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Resting human platelets express cytoplasmic HMGB1, activated platelet release of HMGB1

In our study, we discovered that HMGB1 protein was high expressed in washed human
platelets (WPs). Next, we further confirmed HMGB1 protein were mainly located in the
intracellular when it in the resting state by immunoblotting or ELISA test, rarely
HMGB1 protein exist in the plasm, the resting platelets in pellets contain HMGB1 protein
were 10.27?±?1.01 ng/10
⁹
platelets, the extracellular fluid of platelets contain HMGB1 protein were only 0.19?±?0.13 ng/10
⁹
platelets, however, when added simultaneously with the platelet agonists thrombin
(0.2 U/ml), thrombin-stimulated platelets contain HMGB1 protein in pellets were changed
to 2.39?±?0.70 ng/10
⁹
platelets and extracellular HMGB1 protein levels in the supernatant significantly
increase to 7.86?±?1.10 ng/10
⁹
platelets. Compared to resting platelets, the level of HMGB1 protein both in pellets
and in supernatant had statistically significant difference after thrombin-stimulated
platelet (Fig. 1). The results are consistent with other researcher previous finding 18]. The purpose of releasing of the HMGB1 protein during platelet activation is still
not clear, what kind of role it will play in platelet activation is our next step
focal point.

Fig. 1. Platelets express HMGB1. a Representative immunoblot of HMGB1 expressions in WPs (1?×?10
⁹
/ml) from five different health donors, Western blot analysis of extracts HMGB1 protein
by HMGB1 polyclonal antibodies. b HMGB1 was assessed by ELISA in pellets and supernatant of WPs that either resting
or adding thrombin (0.2 U/ml). After WPs were activated, the concentration of HMGB1
in pellets or in the supernatant signicantly different from the resting WPs group
(?=?5, *P??0.01). c WPs incubated with or without (negative controls) rabbit polyclonal-anti-HMGB1 (blue)
in combination with rabbit-anti-Actin (red), respectively. Images under fluorescent
microscopy, the results indicated that the resting WPs high expression of HMGB1 protein

High conceration of rHMGB1 protein directly induce platelet aggregation and low dose
of rHMGB1 protein potentiates other agnoists-induced platelet aggregation activated
platelets release of HMGB1 protein, which is whether similar to thromboxane A2 (TXA2),
ADP, or Polyphosphates (Polyp) that were released from activation of platelets then
involved in increasing platelet-self activation or enhances blood clotting reactions.so
we first examined the effect of rHMGB1 protein on platelet aggregation, test results
were amazing and revealed that stimulation of WPs with various levels of HMGB1 protein
(0 to 160 ?g/ml) led to a concentration dependent induction of platelet aggregation
(Fig. 2a, b). A few micrograms of (1–5 ?g/ml) rHMGB1 protein alone can not induce platelet aggregation,
while as the protein concentration increasing, compared to control group (ALB), rHMGB1
protein (10 ?g/ml) induce platelet aggregation rate were 10.3 %?±?4.7 % (?=?5, P??0.05), because LPS stimulation also can induce platelet secretion and promote aggregation.
we wish to exclude the possibility that contaminating LPS in the HMGB1 preparation
(500 pg LPS per microgram of HMGB1) contributes to the observed increase platelet
aggregation. Accordingly, an effective inhibitor of LPS, polymyxin B (PMB), was employed
in parallel experiments. when given at a low concentration (500 pg/ml), LPS not direct
induce platelet aggregation, LPS effect completely abrogated after rHMGB1 by co-incubation
with PMB (1 ?g/ml). We found that rHMGB1 (40 ?g/ml)-induced platelet aggregation was
not decreased by PMB (Fig. 2c, d). in contrast, rHMGB1-induced platelet aggregation generation was severely impaired
in the presence of the proteinase K (PK) which dissolves the rHMGB1 proteins and destroy
the native protein structure. When HMGB1 pre-incubated with PK (500 ?g/ml) then the
platelet aggregation drop markedly from 47.4 %?±?5.2 % to 11.7 %?±?5.7 % (?=?5, P??0.05). We acquired the same result that treatment HMGB1 via rreversible thermal
denaturation that changed the protein high-grade structure with high temperature cannot
induce platelet aggregation.

