Age-Related Increases in Plasma Factor VIII and Von Willebrand Factor in a C57BL/6 Mouse Model Are Associated with Increased Factor VIII and Von Willebrand Factor Gene Expression and Reduced Expression of the Clearance Receptor, Stabilin-2

Background: Aging is associated with a state of hypercoagulability, as the result of increased concentrations of plasma coagulation proteins. Plasma levels of Factor VIII (FVIII) and von Willebrand factor (VWF) increase with age in humans, but the potential contribution of increases in gene expression with age has not been studied. These two proteins circulate in a non-covalent complex and are cleared together from plasma, hence, a reduction in the expression of their clearance receptors is also a possible pathogenetic explanation. In contrast, plasma levels of ADAMTS13 have been shown to be reduced in later life in humans, but again the mechanism responsible for this age-related pathophysiology is currently unknown. In this study, we utilized a mouse model in which age-related changes in plasma levels of FVIII, VWF and ADAMTS13 were initially documented. Here, we evaluated age-related changes in the gene expression of VWF, FVIII, ADAMTS13 and the clearance receptors low-density lipoprotein receptor-related protein 1 (LRP1), scavenger receptor class A member 5 (SCARA5) and Stabilin-2 (Stab2).

Methods: Liver, spleen and lung samples were collected from normal C57BL/6 mice at 9- (n=10), 55- (n=8) and 97-weeks of age (n=15). Also, liver and spleen samples were collected at 3-weeks of age (n=5). Total mRNA was isolated from the tissues and gene expression analysis performed through qRT-PCR by a two-step relative quantification against mouse GAPDH. Expression of murine Factor IX (f9) and Protein C (proc) genes were also measured as positive and negative controls, as the developmental expression of these genes has been extensively studied. The 9-weeks old mice were used as a reference, and expression levels in this group were set as 1. Results were expressed as the fold change median and 95% CI from the 9 week standard group. Data was log₁₀ transformed and compared with a Mann-Whitney test. Additionally, plasma levels of murine VWF, FVIII and ADAMTS13 were measured through ELISA, chromogenic assays and ELISA-based activity assays, respectively, in samples obtained at the same time-points examined for gene expression.

Results: Levels of VWF in plasma showed significant increases with age (p<0.0001), reaching a 2-fold increase by 97-weeks. Expression levels increased gradually with age in all three tissues evaluated, reaching a 1.4-fold increase in the lungs (p=0.008), 1.8-fold in the spleen (p=0.01) and 10.3-fold in the liver (p<0.0001) of 97-weeks old mice. When FVIII plasma levels were measured, a similar age-related increase was observed (p<0.0001). Expression levels increased significantly with age in the lungs by 2-fold (1.53-2.68, p=0.002), but no specific age-related changes were observed in liver and spleen. Plasma levels of mouse ADAMTS13 activity showed an opposite pattern to what has been reported for the human protein, with an age-related increase (p<0.0001). When ADAMTS13 gene expression was analyzed in the liver, higher levels were observed in the 3-week old group [1.32 (1.25-1.41), p=0.04], but no significant changes in expression occurred at later time points. Finally, gene expression analysis of LRP1, SCARA5 and Stab2 genes was performed in liver and spleen, the two main organs involved in VWF/FVIII clearance. Expression of these three receptor genes was significantly reduced in both tissues at 3-weeks (<0.04 fold for all estimates). Expression of LRP1 in the liver was an exception to this pattern, with a level that was similar to the 9-week old mice [1.44 (0.96-2.17), p=0.77]. Interestingly, no Stab2 expression was detected in the liver at any point. With aging, no significant changes occurred in SCARA5 and LRP1 gene expression that could be associated with higher plasma levels of VWF/FVIII. However, splenic Stab2 expression significantly decreased with age, reaching a 0.18-fold (0.13-0.25, p=0.02) reduction in the 97-weeks old spleen samples. The positive control gene used (f9) showed no increases in expression with age [1.11 (1.00-1.23), p=0.60], possibly due to strain differences with reported studies, while the negative control gene proc showed no changes [0.87 (0.82-0.93), p=0.28], as expected.

Conclusions: Changes in gene expression with increasing age appear to be contributing to the increases in VWF and FVIII plasma levels. Our studies have shown age-related increases in expression of the VWF and FVIII genes and reduced expression of the clearance receptor Stabilin-2.