Bivalent property of intra-platelet VWF in liver regeneration and HCC recurrence: A prospective multicenter study

Abstract

BACKGROUND AND AIMS:

A striking difference has been observed in structure and functional properties between plasma and platelet von Willebrand factor (VWF). While the existing evidence has revealed a clinical relevance of plasma VWF-Ag in liver regeneration (LR) and different cancers, this study was designed to explore the properties of intra-platelet (IP) and serum VWF-Ag in patients with hepatocellular carcinoma (HCC) undergoing partial hepatectomy.

METHODS:

A total of 40 patients undergoing partial hepatectomy were prospectively recruited from 3 institutions. VWF-Ag concentrations were evaluated mainly in serum and platelet extracts. Patients were followed-up for postoperative liver dysfunction and HCC recurrence.

RESULTS:

We observed a post-resection increase in the concentration of VWF-Ag in serum and platelet. Patients with postoperative liver dysfunction had substantially reduced serum and IP VWF-Ag concentrations. After a 2-year follow-up, patients with higher post-resection serum and IP VWF-Ag concentrations were found to develop early HCC recurrence. Likewise, IP VWF-Ag was able to independently predict post-resection early HCC recurrence.

CONCLUSION:

This multicenter, prospective, pilot study demonstrates a bivalent property of IP VWF in LR and oncological outcome; low preoperative VWF appeared to have a negative association on post-resection liver dysfunction, whereas, patients with higher post-resection VWF-Ag concentrations were found to have early HCC recurrence.

Keywords

Platelet von Willebrand Factor liver regeneration HCC HCC recurrence

1. Introduction

Von Willebrand factor (VWF), a multimeric, multidomain, multifunctional adhesive glycoprotein, predominantly operates its functions in primary hemostasis by anchoring platelet adhesion and aggregation to exposed subendothelium in damaged blood vessels. The biosynthesis of VWF is confined to endothelial cells (EC), where it is stored in organelles known as Weibel-Palade (WP) bodies, and megakaryocytes or platelets, where it is localized in the alpha-granules. Although the major volume of VWF is secreted by EC, platelets also contain a substantial quantity of VWF, accounting for 10–25% of the total amount of VWF-antigen (concentration) present in normal platelet-rich plasma [13, 23, 37]. Interestingly, the functional properties of intra-platelet (IP) VWF markedly differs from that of the circulating (plasma) VWF [20].

Liver regeneration (LR) after partial hepatectomy (PH) triggers a hypercoagulable state associated with a reduction of gene expression of the many coagulation cascade proteins and elevation in the plasma concentrations of factor VIII and VWF [31]. In rodent models, VWF knockout negatively affected the LR [16]. It has been suggested that platelet accumulation and LR both are largely dependent on VWF [16]. Recently, Starlinger et al. were able to demonstrate that an early VWF-Ag burst after PH is crucial for adequate platelet accumulation and concomitant LR. With this first-in-human evidence they have also stressed on the diagnostic potential of preoperative plasma VWF-Ag concentrations to predict poor postoperative outcome after PH [30].

Tumor cells activate coagulation, through the release of tissue factor, microparticles or P-selectin ligands and expression of thrombin that further augment coagulation. Likewise, tumor cells achieve attachments to endothelium via e-selectin [10]. Platelet coating of tumor cells provides a mechanical shield by protecting the circulating tumor cells from immune attacks [9]. The alliance between platelet, endothelial and tumor-cell membrane integrins is now known to facilitate the hematogenous metastasis of tumor [9]. Studies have demonstrated the presence of fibrin deposition and platelet aggregation in and around different tumor specimens, indicating local activation of coagulation [7]. Furthermore, hemostatic abnormalities are found in 60–100% of patients with malignant tumors on laboratory investigations, including those without thrombotic manifestations [25]. Numerous possibilities of utilizing platelet kinetics in the diagnostic and prognostic evaluation of hepatocellular carcinoma (HCC) have already been observed [3].

