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Nihms 1671878
Nihms 1671878
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Semin Thromb Hemost. Author manuscript; available in PMC 2021 September 09.
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Summary/abstract
The liver is unique in its remarkable regenerative capacity, which enables use of liver resection as
a treatment for specific liver diseases, including removal of neoplastic liver disease. After
resection, the remaining liver tissue (i.e, liver remnant) regenerates to maintain normal hepatic
function. In experimental settings as well as patients, removal of up to 2/3rd of the liver mass
stimulates a rapid and highly coordinated process resulting in regeneration of the remaining liver.
Mechanisms controlling the initiation and termination of regeneration continue to be discovered,
and many of the fundamental signaling pathways controlling proliferation of liver parenchymal
cells (i.e., hepatocytes) have been uncovered. Interestingly, while hemostatic complications (i.e.,
bleeding and thrombosis) are primarily thought of as a complication of surgery itself, strong
evidence suggests that components of the hemostatic system are in fact, powerful drivers of liver
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regeneration. This review focuses on clinical and translational evidence supporting a link between
the hemostatic system and liver regeneration, and the mechanisms whereby the hemostatic system
directs liver regeneration discovered using experimental settings.
Hepatectomies are performed for eligible patients based on the resection of all radiologically
and macroscopically detectable tumor, while preserving at least 20–25% of healthy total
liver volume5, 6. In most patients, the liver remnant regenerates to its original size to restore
normal hepatic function. Importantly, despite substantial improvements in surgical
techniques and peri-operative care, postoperative morbidity and mortality remain an
Corresponding authors: Patrick Starlinger, MD, PhD, Department of Surgery, Mayo Clinic, Rochester, MN, 200 1st St SW,
Rochester, MN 55905, Tel: +1 (507) 284-2511, starlinger.patrick@mayo.edu. Dafna Groeneveld, PhD, Pathobiology and Diagnostic
Investigation, Michigan State University, 1129 Farm Lane, 254 Food Safety Toxicology, East Lansing, MI, 48824, Tel: +1
517-884-3484, groene12@msu.edu.
Starlinger et al. Page 2
important concern after liver resection7, 8. In fact, postoperative death after liver resection is
reported to occur in up to 13% of high-risk patients9, 10.
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Failed regeneration of the liver remnant can ultimately contribute to postoperative hepatic
dysfunction and liver failure. Although extensive research has been performed on molecular
mechanisms of liver regeneration, very limited translational research has been performed to
document the relevance of these findings in the human setting, thereby impeding the
development of new therapeutic compounds and strategies. Indeed, there is no treatment
option available to promote liver regeneration for patients developing postoperative liver
failure after liver resection. New surgical strategies to accelerate (postoperative) liver
regeneration have been developed. Portal vein embolization (PVE) and portal vein ligation
(PVL) have been widely used to increase the volume of the future liver remnant. Both
techniques involve occlusion of the portal branches that provide blood to the liver fragments
that will undergo resection, leading to hypertrophy of the future liver remnant11. In fact,
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PVL is one of the first steps in the two-stage surgical hepatectomy model called ALPPS
(Associating Liver Partition and Portal vein ligation for Staged hepatectomy), where rapid
liver regeneration is initiated by a combination of PVL and parenchymal transection. The
more rapid liver regeneration observed after ALPPS has been attributed to a reduction in
portal collateral formation12. A recently proposed method to increase the future liver
remnant is the Liver Venous Deprivation (LVD) technique. It combines embolization of both
the portal and hepatic vein in order to increase the damage of the embolized liver leading to
rapid hypertrophy of the future liver remnant13. The underlying mechanisms triggering liver
regeneration in these models are not completely understood. Previous studies suggest that
one mechanism is an alteration in portal blood flow, which may stimulate liver regeneration
via increased shear stress, increased accessibility of pro-regenerative factors, or reduced
oxygen pressure14. However, even with these surgical advances, postoperative liver failure
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remains a frequent and critical issue without any available pharmacologic strategies for
prophylaxis or treatment9, 10. As postoperative liver dysfunction and failure remains the
main cause of death after liver resection, identifying strategies to promote liver regeneration
is critical. Such therapies ideally should reduce post-operative complications caused by
failed liver regeneration, potentially accelerate patient recovery after liver resection and most
importantly, reduce the incidence of postoperative mortality.
standard PHx model most commonly involves surgical removal of 2/3rd of the rodent liver15.
