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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 5  |  Issue : 1  |  Page : 42-50

Alginate oligosaccharide inhibits platelet activation with minimal impact on bleeding time


1 Department of Cardiology, Zhongshan Hospital, Shanghai Institute of Cardiovascular Diseases, Fudan University, Shanghai, China
2 Department of Cardiology, The Affiliated Hospital of Qingdao University, Qingdao, China

Date of Submission04-Jan-2020
Date of Acceptance09-Mar-2020
Date of Web Publication4-Apr-2020

Correspondence Address:
Dr. Jun-Bo Ge
Department of Cardiology, Zhongshan Hospital, Shanghai Institute of Cardiovascular Diseases, Fudan University, Shanghai
China
Prof. Ying-Nan Bai
Department of Cardiology, Zhongshan Hospital, Shanghai Institute of Cardiovascular Diseases, Fudan University, Shanghai
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cp.cp_2_20

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  Abstract 


Background: Antiplatelet drugs are widely used in the prevention and treatment of arterial thrombotic diseases but are associated with increased risk of bleeding. Alginate oligosaccharide (AOS), a biodegradable polymer extracted from macroalgae, has been shown to inhibit phosphorylation of mitogen-activated protein kinases, which, in turn, are critical for platelet activation. The present study aimed to examine whether AOS possesses antiplatelet and antithrombotic activity, and if so, the underlying mechanisms. Methods: We detected the effects of AOS on human platelet aggregation and adenosine triphosphate (ATP) release induced by thrombin and collagen, as well as platelet clot retraction and spreading. FeCl3-injured mouse mesenteric arteriole thrombosis was evaluated in adult C57BL/6 mice pretreated with either AOS (200 mg/kg/d through gavage for 7 consecutive days) or clopidogrel (30 mg/kg/d for 2 days). The impact of AOS on bleeding time in comparison to clopidogrel was also analyzed. Results: At a range of 0.1–1.0 mg/mL, AOS concentration dependently inhibited human platelet aggregation and ATP release induced by thrombin and collagen, as well as platelet clot retraction and spreading. The final occlusion time injured by FeCl3in mice pretreated with AOS was significantly increased (from 11.9 ± 0.9 min in vehicle control to 17.6 ± 1.0 min, P < 0.01), as well as the first occlusion time (from 4.4 ± 0.5 min in vehicle control to 7.6 ± 0.7 min, P < 0.01). Bleeding time on tail snip was 534 ± 62 s in vehicle control, 581 ± 60 s in mice with AOS pretreatment (P = 0.59 vs. control), and 1260 ± 83 s in mice receiving clopidogrel pretreatment (P < 0.01 vs. control). Preliminary mechanistic investigation using human platelets showed a decreased level of phosphorylated MAP kinases (i.e., p38, Erk1/2, and JNK) by AOS. Conclusions: AOS has antiplatelet and antithrombotic activity, with minimal impact on bleeding time.

Keywords: Alginate oligosaccharide, antiplatelet, antithrombotic, bleeding, mitogen-activated protein kinase


How to cite this article:
Qi ZY, Liu X, Bai YN, Ge JB. Alginate oligosaccharide inhibits platelet activation with minimal impact on bleeding time. Cardiol Plus 2020;5:42-50

How to cite this URL:
Qi ZY, Liu X, Bai YN, Ge JB. Alginate oligosaccharide inhibits platelet activation with minimal impact on bleeding time. Cardiol Plus [serial online] 2020 [cited 2020 May 27];5:42-50. Available from: http://www.cardiologyplus.org/text.asp?2020/5/1/42/281938




  Introduction Top


Thrombosis in the arterial circulation is the principal pathological cause of arterial thrombotic diseases such as acute coronary syndrome (ACS) and ischemic stroke, which are the leading causes of morbidity and mortality worldwide. Platelet activation and accumulation at sites of vascular injury play important roles underlying those arterial thrombotic diseases.[1],[2] Accordingly, antiplatelet drugs represented by aspirin and the P2Y12 antagonists clopidogrel and ticagrelor are effective for prevention and treatment of ACS and ischemic stroke.[3]

Although antiplatelet drugs have been proven to be useful in reducing arterial thrombotic diseases, morbidity and mortality are still high.[4] Moreover, all these antiplatelet drugs have a common side effect, which is causing bleeding to some extent, contributing to the increased mortality and hence limiting their use to increase antithrombotic effects by increasing doses. Moreover, reports of drug resistance have been increasing in recent years.[5],[6] Therefore, to develop novel antiplatelet drugs with better efficacy and safety profile is urgent.

