Introduction MST‐188 (Mast Therapeutics, San Diego, CA.) is a highly purified form of the , triblock copolymer poloxamer 188. It exhibits potent rheologic properties that result in improved blood flow especially under low shear conditions. Initial observations suggested that endogenous fibrinolysis is facilitated by this agent. In this study we investigate the effect of MST‐188 on urokinase induced fibrinolysis. Materials and Methods A dynamic fibrinokinetic assay was used to assess both the assembly and dissolution of fibrin clots. MST‐188 (0 ‐ 10 mg/ml) was added to citrated human plasma with and without urokinase at a concentrations between 0 ‐ 2,500 U/ml (N=10‐15 donors). Clotting was initiated with the sequential addition of and calcium chloride (0.25 uM) and then thrombin (0.5u/ml). Absorbance was monitored to detect the assembly and dissolution of fibrin. Results Control studies of MST‐188 alone had a dose dependent effect on fibrin assembly as reflected by both the rate of change and peak optical density. Urokinase alone showed no effect on clot formation but a dose dependent increase in clot lysis. Supplementation of MST‐188 to urokinase facilitated its lytic profile in a concentration dependent manner accelerating the time to onset of lysis and reducing the overall AUC for clot dissolution. Discussion These results demonstrate that MST‐ 188 increases urokinase mediated fibrinolysis. The increase in the susceptibility of the formed clot to lysis in the presence of MST‐188 may be due to structural modification of the clot. MST‐188 may bind to fibrin strands, thereby modifying their accessibility to the lytic action of generated plasmin.

Introduction: MST-188 (purified poloxamer 188) is a tri-block co-polymer with high affinity to hydrophobic cellular surfaces that inhibits hydrophobic adhesive interactions in the circulation. It also facilitates blood flow by reducing blood viscosity and reportedly exhibits anti-adhesion and anti-inflammatory properties. Currently, this agent is under study in with patients experiencingsickle cell crisis and in patients with acute limb ischemia. Since MST-188 may be administered with other anti-coagulant agents such as heparin its potential interaction to modulate the anti-coagulant effects of this drug require experimental validation. These studies are designed to investigate the potential interactions between heparin and MST-188 in a rat model of tail transection bleeding and jugular vein clamping induced thrombosis model. Materials and Methods: The in vitro interactions between MST-188 were investigated by supplementing this agent to normal rat plasma (NRP) and heparinized rat plasma at a fixed concentrations of 1.25 and 2.50 mg/mL. The concentration of heparin was kept at 1.25 and 2.50 μg/mL. In the in vivo studies individual groups of rats (n=6-8) were administered with saline as a control, heparin in the dosage range of 125-500 ug/kg intravenously and MST-188 at 25 mg/kg IV followed by heparin at the 125-500 ug/kg dosages. Rat tail resection time was measured 5 minutes after administration of heparin and clot occlusion index was measured as number of jugular vein clamping required to occlude the blood vessel. After the completion of the procedure blood samples were obtained through cardiac puncture and used for ex vivo analysis of PT, aPTT, heptest and thrombin time. Results: In the in vitro studies heparin produced a concentration dependent prolongation of the aPTT, heptest and thrombin time. MST-188 did not produce any effects on the aPTT and heptest time, however it decreased the thrombin time. MST-188 at a higher concentration of 2.5 and 5.0 mg/ml produced a shorting of heparins anticoagulant responses, as measured by aPTT and thrombin time. Heparin produced a dose dependent increase in both the bleeding time (p<0.0001) and number of jugular vein clamps to occlude the blood vessel (p<0.0001). MST-188 at dosages of 25 mg/kg produced a significant increase on both bleeding time (p <0.05) and number of clampings required to occlude the blood vessel (p<0.0001). When MST-188 was administered simultaneously with heparin it augmented the bleeding time (p < 0.05) and increased the number of jugular vein clamps required to occlude the blood vessel (p < 0.05). The ex vivo analysis of blood samples collected from rats treated in different regimens did not exhibit any anti-coagulant effects as measured by PT, aPTT, heptest and thrombin time. Conclusion: These studies suggest a differential response of MST-188. While in vitro it exhibits a prohemostatic response as evident by shortening of thrombin time, in the in vivo setting it enhances the anticoagulant effects of heparin as evident by increased bleeding time and increased number of jugular vein clamps required to occlude the vessel. Thus, both of these mechanisms are involved in the mediation of the beneficial effects observed with this agent in vaso-occlusive and thrombotic processes. Disclosures Emanuele: Mast Therapeutics: Employment.