PHx is followed rapidly by a series of cell signaling events coordinating proliferation of the
remaining liver parenchymal cells (i.e., hepatocytes) and reconstitution of hepatic non-
parenchymal cells, ultimately restoring liver mass and function within 7–10 days15–17.
Strong experimental evidence indicates this process is mediated by a complex interplay of
pathways. Hepatocyte proliferation, for example, involves a number of growth factors
including hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF),
Semin Thromb Hemost. Author manuscript; available in PMC 2021 September 09.
Starlinger et al. Page 3
include non-mitogenic cytokines, bile acids, hormones, and other small molecules. The
reader is referred to excellent reviews in which these processes have been detailed21, 22.
There is considerable redundancy in the various pathways to ensure maximal regeneration.
Indeed, there are numerous examples of deficiencies in a single pathway delaying liver
regeneration, but few if any examples where a single deficit fully blocks the regenerative
response. It seems as if the inhibition of multiple critical pathways is needed to block liver
regeneration completely23.
regeneration after PHx. Indeed since the first description that platelets can promote
hepatocyte proliferation in 198224, significant evidence has been generated supporting a
relevant role of platelets in liver regeneration. In particular, the significance of platelets
during liver regeneration is supported by 1) their rapid accumulation (i.e., within 5 min) in
the liver sinusoids after PHx25, 26 and translocation into the space of Disse 26 and 2)
evidence that platelet number is directly associated with successful regeneration after PHx.
Administration of platelet inhibitors or platelet depletion each significantly reduced
hepatocyte proliferation and delayed liver regeneration after PHx25, 27. On the other hand,
increasing circulating blood platelet count significantly promoted liver regeneration after
70% PHx28 and counteracted liver failure induced by extended (90%) PHx29, 30.
Collectively, these studies suggest that platelets play a critical role as one of the earliest
triggers of regeneration after PHx.
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The exact molecular mechanisms whereby platelets contribute to liver regeneration need to
be clarified (Figure 1). It has been proposed that local release of platelet granule contents in
response to liver resection is responsible for platelet-mediated liver regeneration31–34.
Indeed, in vitro studies have shown that platelets induce hepatocyte proliferation by release
of growth factors35, 36. In this context, it is important to differentiate alpha and dense
granules, which store different types of growth factor/molecules that may have divergent
effects on liver regeneration.
Dense granules: Multiple experimental and clinical studies suggest that platelet-derived
serotonin could form a key component of platelet-mediated liver regeneration27, 37–39.
However, there is also conflicting data on the role of serotonin in liver regeneration after
liver resection. Mice lacking platelet serotonin (TPH1−/− mice) show a clear delay in liver
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regeneration after PHx27. However, TPH1−/− mice also have reduced platelet adhesion in
vivo40, most likely due to defects in both von Willebrand factor (VWF)-mediated platelet
adhesion and secondary platelet activation40, 41. As both platelet activation27 and VWF-
dependent platelet accumulation25 are required for platelet-mediated liver regeneration, the
delayed regenerative capacity in TPH1−/− mice may also be explained by these defects in
hemostasis. It is also worth noting that liver regeneration after PHx is unaffected in plasma
membrane serotonin transporter (SERT) deficient rats, despite having a marked reduction in
Semin Thromb Hemost. Author manuscript; available in PMC 2021 September 09.
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platelet serotonin levels42. While experimental and translational evidence is growing, the
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α-granules: Platelets store a wide variety of growth factors, including insulin-like growth
factor (IGF), VEGF, HGF, in their α-granules which are released upon platelet activation.