VWF provides a bridge between collagen and plat-elets inducing rolling of platelets that results into platelet activation accompanied by shape change and exposure of phosphatidyl-serine on the outer membrane, and release of platelet VWF and other growth factors [26]. Clinical studies have reported an increased concentration of plasma VWF antigen in a variety of malignancies including colorectal carcinoma [27, 33], urinary bladder cancer [36], ovarian carcinoma [8], and lymphoblastic leukemia [12]. Likewise, local and plasma VWF concentrations were found elevated in HCC as compared to chronic hepatitis-B patients [18].

In this study, we investigated the kinetics of IP and platelet-derived (serum) VWF-Ag after PH in patients with HCC and followed them for 2 years. Since the properties of IP VWF-Ag differ from the plasma VWF-Ag, in this study, we have focused on the IP and serum VWF-Ag dynamics and their association with LR and post-resection HCC recurrence.

2. Patients and methods

2.1 Prospective study cohorts

Forty patients with primary HCC (all with Child-Pugh class A subgroup) who went on to have liver resection were enrolled in the study from May 2013 to June 2015. This study is registered in UMIN Clinical Trial Registry (UMIN000026380). The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki (6th revision, 2008) as reflected in a priori approval by the institution’s human research committee. Patients were recruited from 3 different institutions; Kagoshima university hospital, Kirishima medical center and Kagoshima medical center, national hospital organization. All three institutional ethics committee approved analyses of blood samples and patients’ data; signed, informed consent was obtained from all patients. All methods were performed in accordance with relevant guidelines and regulations.

All patients underwent a thorough laboratory evaluation, including serum $\alpha$ -fetoprotein (AFP) and protein induced by vitamin K antagonist-II (PIVKA-II). Hepatic functional reserve was assessed by indocyanine green (ICG) clearance test, 99mTc-galactosyl human serum albumin (GSA) scintigraphy and Child-Pugh score. The diagnosis and staging of HCC were established with triple-phase computed tomography (CT) of the abdomen.

2.2 Definition of Postoperative liver dysfunction

Postoperative liver dysfunction (LD) was assessed based on the “50–50 criteria” criteria by Balzan et al. [4]. This study focused on the later phase (delayed) hepatic regeneration and not merely liver failure, thus a serum bilirubin concentration $>$ 2.9 mg/dL or a PT value $<$ 50% recorded on any day within the first postoperative weeks were defined as postoperative LD.

2.3 Follow up

After the first-month follow-up, patients were followed with ultrasonography (USG) every 3 months after resection, and if USG suggested any evidence of tumor, subsequent contrast-enhanced CT or magnetic resonance imaging (MRI) of the abdomen and non-contrast CT of the chest were performed. The diagnosis of intrahepatic recurrence was made on the basis of imaging alone if the tumor exhibited the typical enhancement characteristics. A 2-year follow-up was described as the end point for early HCC recurrence in this study.

2.4 Sample preparation

2.4.1 Serum and plasma

Venous blood was collected preoperatively (PRE OP) and after four weeks post-resection (POST OP).

Whole blood was collected in a serum separating tube and 3 citrate tubes, containing 0.5 ml of sodium citrate (for plasma and platelet preparations), an EDTA-2k tube (Venoject II, Terumo Corp., Tokyo, Japan) was used to collect blood for cell count. Serum tube was incubated at room temperature for 30 minutes to allow clotting. Serum and plasma tubes were centrifuged at 1710 $\times$ g for 10 minutes. The top 75% of the resultant supernatant was gently pipetted to avoid contamination.