Fig. 2. HMGB1 directly induces platelet aggregation, results are expressed as mean?±?SE. The
data shown here are representatives of at least five experiments from different donor.
a Various concentrations of HMGB1 protein induced platelet aggregation curves, albumin
(ALB) is negative control, (b). Record data are means the maximum platelet aggregation rate induced by HMGB1 (?=?5, *P??0.05 vs. ALB group). c and d The effects of PMB or proteinase K on HMGB1-induced platelet aggregation curves.
HMGB1 (40 ?g/ml) were pretreatmented with PMB (1 ?g/ml) or proteinase K (500 ?g/ml
PK) for 10 min at RT, or thermal denaturation of HMGB1 (TD) at 100 °C for 10 min respectively,
rHMGB1-induced platelet aggregation were obvious inhibited by PK or treatment of thermal
denaturation, but not affected by PMB. (?=?5, *P??0.05; **P??0.05. vs HMGB1 group)

Hatada et al. from Japan reported that disseminated intravascular coagulation (DIC)
was associated with significantly high plasma HMGB1 and the highest plasma levels
of HMGB1 can be achieved 16.58?±?11.01 ng/ml in non-survivors patients with organ
failure 21]. Accordingly this reports, we speculated that the highest plasma levels of HMGB1
protein in local inflammation, necrotic tissue or worn-out organs may achieve a few
micrograms. now that low dose of HMGB1 protein (1 ?g/ml) which alone incapable of
inducing platelet aggregation, it was unknown that low level of HMGB1 protein together
with other agonists whether had best cooperation effects. Therefore, when adding simultaneously
with subthreshold concentrations of agonists thrombin (0.2 U/ml) or collagen (0.3 ?g/ml),
we discovered that HMGB1 increased the maximum platelet aggregation rate induced by
subthreshold level of thrombin or collagen (Fig. 3a, b). These results were further confirmed by thromboelastogram (TEG) analyzes. when
the whole blood were pretreatmented with HMGB1, the Maximum Amplitude (MA) in TEG,
which represents fibrin and platelet aggregation, was significantly enhanced from
61.7?±?6.8 mm to 75.6?±?7.5 mm (?=?5, P??0.05) (Fig. 3c, d). Meanwhile, from the the result of TEG, we found that the cloting reacting time
(R min), which represents the rate of initial fibrin formation and is related to plasma
clotting factors and circulating inhibitory activity, gotten slightly extended but
between of two have no statistical differences (data not show). These result suggested
that HMGB1 incapable of initiating coagulation through directly activate coagulation
factor.

Fig. 3. HMGB1 enhanced agonists-induced platelet aggregation. a A subthreshold concentration of thrombin (0.2 U/ml) or collagen (0.3 ?g/ml) was added
to WPs which were preincubeted with HMGB1 (1 ?g/ml) or ALB for 10 min at 37 °C, above
is representive platelet aggregation curve. b Data are expressed as mean?±?SE, HMGB1 (1 ?g/ml) that significantly enhancing thrombin
or collagen-induced platelet aggregation rate (?=?5, *P??0.05. vs collagen?+?ALB or thrombin?+?ALB group respectively). c Representative map of thromboelastogram (TEG) analyzes, the whole blood prior stimulated
with HMGB1 (1 ?g/ml) for 10 min at 37 °C, this one Maximum Amplitude (MA) was 77.1 mm.
d Analyzing quantitative data from C, the MA of the HMGB1 group were enhanced (?=?5, *P??0.05. vs HMGB1?+?ALB)

Measurement of ATP release and expression of P-selectin

Platelet secretion is critical in amplifying platelet activation and in stabilizing
thrombus. It is well established that activation platelets secreted ATP from dense
granules. In addition, P-selectin which is a member of selectin family of cell surface
receptor and also known as platelet activation dependent granule external membrane
protein. To determine HMGB1simulated platelet secretion, we first measured the release
of ATP in the supernatant in HMGB1 stimulated WPs (Fig. 4a). The results indicated that a few micrograms of HMGB1 alone was suficient to induce
platelets release of ATP, when WPs incubated with HMGB1 protein (1 ?g/ml), measured
ATP in the supernatant were 0.70?±?0.08 nmol/mL, while the control (add Buffer) were
only 0.53?±?0.07 nmol/ml, ATP secretion was sinificantly enhanced in HMGB1-stimulated
WPs. Similarly, in order to exclude the possibility that contaminating LPS contributes
to the observed increase platelet secretion. LPS (500 pg) was able to slightly increase
ATP in WPs (P??0.05), co-incubation with PMB (1 ?g/ml) completely blocked LPS-mediated
platelet secretion, but did not change rHMGB1-induced platelet secretion (Fig. 4d).