Most of these are potent inducers of liver regeneration. However, thrombospondin-1
(TSP-1), an inhibitor of regeneration, is also stored in this granule subtype. Clear evidence
supports a pro-regenerative role for platelets, and yet the cargo of α-granules would be
anticipated to have dichotomous effects. One potential explanation for this is the distinct
packaging and agonist-dependent α-granule subset release, hypothesized by Italiano et al.43,
which would allow for distinct platelet responses to specific stimuli43–48. Further studies are
required to evaluate this possibility after liver resection, although our recent studies suggest
a connection between levels of pro- and anti-proliferative growth factors and outcome in
patients after liver resection33. Indeed, platelets from patients with postoperative liver
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dysfunction lacked capacity to secrete VEGF stored in their α-granules49. It should be noted
that not all studies align with this hypothesis. Kirschbaum et al. found no evidence of release
and consumption of platelet-derived growth factors after partial hepatectomy in humans50.
One possible difference between these studies is differences in sample collection
strategies51, highlighting both the need for standardized collection techniques and for more
studies to evaluate the contribution of α-granule content to liver regeneration. Similarly, it is
also important to consider that there may be general effects of major abdominal surgery on
growth factor release and/or expression50.
In addition to release of platelet growth factors, another mechanism whereby platelets could
stimulate liver regeneration is via transfer of platelet RNA to hepatocytes31. In vitro studies
have shown that platelets stimulate proliferation of hepatocytes, which required direct
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contact between platelets and these cells52. Not only was direct contact required, but
platelets were also internalized by hepatocytes53. Although platelets do not contain a
nucleus, they do contain a wide selection of circulating RNAs54. In recent years it has been
demonstrated that platelets are capable of RNA transfer to recipient cells, including
monocytic and endothelial cells55. The Lisman group has demonstrated that RNA transfer
from platelets to hepatocytes partly drives hepatocyte proliferation in vitro53. However,
whether functional RNA transfer also occurs in vivo during liver regeneration has yet to be
demonstrated.
Although platelets seem to be able to stimulate liver regeneration directly, other mechanisms
involve their interaction with liver cell types to indirectly support liver regeneration. Platelets
have been shown to stimulate liver sinusoidal endothelial cells (LSECs) to release potent
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hepatocyte growth factors such as HGF, VEGF and IL-652. This is of importance as
immediately after induction of liver regeneration, LSECs are likely to contact platelets56.
Indeed, 2 hours after induction of liver regeneration in humans, abundant interaction
between LSECs and platelets has been documented56. Moreover, there is evidence to suggest
a role for Kupffer cells in platelet-dependent liver regeneration, although thrombocytosis
also promotes liver regeneration even after Kupffer cell depletion57, 58. The possibility that
platelets could stimulate liver regeneration via non-liver cell dependent mechanisms has
been largely unexplored. However, one alternative hypothesis involves platelet-dependent
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facilitation of the inflammatory response. Leukocytes accumulate in the liver remnant after
PHx and liver regeneration is delayed in leukocytopenic mice59. Platelets are well known to
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recruit inflammatory cells60, and thus, it seems plausible that platelets could promote liver
regeneration by facilitating leukocyte recruitment to the remnant liver. The interaction
between platelets and the inflammatory response in the setting of PHx has not been
investigated extensively, but recent studies in other models have demonstrated that platelets
play an important role in neutrophil accumulation to sites of inflammation and tissue
repair61, 62.
very rapid after PHx, peaking within 30 minutes25, 63, and platelet accumulation in the liver
shortly after PHx is most impactful with respect to liver regeneration. Indeed, timing seems
to be of critical relevance as platelet depletion prior to PHx significantly reduced hepatocyte
proliferation, whereas antibody-mediated thrombocytopenia initiated 2 hours after PHx had
much less impact25. These studies imply that hepatic platelet accumulation in the first
several hours after PHx is critical for liver regeneration. The mechanism driving platelet
accumulation in the liver after PHx is likely multifactorial. Kirschbaum et al. found that
administration of an anti-VWF antibody reduced hepatic platelet accumulation 30 min after
PHx, and that hepatocyte proliferation was reduced in VWF-deficient mice after PHx25.
