Enhancement of Pig Embryonic Implants in Factor VIII KO Mice: A Novel Role for the Coagulation Cascade in Organ Size Control Anna Aronovich1, Dalit Tchorsh1, Elias Shezen1, Chava Rosen1, Yael Klionsky1, Sivan Cohen1, Orna Tal1, Uri Martinowitz2, Helena Katchman1, Smadar Eventov-Friedman1, Ninette Amariglio3, Jasmine Jacob-Hirsch3, Gideon Rechavi3, Yair Reisner1* 1 Department of Immunology, Weizmann Institute of Science, Rehovot, Israel, 2 The Israel National Hemophilia Center, Sheba Medical Center, Tel Hashomer, Israel, 3 Cancer Research Center, Sheba Medical Center, Tel Hashomer, Israel Abstract Very little is known about the mechanisms that contribute to organ size differences between species. In the present study, we used a mouse model of embryonic pig tissue implantation to define the role of host Factor VIII in controlling the final size attained by the implant. We show here that pig embryonic spleen, pancreas, and liver all grow to an increased size in mice that are deficient in the Factor VIII clotting cascade. Similar results were obtained using the transplantation model after treatment with the low molecular weight heparin derivative Clexane which markedly enhanced transplant size. Likewise, enhanced size was found upon treatment with the direct thrombin inhibitor Dabigatran, suggesting that organ size regulation might be mediated by thrombin, downstream of Factor VIII. Considering that thrombin was shown to mediate various functions unrelated to blood clotting, either directly by cleavage of protease-activated receptors (PARs) or indirectly by cleaving osteopontin (OPN) on stroma cells, the role of PAR1 and PAR4 antagonists as well as treatment with cleaved form of OPN (tcOPN) were tested. While the former was not found to have an impact on overgrowth of embryonic pig spleen implants, marked reduction of size was noted upon treatment with the (tcOPN). Collectively, our surprising set of observations suggests that factors of the coagulation cascade have a novel role in organ size control. Citation: Aronovich A, Tchorsh D, Shezen E, Rosen C, Klionsky Y, et al. (2009) Enhancement of Pig Embryonic Implants in Factor VIII KO Mice: A Novel Role for the Coagulation Cascade in Organ Size Control. PLoS ONE 4(12): e8362. doi:10.1371/journal.pone.0008362 Editor: Martin Pera, University of Southern California, United States of America Received September 22, 2009; Accepted November 23, 2009; Published December 21, 2009 Copyright: 2009 Aronovich et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Mrs. Erica Drake and the Gabriella Rich Center for Transplantation Biology Research. The funding supported mice and other supplies. There was no other funding. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: yair.reisner@weizmann.ac.il Introduction Despite great progress in the molecular dissection of embryo- genesis, the mechanisms controlling the size of tissues and organs remain a mystery. In general, it is thought that extrinsic control mechanisms are associated with nutrition or systemic growth signaling factors, such as those operating through the insulin/ PI3K or the TOR pathways, while intrinsic mechanisms are likely linked to patterning morphogens and apoptosis-signaling com- plexes, as well as to stem cell numbers[1–4]. Some of the early insights in this area of investigation came from transplantation experiments in which infant rat hearts or kidneys transplanted into adult rats grew and attained the size of an adult organ[5]. Similar results were also found by Metcalf et al. in early transplantation experiments with fetal mouse organs. For example, when multiple fetal mouse thymus glands were transplanted into an adult mouse, each was found to grow to its normal adult size[6]. However, when the same experiment was performed using fetal spleens, the total mass of the transplanted spleen tissue attained the mass of only a single normal adult spleen, suggesting that the growth of these fetal tissues is limited by extrinsic factors, outside the spleen[7]. Very recently, we defined optimal ‘windows’ of gestation of human and pig embryonic precursor tissues that afford optimal growth and development of liver, pancreas and spleen upon implantation into NOD-SCID mice[8]. Our studies enabled not only a rational selection of fetal tissue for transplantation but, among many parameters, also provide a system in which to manipulate and study organ size control. Using this system, we have now found, surprisingly, that a difference in the functionality of one gene in the recipients, namely coagulation Factor VIII, has a major impact on the final size attained by different embryonic pig tissues upon transplantation into mice. Interrogation of other factors along the coagulation cascade that are triggered by Factor VIII, such as Factor Xa and thrombin, suggests that thrombin, which is the terminal enzyme of the hemostatic system, is likely to be the actual mediator of this novel checkpoint of organ size control. Furthermore, our ability to reverse the enhancement of implant size by treatment with the cleaved form of OPN that is released upon treatment with thrombin, strongly indicates that thrombin likely exerts its regulatory activity through interaction with OPN on stroma cells, as previously shown for maintenance of the blood tissue size under normal steady state hoemostasis[9,10]. PLoS ONE | www.plosone.org 1 December 2009 | Volume 4 | Issue 12 | e8362