Alginate oligosaccharide (AOS), produced by depolymerizing alginate, is an acidic polysaccharide that is extracted from marine brown algae, using different degradation methods such as enzymatic degradation, acid hydrolysis, and oxidative degradation.[7] AOS is composed of 1,4-linked β-d-mannuronic acid (M) and α-l-guluronic acid (G) residues.[8] It is nonimmunogenic, nontoxic, and biodegradable polymer; therefore, it is an attractive candidate for biomedical application.[9] We have demonstrated that AOS could prevent acute doxorubicin cardiotoxicity [10] and alleviate myocardial reperfusion injury.[11] In addition, AOS has been reported to exert several other pharmacological benefits in recent years, such as anti-inflammatory,[12] antiapoptotic,[13] and antiproliferative effects.[14] In PC12 cells, AOS attenuates H2O2-induced phosphorylation of mitogen-activated protein kinases (MAPKs) in a concentration-dependent manner.[13] Several previous studies using selective inhibitors or knockout mice demonstrated that MAPKs are critical regulators of platelet activation.[15],[16],[17],[18] Platelets are also considered to be essential in inflammation.[19] However, whether AOS affects platelet activation and its pro-inflammatory role is not clear, and the underlying mechanisms also need to be elucidated. Here, the aim of the present study was to investigate the antiplatelet and antithrombotic effects of AOS and the underlying mechanisms, as well as its anti-inflammatory activity.


  Methods Top


Preparation of alginate oligosaccharide

AOS, a gift from Qingdao BZ Oligo Biotech Co., Ltd. (Qingdao, China), was produced by enzymatic degradation as previously described.[20] Detailed information is available in our previous study.[10] The chemical structure of AOS acquired through enzymatic degradation is shown in [Figure 1]. The relative molecular weight of AOS is 1.2 kDa.
Figure 1: Schematic representation of the molecular structure of alginate oligosaccharide prepared by enzymatic degradation

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Mice

C57BL/6 mice (6–8 weeks of age) were obtained from the Department of Laboratory Animal Science, Fudan University, Shanghai, China. All experiments were performed in accordance with institutional guidelines and approved by the Fudan University Animal Care and Use Committee.

Preparation of human platelets

All experiments using human subjects were performed in accordance with the Declaration of Helsinki and approved by the Institutional Review Board, Fudan University. Platelets were from healthy volunteers without taking any antiplatelet drugs or anti-inflammatory drugs for at least 14 days, and informed consent was obtained before blood collection. Washed platelets were prepared as described previously.[21],[22],[23]

Platelet aggregation and platelet secretion studies

Platelet aggregation in response to agonists or antagonists was analyzed using a Lumiaggregometer (Model 400 vs. Chrono-Log) under stirring conditions (900 rpm) at 37°C as described previously.[21],[22],[23] Washed platelets (3×108/mL) in Tyrode buffer were stimulated with thrombin (Chrono-Log, cat# 386, Havertown, PA) and collagen (Chrono-Log, cat# 385, Havertown, PA). Thrombin and collagen were added to initiate aggregation with or without preincubation with AOS for 5 min. The chart recorder (Model 707, Chrono-Log) was set for 1 cm/min. The baseline was set using Tyrode's buffer or platelet-poor plasma as blank. Platelet secretion was monitored in parallel with aggregation as adenosine triphosphate (ATP) release with the addition of CHRONO-LUME reagent (Chrono-Log) to the platelet suspension simultaneously.

Cell culture

The human Meg-01 megakaryocytic cells were cultured in RPMI-1640 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum and 20-mM L-glutamine. Cells were preincubated with AOS of varying concentrations for 2 h prior to exposure to 1-μg/mL lipopolysaccharide (LPS) for 24 h. Cells were then collected and processed for protein extraction. Conditioned media were used for the quantification of pro-inflammatory cytokines by enzyme-linked immunosorbent assay kits.

