Expression of Blood Coagulation Factor X Is Not Liver-Restricted.

Abstract Factor X (FX) is a vitamin K-dependent clotting factor that plays a critical role in blood coagulation by catalyzing the conversion of prothrombin to thrombin. Although predominantly found in liver, expression of FX is not liver-restricted as shown in previous studies (JBC 271:2323–2331, 1996; EMBO J. 11:467–472, 1992). Expression of FX was also detected in whole mouse embryo extracts by RT-PCR as early as E7.5, prior to the formation of a liver bud (TH 84:1023–1030, 2000). Other studies have suggested additional biological functions for FX that are independent of its role in blood coagulation, including stimulation of mitogenic activity in endothelial cells, enhancement of platelet-derived growth factor (PDGF) release from vascular smooth muscle cells, induction of cytokine production, and up-regulation of the early growth response-1 (egr-1) gene transcription. FX-knockout [FX (−/−)] mice generated by our group as well as by Dewerchin et al. showed partial embryonic lethality beginning as early as E10.5 and fatal perinatal bleeding in the remaining FX (−/−) mice surviving to term. However, as for several other coagulation-related knock-out mice, the exact cause of embryonic lethality observed in some FX (−/−) mice remains to be deciphered. In light of these observations, we sought to determine the spatial and temporal patterns of FX expression in both developing and adult mice. Our preliminary studies, utilizing the combined techniques of Northern blot analysis, immuno-histochemistry, and in situ hybridization revealed the following. Northern blot analysis of mRNA isolated from different tissues of wild-type (+/+) adult mice showed FX transcript in multiple tissues including liver, stomach, spleen, lung, colon, ovaries, placenta, and heart (in decreasing levels of FX expression). Results of immuno-histochemistry on selective adult mouse tissues were similar to the results of Northern blot analysis with the exception of kidney, in which we found FX protein in the cortical, but not in the medullary region. Specifically, we noted expression of FX protein in the bronchi/bronchioles of the lung, and selective cells in the myocardium and in pancreas. However, despite detection of FX transcript in spleen and placenta, we failed to detect FX protein in either of these tissues. In developing embryos, immuno-histochemistry revealed expression of FX protein in liver, small intestines, and thymus for wild-type E14.5 paraffin-embedded sagittal sections, and expression of FX protein in liver and selective cells within the brain for E15.5 sagittal sections. Additionally, we carried out in situ hybridization of paraffin-embedded sagittal sections, using digoxigenin-labeled Factor X antisense riboprobe constructed from a 1 kb fragment of the 5′ end of the murine FX cDNA (identical to the probe used in Northern blot). For E12.5 embryos, FX transcript was found predominantly in the liver. For E14.5 embryos, we detected FX transcript not only in the liver, but also in the kidney (specifically in the primitive glomeruli). For E15.5 embryos, we noted FX transcripts in liver, lung, and selective cells in the brain. Already in progress, additional studies including both FX (+/+) and FX (−/−) embryos at earlier stages of development (for example E9.5 to E13.5) and additional adult tissues should provide a more complete delineation of the spatial and temporal patterns of FX expression in mouse development and adulthood. In conclusion, expression of murine FX is not restricted to liver during embryonic development or during adulthood.