2.4.2 Platelet isolation

Citrate tubes containing venous blood were centrifuged at 90 $\times$ g for 15 minutes. About 75% of the top resultant platelet rich plasma (PRP) was pipetted to avoid contamination. The PRP was further centrifuged at 2810 $\times$ g to isolate platelets. The supernatant, platelet poor plasma (PPP), was collected precisely and decanted for the complete removal of the plasma from the pellets. Platelet pellets isolated from each 200 $\mu$ l of PRP were suspended in 220 $\mu$ l of lysis buffer (150 mM sodium chloride, 25 mM Tris-HCl pH 7.6, 1% Tergitol-type NP-40 and 0.1% sodium dodecyl sulfate, 1% sodium deoxycholate in distilled water to make 100 ml solution); after incubating for 20 minutes, the lysate solution was pipetted and vortexed until the pellets were completely dissolved in the solution. CBC was carried out in 3 preparations: whole blood, PRP and PPP.

2.4.3 Quantification of cytokines

Serum and platelet extracts were analyzed together by commercially available enzyme-linked immunosorbent assay (ELISA) tests for human Von Willebrand factor (Abcam, Waterloo, Australia, ab108918) according to the manufacturer’s guidelines.

2.4.4 Calculation of IP VWF concentration

Platelet content of VWF-Ag per 10 [6] platelets was calculated using the previously described equation [2]. Briefly, 220 $\mu$ l of lysis buffer was added in platelets isolated from each 200 $\mu$ l of PRP. Platelet count along with FBC was carried out in the isolated PRP.

(220 $\mu$ l $\times$ VWF-Ag (mU/ml) in platelet lysate solution/1000 $\times$ 10 ${}^{6}$ platelets/ $\mu$ l)/(200 $\mu$ l PRP $\times$ platelet count in PRP (10 ${}^{6}$ platelets/ $\mu$ l).

Table 1
Clinico-pathological data at the time of resection of primary HCC and after 2-years follow-up

Variable demographical and

clinical data (N

=

40)

All patients (N

=

40)

Post-resection recurrence (

n=

15)

No recurrence (

n=

25)

Age, median (SD)

(8.43)

71.0

(8.42)

74.0

(8.59)

Sex,

n

(%)

Male

(75%)

(60%)

(84%)

Female

(25%)

(40%)

(16%)

Etiology,

n

(%)

HBV

(30.0%)

(26.66%)

(32%)

HCV

(32.5%)

(46.66%)

(20%)

None

(37.5%)

(26.66%)

(48%)

Fibrosis,

n

(%)

(10%)

(0%)

(16%)

1–2

(45%)

(40%)

(25%)

3–4

(45%)

(60%)

(36%)

Total bilirubin, mg/dl

Median (SD)

0.87

(0.37)

0.80

(0.28)

0.72

(0.42)

\geqslant

1.0

1.31

(0.40)

1.3

(0.21)

1.2

(0.51)

INR

Median (SD)

1.11

(0.24)

1.09

(0.36)

1.02

(0.09)

\geqslant

1.0

1.13

(0.26)

1.09

(0.36)

1.04

(0.83)

AFP, ng/ml

Median (SD)

835.2

(2227)

24.30

(2072)

15.7

(2344)

\geqslant

2076

(3186)

102

(2703)

914.8

(3671)

Platelet count,

\times

{}^{3}

/ul

Median (SD)

156.2

(65.13)

134

(58602.12)

156

(67503.50)

<

135

79.3

(11.11)

(12864.68)

76.5

(12864.68)

Extent of resection,

n

(%)

Minor hepatectomy

(67.5%)

(73.33%)

(60%)

Major hepatectomy

(32.5%)

(26.66%)

(40%)

Tumor stage,

n

(%)

I–II

(70%)

(73.33%)

(68%)

III–IV

(30%)

(26.66%)

(32%)

Tumor size,

n

(%)

<

5 cm

(80%)

(73.33%)

(72%)

\geqslant

5 cm

(20%)

(26.66%)

(28%)

Histological grade,

n

(%)

Well to moderate

(80%)

(86.66%)

(76%)

Poor

(20%)