Western blot analyses

After incubation with or without different concentrations of AOS for 5 min, human washed platelets were stimulated with or without agonists for 5 min, lysed, and subjected to Western blot analysis. Effects of AOS on proteins implicated in the inflammatory process were examined in Meg-01 cells.

Platelet clot retraction

Human platelets were processed as previously described.[24] Briefly, 2 mg/mL fibrinogen (Sigma-Aldrich, cat# F3879, St Louis, MO) was added to human platelets suspended in Tyrode buffer and dispensed in 0.3 mL aliquots into cuvettes. Clot retraction was induced by stimulation with thrombin (1.0 U/mL) at 37°C and monitored by taking photographs at indicated time points using a digital camera. Clot surface area was quantified using NIH ImageJ software. The results were expressed as percentage of retraction (% = areat/areat0× 100%).

Platelet spreading

The analysis of platelet spreading on immobilized fibrinogen was done as described previously.[24] Human washed platelets in Tyrode's solution were preincubated with AOS for 5 min, transferred onto Lab-Tek Chamber Slides (Nalge Nunc International, Rochester, NY) precoated with 20 μg/mL of fibrinogen, and then allowed to adhere for 15–60 min at 37°C. After washing with phosphate-buffered saline, the attached platelets were fixed, permeabilized, and then stained with fluorescein isothiocyanate (FITC)-labeled phalloidin. Adherent platelets were viewed using a Leica SPE confocal microscope.

FeCl3-injured thrombus formation

FeCl3-injured thrombus formation in mouse mesenteric arteriole was examined using intravital microscopy, as described previously.[25],[26] Briefly, calcein-labeled platelets were injected via the femoral vein in mice pretreated with AOS (200 mg/kg/d, 7 days), clopidogrel (30 mg/kg/d, 2 days, Sanofi-Aventis, Hangzhou, China), or vehicle by gavage. Thrombosis was induced by 10% FeCl3 and recorded with intravital microscopy. Drug dosage was based as described previously.[10],[11],[25]

Bleeding time

Bleeding time was measured as described previously.[26] Briefly, the mouse tail was clipped (for 5 mm) and the tail was immersed in 10-mL saline at 37°C. Bleeding time was measured.

Statistical analysis

All data were expressed as mean ± standard error of mean. Differences between the groups were analyzed by one-way analysis of variance followed by a Newman–Keuls test using GraphPad Prism version 7.0 (GraphPad Inc., San Diego, CA, USA). P < 0.05 was considered to be statistically significant.


  Results Top


Alginate oligosaccharide inhibits platelet aggregation and adenosine triphosphate release

At 0.1–1.0 mg/mL, AOS concentration dependently inhibited platelet aggregation and ATP release [Figure 2]. The rate of platelet aggregation induced by 0.1 U/mL thrombin was 65.3% ± 3.5% in the vehicle control, 52.7% ± 2.4% at 0.1 mg/mL, 32.7% ± 2.4% at 0.5 mg/mL, and 12.7% ± 1.8% at 1.0 mg/mL AOS. The rate of platelet ATP release induced by 0.1 U/mL thrombin was 36.7% ± 4.1% in the vehicle control, 22.0% ± 1.2% at 0.1 mg/mL, 12.7% ± 1.8% at 0.5 mg/mL, and 0.7% ± 0.6% at 1.0 mg/mL. AOS also concentration dependently inhibited platelet aggregation and ATP release induced by thrombin at a lower concentration of 0.05 U/mL and by collagen (1.0 and 0.6 μg/mL).
Figure 2: Alginate oligosaccharide inhibited platelet aggregation and adenosine triphosphate release induced by thrombin and collagen. (a) Alginate oligosaccharide concentration dependently inhibited platelet aggregation and adenosine triphosphate release induced by 0.1 U/mL and 0.05 U/mL thrombin. (b) Alginate oligosaccharide concentration dependently inhibited platelet aggregation and adenosine triphosphate release induced by 1.0 μg/mL and 0.6 μg/mL collagen. (c and d) Quantification data of platelet aggregation and adenosine triphosphate release in (a) and (b), respectively (n = 3). *P < 0.05 versus the control group; **P< 0.01 versus the control group

