טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTatour Mifleh
SubjectInvolvement of white blood cells heparanase in activation
of the coagulation system
DepartmentDepartment of Medicine
Supervisor Dr. Yona Nadir
Full Thesis textFull thesis text - English Version


Abstract

Heparanase is an endo-β-D-glucuronidase that is capable of cleaving heparan sulfate (HS) side chains of HS proteoglycans (HSPG) on cell surfaces and the extracellular matrix, activity that is strongly implicated in tumor metastasis and angiogenesis. Apart of its well characterized enzymatic activity, heparanase was noted to exert also enzymatic-independent functions.

Heparanase was also demonstrated to serve a major role in the hemostatic system. Heparanase over-expression in human leukemia, glioma and breast carcinoma cells resulted in marked increase in tissue factor (TF) levels verified by immunoblot and real-time PCR analyses. Likewise, TF was induced by exogenous addition of recombinant heparanase to tumor cells and primary endothelial cells, induction that was mediated by p38 phosphorylation and correlated with enhanced procoagulant activity. Notably, TF induction was independent of heparanase enzymatic activity, further expanding the multiple non-enzymatic effects of heparanase in normal and pathological processes. Furthermore, heparanase over-expression or exogenous addition induced 2-3 fold increase of TF pathway inhibitor (TFPI) expression, evident by immunoblotting and real-time PCR analyses. Similarly, heparanase stimulated accumulation of TFPI in the cell culture medium. A physical interaction between heparanase and TFPI was demonstrated, suggesting a mechanism by which secreted heparanase interacts with TFPI on the cell surface, leading to dissociation of TFPI from the cell membrane and increased coagulation activity, thus further supporting the local pro-thrombotic function of heparanase. Heparanase main storage in the body is located to the platelets, neutrophils and monocytes. Thus, most of the heparanase is circulation in our blood. Taking into account the pro-coagulant, pro-inflammatory and pro-metastatic properties of heparanase, we wished to explore the neutrophils heparanase regarding the mechanism of release and effect in the coagulation system. We hypothesized that neutrophils heparanase is released upon activation of the coagulation system and enhances the processes of coagulation and inflammation activation. In the present work we demonstrated that thrombin is a strong inducer of heparanase release. Other molecules including ATP, ADP, as well as lipopolysaccharide (LPS) immediately or after 4 hours incubation, had no effect on heparanase release. Effect of thrombin was found to be via protease-activated receptor 1 (PAR-1). PAR-2 and PAR-4 were not found to be significantly involved in the heparanase release. ERK intracellular pathway 2 was shown to convey the signal inside the neutrophil while other pathways including Src, PI3K and p38, were not found to be involved. A quantitative analysis of heparanase amount revealed that in a single platelet cell there is a larger amount of heparanase compared to one neutrophil and in a single neutrophil there is a larger amount compared to a monocyte and a lymphocyte. Considering the large number of platelets in the circulation compared to neutrophils, and the immediate release of both of them induced by thrombin, may imply that the main source of heparanase in the blood is the platelets. As neutrophils are the first to infiltrate injured tissues, local effect of heparanase released from neutrophils on wound healing regarding hemostasis and angiogenesis is to be further investigated.