(13.33%)

(24%)

2.4.5 Statistical analysis

Statistical analyses were conducted with SPSS software (version 25.0.0; SPSS, Inc., Chicago, IL) and Graph Pad Prism (version 6.0d for Mac OS X, USA, GraphPad Software, San Diego California, USA), and were based on nonparametric tests (Mann-Whitney’s U test, Wilcoxon’s signed rank test, and Spearman’s correlation). The Fisher’s exact test was used to evaluate frequencies between categorical variables. Patients were stratified into lower and higher postoperative serum and IP VWF-Ag groups based on the cut-off point (median value) obtained from the non-recurrent group; on this basis, Cox’s proportional hazards regression model was used for the univariable (UVA) and multivariable analyses (MVA) to determine the variables independently associated to recurrence. Due to the collinearity between serum and IP VWF, MVA was separately run for these two variables. Receiver operating characteristic (ROC) analysis was performed to assess the specificity and sensitivity of Serum and IP VWF levels to predict recurrence. Two-tailed P values of less than 0.05 were considered statistically significant. IP VWF-Ag was expressed per 10 ${}^{6}$ platelets.

2.5 Data availability statement

All data generated or analyzed during this study are included in this published article (and its Supplementary Information files). The additional datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

3. Results

A total of 40 patients diagnosed of primary HCC who went to have PH between May 2013 and June 2015 from 3 different institutions (Kagoshima university hospital, Kirishima medical center and Kagoshima medical center national hospital organization) were included in our prospective study cohort. This multicenter approach was advocated to maintain the high homogeneity in the patients’ characteristics and clinical validation in this early pilot study. Table 1 summarizes the clinico-pathological characteristics of the patients at the time of resection and on the basis of post-resection HCC recurrence after a 2-year follow-up study. During this 2-year follow-up study, 15 patients developed post-resection HCC recurrence.

3.1 Intra-platelet VWF-Ag is elevated after partial hepatectomy and tended to associate with the postoperative liver dysfunction

We initially evaluated the perioperative VWF-Ag concentrations in serum and platelet extracts. To exclude the biases of platelet activation elicited by surgery-related injuries, we measured the growth factors four weeks after PH, when the surgical wounds were already healed. Serum and IP VWF-Ag concentrations revealed a distinct and significant increase after PH (median serum VWF-Ag: PRE OP, 877.6 mU/ ml; POST OP, 2179 mU/ml, $P<$ 0.00010, and median IP VWF-Ag $\times$ 10 ${}^{6}$ platelets: PRE OP, 0.81 mU; POST OP, 1.17 mU, $P<$ 0.05; Fig. 1a and b).

Figure 1.

VWF-Ag concentrations before (PRE OP) and post-resection (4 weeks after PH) (POST OP) in: serum (a) and IP (b). IP VWF concentration expressed per 10 ${}^{6}$ platelets. Wilcoxon matched-pairs signed rank test. ${}^{*}P\leqslant$ 0.05; ${}^{****}P\leqslant$ 0.0001.

We then assessed the preoperative serum and IP VWF-Ag concentrations on the basis of postoperative liver dysfunction (LD). Patients with LD tended to have less preoperative serum and IP VWF-Ag concentrations (median serum VWF-Ag: No LD, 1034 mU/ ml; LD, 729.8 mU/ml, $P<$ 0.05, and median IP VWF $\times$ 10 ${}^{6}$ platelets: No LD, 0.83 mU; LD, 0.65 mU, $P>$ 0.05; Fig. 2a and b).

Figure 2.

Preoperative VWF-Ag concentrations in patients with and without liver dysfunction (LD) in: serum (a) and IP (b). IP VWF-Ag concentration expressed per 10 ${}^{6}$ platelets. Mann-Whitney test. ${}^{*}P\leqslant$ 0.05.

