Organ Size Control Figure 4. H&E staining of an E42 pig spleen implant 12 weeks following transplantation. Oversized spleen implant is shown in RAG2/2 FVIII KO compared to its non-hemophilic RAG2/2 counterpart. Bar indicates 2 mm. doi:10.1371/journal.pone.0008362.g004 endothelial cells, vascular smooth muscle cells (VSMC), mono- cytes, T lymphocytes and fibroblasts have been suggested[13–15]. Cellular effects of thrombin are mediated by PARs, members of the G protein-coupled receptors that carry their own ligand which remains cryptic until unmasked by proteolytic cleavage by thrombin[15,16]. Thrombin can also affect cells indirectly by cleaving OPN which is a secreted, multifunctional glycoprotein that exists either as a full-length molecule or as proteolytically cleaved fragments. A unique N-terminal fragment of OPN (trOPN), produced by thrombin cleavage has previously been shown to be present in plasma and milk and may have physiologically distinct roles[17-19]. In particular, It has been previously demonstrated by Nilsson et al. [20] that trOPN acts as a negative regulator of both human and mouse hemopoietic stem and progenitor cell (HSC/HPC) proliferation and differentiation in vitro. Recently this group had expanded their study and has shown that in the bone marrow OPN exists predominantly in its thrombin-cleaved forms. Notably, the N-terminal cleavage fragment binds to both alpha9beta1 and alpha4beta1 integrins, which are expressed on HSC/HPC and by that plays a key role in the attraction, retention, regulation, and release of hematopoietic stem and progenitor cells to, in, and from their BM niche [21]. Thus, it was of interest to define whether thrombin enhances implant size through PAR signaling or via its effect on OPN. In order to investigate thrombin’s receptors PAR signaling we have used specific PAR-1 and PAR-4 antagonists which were previously described in the literature as potent agents in blocking thrombin activation[22–25]. As can be seen in Fig. 6, treatment of non- hemophilic recipients with PAR1 or PAR4 antagonists did not reveal any enhancement of pig spleen implants. In contrast, treatment of Factor VIII KO recipients of pig spleen implants with trOPN exhibited a significant inhibition of implant size compa- rable to that found in the non-hemophlic recipients, suggesting that the regulatory role of thrombin is mediated through trOPN, cleaved by thrombin from OPN. Discussion Organ size control during embryonic development or in tissue regeneration, involves a fine balance between cell growth, proliferation and death, maintained by extrinsic and intrinsic factors[1–4]. Our assay using heterologous embryonic transplantation provides an additional tool in the study of growth control, in that it enables investigation of the cross-talk between extrinsic host factors and intrinsic mechanisms relevant to size control in the implant. In particular, this assay enables pinpointing the role of different extrinsic genes by using mutated or KO host mice. Thus, we demonstrate, for the first time, that host Factor VIII plays a critical role as an extrinsic factor in controlling the final size of pig-derived organs. The lack of any effect on mouse embryonic transplants, regardless of whether the host or the donor is derived from a hemophilic or non-hemophilic mouse, suggests that the role of Factor VIII is likely limited to oversize growth checkpoint that operates upon implantation of tissue from a larger animal, such as the pig. Thus, if we assume that mouse and pig embryonic implants are endowed with stem cell pools of different size prior to transplantation, they are likely to attain different organ sizes upon completion of growth and differentiation in the mouse recipients. Consequently, in hemophilic mice lacking the potential inhibitory activity (i.e. overgrowth checkpoint) mediated by Factor VIII, the size of the pig implants is likely to be larger, while mouse implants growing to their normal expected size will not exhibit excessive growth, and therefore will not be subject to Factor VIII control. Further interrogation of other factors downstream of Factor VIII in the coagulation cascade revealed that thrombin is probably PLoS ONE | www.plosone.org 6 December 2009 | Volume 4 | Issue 12 | e8362