Organ Size Control Figure 5. Direct involvement of coagulation cascade factors in enhancement of growth of pig embryonic transplants. A. Coagulation cascade scheme. B. Enhancement of embryonic pig liver growth by Clexane administration in Rag2/2 mice C. Enhancement of embryonic pig liver growth by Dabigatran administration in Rag2/2 mice. Pig albumin serum levels, detected by specific ELISA, are shown at 7, 14 and 21 days after implantation of E42 pig liver precursor tissue in the presence or absence of Dabigatran administration. The results are compared to those obtained in hemophilic Rag2/2 factor VIII KO mice. D. E42 pig spleen transplant with and without Dabigatran administration in hemophilic Rag2/2 factor VIII KO mice. doi:10.1371/journal.pone.0008362.g005 the Factor VIII downstream factor that actually mediates regulation of oversize control in our models. Thus, as outlined schematically in Fig. 7, interference with steady state levels of factors of the coagulation cascade, such as occurs in Factor VIII KO mice or upon blockade of Factor Xa or thrombin, also overrules this checkpoint, thereby leading to oversized organs. The suggestion that thrombin is a likely candidate for the actual inhibitory activity leading to oversize control, is supported by its terminal position in the hemostatic system, acting downstream of Factor VIII and Factor Xa. The possibility that thrombin is indeed the bridge between the coagulation system and the checkpoint of organomegaly, is in line with a substantial body of literature demonstrating involvement of thrombin in various functions unrelated to blood clotting. Thus, thrombin is capable of signaling through PARs[26], which are expressed throughout the body and known to be involved in vascular responses[27], embryonic development[28], and malig- nancies[29]. Interestingly, several of these activities, including platelet activation and stimulation of tumor cell prolifera- tion[30,31], are associated with promotion of growth, which is in opposition to our suggestion that inhibition of thrombin leads to massive overgrowth. Indeed, we found that treatment with PAR1 and PAR4 antagonists did not affect implants size in our assay. While PAR signaling pathways might control stem/progenitor cells directly, another major pathway could potentially be mediated non-autonomously by stromal cells in the stem cell niche, through the cleavage of osteopontin (Spp1, OPN) by thrombin. Osteopontin (Spp1, OPN) is a highly acidic phospho- protein with pleiotropic effects, including regulation of inflamma- tion, cell adhesion and angiogenesis. OPN has recently been demonstrated to be a pivotal molecule in limiting the size of the HSC pool[32]. Interestingly, it has been shown that the thrombin- cleaved form of OPN promotes the quiescence of HSCs by PLoS ONE | www.plosone.org 7 December 2009 | Volume 4 | Issue 12 | e8362