3.2 Patient with post-resection HCC recurrence showed higher concentrations of post-resection intra-platelet VWF-Ag

After a 2-year follow-up, we stratified the platelet VWF-Ag concentration on the basis of post-resection HCC recurrence. We observed that the patients with early recurrence had higher post-resection serum and IP VWF-Ag compared to patients with no recurrence (POST OP median serum VWF-Ag: no recurrence, 1356 mU/ml; recurrence, 3069 mU/ml, $P<$ 0.05; and POST OP median IP VWF $\times$ 10 ${}^{6}$ platelets no recurrence, 0.91 mU; recurrence, 1.62 mU, $P<$ 0.01 Fig. 3a and b).

Figure 3.

Post-resection VWF-Ag concentration in patients with and without HCC recurrence in: serum (a) and IP (b). IP VWF-Ag concentration expressed per 10 ${}^{6}$ platelets. Mann-Whitney test. ${}^{*}P\leqslant$ 0.05; ${}^{**}P\leqslant$ 0.01.

In our investigations, we have been focusing on the post-resection recurrence, which represents a different continuum of tumourigenesis than that of the primary cancers, thus, our studies stress on the post-resection markers. However, the preoperative serum and IP VWF-Ag were in accordance with the post-resection results (PRE OP median serum VWF: no recurrence, 830 mU/ml; recurrence, 1220 mU/ml, $P<$ 0.05; and PRE OP median IP VWF $\times$ 10 ${}^{6}$ platelets: no recurrence, 0.79 mU; recurrence, 1.01 mU, $P>$ 0.05 Supplementary Fig. 1a and b).

3.3 Post-resection intra-platelet VWF-Ag independently predicted early HCC recurrence

Since we observed a distinct difference in concentrations of platelet VWF-Ag in patients with and without early post-resection recurrence, we further evaluated if serum and IP VWF-Ag could predict HCC recurrence. The univariable (UVA) and multivariable analyses (MVA) with the Cox regression hazard model showed that the post-resection IP VWF-Ag could independently predict HCC recurrence (hazard ratio $=$ 0.242, CI $=$ 0.07–0.76, $P<$ 0.05). Considering the collinearity of platelet count with the platelet VWF, platelet count was excluded in the MVA. The detailed outcome of univariable and multivariable analyses has been demonstrated in Table 2.

Table 2
Univariable and multivariable analyses by cox regression hazard model to predict post-resection early HCC recurrence

	Univariate				Multivariate
Variables	B	Exp(B)	95% CI	$P$	B	Exp(B)	95% CI	$P$
Age at resection	0.006	1.00	0.94–1.06	0.83
Sex	$-$ 0.03	0.97	0.30–3.03	0.95
Etiology (viral/non viral)	0.34	1.40	0.55–3.58	0.47
Fibrosis grade (0–2/3–4)	$-$ 0.68	0.50	0.17–1.41	0.19
Tumor size, cm	$-$ 0.82	0.92	0.75–1.12	0.42
Tumor stage (I–II/III–IV)	$-$ 0.38	0.96	0.30–3.02	0.94
Tumor multiplicity	0.48	1.61	0.51–5.09	0.41
Microvascular Invasion	$-$ 0.50	0.60	0.13–2.69	0.51
Histological differentiation	0.48	1.63	0.36–7.22	0.52
Prior TACE/RFA	1.36	3.91	1.24–12.32	0.02	$-$ 1.35	0.25	0.81–0.81	0.02
Type of hepatectomy	$-$ 0.38	0.682	0.21–2.14	0.51
INR $\geqslant$ 1.0	1.77	5.90	0.77–44.94	0.09
AFP $>$ 20 ng/mL	0.60	1.83	0.66–5.06	0.24
PIVKA II $>$ 40, mAU/L	$-$ 0.25	0.77	0.28–2.14	0.62
AST, U/L	0.20	1.01	0.99–1.04	0.09
ALT, U/L	$-$ 0.005	0.99	0.97–1.01	0.61
Albumin, g/L	0.20	1.23	0.46–3.24	0.67
TB, mg/dL	0.14	1.15	0.34–3.92	0.81
PRE OP platelet count $\times$ 10 ${}^{3}$ / $\mu$ L	$-$ 0.08	0.92	0.83–1.01	0.10
POST OP platelet count $\times$ 10 ${}^{3}$ / $\mu$ L	$-$ 0.15	0.85	0.74–0.99	0.04
POST OP serum VWF-Ag, mU/mL	$-$ 1.53	0.21	0.04–0.9	0.04	$-$ 1.23	0.29	0.06–1.33	0.11
POST OP IP VWF-Ag, mU/ml $\times$ 10 ${}^{6}$ platelets	$-$ 1.42	0.24	0.07–0.76	0.01	$-$ 0.14	0.24	0.07–0.7	0.01
Clavien-dindo severe morbidity	$-$ 0.77	0.46	0.06–35.0	0.45

