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. 2006 May 15;107(10):3912-21.
doi: 10.1182/blood-2005-08-3130. Epub 2006 Jan 24.

Roles of protease-activated receptors in a mouse model of endotoxemia

Eric Camerer  1 , Ivo Cornelissen, Hiroshi Kataoka, Daniel N Duong, Yao-Wu Zheng, Shaun R Coughlin
Affiliations

Roles of protease-activated receptors in a mouse model of endotoxemia

Eric Camerer et al. Blood. .

Abstract

Endotoxemia is often associated with extreme inflammatory responses and disseminated intravascular coagulation. Protease-activated receptors (PARs) mediate cellular responses to coagulation proteases, including platelet activation and endothelial cell reactions predicted to promote inflammation. These observations suggested that PAR activation by coagulation proteases generated in the setting of endotoxemia might promote platelet activation, leukocyte-mediated endothelial injury, tissue damage, and death. Toward testing these hypotheses, we examined the effect of PAR deficiencies that ablate platelet and endothelial activation by coagulation proteases in a mouse endotoxemia model. Although coagulation was activated as measured by thrombin-antithrombin (TAT) production and antithrombin III (ATIII) depletion, Par1(-/-), Par2(-/-), Par4(-/-), Par2(-/-):Par4(-/-), and Par1(-/-):Par2(-/-) mice all failed to show improved survival or decreased cytokine responses after endotoxin challenge compared with wild type. Thus, our results fail to support a necessary role for PARs in linking coagulation to inflammation or death in this model. Interestingly, endotoxin-induced thrombocytopenia was not diminished in Par4(-/-) mice. Thus, a mechanism independent of platelet activation by thrombin was sufficient to cause thrombocytopenia in our model. These results raise the possibility that decreases in platelet count in the setting of sepsis may not be caused by disseminated intravascular coagulation but instead report on a sometimes parallel but independent process.

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Figures

Figure 1.
Figure 1.
Sex differences in survival after endotoxin. N5 C57BL/6 females (solid lines) and males (dotted lines) from the colonies used for the studies in Figures 2, 3, 4, 5, 6 were injected intraperitoneally with high-dose LPS (60 mg/kg; left) or low-dose LPS (30 mg/kg for females, 20 mg/kg for males; right) and monitored for 26 and 72 hours, respectively. P values as determined by log-rank test are shown only when less than .05.
Figure 2.
Figure 2.
Characterization of disseminated intravascular coagulation after endotoxin. (Left) C57BL/6 females were injected intraperitoneally with 30 (low dose) or 60 (high dose) mg/kg LPS. At the indicated time, blood was collected and platelet counts, TAT levels, and ATIII levels determined (mean ± SD, n = 6). (Right) C57BL/6 females were injected intraperitoneally with 60 mg/kg LPS with or without hirudin (5 mg/kg) treatment as described in “Materials and methods”; untreated control mice were processed in parallel. At 8 hours, mice were perfusion fixed and tissues processed for immunohistochemistry. Antifibrin immunostaining of paraffin sections of liver is shown. Note fibrin deposition in hepatic microvessels of LPS-treated mice and its prevention by hirudin.
Figure 3.
Figure 3.
Survival of PAR-deficient mice after endotoxin challenge: single knockouts. (A) Par1–/– (top panels), Par2–/– (middle panels), and Par4–/– (bottom panels) mice and strain- (about 97.5% C57BL/6, about 2.5% 129SVJae), age-, and sex-matched controls were injected intraperitoneally with LPS and monitored for 26 (high-dose LPS) or 72 (low-dose LPS) hours. (B) Comparisons that showed trends toward differences in panel A were repeated in littermate-controlled studies. These were in the same strain background as in panel A, except for PAR2, which was 50% C57BL/6, 50% 129SVJae. (C) Pooled low-dose data from panels A-B. The top panel compares wild-type responses. In all panels, high-dose LPS was 60 mg/kg for both sexes; low dose was 30 mg/kg for females and 20 mg/kg for males. These dosing conventions were maintained for all subsequent studies. P values as determined by log-rank test are shown when < .05.
Figure 4.
Figure 4.
Protease signaling in endothelial cells from Par1–/–:Par2–/– mice. Dermal microvascular endothelial cells isolated from Par1–/–:Par2–/– neonates or matched controls were serum-starved, loaded with 3H-inositol, and exposed to vehicle, protease, or agonist peptide as indicated. To exclude nonspecific responses to contaminating thrombin, hirudin was included with all proteases except thrombin. Results from 2 independent experiments are shown. Each bar represents 1 experiment done in triplicate; error bars show standard deviation. Par1–/–:Par2–/– cells did respond to nonprotease agonists such as serum and lysophosphatidic acid; h indicates human; m, mouse.
Figure 5.
Figure 5.
Survival of PAR-deficient mice after endotoxin challenge: double knockouts. (A) Par1–/–:Par2–/– (50% C57BL/6, 50% 129SVJae) mice and strain-, age-, and sex-matched controls were injected intraperitoneally with LPS and survival monitored as indicated. (B) Par2–/– (about 97.5% C57BL/6, about 2.5% 129SVJae) females and littermate controls were injected intraperitoneally with hirudin (1 mg/kg) 30 minutes before and 30 minutes, 2 hours, and 4 hours after intraperitoneal injection of low-dose LPS.
Figure 6.
Figure 6.
Endotoxin-induced thrombocytopenia in Par4–/– mice. Par4–/– males or littermate control males were injected intraperitoneally with 20 mg/kg LPS. Twelve hours later, mice were bled for platelet counts and perfused for immunohistochemistry. (Top left, mean ± SD) Platelet counts in Par4–/– and littermate controls before and after LPS treatment. (Top right and bottom) Antiplatelet immunostaining of paraffin sections of liver from an LPS-treated Par4–/– or Par4+/+ mouse or a saline-treated Par4+/+ control. Note immunostaining for platelets in microvessels of LPS-treated mice regardless of genotype.
Figure 7.
Figure 7.
Effect of inhibition of thrombin, fibrin(ogen), or platelet function upon endotoxin mortality. Effects of thrombin inhibition (A) or lack of fibrinogen (B) or platelets (C) on endotoxin mortality. Wild-type C57BL/6 female mice treated with hirudin (5 mg/kg intraperitoneally), antiplatelet serum, and/or FTY720 (0.1 mg/kg intraperitoneally) and Fib–/– or Nf-E2–/– (both 50% C57BL/6, 50% 129SVJ) mice and appropriate vehicle- or nonimmune serum-injected or littermate controls were injected intraperitoneally with high- or low-dose LPS and monitored for 26 or 72 hours as indicated. P values as determined by log-rank test are shown when less than .05. For littermate studies, P value refers to comparison between knockouts and wild types.
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