A Novel Role For Factor Viii And Other Coagulation Factors In Organ Size Control

Abstract Despite great progress in molecular genetics and embryogenesis, size control of tissues and organs remains a mystery. In general, it seems that extrinsic mechanisms are associated with nutrition or systemic growth factors signaling such as the Insulin/PI3K or the TOR pathways, while intrinsic mechanisms are likely linked to patterning morphogens and apoptosis signaling complexes. Very recently we defined optimal ‘windows’ of gestation of human and pig embryonic precursor tissues which afford optimal growth and development of liver, pancreas and spleen upon implantation into NOD-SCID mice. This type of study enables not only a rational selection of tissue for transplantation but, among many parameters, it also provides a system and ability to manipulate organ size control. Using this system we surprisingly found that pig embryonic implants grow to a significantly larger size in Factor VIII Knock Out (KO) NOD-SCID mice. The average size of E42 pig spleen implant grown for 3 months in Factor VIII KO SCID recipients was 6.78 ± 2.16gr compared to 2.46 ± 0.82gr in non-hemophilic NOD-SCID recipients (p<0.05). Histological examination of the growing spleen implants revealed normal growth, development and vascularization patterns comparable to those found in the corresponding Factor VIII wild type mice, ruling out potential induction of a malignant process. A similar enhancement of implant size was exhibited by pig embryonic pancreas precursor tissue. The growth of pancreatic tissues was determined by monitoring blood levels of pig insulin using a specific ELISA, previously shown to correlate with implant size. Likewise, the total volume of insulin positive cells was 0.68 ± 0.21mm3 in Factor VIII NOD-SCID versus 0.23 ± 0.18mm3 in non-hemophilic NOD-SCID recipients, respectively, suggesting an overall enhancement of implant size by a factor of three (p<0.05). A significant enhancement, by at least a factor of two of implant size, was also found upon implantation of embryonic pig liver in Factor VIII KO NOD-SCID mice, as evaluated by ELISA for pig albumin blood levels. In order to rule out potential artifacts due to genetic abnormalities in NOD-SCID FVIII KO mice, we attempted to introduce the Factor VIII KO mutation into a different SCID mouse, namely, RAG−/−. Thus, a new RAG−/− FVIII KO colony was established and similar results of oversized implants were achieved. In contrast to the pig implants, organ size was not enhanced upon implantation of mouse embryonic tissue into Factor VIII KO SCID mice. The lack of any effect on mouse embryonic transplants suggests that the role of Factor VIII is likely limited to a checkpoint of excessive growth (i.e. overgrowth checkpoint) which only operates upon implantation of tissue from a larger animal, such as the pig. To test the potential control by Factor VIII of oversized organs in a more physiological setting, we further tested its role in controlling G-CSF mediated splenomegaly. Thus, treatment with G-CSF of Factor VIII KO C57BL/6 mice was associated with significantly enhanced splenomegaly, compared to that found in the non-hemophilic counterparts, upon infusion of the same doses of G-CSF. Average spleen weight in the former group was 271.03 ± 154.86 mg, compared to 182.5 ± 58.43 mg in the latter group, p = 0.022). Furthermore, continuous administration by osmotic pumps of exogenous Factor VIII inhibited the enhanced splenomegaly in SCID FVIII KO mice under G-CSF stimulation (P<0.033)). Considering that Factor VIII KO could potentially affect the activity of other factors along the coagulation cascade, we further interrogated the role of other factors in the two models for organ size control. Thus, we found that enoxaparin, which inhibits the activity of both factor Xa and thrombin, markedly enhances G-CSF splenomegaly (p = 0.0017), as well as the size of pig embryonic liver implants in SCID mice with wild type Factor VIII (p<0.05). Likewise, a direct thrombin inhibitor (dabigatran) was found to significantly enhance G-CSF splenomegaly (p=0.0004), suggesting that it might be mediated by thrombin downstream of Factor VIII. Indeed, apart from its role in clot formation, thrombin has been shown to be capable of signaling through Protease-Activated Receptors (PARs), which are expressed throughout the body and known to be involved in vascular responses, embryonic development and malignancies. Collectively, our surprising set of observations suggests a yet unknown role in organ size control for factors of the coagulation cascade.