3.4 Exploration of the cut-off value for post-resection serum and IP VWF-Ag in early HCC recurrence and their relevance in estimating disease-free interval

Figure 4.

ROC-curve analysis of serum and IP VWF-Ag to define a cut-off value to predict post-resection HCC recurrence. IP VWF-Ag concentration expressed per 10 ${}^{6}$ platelets.

As we observed a significantly higher concentration of post-resection serum and IP VWF along with predictive potential in patients with early HCC recurrence, we plotted receiver operating characteristic (ROC) curves to assess their diagnostic relevance (Fig. 4). The Youden’s index from ROC curves revealed a cut-off of 1428 mU/ml for serum and 1.11 mU for IP VWF-Ag $\times$ 10 ${}^{6}$ platelets to identify patients with early post-resection HCC recurrence with sensitivity of 86.70% for serum VWF-Ag and 80% for IP VWF-Ag; and specificity of 56% for serum and 64% for IP VWF-Ag. This yielded an area under curve (AUC) of 0.72; 95% CI (confidence interval), 0.56–0.88; $P<$ 0.05 for serum, and AUC of 0.76; 95% CI, 0.61–0.91; $P<$ 0.01 for IP VWF-Ag.

On the basis of the cut-off values obtained from the ROC curves, patients were classified in high and low concentrations of VWF-Ag in serum and IP [Post serum: low group (N) $=$ 16, recurrence occurred (n) $=$ 2; high group (N) $=$ 24, recurrence occurred (n) $=$ 13] [Post IP VWF: low group (N) $=$ 19, recurrence occurred (n) $=$ 3; high group (N) $=$ 21, recurrence occurred (n) $=$ 12]. Subsequently, a log-rank test was run to determine the differences in disease-free interval (DFI) distribution between groups with high and low VWF-Ag. The patients with higher serum [ $\chi$ 2 (2) $=$ 7.41, $P<$ 0.01] or IP [ $\chi$ 2 (2) $=$ 6.90, $P<$ 0.01] VWF-Ag were found to have shorter DFI (Fig. 5a and b).

Figure 5.

Kaplan-Meier disease-free interval curves according to high and low post- resection serum (a) and IP (b) VWF-Ag obtained from ROC curves.

4. Discussion

VWF synthesized in platelets is predominantly stored in $\alpha$ -granules and only a tiny portion of it is secreted in the plasma. Certainly, plasma VWF is the main determinant of the primary hemostasis whereas IP VWF partially contributes to it [15]. Less is known about the precise role of IP VWF. Albeit some existing evidence on plasma VWF in LR and liver disease, investigation on the role of IP VWF in liver pathophysiology has remained an untouched area. In this study, we were able to show the first human evidence on a bivalent property of IP VWF-Ag in LR after PH and early HCC recurrence.

