Controlling thrombosis is central to management of various pathologies. Studies proposed here will identify a new, unexpected path to thrombosis, involving the enzyme Lysyl Oxidase (LOX). The proposed work looks at LOX effects on platelet function and thrombosis in primary myelofibrosis (PMF), a pathology hallmarked by proliferation and clustering of megakaryocytes, and myelofibrosis in bone marrow. Data from the Swedish Cancer Register from 1980 to 2009 showed that in a cohort of 11,155 patients with myeloproliferative neoplasms and 44, 620 matched healthy controls, the risk of arterial thrombosis was significantly 4.9-fold increased (4.8-5.0 p<0.001) in the patients compared to matched controls, highlighting the importance of studying regulators implicated in controlling thrombosis associated with this pathology. LOX is known to oxidize peptidyl lysines, leading to cross-linked matrix proteins in the bone marrow niche. Our published studied identified a link between LOX upregulation in megakaryocytes and bone marrow fibrotic progression in a mouse model of myelofibrosis. Importantly, more recently we found LOX expression to be vastly upregulated in platelets of patients with PMF (of which the majority tested carry the JAK2V617F or calreticulin gene mutations), compared to matching controls, and platelets isolated from PMF patients have greater adhesion to type I collagen, compared to controls. Further, transgenic mouse platelets engineered to overexpress LOX, at a level similar to cells from a myelofibrotic mouse model, show increased adhesion to monomeric collagen, and significantly greater propensity for arterial thrombosis in vivo. Considering LOX known activity, we hypothesized that it influences platelets through lysine oxidation of at least one of the collagen receptors, further supported by preliminary studies involving Oxy-Western blot analysis. Thus, we propose two specific aims of research:
Aim 1. To explore the mechanism by which LOX affects platelet response to collagen using proteomics approaches, and mouse and human MPN samples;
Aim 2. To determine the relevance of LOX- regulated thrombosis in vivo in myelofibrotic mice, and the role of collagen receptors in this effect. Pursuing these aims will be facilitated by: 1) a new transgenic mouse line produced in our lab, in which platelet LOX level is upregulated under wild type background (free of MPN), and 2) the availability of a new LOX inhibitor, and Lox gene loss of function studies. This work is innovative in that we are the first to reveal a link between LOX and collagen receptors activation. Further, these receptors were not suspected before to be regulated by oxidation. Our research is significant in that it will shed new light on basic mechanisms of platelet activation, in general, and implicate LOX in thrombotic events related to PMF, thus, recognizing the LOX pathway as new potential target for future anti-thrombosis drug development.
Controlling thrombosis is central to management of various pathologies. Studies proposed here will identify a new, unexpected path to thrombosis, involving the enzyme Lysyl Oxidase (LOX; a cross linking enzyme), with significant bearing on understanding one of the mechanisms by which thrombosis is augmented in states of primary myelofibrosis, a disease hallmarked by replacement of normal structures in the bone marrow by scar tissue, characterized by the excess deposition and cross-linking of matrix proteins. Considering that LOX is also overexpressed in other pathologies associated with increased thrombosis, such as chronic kidney disease, or arterial stenosis, our proposed focus on elucidating mechanisms of LOX effect on platelet function has far reaching implications. !
|Leiva, Orly; Leon, Catherine; Kah Ng, Seng et al. (2018) The role of extracellular matrix stiffness in megakaryocyte and platelet development and function. Am J Hematol 93:430-441|