not been fully defined. During thrombosis, the recruitment of neutrophils to activated platelets is considered an important step linking thrombosis to inflammatory responses. This phenomenon is highly regulated and influenced by the cross-talk between platelets and neutrophils. This cross-talk involves a variety of mediators including adhesion molecules, chemokines, shed proteins and various proinflammatory lipids. The initial studies presented in this thesis have examined the impact of platelet shed proteins on neutrophil adhesion and activation. These studies demonstrated that the most extensive surface protein shedding was observed in the procoagulant phenotype of platelets (PS positive) so-called SCIP (Sustained Calcium-Induced Platelet morphology) suggesting that ectodomain shedding is influenced by different platelet activation states. I have found that these shed proteins can negatively regulate the adhesive function of platelets, by reducing integrin alphaIIb beta3 engagement of immobilized fibrinogen, leading to decreased platelet adhesion and spreading. However, this platelet inhibitory activity was spread out over at least 4 distinct chromatographic peaks, making purification of the biological active molecules challenging. Notably, shed material from the surface of platelets appeared to have limited capacity to regulate neutrophil adhesion or activation, with most of the neutrophil activating activity residing within the granule component of activated platelets. Studies presented in chapter 4 have focused on the identification and characterization of the major platelet released product(s) regulating neutrophil adhesive function. Initial studies excluded a major contribution for released small molecules (<5kDa) in inducing neutrophil activation. Through the use of different chromatographic procedures I was able to identify the alpha-granule protein NAP-2 (Neutrophil Activating Protein-2) as the principal component released by platelets regulating neutrophil adhesive function. This was somewhat unexpected, given previous findings for an important role for soluble P-selectin and shed CD40L in regulating neutrophil activation. Subsequent studies in chapter 5 investigated the distribution of neutrophil activating activity in various platelet fractions and revealed that the majority of activity resided on/within intact platelets (~70%), with ~20% found in the microparticle (MP)-free releasate and ~10% in MP. In addition to the unexpected activity distribution, the activation status of platelets also had a major influence on their ability to modulate neutrophil reactivity. Platelet degranulation and P-selectin expression was critical for inducing heterotypic platelet-neutrophil adhesion and for an increase in Mac-1 expression/activation, a process enhanced by platelet conversion to SCIPs. Analysis of the key proinflammatory mediators promoting Mac-1 activation revealed an important co-operative role for PAF, leukotrienes and NAP-2 in promoting neutrophil activation. Taken together, these studies suggest an important role for transcellular lipid metabolism between platelets and neutrophils in enhancing subsequent neutrophil activation. The studies presented in chapter 6 reveal an important feedback activation mechanism operating between platelet and neutrophils, that serves to enhance the procoagulant function of platelets. These studies reveal that the aggregation of neutrophils with TRAP-stimulated platelets not only enhances Mac-1 activation, but also feeds back on platelets to enhance cytosolic calcium flux. This feedback function of neutrophils was dependent on Mac-1 ligand engagement, P-selectin and PAF-generation and was associated with the conversion of platelets from a PS negative to a PS positive phenotype, leading to enhanced platelet procoagulant function. The exact molecular mechanism by which neutrophils induce platelet procoagulant formation remains to be fully delineated, however given the importance of these cells in promoting innate immune and inflammatory functions, such mechanisms may have pathophysiological importance. In summary, the results from my PhD thesis have provided new insights into the complex regulation of neutrophil activation by various components of activated platelets. The finding that neutrophils can provide feedback regulation of platelets to promote procoagulant function may provide new insight into the role of transcellular activation mechanisms in promoting inflammatory and thrombotic responses in a variety of human diseases.

History

Principal supervisor

Shaun Jackson

Year of Award

2009

Department, School or Centre

Australian Centre for Blood Diseases

Campus location

Australia

Course

Doctor of Philosophy

Degree Type

DOCTORATE

Faculty

Faculty of Medicine Nursing and Health Sciences