Besides VWF, components of the coagulation cascade may attract platelets to the liver after
PHx. For example, we have shown that depletion of plasma fibrinogen with ancrod also
prevented hepatic platelet accumulation63. This suggests that in addition to adhesive proteins
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like VWF, coagulation factors are critical drivers of platelet accumulation in the liver after
PHx, a potential basis for prior studies connecting blood coagulation to liver
regeneration63, 64.
reported that hepatic fibrin(ogen) deposition was increased in mice even 48 hours after
PHx64, suggesting a persistent response. In agreement with observations in liver TF-
deficient mice63, administration of the thrombin inhibitor lepirudin reduced hepatic
fibrin(ogen) deposition64, providing further evidence that thrombin drives fibrin(ogen)
deposition in this setting. Beier et al. also discovered that plasminogen activator inhibitor-1
deficiency, anticipated to increase plasmin activity, reduced hepatic fibrinogen deposition
after PHx64. Indeed, prior studies show that activation of fibrinolysis follows shortly after
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fibrin deposition in mice after PHx65. Notably, interpretation of these studies is complex as
prior studies suggest a pro-regenerative role for plasmin(ogen) in liver regeneration66–68,
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Importantly, not all proteolytic targets of thrombin contribute to regeneration. For example,
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hepatocyte proliferation was unaffected after PHx in mice lacking protease activated
receptor-4 (PAR-4)63, the primary receptor for thrombin on mouse platelets70. It is also
important to note that fibrin(ogen) may contribute to liver regeneration through multiple
mechanisms, as it engages not only platelet integrins but also β2 integrins expressed by
leukocytes, another cell type known to play a key role in regeneration after PHx59, 71, 72.
Further studies are needed to identify specific mechanisms underlying fibrin(ogen)-directed
platelet accumulation in the liver after PHx. Notably, platelets themselves amplified hepatic
fibrin(ogen) deposition after PHx63, highlighting clear cross-talk contributing to
amplification of intrahepatic coagulation after PHx. Similarly, additional studies are required
to determine how fibrin(ogen) and VWF collectively promote hepatic platelet accumulation
after PHx. Analogous to PHx in mice, an increase in hepatic fibrin(ogen) deposition was
also evident in liver biopsies from liver resection patients taken 2 hours after ligation of the
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Semin Thromb Hemost. Author manuscript; available in PMC 2021 September 09.
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The sudden increase in portal pressure after resection exposes the endothelial cells to
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excessive shear forces. Indeed, immediate (i.e. within 10 minutes) ultrastructural changes in
the endothelial lining have been observed following PHx77. It is reasonable to speculate that
platelets respond to these changes as they are known to adhere to LSECs78. In this context,
potential VWF release from activated endothelial cells in response to increased shear stress
may play a role in recruiting platelets. Indeed, VWF plasma levels increase rapidly after
partial hepatectomy56, 79. Although these changes may be a general effect of major
abdominal surgery79, 80, VWF plasma levels increased within two hours after induction of
liver regeneration in the liver vein of patients undergoing partial hepatectomy56.
Furthermore, this rapid VWF increase after induction of liver regeneration seemed to be
required for platelet accumulation and liver regeneration in patients56.
Although our previous studies identified that the rapid procoagulant response after PHx is
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initiated by hepatic TF, the trigger for hepatocyte TF activation after PHx has yet to be
discovered. Upon vascular injury, subendothelial TF is exposed to its ligand factor VIIa
(FVIIa), initiating coagulation. However, hepatocytes expresses a TF:FVIIa complex that is
an encrypted state and requires activation via decryption to activate coagulation81.
Phosphatidylserine (PS) externalization triggered by apoptosis is one potential mechanism
of TF decryption in the injured/diseased liver82–84. However, apoptosis after partial
hepatectomy is minimal due to activation of the anti-apoptotic Akt pathway85. It seems
plausible that the immediate change in portal blood flow could activate intrahepatic
coagulation. Indeed, in other experimental settings, it has been shown that shear stress
increased TF expression and activity86, 87. Whether hemodynamic changes after partial
hepatectomy activates coagulation remains to be determined.
