Role of procoagulant microparticles in mediating complications and outcome of acute liver injury/acute liver failure

Abstract

Microparticles (MPs), membrane fragments of 0.1-1.0 μm, are derived from many cell types in response to systemic inflammation. Acute liver failure (ALF) is a prototypical syndrome of systemic inflammatory response syndrome (SIRS) associated with a procoagulant state. We hypothesized that patients with ALF develop increased procoagulant MPs in proportion to the severity of systemic complications and adverse outcome. Fifty patients with acute liver injury (ALI), 78% of whom also had hepatic encephalopathy (HE; ALF), were followed until day 21 after admission. MPs were characterized by Invitrox Sizing, Antigen Detection and Enumeration, a light-scattering technology that can enumerate MPs as small as 0.15 μm, and by flow cytometry. Procoagulant activity was assessed by a functional MP-tissue factor (MP-TF) assay. Sixteen patients (32%) died and 27 (54%) recovered without liver transplantation (LT). Total MPs (0.15-1.0 μm) were present in nearly 19-fold higher concentrations in ALI/ALF patients, compared to healthy controls (P < 0.0001). MP-TF assays revealed high procoagulant activity (9.05 ± 8.82 versus 0.24 ± 0.14 pg/mL in controls; P = 0.0008). MP concentrations (0.28-0.64 μm) were higher in patients with the SIRS and high-grade HE, and MPs in the 0.36-0.64-μm size range increased in direct proportion to SIRS severity (P < 0.001) and grade of HE (P < 0.002). Day 1 MPs (0.28-0.64 μm) correlated with laboratory predictors of death/LT (higher phosphate and creatinine; lower bicarbonate), and day 1 and 3 MPs were higher in patients who died or underwent LT, compared to spontaneous survivors (P ≤ 0.01). By flow cytometry, 87% of patients had circulating CD41(+) MPs, indicating platelet origin.

Conclusion: Highly procoagulant MPs of specific size ranges are associated with the SIRS, systemic complications, and adverse outcome of ALI/ALF. MPs may contribute to the multiorgan system failure and high mortality of ALF.

Fig. 1

MP charcterization by ISADE. (A) Demonstration of the ability of ISADE to resolve a mixture of polystyrene beads with sizes of 0.2, 0.24, 0.3, 0.35, 0.4, and 0.5 μm. Both the size and number of beads were accurately reported. The low counts noted on either side of each bead size results from a small variation in bead size, confirmed by SEM. (B) Enumeration and sizing of MPs in PPP from a patient with ALF from disulfuram. Tracings from two different freeze-thawed aliquots are shown, demonstrating the small intersample variability of the results. Three distinct size ranges were identified, separated by solid black lines. Mid-sized MPs of 0.28–0.64 μm were most closely associated with ALF complications, laboratories, and outcome.

Fig. 2

MP concentration and procoagulant activity in patients with ALI/ALF and normal healthy controls. (A) Total log₁₀MP/mL of size range 0.15–1.0 μm in 50 ALI/ALF patients on admission to the hospital and 13 healthy controls of similar age and gender distribution (P < 0.0001). (B). MP-TF activity in 34 ALI/ALF patients on admission to the hospital and 13 healthy controls (P = 0.0008). Error bars indicate mean ± SD.

Fig. 3

Relationship of MP concentration to number of positive SIRS components on admission for ALI/ALF and grade of HE. (A) Log₁₀MP/mL (0.36–0.64 μm) versus number of positive SIRS components on admission (P = 0.0002). (B) Log₁₀ MP/mL (0.36–0.64 μm) versus maximal grade of HE during the first 7 days of admission (P = 0.0015). Error bars indicate mean ± SD.

Fig. 4

Relationship of MP concentration (0.28–0.64 μm) to outcome of ALI/ALF. (A) Log₁₀MP/mL on day 1 of admission according to outcome by day 21, TFS, or death/LT (P = 0.006). (B) Log₁₀MP/mL on day 1 according to overall survival by day 21 (P = 0.010). (C) Log₁₀MP/mL on day 3 of admission according to TFS versus death/LT by day 21 (P = 0.0002). (D) Log₁₀MP/mL on day 3 according to overall survival by day 21 (P < 0.05). The range of MP concentration on day 3 was lower than the range on day 1 samples as a result of early mortality of 3 patients with high day 1 MP concentrations. Error bars indicate mean ± SD.

Fig. 5

Prevalence of MP phenotypes in plasma of patients with ALI/ALF by flow cytometry.