Systemic hypothermia increases PAI-1 expression and accelerates microvascular thrombus formation in endotoxemic mice

Abstract

Introduction: Hypothermia during sepsis significantly impairs patient outcome in clinical practice. Severe sepsis is closely linked to activation of the coagulation system, resulting in microthrombosis and subsequent organ failure. Herein, we studied whether systemic hypothermia accelerates microvascular thrombus formation during lipopolysacharide (LPS)-induced endotoxemia in vivo, and characterized the low temperature-induced endothelial and platelet dysfunctions.

Methods: Ferric-chloride induced microvascular thrombus formation was analyzed in cremaster muscles of hypothermic endotoxemic mice. Flow cytometry, ELISA and immunohistochemistry were used to evaluate the effect of hypothermia on endothelial and platelet function.

Results: Control animals at 37 degrees C revealed complete occlusion of arterioles and venules after 759 +/- 115 s and 744 +/- 112 s, respectively. Endotoxemia significantly (p < 0.05) accelerated arteriolar and venular occlusion in 37 degrees C animals (255 +/- 35 s and 238 +/- 58 s, respectively). This was associated with an increase of circulating endothelial activation markers, agonist-induced platelet reactivity, and endothelial P-selectin and plasminogen activator inhibitor (PAI)-1 expression. Systemic hypothermia of 34 degrees C revealed a slight but not significant reduction of arteriolar (224 +/- 35 s) and venular (183 +/- 35 s) occlusion times. Cooling of the endotoxemic animals to 31 degrees C core body temperature, however, resulted in a further acceleration of microvascular thrombus formation, in particular in arterioles (127 +/- 29 s, p < 0.05 versus 37 degrees C endotoxemic animals). Of interest, hypothermia did not affect endothelial receptor expression and platelet reactivity, but increased endothelial PAI-1 expression and, in particular, soluble PAI-1 antigen (sPAI-Ag) plasma levels.

Conclusion: LPS-induced endotoxemia accelerates microvascular thrombus formation in vivo, most probably by generalized endothelial activation and increased platelet reactivity. Systemic hypothermia further enhances microthrombosis in endotoxemia. This effect is associated with increased endothelial PAI-1 expression and sPAI-Ag in the systemic circulation rather than further endothelial activation or modulation of platelet reactivity.

Figure 1

Microvascular thrombus formation in vivo. Occlusion times of arterioles and venules upon ferric chloride-induced thrombus formation in 37°C saline controls (10 ml/kg body weight NaCl; -24 h intraperitoneally; N = 10 preparations) and 37°C, 34°C and 31°C endotoxemic animals (10 mg/kg body weight lipopolysacharide (LPS); -24 h intraperitoneally; N = 8 preparations per group). Values are given as means ± standard error of the mean; *p < 0.05 versus 37°C saline controls; ^#p < 0.05 versus 37°C endotoxemic animals.

Figure 2

Soluble plasminogen activator inhibitor-1 antigen (sPAI-Ag) concentrations. Plasma concentrations of circulating sPAI-Ag in 37°C saline controls (10 ml/kg body weight NaCl; -24 h intraperitoneally; n = 5 animals) and 37°C, 34°C and 31°C endotoxemic animals (10 mg/kg body weight lipopolysacharide (LPS); -24 h intraperitoneally; n = 4 animals per group). Values are given as means ± standard error of the mean; *p < 0.05 versus 37°C saline controls; ^#p < 0.05 versus 37°C endotoxemic animals.

Figure 3

Circulating endothelial activation markers. Plasma concentrations of circulating (a) soluble (s)P-selectin, (b) sE-selectin, (c) intercellular adhesion molecule (sICAM)-1 and (d) vascular cell adhesion molecule (sVCAM)-1 in 37°C saline controls (10 ml/kg body weight NaCl; -24 h intraperitoneally; n = 5 animals) and 37°C, 34°C and 31°C endotoxemic animals (10 mg/kg body weight lipopolysacharide (LPS); -24 h intraperitoneally; n = 4 animals per group). Values are given as means ± standard error of the mean; *p < 0.05 versus 37°C saline controls; ^#p < 0.05 versus 37°C endotoxemic animals; ^§p < 0.05 versus 34°C endotoxemic animals.

Figure 4

Endothelial P-selectin and plasminogen activator inhibitor (PAI)-1 expression. Analysis of the endothelial expression of (a) P-selectin and (b) PAI-1 in 37°C saline controls (10 ml/kg body weight NaCl; -24 h intraperitoneally; n = 10 tissue specimen) and 37°C, 34°C and 31°C endotoxemic animals (10 mg/kg body weight lipopolysacharide (LPS); -24 h intraperitoneally; n = 8 tissue specimens per group). Values are given as means ± standard error of the mean. *p < 0.05 versus 37°C saline controls.