Functional aspects of prothrombin in bone

Prothrombin (PT) is the inactive zymogen to the serine protease α-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 αthrombin 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 α-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 αthrombin 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. POPULARVETENSKAPLIG SAMMANFATTNING I avhandlingen har blodkoagulationsproteinet protrombins (PT) aspekter pa ben studerats. Koagulationen initieras nar PT konverteras till proteaset α-trombin vilket sker i det cellbundna protrombinaskomplexet. Darefter omvandlar α-trombin fibrinogen till fibrin, som tillsammans med blodplattar bildar en propp. Protrombins och α-trombins roller i ben har tidigare studerats och det har pavisats samband mellan koagulation och olika inflammatoriska sjukdomstillstand dar en okad cellmedierad nedbrytning av benet sker. Osteoklasten ar den bennedbrytande cellen i kroppen vilken bildas da forstadieceller kommer i kontakt med proteinet RANKL. Darefter fuserar flera enkarniga celler ihop till en flerkarnig cell och i samband med detta borjar den flerkarniga osteoklasten att uttrycka proteiner sasom Tartrat resistent surt fosfatas (TRAP), Matrixmetalloproteas 9 och Katepsin K. Resultaten i avhandlingen visar att PT tillverkas av osteoklaster och att PT finns i narheten av osteoklaster i den nybildade benmatrixen. Dessutom pavisades att protrombinasaktiviteten i RANKL-stimulerade pre-osteoklastkulturer okade i samband med osteoklastens mognadsgrad. Osteoklastens mojlighet att omvandla PT till α-trombin hade samband med ett okat genuttryck samt en okad proteinniva av koagulationsfaktorn Vavnads faktor (TF) och av en okad proteinniva av koagulationsfaktorn FXa. Genom adhesion till PT eller osteopontin (OPN) isolerades tva olika populationer av flerkarniga TRAP-positiva osteoklastlika celler fran rattben. Tidigare okanda aspekter avslojades vid jamforelser mellan de PT-adherenta och OPN-adherenta cellerna. De OPN-adherenta cellerna uppvisade typiska osteoklastegenskaper och resorberade mineraliserat ben. Analyser av genuttryck, fagocytos, cellmigration samt immunolokalisering visade att de PT-adherenta cellerna ar flerkarniga benmargsmakrofager med mojliga roller i det medfodda immunsystemet, jarn-homeostas samt vid bildning av erytrocyter. Sammanfattningsvis visar resultaten i avhandlingen att osteoklasten ar en del av det externa koagulationssystemet samt att en subpopulation av benmargsmakrofager ar flerkarniga, TRAP-positiva och uttnytjar PT som en adhesions-ligand. LIST OF PUBLICATIONS I. Localization and Expression of Prothrombin in Rodent Long Bones. Erik Karlstrom, Maria Norgard, Kjell Hultenby, Eszter Somogyi-Ganss, Rachael Sugars, Goran Andersson and Mikael Wendel. Submitted manuscript. II. RANKL induces components of the extrinsic coagulation pathway in osteoclasts. Erik Karlstrom, Barbro Ek-Rylander, Mikael Wendel and Goran Andersson. Biochemical and Biophysical Research Communications, 394(3):593-9; 2010. III. Osteoclast size heterogeneity in rat long bones is associated with differences in adhesive ligand specificity. Yingwei Hu, Barbro Ek-Rylander, Erik Karlstrom, Mikael Wendel and Goran Andersson. Experimental Cell Research, 314(3):638-50; 2008. IV. Isolation and Characterization of a Multinucleated Tartrate-Resistant Acid Phosphatase-Positive Bone Marrow Macrophage. Erik Karlstrom, Barbro Ek-Rylander, Mikael Wendel and Goran Andersson. Submitted manuscript. Additional publications not included in the thesis ADAMTS-1 increases the three-dimensional growth of osteoblasts through type I collagen processing. Anders P. Rehn, Mark A. Birch, Erik Karlstrom, Mikael Wendel and Thomas Lind. Bone, 41(2):231-8; 2007. Expression of HMGB1 during tooth development. Rachael Sugars, Erik Karlstrom, Cia Christersson, Marie-Louise Olsson, Mikael Wendel and Kaj Fried. Cell and Tissue Research, 327(3):511-9; 2007.