Organ Size Control Figure 3. Enhancement of pancreas and liver size and function in Factor VIII KO- SCID mice. (A) Pig insulin serum levels following implantation of E42 pig pancreas into Factor VIII KO SCID and NOD-SCID mice. Data represent averages of four independent experiments (P,0.005). (B) Morphometric analysis of E42 pancreas implants growth. Both recipients were evaluated for graft volume (left column) and beta cells volume (middle column) for pig insulin positive beta cell volume (right column), 3 months after transplantation (n = 5). The specific difference in volume was not associated with a significant increase in b cell number. (C) Normal histological findings following E42 pig pancreas transplantation under the kidney capsule of NOD-SCID and Factor VIII KO-SCID mice. Pig insulin appears red, while glucagon is stained green. (D) Pig albumin serum levels following implantation of E42 pig liver precursor tissue into Factor VIII KO SCID and NOD-SCID mice. Data represent three independent experiments (P,0.05). Illustration of increased growth and retained functionality of the liver grafts in Factor FIII KO-SCID recipient, demonstrated by H&E staining (E), and immunohistological staining of pig albumin (F), and PAS (G). doi:10.1371/journal.pone.0008362.g003 thrombin is activated by Factor Xa (Fig. 5A). Thus, either of these downstream factors could potentially mediate the observed size enhancement in our assays. One approach to dissect the role of these factors was to use the anti-coagulant, Clexane (a low molecular weight heparin deriva- tive) which blocks Factor Xa and, to a lesser degree, thrombin. We evaluated organ size in the pig liver transplantation model in the presence or absence of Clexane administration. Implantation of pig embryonic liver provides a rapid assay, as growth can be monitored by the appearance of pig albumin (detectable by specific ELISA) in the mouse serum as early as 7 days post transplant. As can be seen in Fig. 5B, Clexane administration in non-hemophilic recipients induced marked enhancements of pig albumin blood levels on days 7, 14 and 21 post transplant compared to control recipients, not receiving Clexane. Since Clexane could potentially block not only Factor Xa, but also inhibits thrombin to some extent, further analysis was performed using a more specific inhibitor, namely, Dabigatran, which blocks only thrombin. As can be seen in Fig. 5C, Dabigatran administration led to marked enhancement of pig embryonic liver implants, similar to that exhibited by Clexane. Representative E42 spleen transplant with and without Dabiga- tran administration is shown in Fig. 5D. Those results identify thrombin as a potential direct player in the enhancement of pig transplants growth. The Role of PAR’s and OPN Cleavage by Thrombin Thrombin is a serine protease that transforms fibrinogen into fibrin and activates blood platelets. However in addition to its role in coagulation, multiple effects on a variety of cell types including PLoS ONE | www.plosone.org 5 December 2009 | Volume 4 | Issue 12 | e8362