Age-dependent vulnerability to endotoxemia is associated with reduction of anticoagulant factors activated protein C and thrombomodulin

Abstract

The protein C (PC) pathway is an important anticoagulant mechanism that prevents thrombosis during the systemic inflammatory response. Thrombomodulin (TM), an endothelial cell membrane receptor, accelerates the conversion of PC to activated protein C (APC), which leads to the down-regulation of thrombin production and fibrin formation. Induction of acute endotoxemia in young and aged mice with a low dose of bacterial endotoxin lipopolysaccharide (LPS, 2.5 mg/kg) caused a high mortality rate in aged (80%) but not young (0%) mice. After injection with this dose of LPS, fibrin formation was significantly elevated only in aged mice, plasma APC levels were increased only in young mice, and TM expression was profoundly depressed in the aged. The increased thrombosis, suppressed APC level, and decreased TM expression were not observed in young mice receiving a higher dose of LPS (20 mg/kg), which resulted in a mortality rate (78%) equivalent to that seen in aged mice with the low-dose LPS. Mutant mice with reduced TM showed significantly less plasma APC and increased fibrin formation compared with wild-type mice after LPS. These results demonstrate that PC pathway activation is suppressed with aging and is partly responsible for age-associated thrombosis and high mortality during endotoxemia.

Figure 1

Age-dependent mortality during endotoxemia. (A) A survival study demonstrating age-dependent mortality during endotoxemia. Young (4 months) and aged (24 months) male C57BL/6 mice received an intraperitoneal injection with LPS at various doses, and survival was monitored for 7 days. For each dose point, 8 to 14 mice were studied. No mortality was found in control mice with saline injection (not shown here). (B) Seven-day survival curves from the same experiment comparing mortality rates of young mice (LPS 2.5 mg/kg or 20 mg/kg) and aged mice (LPS 2.5 mg/kg). There was no statistical difference between the survival curve patterns of aged mice with 2.5 mg/kg LPS and young mice with 20 mg/kg LPS (P = .52). (C) Degree of hypothermia 12 hours after LPS injection was compared among young mice (LPS 2.5 mg/kg or 20 mg/kg) and aged mice (LPS 2.5 mg/kg). ***Statistical significance (P < .001). There was no statistical difference between body temperatures of aged mice with 2.5 mg/kg LPS and young mice with 20 mg/kg LPS (P = .54).

Figure 2

Age-dependent coagulation during endotoxemia. (A) Western blot analysis of a time course experiment examining fibrin formation in lung, kidney, and liver of young (4 months) and aged (24 months) mice that were sacrificed 6 and 12 hours after LPS injection (2.5 mg/kg, intraperitoneal). The control mice without LPS injection are represented by 0 h. Each lane contained 40 μg of protein derived equally from 5 individual mice. Each membrane was reprobed with anti–β-actin antibody to assure equal protein loading. (B) Densitometric analysis of panel A. (C) Fibrin formation 12 hours after LPS injection was further compared among individual samples, which were pooled in panel A. Each lane contained 40 μg of protein from an individual mouse. (D) Densitometric analysis of panel C. (E) Western blot analysis comparing thrombosis in lungs from young mice with a high dose of LPS (20 mg/kg) and aged mice with a low dose of LPS (2.5 mg/kg; n = 4 in each group). Each lane represents a protein sample from an individual mouse. (F) Densitometric analysis of panel E. (D-F) Data are mean ± SD. *Statistical significance (P < .05). **Statistical significance (P < .01).

Figure 3

Age-associated difference in plasma APC levels during endotoxemia. (A) Young (4 months, n = 6) and aged (24 months, n = 4) C57BL/6 mice were sacrificed 12 hours after LPS injection (2.5 mg/kg, intraperitoneal). Plasma APC levels were determined by immunocapture assay. For controls, plasma samples from young and aged mice (n = 4, each group) without LPS injection were assayed for APC. (B) Plasma APC levels were compared 12 hours after LPS injection in young mice with a high dose (20 mg/kg) versus aged mice with a low dose (2.5 mg/kg; n = 4 in each group). Data are mean ± SD. *Statistical significance (P < .05). **Statistical significance (P < .01).

Figure 4

Age-dependent loss of TM in lungs during endotoxemia. (A) Western blot analysis of a time course experiment assessing TM levels in lungs of young (4 months) and aged (24 months) mice that were sacrificed 6 and 12 hours after LPS injection (2.5 mg/kg, intraperitoneal). The control mice without LPS injection are represented by 0 h. Each lane contained 40 μg of protein derived equally from 5 individual mice. The membrane was reprobed with anti–β-actin antibody to assure equal protein loading. (B) Densitometric analysis of panel A. (C) TM levels at 12 hours after LPS injection were further compared among individual samples, which were pooled in panel A. Each lane contained 40 μg of protein from an individual mouse. (D) Densitometric analysis of panel C. (E) TM levels in lungs were compared in young mice with a high dose of LPS (20 mg/kg) versus aged mice with a low dose of LPS (2.5 mg/kg) 12 hours after injection (n = 4 in each group). (F) Densitometric analysis of panel E. (D-F) Data are mean ± SD. *Statistical significance (P < .05). **Statistical significance (P < .01).

Figure 5

Age-dependent loss of TM in pulmonary vascular cells during endotoxemia. Immunohistochemical analysis of TM was performed on lung sections from young (4 months) and aged (24 months) mice that were sacrificed 12 hours after LPS injection (2.5 mg/kg, intraperitoneal). Strong positive immunostaining for TM is seen in young control (A), aged control (B), and young mice with LPS (C), but not in aged mice with LPS (D). Control mice received no LPS. TM-positive cells in capillaries and large vessels (V) are indicated by arrowheads and arrows, respectively (original magnification ×400).

Figure 6

Augmented coagulation in TM^pro mice during endotoxemia. (A) Western blot analysis confirming TM levels in lung protein samples from TM^pro mice and wild-type mice (both 4-5 months old) that were sacrificed 6 hours after LPS injection (5 mg/kg, intraperitoneal). Control mice received no LPS (indicated as LPS−). Each lane contained 40 μg of protein from an individual mouse. (B) Desitometric analysis of panel A. ***Statistical significance (P < .001). (C) Western blot analysis demonstrating fibrin formation in kidney and liver from the same animals. Each lane contained 40 μg of protein from an individual mouse. (D) Densitometric analysis of panel C. Data are mean ± SD. *Statistical significance (P < .05). **Statistical significance (P < .01). This experiment was repeated once with similar results.

Figure 7

Low plasma APC levels in TM^pro mice during endotoxemia. Both TM^pro mice (n = 4) and wild-type (WT, n = 5) mice (6 months old) were sacrificed 6 hours after LPS injection (5 mg/kg, intraperitoneal). Control mice received no injection (n = 4 in each group). The plasma APC levels were determined by immunocapture assay. Data are mean ± SD. *Statistical significance (P < .05). ***Statistical significance (P < .001).