Functional aspects of prothrombin in bone

Abstract: Prothrombin (PT) is the inactive zymogen to the serine protease alpha-thrombin which in the coagulation cascade is responsible for clot formation through converting fibrinogen to fibrin. PT has been shown to be present in calcified bone matrix, and alpha-thrombin has been implicated in various bone resorbing disorders of inflammatory origin, such as rheumatoid arthritis and periodontitis. Bone resorption is mediated by the multinucleated osteoclast, differentiated from the monocyte/macrophage lineage in the presence of the cytokine RANKL, which induces expression of osteoclastic genes such as tartrate resistant acid phosphatase (TRAP), matrix metalloproteinase 9 and cathepsin K. The thesis is divided into studies of the localization and expression of PT in bone, conversion of PT to alpha-thrombin by osteoclasts, adhesion of multinucleated TRAP positive bone cells to PT and osteopontin (OPN), and phenotypical heterogeneities between multinucleated cells adhering to PT or OPN. The results show that PT is synthesized by osteoclasts and located in close association to osteoclasts predominantly in the newly formed bone matrix of the metaphysis. Furthermore, the prothrombinase activity in RANKL stimulated pre-osteoclast cultures was increasing during osteoclast differentiation. The prothrombinase activity correlated to enhanced mRNA and protein levels of tissue factor as well as protein levels of coagulation factor Xa. Moreover, novel aspects were found in comparison between two TRAP-positive multinucleated bone cells isolated from neonatal rat long bone through adhesion to PT or OPN. In comparison to PT-adherent cells, OPN-adherent cells displayed osteoclast characteristics and efficiently resorbed mineralized bone. Gene expression, immunolocalization, TRAP promoter utilization, and phagocytosis showed that the PTadherent cell is a multinucleated bone marrow macrophage with a putative role in innate immune responses, iron homeostasis and erytropoiesis. In addition, in the presence of M-CSF the PT-adherent cell enhanced its migration on PT. In contrast, MCSF had an inhibitory effect on OPN-adherent cell migration on OPN. In conclusion, the results in the thesis demonstrate that osteoclasts convert PT to alpha- thrombin by utilizing components of the extrinsic coagulation pathway and that a TRAP-positive multinucleated subpopulation of bone marrow macrophages utilizes PT as an adhesive ligand.