Studies of Heparin Variants in Sepsis-Associated Immunothrombosis

Abstract

Sepsis is a life-threatening disease characterized by a dysregulated host response to infection, often leading to shock, organ failure, and death. It remains a global emergency, causing 6 to 8 million deaths annually. A hallmark of sepsis is immunothrombosis, which refers to infection-induced clotting. Although many clinical trials have failed to yield new therapies for sepsis, there is renewed interest in heparin. In COVID-19 trials, therapeutic-dose heparin improved outcomes in moderately ill, but not critically ill patients, suggesting that treatment timing may influence the host response. Heparin also has anti-inflammatory properties and can neutralize histone-mediated cytotoxicity. Histones are major components of neutrophil extracellular traps which exert procoagulant and cytotoxic effects observed in immunothrombosis. However, it remains unclear whether heparin’s protective effects are due to its anticoagulant activity or its size, which enables its anti-inflammatory functions. We compared the therapeutic potential of 3 heparin variants: LMWH, Vasoflux (low-anticoagulant-LMWH), and fondaparinux (ultra-small-LMWH). We used the 48-hour FIP model of abdominal sepsis. In this model, C57BL/6J male and female mice (10-12 weeks old) received an intraperitoneal injection of rat fecal slurry to induce sepsis. Heparins were injected in therapeutic doses at various time points (2/4 hr, 8 hr, 12 hr, and 16 hr post-slurry injection). We assessed survival, physiological and hematology parameters, bacterial burden, blood biomarkers of immunothrombosis, and organ histology. We found LMWH’s protective effects are time dependent, with treatment between 8-12 hours post-slurry injection improving survival, immune function, and reducing inflammation and cell death. Vasoflux showed a similar survival benefit by attenuating immunothrombosis through anti-inflammatory mechanisms, whereas fondaparinux didn’t, suggesting heparin’s benefits are likely independent of anticoagulation and mediated by anti-inflammatory properties. These studies identify the optimal time of treatment and provide new insight into which part of the heparin molecule improves survival in sepsis, informing safer, more targeted therapeutic strategies.