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Abstract
BackgroundDuring gram-negative sepsis, lipopolysaccharide (LPS) induces tissue factor expression on monocytes. The resulting disseminated intravascular coagulation leads to tissue ischemia and worsens the prognosis of septic patients. There are indications, that fever reduces the mortality of sepsis, the effect on tissue factor activity on monocytes is unknown. Therefore, we investigated whether heat shock modulates LPS-induced tissue factor activity in human blood.MethodsWhole blood samples and leukocyte suspensions, respectively, from healthy probands (n = 12) were incubated with LPS for 2 hours under heat shock conditions (43°C) or control conditions (37°C), respectively. Subsequent to further 3 hours of incubation at 37°C the clotting time, a measure of tissue factor expression, was determined. Cell integrity was verified by trypan blue exclusion test and FACS analysis.ResultsIncubation of whole blood samples with LPS for 5 hours at normothermia resulted in a significant shortening of clotting time from 357 ± 108 sec to 82 ± 8 sec compared to samples incubated without LPS (n = 12; p < 0.05). This LPS effect was mediated by tissue factor, as inhibition with active site-inhibited factor VIIa (ASIS) abolished the effect of LPS on clotting time. Blockade of protein synthesis using cycloheximide demonstrated that LPS exerted its procoagulatory effect via an induction of tissue factor expression. Upon heat shock treatment, the LPS effect was blunted: clotting times were 312 ± 66 s in absence of LPS and 277 ± 65 s in presence of LPS (n = 8; p > 0.05). Similarly, heat shock treatment of leukocyte suspensions abolished the LPS-induced tissue factor activity. Clotting time was 73 ± 31 s, when cells were treated with LPS (100 ng/mL) under normothermic conditions, and 301 ± 118 s, when treated with LPS (100 ng/mL) and heat shock (n = 8, p < 0.05). Control experiments excluded cell damage as a potential cause of the observed heat shock effect.ConclusionHeat shock treatment inhibits LPS-induced tissue factor activity in human whole blood samples and isolated leukocytes.
Highlights
During gram-negative sepsis, lipopolysaccharide (LPS) induces tissue factor expression on monocytes
While active tissue factor is absent in the peripheral blood under physiological conditions, the activation of hemostasis during sepsis is mediated by the expression of tissue factor on the surface of monocytes [5,6]
We investigated the effects of LPS on clotting time: incubation of whole blood samples with LPS (100 μg/mL) shortened clotting time from 357 ± 108 sec to 82 ± 8 sec (n = 12; p < 0.05) when samples were incubated at 37°C for 5 hours (Figure 1)
Summary
During gram-negative sepsis, lipopolysaccharide (LPS) induces tissue factor expression on monocytes. The resulting disseminated intravascular coagulation leads to tissue ischemia and worsens the prognosis of septic patients. We investigated whether heat shock modulates LPS-induced tissue factor activity in human blood. Gram-negative sepsis is mediated by lipopolysaccharide (LPS), a bacterial membrane constituent, which activates toll like receptor 4 (TLR-4). The resulting complex biological responses include an activation of the immune, inflammatory and coagulation systems [1,2,3,4]. Intravascular tissue factor expression is of striking pathophysiological importance: the resulting activation of coagulation leads to disseminated intravascular coagulation, intravascular fibrin deposition, tissue ischemia and cell damage [7,8]. We investigated whether heat shock affects LPS-induced activation of coagulation via a reduction of tissue factor expression
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