Fig. 4. a ATP release was determined (by ATP Assay Kit) in rHMGB1-incubated WPs (1?×?10
⁹
/ml), WPs were treated with rHMGB1 (1-80 ?g/ml) in an aggregometer (1200 rpm) for
5 min at 37 °C (?=?6, *P??0.05 vs. rest group). b WPs were pre-incubated with various concentrations of HMGB1 protein for 10 min, fixed
with paraformaldehyde, then incubated with HMGB1 in the presence of a monoclonal anti-human
P-selectin Ab or control mouse IgG (Rest) for 30 min at room temperature. (?=?6, *P??0.05; **P??0.05, vs. rest group). c Flow cytometric determination of P-selectin expression level. d The effects of LPS and PMB on rHMGB1-induced ATP in WPs. (?=?6, *P??0.05; **P??0.05, vs. rest group)

During platelet activation, P-selectin is translocated from intracellular granules
(?-granules) to the external membrane, it expression on platelets determines size
and stability of platelet aggregates 22]. HMGB1 inducing platelets P-selectin expression and the results suggested that HMGB1
also stimulated platelets ? granule secretion (Fig. 4b, c). measured P-selectin expression level in HMGB1-incubated (1 ?g/ml) WPs were 14.0 %?±?5.3 %,
although P-selectin expression level had a slightly increase while the results between
the two (control group, 6.4 %?±?4.8 %) had no statistical difference, higher dose
of HMGB1 (10 ?g/ml) produced a significant increase P-selectin-positive platelets.
in a words, these results revealed that HMGB1 stimulate platelet both dense and ?
granule.secretion.

HMGB1-induced platelet aggregation depends on GPIIb-IIIa complex activation

The linking of the platelets via fibrinogen brings about platelet aggregation. The
glycoprotein (GP) layer of the platelet, such as GPIb, GPIIb-IIIa or GPIa-IIa and
so on, plays a very important role in platelet function including adhesion and aggregation
23]. Normal primary platelet aggregation mainly requires agonist-mediated activation
of membrane GPIIb-IIIa, which directly interact with fibrinogen mediated platelet-platelet
interactions, may be involving other GP. We unknown whether HMGB1-induced platelet
aggregation also mediated via GPIIb-IIIa which triggered an outside-in signaling cascade.
Next, we found a patients with Glanzmann thrombasthenia (GT), who was first diagnosed
and molecular biological analyses showed a severe reduction in surface GPIIb-IIIa
receptors in platelets 20]. (Figure 5a) (the details of diagnosis information we published in Blood, 1996). The most common
of GT patients, was characterized by a defective platelet aggregation in response
to agonists that depended GPIIb-IIIa receptors. results from we found that HMGB1 was
incapable of inducing platelet aggregation in patients with GT, as positive control,
ristocetin can stimulate GT patients platelet aggregation via characterizing the interaction
of von Willebrand factor (VWF) with platelets GPIb 24]. Furthermore, to obtain more direct evidence for such a linkage that the activated
conformation of GPIIb-IIIa formation on HMGB1-mediated platelets. We confirmed PAC-1
expression on HMGB1-stimutited platelets by flow cytometry. The results indicated
that a significant increase of PAC-1 expression on HMGB1-stimulated platelets. All
these results indicated that signaling in HMGB1-induced platelet aggregation also
depending an increased affinity of integrin GP IIb/IIIa.

Fig. 5. HMGB1-induced platelet aggregation depends on GPIIb-IIIa. a the results of the flow cytometric analysis of a patients with Glanzmann thrombasthenia
(GT), CD41a and CD61 only expressed 0.7 % and 0.5 % respectively. b HMGB1 was incapable of promoting platelet aggregation in patients with GT, as positive
control, Ristocetin (1.25 mg/ml) can promote platelet aggregation. c Representative graph of Flow cytometry analysis of PAC-1. d HMGB1 up-regulated the expression of platelet surface Pac-1 (?=?5, *P??0.01 vs. ALB)

