Sepsis-induced systemic inflammation results in coagulation abnormalities that may be different in gram-positive and gram-negative infections. We used ciprofloxacin to induce a predominantly gram-positive Enterococcus faecalis polymicrobial sepsis in rats. Ciprofloxacin-untreated rats exhibited a predominantly gram-negative polymicrobial sepsis. Rats were subjected to 30% body surface area burn (B), cecal ligation puncture (CLP) with a 22-gauge needle, and B + CLP. Ciprofloxacin-treated B + CLP rats showed a significant decrease in plasma thrombin activatable fibrinolysis inhibitor (TAFI) levels compared with sham rats. However, plasma tissue factor pathway inhibitor (TFPI) levels decreased significantly in B, CLP, and B + CLP groups compared with sham rats. The ciprofloxacin-untreated group showed a significant decrease in plasma TAFI levels in CLP and B + CLP and plasma TFPI levels decreased in all 3 groups compared with sham rats. Histological changes in the liver and kidney included vascular congestion and parenchyma bleed following B + CLP in ciprofloxacin-untreated rats. These results suggest that plasma TAFI and TFPI levels differ depending on the type of bacteria involved in the septic process.
Background: The pathogenesis of thrombosis involves both cellular and humoral processes. Most antithrombotic drugs exhibit either anti-protease or anti-platelet effects. A combination of anti-protease and anti-platelet drugs provides better efficacy in the management of thrombotic disorders. A series of synthetic low molecular weight serine protease inhibitors with varying anti-platelet effects (Medicure Inc.) are being assessed for antithrombotic properties.
Materials and Methods: This investigation reports on a compound with low antithrombin/high anti-platelet activity MC 45301 (A) and a compound with high antithrombin/low anti-platelet activity MC 45308 (B) activity in in-vitro and in-vivo settings used to profile antithrombotic drugs.
Results: A exhibited strong anti-platelet actions as measured using ADP as an agonist (IC50=1.1 g/ml), whereas B had a higher IC50 (9.4 g/ml). In the antithrombin titration assay A (>100 μg/ml) showed a relatively higher IC50 than B (45 μg/ml). In the global anticoagulant assays, A exhibited somewhat weaker effects than B. In the Xa generation assay, both compounds exhibited similar effects. However, in the thrombin generation assays B exhibited stronger effects. In whole blood assays both compounds produced anticoagulant and anti-platelet effects. Intravenous administration of these compounds to rabbits over a dose range of 50–500 g/kg produced strong dose dependent antithrombotic actions. In comparison to direct antithrombin agents such as argatroban, at a comparable dose, B produced identical antithrombotic actions, which were disproportional to the systemic anticoagulant effects. A produced modest antithrombotic actions with minimal ex vivo clotting effects. This data is highly suggestive that compounds with dual targets are able to produce stronger antithrombotic actions relative to monotherapeutic agents. Additional studies in arterial thrombosis may provide newer insights into the antithrombotic actions of compounds with dual sites of action. Moreover, these agents may be more effective in thrombotic conditions where both platelets and the coagulation system are involved.
Thrombin activatable fibrinolytic inhibitor (TAFI) acts as an inhibitor of fibrinolysis by cleaving the arginine and lysine amino acid residues from the carboxy terminus of fibrin, rendering it resistant to digestion by plasmin. This study determines the effect of direct thrombin inhibitors on TAFI functionality and if there is a correlation to their anti‐IIa effects. The following thrombin inhibitors were tested: argatroban, melagatran, angiomax, LU 208791, hirudin and dabigatran. All agents were supplemented to normal human pooled plasma in various concentrations (10–0.16 μg/ml). The functional TAFI levels were determined using a chromogenic substrate based method developed by Pentapharm Inc. (Basel, Switzerland), the Pefakit® TAFI. The anti‐IIa effects were determined using a chromogenic substrate based method by American Diagnostica (Stamford, CN). All of the thrombin inhibitors were capable of inhibiting TAFI functionality; LU 208791 was the strongest inhibitor with an IC50 of 0.09 μg/ml while angiomax was the weakest with an IC50 of 6.6 μg/ml. These results for the most part corresponded to the anti‐IIa results. LU 208791 had the strongest anti‐IIa effects (IC50=0.39 μg/ml), while angiomax had an IC50 >10 μg/ml. The IC50s for TAFI inhibition by argatroban and hirudin were not significantly different (1.3 and 1.2 μg/ml); however, their IC50s for thrombin inhibition were >10 and 3.0 μg/ml respectively. This indicates that argatroban may inhibit TAFIa directly. These results show that the bleeding complications observed when using thrombin inhibitors may be due to the indiscriminate inhibition of TAFI. Persistent inhibition of TAFI by thrombin inhibitors may compromise both their safety and efficacy.
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