The Consumption of Plasminogen Following Severe Burn and Its Implications in Muscle Calcification

INTRODUCTION Burn and blast injuries present with serious risk for complications including fibrosis and calcification of the soft tissues. Plasmin, the active form of plasminogen, is the key serine protease responsible for the degradation of fibrin (fibrinolysis). Our lab has previously shown that plasmin is essential for the prevention of muscle fibrosis and calcification following injury. We hypothesized that 1) plasminogen is consumed in response to severe burn, and 2) this transient plasminogen deficiency impairs proper muscle regeneration. METHODS To test our first hypothesis in humans, we collected blood from 10 control subjects at one time‐point and 20 burn patients with burns ranging from 25–75% total body surface area (TBSA) at 0, 3, 7, and 14 days post‐admission. Plasma plasminogen levels were assessed by human plasminogen ELISA. Methods including human subjects were pre‐approved by Vanderbilt's IRB. Because our lab has demonstrated that plasmin activity prevents muscle calcification, we used the formation of dystrophic calcification and as an output measure for impaired muscle regeneration. To assess muscle regeneration following severe burn in mice, we induced bilateral muscle injuries in the mice by injecting cardiotoxin (CTX) into the calf muscles and then we administered a 30% TBSA burn to the dorsum by scalding in 100°C water. Control groups received either the burn alone or the CTX injection alone. Another group of mice received the same injury and burn concurrent with a subcutaneous injection of either an antisense oligonucleotide (ASO) against plasmin's inhibitor, α2‐antiplasmin, or a control ASO, beginning 2 weeks prior to injury. Following the burn injury, weekly radiographs of the lower extremities were taken up to 28 days post‐injury (DPI) to assess dystrophic calcification. Following sacrifice at 28 DPI, ex vivo micro‐computed tomography (μCT) was performed on the lower extremities to further assess muscle calcification. To assess plasminogen consumption following severe burn in mice, we collected blood from animals with either CTX‐induced injury, injury with burn (as described above) or no injury following sacrifice at 3 DPI. Plasma plasminogen levels were assessed by mouse plasminogen ELISA. Vanderbilt IACUC pre‐approved all animal experiments. RESULTS Burn patients exhibited a significant plasminogen deficit compared to control subjects at 0 and 3 days post‐admission, followed by a return to control plasminogen levels between 7 and 14 days ( Figure 1 ). Consistent with the human subjects, mice exhibited a significant deficit in circulating plasminogen following burn when compared to both uninjured mice and mice given CTX injection alone. In the animal models assessing muscle regeneration, following the CTX‐induced injury alone, mice did not form significant dystrophic calcification in the injured muscles, but the mice that received the CTX injection and the burn developed dystrophic calcification in the injured muscle at 7 DPI. This phenotype in the burn mice was rescued by a systemic increase in plasmin activity through weekly administration of α2AP ASO ( Figure 2 ). DISCUSSION While many response mechanisms following severe burn have been implicated in impaired healing, the loss of plasminogen and the consequences of this have not been well characterized. Conclusively, this study suggests that severe burn increases the risk of the formation of calcification in injured muscle due to consumption of plasminogen. Support or Funding Information NIH T32 AR059039‐06A1 Rheumatology Training Grant, Vanderbilt Department of Orthopaedics and Rehabilitation, Caitlin Lovejoy Fund