HMGB1 induced platelet activation depended TLR4

At present, it is very clear that HMGB1 transmembrane signaling pathways mainly depend
TLR-4, TLR-2, and RAGE 10], 11]. To evaluate the potential roles of these receptors in HMGB1-induced platelet activation,
the involvement of TLR2, TLR4 and RAGE in HMGB1-induced platelets aggregation or secretion
was investigated by specific neutralising antibodies. WPs were preincubated with various
concentration of blocking Abs directed against these receptor for 20 min respectively
and then rHMGB1 (40 ?g/ml) was added to platelet aggregation test. Our results suggested
that HMGB1- induced platelet aggregation was obviously decreased after in the presence
of increasing concentrations of anti-TLR4 Abs. in contrast, the irrelevant control
antibody (IgG2a) or anti-TLR2 Abs and anti-RAGE Abs did not alter HMGB1-induced platelet
aggregation even when given the same concentrations. Similarily, change in expression
of P-selectin was consistent with above observations. WPs pretreatmented with anti-TLR4
(50 ?g/mL) for 20 min and then HMGB1 (40 ?g/mL) was added to measure P-selectin expression,
the exposure of P-selectin were reduced from 52.8 %?±?5.6 % to 24.7 %?±?6.1 % in HMGB1-simulated
WPs (*P??0.05) (Fig. 6f, g), meanwhile, we examined the effect of the simultaneous with all neutralizing antibodies
and the results indicated incapable of any further enhance the inhibition effect (data
not shown). In conclution, above results suggested that the stimulatory effect of
HMGB1 on platelet activation is, at least in part, TLR4 dependent.

Fig. 6. HMGB-induced platelet activation dependent on TLR4, the data shown here are representatives
of at five experiments from different donors. Results are Means?±?SD. Prior to stimulation
with HMGB1 (40 ?g/ml), WPs were pretreatmented with various conceration of anti-TLR4
Abs, anti-TLR2 Abs, anti-RAGE Abs, or irrelevant control concerationg IgG2a in an
aggregometer (1200 rpm) for 20 min respectively. Representative aggregation curves
with anti-TLR4 (a), anti-TLR2 (b), anti-RAGE (c) or IgG2a (d) are shown. e, f Statistical analysis of the maximum platelet aggregation rata (?=?5, *P??0.05, **P??0.01, vs IgG2a) and P-selectin expression data (?=?5, *P??0.05 vs IgG2a). g Representative flow cytometric data showed that reduced HMGB1-induced P-selectin
expression

HMGB1 stimulates platelet aggregation by blocking the activity of nitric oxide/cGMP
signaling

cAMP and cGMP were an important secondary messenger in platelet, cAMP-dependent protein
kinase A and cGMP-dependent protein kinase substrates translate prostacyclin and nitric
oxide signals into a block of platelet adhesion, granule release, and aggregation
25] to investigated intracellular cAMP and cGMP levels in HMGB1-stimulated WPs by immunoassay
kit, we measured cAMP and cGMP level in HMGB1-stimulated WPs and interesting found
cGMP levels decreased in a dose-dependent manner after presence of serum, however,
the changed of cAMP levels had no statistical difference (Fig. 7a). The reduced of cGMP levels play what a role in HMGB1-stimulated platelet was unknow,
so next, we prior adding cGMP analogs 8-pCPT-cGMP or cGMP-elevating NO donor SNP WPs
(sodium nitroprusside) to WPs respectively. The result indicated that HMGB1 (40 ?g/ml)-
induced the maximum platelet aggregation rate was significantly reduced after platelets
incubation with 8-pCPT-cGMP, or SNP (ALB used as a negative control) (Fig. 7b, c). Above results suggested that nitric oxide/cGMP signaling play a suppressing role
in HMGB1-stimulated platelets activation. On the other hand, we previous studies indicated
that HMGB1 signals by binding to TLR4 to induce platelets activation, to investigate
whether TLR4, TLR2 and RAGE receptors may be upstream of the cGMP signaling pathway.
We examined the effect of anti-TLR4 Abs, anti-TLR2 Abs and anti-RAGE Abs on HMGB1-induced
cGMP production respectively, our studies revealed that HMGB1-induced cGMP level change
was almost completely abolished by anti-TLR4 Abs, there were also slightly inhibited
by anti-TLR2 and anti-RAGE, but both of them have no statistical differences (Fig. 7d). Together with previous found showed that HMGB1 induced human platelets activation
main depending on TLR4 /cGMP axis and the results futher supported our previous discover.