Strong evidence from experimental studies links changes in the hemostatic system with liver
regeneration. Moreover, clinical studies have provided corresponding evidence in patients,
wherein fibrinogen or platelet-centered measurements associate with development of liver
dysfunction and outcome after liver resection. Thus, with further study it may be possible to
identify strategies to accelerate liver regeneration in patients after liver resection.
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Starlinger et al. Page 8
Although many of the pro-regenerative factors released by platelets have very short half-
lives when circulating free in blood, storage in platelets serves to protect these factors51. In
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this way, platelets represent very sensitive sentinels that rapidly respond to injury by
releasing their cargo. Platelet activation and aggregation leads to site-specific release of
granule content, which produces locally high concentrations of platelet-derived factors
important for liver regeneration. Rapid availability of these growths factors is a prerequisite
for functional liver regeneration processes. Thrombopoietin (TPO) has been shown to
promote liver regeneration in mice, presumably by increasing platelet number88. Therefore,
TPO might represent a potential therapeutic target as TPO mimetics are approved and in
clinical use. However, elevating platelet number might increase the risk for (portal vein)
thrombosis, mortality, and cancer recurrence89–91. In addition to modulation of platelet
counts, pharmacological modulation of perioperative hepatic hemodynamics, via its impact
on intrahepatic platelet accumulation and perhaps coagulation activation, might represent a
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Identifying mechanisms driving hepatic platelet accumulation seems likely to reveal novel
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Semin Thromb Hemost. Author manuscript; available in PMC 2021 September 09.
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Furthermore, our results indicate that the increase in VWF after liver resection is limited in
patients with underlying liver disease56. As an alternative, administration of these proteins to
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augment the early hepatic platelet response after liver resection seems plausible and would
circumvent possible endothelial exhaustion. Both VWF and fibrinogen are available as
concentrates and used clinically (i.e., Humate-P/Haemate-P, RiaSTAP/Haemocomplettan P).
It seems plausible that in patients where fibrinogen or VWF levels are deemed too low to
support regeneration, this defect could easily be corrected intraoperatively, or even modestly
increased to further promote regeneration. It is worth noting that liver resection patients
commonly receive anticoagulants approximately 6 hours after surgery to reduce the risk of
thrombosis. Hepatic platelet and fibrin(ogen) deposits responsible for driving liver
regeneration occur prior to this63, making it unlikely that postoperative anticoagulation
interferes with this mechanism of regeneration. This also keeps with the plausibility of using
intraoperative interventions to promote platelet-directed liver regeneration while limiting the
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Acknowledgments
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Research support: This research was supported by grants from the National Institutes of Health (NIH) to JPL (R01
ES017537, DK120289), support from the USDA National Institute of Food and Agriculture, and an EHA Research
Grant from the European Hematology Association (EHA) to DG. The content is solely the responsibility of the
authors and does not necessarily represent the official views of the NIDDK or the NIH.
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Figure 1:
Mechanisms linking hemostatic factors to liver regeneration: Platelets rapidly accumulate in
the liver remnant after partial hepatectomy. Accumulation of platelets in the liver appears to
depend on multiple factors including von Willebrand factor (VWF), and on fibrin deposits
formed as a result of intrahepatic coagulation. Experimental evidence suggests that platelets
contribute to liver regeneration through multiple mechanisms including 1) degranulation and
release of serotonin and other growth factors, 2) transfer of RNA by direct interactions with
hepatocytes and 3) potentially through cell-cell interactions with leukocytes or other non-
parenchymal cells. Among the gaps in knowledge include the source of VWF responsible
for driving platelet accumulation (plasma, endothelial cells or platelets themselves), and
whether fibrin drives regeneration through mechanisms independent of its role in platelet
accumulation.
TF = Tissue factor
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