Figure 6. The role og PARS and OPN cleavage by thrombin in defining size of E42 pig embryonic spleen implants. E42 pig spleen tissue was implanted into NOD-SCID and FactoorVIII KO NOD - SCID mice and final size of the implant was defined 4 weeks after transplantation. To define the effect of PARs cleavage by thrombin, PAR-1 and PAR-4 antagonists were administered to NOD-SCID recipients as described in Methods. To evaluate the role of OPN cleavage, trOPN was administered to Factor VIII NOD-SCID recipients as described in Methods. Each group comprised 7 mice. Enhancement of growth of E42 spleen tissue in Factor VIII NOD-SCID mice compared to NOD-SCID mice was statistically significant (t test, p,0.005)). This enhancement was significantly inhibited upon treatment with trOPN (p,0.05). doi:10.1371/journal.pone.0008362.g006 exerting a profound suppression of proliferation of HSCs, without inducing apoptosis[9]. OPN is a negative regulatory element of the stem cell niche that limits the size of the stem cell pool and may provide a mechanism for restricting excess organ growth. Thrombin is the major serine protease that cleaves OPN, giving rise to a 24-kDa and a 45-kDa fragment. The 45-kDa fragment has multiple functional advantages in processes such as cell attachment, migration, and spreading, through binding to a9b1 and a4b11. Our present finding that treatment of factor VIII KO recipients of pig embryonic spleen exhibit a deuced implant size, Organ Size Control similarly to that found in the non-hemophilic recipients, strongly indicates that thrombin likely exerts its regulatory effect through cleavage of OPN . While studies on the HSC niche in the bone marrow are relatively well advanced[33-35]very little is known regarding the stem cell niche in the embryonic pig tissue used for transplantation in our assays. The new insights on the role of thrombin and tcOPN as well as the well characterized binding of the latter to the integrins a9b1 and a4b11, could facilitate our ability to identify and localize stem cell - niche interactions which might occur in non –hematopoietic organs and tissues. However, other possibilities that might underline the inhibitory activity exerted by tcOPN, including potential effect on the vasculature should be considered. In conclusion, our data suggest a novel role for factors of the coagulation cascade in organ size control. In particular, we were able to pinpoint thrombin and its substrate OPN as the direct players. This surprising finding may not only offer new means to enhance or reduce the final size of implanted xenogeneic embryonic tissues, but might also adds a novel insight into the mysterious question of organ size control in mammals. Clearly, further studies investigating the role of the coagulation cascade factors in more physiological settings, and in particular their potential ability to affect normal tissue stem cells under extreme growth stimuli, are warranted. Materials and Methods Animals All animals were maintained under conditions approved by the Institutional Animal Care and Use Committee at the Weizmann Institute. The study protocol was approved by the ethics committees at Kibbutz Lahav and the Weizmann Institute. In these experiments, 8–10 week old immune deficient NOD-SCID mice and NOD-SCID Factor VIII KO mice (Weizmann Institute Animal Breeding Center, Rehovot, Israel) or RAG2/2 mice and RAG2/2 FVIII KO mice were used as hosts for the transplantation studies. To obtain immunodeficient hemophilic mice (designated Factor VIII KO–SCID), FVIII-deficient mice Figure 7. Overgrowth stimulus regulation by factors in the coagulation cascade. doi:10.1371/journal.pone.0008362.g007 PLoS ONE | www.plosone.org 8 December 2009 | Volume 4 | Issue 12 | e8362