Review for 10.1186/s12876-025-03739-9 (2025)

Title: Phosphatidylserine induce thrombotic tendency and liver damage in obstructive jaundice
Version: 1 Date: 27 Jan 2025
Reviewer's report:
This is a well-executed study demonstrating that platelets, neutrophils, and endothelial cells
from patients with obstructive jaundice (OJ) exhibit increased phosphatidylserine (PS) exposure
and shed PS+ microparticles (MPs). The authors further show that these prothrombotic surfaces
contribute to increased thrombotic activity and that blocking these surfaces with lactadherin
reduces liver damage in a bile duct ligation (BDL) mouse model. The study provides valuable
insight into the prothrombotic tendencies of OJ patients and offers a potential therapeutic avenue
for mitigating liver damage.
While the experimental design and results are robust, some conclusions drawn from the data are
not fully substantiated and require additional evidence or clarification. Below, I outline major
and minor concerns that should be addressed to strengthen the manuscript.
Major Concerns:
The authors used unconjugated bilirubin to demonstrate PS induction in blood cells and
HUVECs. However, in OJ patients, the majority of circulating bilirubin is conjugated, raising
concerns about the relevance of the experimental model. Conjugated bilirubin may not induce PS
exposure in the same way as unconjugated bilirubin. Moreover, bilirubin may be a confounder,
as its elevation in plasma is accompanied by increases in other components that could induce
PS exposure.
For instance, rising bile acids in OJ have been shown to promote prothrombotic tendencies
(Baker et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 2019) and can directly induce
PS+ platelets (Unsworth et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 2017). To
definitively determine bilirubin’s role as the deleterious effector, the authors should consider
depleting bilirubin from patient plasma and repeating the incubation experiments with blood
cells and HUVECs.
While decreased fibrin deposition in the livers of BDL mice after lactadherin treatment supports
the protective role of blocking PS+, the data do not conclusively link this effect to PS+ platelets,
neutrophils, or MPs identified in vitro. As the authors acknowledge in the discussion, PS
+ exposure in the liver may arise from hepatocytes or liver-resident cells secondary to bile
accumulation.
This limitation does not undermine the therapeutic relevance of targeting liver-specific PS+, but
it does raise questions about whether the PS+ cells evaluated in vitro (blood cells and HUVECs)
directly contribute to the observed in vivo effects. Further clarification or additional experiments
would strengthen this link.
Minor Concerns:
The abstract should be revised to provide a clearer explanation of the experimental settings in
which the respective results were obtained. This would help readers understand the study design
and its implications more effectively.
The sentence, "We found that OJ patients exhibited elevated levels of PS+ BCs, sera-cultured
ECs and associated MPs compared to the controls, which partly due to elevated unconjugated
bilirubin," is difficult to understand and should be rephrased for clarity.
The authors state that OJ patients exhibited lower platelet counts, but Table 1 shows no
significant difference. This discrepancy should be addressed, either by revising the statement or
explaining why the reported difference is relevant despite the lack of statistical significance.
Figure 2C: The observed reduction in extrinsic Xa after incubation with anti-TF raises questions,
as platelets do not express tissue factor. The authors should provide a plausible explanation for
this result or consider revising their interpretation.
"Davide Povero et al. observed a rise in the number of extracellular vesicles derived from
hepatocytes and cholangiocytes in the sera of cholestatic liver disease patients, however, these
results unable to reflect alterations in circulation cells." This sentence is missing a verb.
"However, whether PS exposure on hepatocyte in OJ still unclear." This sentence is also missing
a verb.