Fig. 7. cGMP levels decreased in HMGB1-induced WPs (a) WPs (10
⁸
/ml) under high shear (1200 rpm) in an aggregometer and stimulated with HMGB1, 37 °C
10 min, cGMP and cAMP concentrations were determined by using a immunoassay kit (*P??0.01, **P??0.05 vs control). b, c WPs were pretreatmented with 8-pCPT-cGMP (200 ?M), or SNP (100 ?M) in an aggregometer
(1200 rpm) at 37 °C for 10 min and then addition of HMGB1 (40 ?g/mL), platelet aggregation
generation was then evaluated (?=?5, *P??0.05 vs ALB). d WPs were preincubated with mouse IgG2a or anti-TLR4, anti-TLR2 mAbs or anti-RAGE
mAbs (50 ?g/ml) at 37 °C for 10 min and then incubated with HMGB1 (40 ?g/mL) for 10 min.
cGMP level was evaluated (?=?5, *P??0.05 vs IgG2a). Results are the Means?±?SD of at least 5 experiments

NF-?B inhibitors impair HMGB1-induced platelet activation responses

NF-?B is a major transcriptional regulator of genes involved in survival, proliferation
and inflammation of cell. HMGB1 itself may signal through TLR4 and TLR2, and via RAGE
activation of these receptors results in the activation of NF-?B, which involved in regulation of inflammation and activation of immune cells 11], to evaluated the role of NF-?B in HMGB1-induced platelet activation. WPs preincubated
with various conceration of NF-?B inhibitors BAY 11-7082 before presented of HMGB1.
the results suggested that HMGB1 mediated maxim aggregation rate in WPs was markedly
inhibited by BAY 11-7082 in a dose-dependent manner (Fig. 8a). It was meaning that NF-?B activation playing a stimulatory role in HMGB1-induced
platelets activation. Furthermore, Fig. 8b shows that HMGB1- induced WPs activation resulted in I?B? phosphorylation and more
than a 70 % degradation of I?B? (Fig. 2b). both events were prevented by platelets preincubation the with BAY 11-7082, a specific
inhibitor of NF-?B activation that prevents I?B? phosphorylation 26] meanwhile, it was interesting discovered that the cGMP level significantly elevate
in WPs that pre-treatmented with increasing conceration of BAY11-7082 before adding
HMGB1. (Fig. 8c, d) in order to support above results, we estimated the levels of PAC-1 expression by
flow cytometry, HMGB1-mediated PAC-1 expression were significantly decreased in BAY
11-7082-treated platelets (data not show), In addition, we futher confirmed the TLR4/NF-?B
signal passway and show that anti-TLR4 similar to BAY 11-7082 also showed a similar
inhibitory activity against I?B? phosphorylation.however, guanylate cyclase (sGC)
inhibitor ODQ can increase I?B? phosphorylation that impaired by BAY 11-7082 in HMGB1
stimulated platelets (Fig. 8f). Together, these observations suggest that TLR4/ NF-?B axis is involved in the regulation
of “inside out” signal depending cGMP signal during in HMGB1-stimulated platelet activation.

Fig. 8. NF-?B inhibitors impair HMGB1-induced platelet activation responses and elevated cGMP
level. a, b NF-?B inhibitors impair platelet aggregation induced by HMGB1, WPs were treated with
the indicated concentrations of BAY11-7082 in an aggregometer (1200 rpm) at 37 °C
for 5 min and then stimulated with HMGB1 (?=?5, *P??0.01 vs ALB). c HMGB1 induce I?B? phosphorylation and degradation, WPs were treated with various
dose of HMGB1 for 5 min 37 °C, then Lysates were immunoblotted with an anti- I?B?
Ab. d WPs preincubation with BAY 11-7082 under high shear (1200 rpm) in an aggregometer
for 5 min and then stimulated with HMGB1 (40 ?g/ml) for 5 min, HMGB1 induce I?B? phosphorylation
can be blocked by BAY 11-7082. (?=?4, P??0.01 vs control). e Effects BAY 11-7082 on HMGB1-induced cAMP and cGMP, WPs pretreatment with BAY11-7082
in an aggregometer and stimulated with HMGB1 (40 ?g/ml) for 5 min before termination
of the reaction and then cAMP and cGMP enzyme immunoassays were performed. BAY11-7082
significantly increased cGMP accumulation in a dose dependent manner. (?=?4, *P??0.01 vs. control). f Effects of a-TLR4, SNP, BAY 11-7082 or ODQ on HMGB1-induced I?B? phosphorylation.
WPs were stirred in an aggregometer with above materials for 5 min prior to the addition
of HMGB1 (40 ?g/ml). a-TLR4 (50 ?g/ml), SNP (100 ?M) ODQ (20 ?M) and BAY 11-7082 (40 ?M),
measured I?B? phosphorylation level, all immunoblots are representative of 3 to 4
similar experiments