Naive haemophilia mice displayed different pattern of cytokine profiles of cytokine profiles changes might be associated with subclinical bleeding

Subclinical bleeding is a haemorrhage event not clinically detected in haemophilia, and no reliable method is available for predicting subclinical bleeding. We investigated whether haemophilia mice have subclinical haemorrhage and evaluated potential biomarkers including multiple cytokine changes to predict subclinical haemorrhage. Plasma from naive FVIII-/- and FIX-/- mice and their wild-type counterparts (FVIII WT and FIX WT, respectively) were measured for prothrombin fragment 1 + 2 (F1 + 2) and multiple cytokines. Haemophilia mice with induced hemarthrosis were used as positive clinical bleeding controls. Naive haemophilia mice that displayed higher levels than positive bleeding control were counted. Univariate and multivariate analyses of cytokines were performed. Compared with wild-type mice (FVIII WT 1.1-6.2 vs. FIX WT 2.7-6.7 pmol/l), F1 + 2 widely varied in both haemophilia mouse strains (FVIII-/- 3.7-25.7 vs. FIX-/- 2.7-15.7 pmol/l). Each cytokine varied widely in both naive haemophilia A and B mice, but not significantly, for most cytokines. In comparison to haemophilia mice with hemarthrosis bleeding challenge, naive FVIII-/- mice had elevated pro-inflammatory cytokines and FIX-/- mice had elevated anti-inflammatory cytokines. In addition, interleukin (IL)-4, followed by IL-1, IL-6, TNF-α and MIP-1α in FVIII-/- mice and MIP-1α, followed by IL-1, IL-10 in FVIII-/- mice exhibited significant differences potentially associated with potential subclinical bleeding. Naive haemophilia mice showed elevated pro-inflammatory cytokines with different patterns, represented by pro-inflammatory cytokine elevation in more naive FVIII-/- mice and more anti-inflammatory cytokines in FIX-/- mice.