Evidence stem from rodent and human investigations have now established the pivotal role of platelets in LR [17, 19, 28, 29, 30]. The hemodynamic alteration led by PH triggers platelet accumulation in hepatic sinusoids; this is followed by a sequential interaction with liver cells contributing to the initiation and promotion of LR [22]. Kirschbaum et al. recently demonstrated that platelet influx after PH and its contribution to LR is largely dependent on VWF [16]. The inference of their study was translated into clinical settings in another study conducted by Starlinger et al. suggesting an influential role of VWF-Ag in LR and postoperative liver function recovery after liver resection [30].

Studies suggest some striking differences in properties of EC-derived and IP VWF. A contrasting dissimilarity observed between EC-derived VWF and IP VWF is that the later one does not bind to factor VIII [35]. Another in vitro study highlights the difference in the glycosylation profile between the EC and platelet-derived VWF [21]. Likewise, IP VWF is known to be resistant to ADAMTS 13 cleavage making platelets enriched in ultra-larger VWF multimers [20]. IP VWF-Ag enhances spreading of contact platelets on the subendothelium and is presumed to act on high shear stress by establishing an intercellular bridge between platelets. Liver resection or PH elicits a high shear stress with resultant platelet influx [22]; therefore, the role of IP VWF could be of highly influential during LR. An adequate increase in plasma VWF-Ag concentration following liver resection appeared beneficial for sufficient postoperative hepatic regeneration [30], likewise, we were able to demonstrate that an increase in IP VWF-Ag after PH was associated with post-resection liver function recovery. While the importance of plasma VWF-Ag in LR has already been acknowledged, the relevance of IP VWF in LR is yet to be explored. One issue that can be argued in the current study could be the kinetics of IP VWF that was not evaluated immediately after PH, during the early phase of LR. The elevation of IP VWF-Ag, observed in the late phase of LR in our study, is in accordance with the study conducted by Starlinger et al. demonstrating the elevation of plasma (circulating) VWF-Ag immediately after PH [30]. Although the low statistical power may undermine our findings on the importance of IP VWF-Ag on LR, this first evidence has urged for a more mechanistic investigation to explore the explicit role of IP VWF-Ag in LR. Concurrently, VWF is a well-known acute phase inflammatory marker, and is therefore difficult to establish exactly whether raised VWF-Ag levels immediately after PH is causal or simply an epiphenomenon of (surgical) injury-associated inflammatory process; the current study has precluded this bias to some extent. Besides, the elevated plasma VWF-Ag levels may mechanistically associate with the tight regulation by ADAMTS13. Regenerating liver was found to temporary suppress AMAMTS13 expression, provoking the elevation of plasma VWF-Ag during LR [31]. Our findings of elevation of IP VWF-Ag, which has less to do with suppressed circulating ADAMTS13, indicate a more complex phenomenon on the diverse kinetics of VWF in LR.

Cancer cells are known to induce platelets to produce growth factors and matrix metalloprotease-2 that promulgate inflammation [14]. Tumor-cell interaction with platelets is mediated by integrins and their ligands; however, the exact molecule facilitating this interaction has merely remained as an equivocal assumption. VWF is also one of the major platelet ligands that could potentially participate in platelet-tumor cells interaction. An elevated concentration of plasma VWF-Ag is not only associated with the malignancy-related thrombosis but also correlated with cancer progression in a variety of malignancy [18, 32, 34]. The multimeric nature of VWF enables it to bind several ligands simultaneously and direct interaction with the tumor cells. These include interaction with different receptors such as GPIb, or the integrins aIIbb3 and aVb3 [32]. While most of the aforementioned studies focused on the association between plasma VWF and oncological outcome, we have, for the first time, shown the correlation and predictive potential of IP VWF-Ag in HCC recurrence.

