Small Annexin V-Positive Platelet-Derived Microvesicles Affect Prognosis in Cirrhosis: A Longitudinal Study

Abstract

Introduction: Microvesicles (MVs) with procoagulant properties may favor liver parenchymal extinction, then cirrhosis-related complications and mortality. In a longitudinal cohort of cirrhotic patients, we measured plasma levels of platelet-derived MVs (PMVs), endothelial-derived MVs, and red blood cell-derived MVs, expressing phosphatidylserine (annexin V-positive [AV+]) or not, and evaluated their impact on Model for End-Stage Liver Disease (MELD) score and transplant-free survival.

Methods: MVs were quantified using flow cytometry in plasma from 90 noninfected cirrhotic patients and 10 healthy volunteers matched for age and sex. Impact of plasma microvesicle levels on 6-month transplant-free survival was assessed using log-rank tests and logistic regression.

Results: Microvesicle levels, mostly platelet-derived, were 2.5-fold higher in healthy volunteers compared with cirrhotic patients. Circulating small AV+ PMV levels were lower in cirrhotic patients (P = 0.014) and inversely correlated with MELD scores (R = -0.28; P = 0.0065). During 1-year follow-up, 8 patients died and 7 underwent liver transplantation. In the remaining patients, circulating microvesicle levels did not change significantly. Six-month transplant-free survival was lower in patients with low baseline small AV+ PMV levels (72.6% vs 96.2%; P = 0.0007). In multivariate analyses adjusted for age, ascites, esophageal varices, encephalopathy, clinical decompensation, total platelet counts, MELD score, and/or Child-Pugh C stage, patients with lower small AV+ PMV levels had a significant 5- to 8-fold higher risk of 6-month death or liver transplant. Other PMV levels did not impact on survival.

Discussion: Decreased circulating small AV+ PMV levels are associated with significantly lower transplant-free survival in cirrhotic patients independently of MELD score and platelet counts.

Figure 1.

Baseline plasma levels of all distinguished subtypes of MVs in cirrhotic patients and HVs. The subtypes of MVs were distinguished according to their cellular origin (PMVs, EMVs, RMVs, or unknown MVs), their size (small < 0.5 μm or large MVs ≥ 0.5 μm), and their expression of phosphatidylserine (AV⁺ MVs) or not (annexin V–negative). Because at least 1 marker is mandatory to detect MVs, annexin V–negative MVs of unknown origin could not be detected. In the 2 groups, PMVs are largely predominant, but plasma PMV levels were significantly higher in HVs compared with cirrhotic patients (5,086 vs 2,372/µL, P = 0.003), and significant differences were observed between cirrhotic patients and HVs for all subcategories of PMVs plasma levels of small AV⁺ RMVs were higher in cirrhotic patients (34 vs 18/µL, P = 0.015). No significant differences were observed between cirrhotic patients and HVs for EMVs, which accounted for a low proportion of detectable MVs, and for AV⁺ MVs with no specific cellular origin, which may include hepatocyte-derived AV⁺ MVs. AV⁺, annexin V–positive; EMVs, endothelial-derived microvesicles; HVs, healthy volunteers; PMVs, platelet-derived microvesicles; RMVs, red blood cell–derived microvesicles.

Figure 2.

Plasma levels of small AV⁺ PMVs according to CP stage (a) and MELD score (b). Plasma levels of small AV⁺ PMVs decrease in parallel with the severity of CP stage (a, 503 MVs/µL in CP-A; 378 in CP-B; 264 in CP-C; Kruskal-Wallis test, P = 0.029) and are inversely correlated with MELD score (R = −0.28, P = 0.0065; b). (a) Boxes show IQRs and horizontal lines denote median values. The ends of the whiskers represent the lowest and the highest datapoints within 1.5 × IQR of the lower and upper quartiles. (b) The regression line and its confidence limits (gray zone) are given. AV⁺, annexin V–positive; CP, Child-Pugh; IQR, interquartile ranges; MELD, Model for End-Stage Liver Disease; MVs, microvesicles; PMVs, platelet-derived microvesicles.

Figure 3.

Six-month transplant-free survival according to baseline level of small AV⁺ PMVs in cirrhotic patients. Small AV⁺ PMVs are the only subtype of MVs that impact on prognosis. The area under the receiver operating characteristic curve (AUROC) of small AV⁺ PMVs to predict death or liver transplant at 6 months was 0.723 (95% CI: 0.563–0.831), and the best discriminant value given by the Youden index was 286/µL. Low (solid line) and high (dotted line) levels of small AV⁺ PMVs were defined according to this value. Transplant-free survival was significantly lower in patients with plasma levels of small AV⁺ PMVs <286/µL. Because low plasma values of small AV⁺ PMVs are associated with poor prognosis, we can hypothesize that these MVs can be captured in a phosphatidylserine-dependent manner and that this phenomenon promotes mortality. AV⁺, annexin V–positive; PMVs, platelet-derived microvesicles; 95% CI, 95% confidence interval.