The effect of anticoagulants and the role of thrombin on neutrophil-endothelial cell interactions in septic shock.

To examine the effects of anticoagulants and the role of thrombin on neutrophil-platelet-endothelial cell interactions in septic shock. Controlled experiments using phase-contrast microscopy to study neutrophil, platelet, and endothelial cell interactions in flowing cell suspensions under simulated physiologic conditions. University research laboratory. Adult patients with septic shock and normal volunteers. Microslides were coated with human umbilical vein endothelial cells. Neutrophils and platelets removed from control subjects were stimulated with plasma from patients in septic shock and perfused over endothelial cells. Heparin (H), argatroban (A), antithrombin III (ATIII), and recombinant human activated protein C (rhAPC) with and without thrombin were added to cells suspended in septic plasma and normal plasma. The number of neutrophils adherent to endothelial cells, neutrophil rolling velocity, and the number of neutrophils in aggregates were determined. Flow cytometric analysis of cells was used to identify cell activation and the formation of platelet-neutrophil aggregates. Heparin, A, ATIII, rhAPC all significantly decreased neutrophil adhesion and aggregation, and increased rolling velocity of neutrophils suspended in septic plasma. These results are similar to those observed with normal plasma but present greater absolute changes. Platelet-neutrophil aggregation, platelet activation, and neutrophil activation were significantly decreased by each of the anticoagulants. The addition of thrombin to cell suspensions containing anticoagulants reversed the effects of H, A, ATIII, rhAPC on neutrophil adhesion, adherence, and rolling velocity. In addition, thrombin attenuated the effects of each of these agents on platelet-neutrophil aggregation, platelet activation, and neutrophil activation. These data suggest that H, A, ATIII, and rhAPC decrease sepsis-induced neutrophil-endothelial cell interactions. The reversal of this effect by thrombin suggests that these agents alter neutrophil-endothelial interactions through their anticoagulant effects and the resulting decrease in thrombin activity.

Effect of recombinant activated protein C and low-dose heparin on neutrophil-endothelial cell interactions in septic shock.

OBJECTIVE: To examine the effects of recombinant activated protein C (rhAPC) and low-dose heparin on neutrophil-platelet-endothelial cell interactions in septic shock. DESIGN: Controlled experiments using phase contrast microscopy to study neutrophil, platelet, and endothelial cell interactions in flowing cell suspensions under simulated physiologic conditions. SETTING: University research laboratory. PATIENTS: Adult patients with septic shock and normal volunteers. INTERVENTIONS: Neutrophils and platelets removed from control subjects were stimulated with plasma and serum from 21 patients in septic shock and perfused over endothelial cells. Activated protein C, low-dose heparin, and low-dose heparin with rhAPC were added to cells suspended in septic plasma. Neutrophil rolling velocity and the number of neutrophils adherent to endothelial cells and in aggregates were determined. Flow cytometric analysis of CD11b/CD63 cells was used to identify platelet-neutrophil aggregates. MEASUREMENTS AND MAIN RESULTS: Activated protein C significantly decreased neutrophil adhesion and aggregation and increased rolling velocity in cells stimulated with both septic serum and septic plasma. Significant decreases in platelet-neutrophil aggregates induced by septic plasma were also observed. Low-dose heparin alone had no effects on these variables. The addition of low-dose heparin to cells suspended in septic plasma and rhAPC attenuated the benefits observed with rhAPC alone in each of these variables. CONCLUSIONS: These data suggest that the in vitro addition of rhAPC decreases sepsis-induced interactions between isolated platelets, neutrophils, and endothelial cells. Low-dose heparin attenuates the benefits observed with rhAPC. The changes in neutrophil-endothelial cell interactions demonstrated with rhAPC may play a role in preserving microvascular patency in patients with septic shock.