The paradoxical properties of platelet and its growth factors’ kinetics affect the liver pathophysiology in a very disease-specific, stage-specific, and site-specific manner [5]. A bivalent property of IP serotonin was observed after PH; immediate post-operative serotonin, which appeared to be beneficial for LR tended to be associated with liver cancer recurrence [14]. We observed an altered response of platelet vascular endothelial growth factor (VEGF) in different pathophysiology [1, 11]; in one study, we found a positive correlation of IP VEGF with the tumor characters preoperatively, while the post-resection IP VEGF appeared to adjust its concentration in relation to the future remnant liver volume in patients undergoing PH [1]. Surprisingly, in another study, we observed an exhaustive pattern of some IP growth factors in HCC recurrence [2]. The positive correlation of IP VWF-Ag observed in the current study thus suggests how platelets differentially regulate the synthesis and release of its individual growth factor during LR and cancer recurrence. IP VWF-Ag might impact LR after resection but it might also add to the pathobiology of tumor recurrence.

Collectively, our study provides the evidence for the prognostic and predictive relevance of perioperative IP VWF-Ag; evaluation of IP VWF-Ag thus seems to offer a dual advantage of characterizing the operative and oncological outcome. The perioperative kinetics of IP VWF observed in our study is in accordance with a previous study on plasma VWF that was conducted in a larger cohort [30]. Despite a multicenter, prospective nature of the current study, these results need validation from much larger or randomized cohort to utilize the clinical benefit of perioperative IP VWF-Ag. Hepatic conditions, like portal hypertension, advance liver diseases, influence the plasma VWF-Ag concentrations [6]; however, this study has not been able to address if IP VWF-Ag is associated with the underlying liver conditions.

In conclusion, this study has demonstrated higher serum and IP VWF levels in those patients who developed cancer recurrence, which fits the idea that VWF is involved in regeneration of healthy and growth of malignant liver tissue. However, whether the VWF pool in platelets in really responsible is unclear, further studies are deemed essential to delineate not only the precise dynamics of VWF-Ag but also the detail activities of platelet VWF-Ag during hepatic regeneration and cancer recurrence. It would be too early to argue on the clinical implications of IP VWF-Ag in LR and HCC recurrence, however, this study has been able to seed a fresh insight on a bivalent property of IP VWF-Ag in LR and oncological outcome.

Footnotes

Acknowledgments

Dr. Bibek Aryal is a fellow of Japan society for the promotion of science (JSPS). This study is supported by JSPS KAKENHI Grant Numbers: JP18F16420, JP16H05229 and JP26293130. UMIN clinical Trial Registry (Registration identification number: UMIN000019145).

Conflict of interest

No conflict of interest to disclose.

Supplementary data

Supplement 1.

Preoperative VWF-Ag concentration in patients with and without HCC recurrence in: serum (a) and IP (b). IP VWF concentration expressed per 10 ${}^{6}$ platelets. Mann-Whitney test . ${}^{*}P\leqslant$ 0.05.

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Bivalent property of intra-platelet VWF in liver regeneration and HCC recurrence: A prospective multicenter study

Abstract

BACKGROUND AND AIMS:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Patients and methods

2.1 Prospective study cohorts

2.2 Definition of Postoperative liver dysfunction

2.3 Follow up

2.4 Sample preparation

2.4.1 Serum and plasma

2.4.2 Platelet isolation

2.4.3 Quantification of cytokines

2.4.4 Calculation of IP VWF concentration

Table 1 Clinico-pathological data at the time of resection of primary HCC and after 2-years follow-up

2.5 Data availability statement

3. Results

3.1 Intra-platelet VWF-Ag is elevated after partial hepatectomy and tended to associate with the postoperative liver dysfunction

Table 2 Univariable and multivariable analyses by cox regression hazard model to predict post-resection early HCC recurrence

Footnotes

Acknowledgments

Conflict of interest

Supplementary data

References

Table 1
Clinico-pathological data at the time of resection of primary HCC and after 2-years follow-up

Table 2
Univariable and multivariable analyses by cox regression hazard model to predict post-resection early HCC recurrence