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Alginate oligosaccharide inhibits platelet clot retraction

Platelet clot retraction is controlled by late integrin αIIbβ3-mediated outside-in signaling.[27] At a concentration of 0.1 mg/mL, AOS inhibited clot retraction [Figure 3]. At 20 min, the clot size was 85.0% ± 2.6% in the AOS group versus 69.1% ± 5.1% in the vehicle control (P < 0.05). At 40 min, the clot size was 58.6% ± 2.1% in the AOS group versus 33.0% ± 2.9% in the vehicle control (P < 0.01). At 60 min, the clot size was 35.8% ± 1.9% in the AOS group versus 22.5% ± 1.6% in the vehicle control (P < 0.01). Effects of AOS at a higher concentration of 0.5 mg/mL were more robust.
Figure 3: Alginate oligosaccharide concentration dependently inhibited platelet clot retraction. (a) Results shown are representative of three experiments using platelets from different donors. Alginate oligosaccharide 0.1 mg/mL and 0.5 mg/mL were used. (b) Quantification of clot retraction at 0, 20, 40, and 60 min expressed as mean ± standard error of mean, (n=3). *P < 0.05 versus the control group; **P < 0.01 versus the control group

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Alginate oligosaccharide inhibits platelet spreading

Platelet spreading is an early-phase outside-in signaling event downstream of platelet αIIbβ3 integrin activation.[27] On GPIIb/IIIa binding to fibrinogen, it triggers outside-in signaling, causing platelet spreading which plays a crucial role in thrombosis. As shown in [Figure 4], AOS inhibited platelet spreading on immobilized fibrinogen, as evidence by the decreased formation of platelet filopodia and lamellipodia.
Figure 4: Alginate oligosaccharide inhibited platelet spreading on fibrinogen-coated surface. (a) Human washed platelets were seeded on fibrinogen-coated cover slips in the presence of different concentrations of alginate oligosaccharide or vehicle control. Alginate oligosaccharide concentration dependently inhibited platelet spreading. Data shown are representative of three experiments using platelets from different donors (b)

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Alginate oligosaccharide impairs FeCl3-induced thrombus formation, with minimal impact on bleeding time versus clopidogrel

To explore the role of AOS in thrombus formation in vivo, we evaluated the FeCl3-injured thrombus formation in mesenteric arteriole in mice with or without AOS and clopidogrel using intravital microscopy. As shown in [Figure 5]a and [Figure 5]b, in untreated mice, multiple thrombi more than 20 μm were observed in the mesenteric arteriole at 6 min after FeCl3 injury. At 12 min, the vessel lumen was totally blocked by a stable bulky thrombus. In contrast, mice pretreated with AOS by gavage before FeCl3 injury prevented thrombus formation over 12 min. Further analysis revealed that the time to final occlusion was 17.6 ± 1.0 min (n = 7) in mice receiving AOS versus 11.9 ± 0.9 min in the vehicle control (n = 7) (P < 0.01). Moreover, the time to thefirst thrombus (>20 μm) was 7.6 ± 0.7 min in mice receiving AOS versus 4.4 ± 0.5 min in the vehicle control (P < 0.01). Clopidogrel is currently considered to be the gold standard to evaluate the effects of novel antiplatelet drugs, and thus, it is of interest to compare the antithrombotic role of AOS with clopidogrel. Under our conditions, compared with mice with AOS pretreatment, clopidogrel prevented FeCl3-induced thrombosis over 20 min, both of which markedly prolonged thrombus formation time.
Figure 5: Alginate oligosaccharide inhibited FeCl3-injured thrombus formation in mouse mesenteric arterioles with minimal impact on bleeding time in comparison to clopidogrel. (a) Representative images of thrombus formation at baseline and 6 and 12 min after 10% FeCl3-induced vascular injury in mouse mesenteric arterioles. (b) Statistical analysis of FeCl3-induced thrombus formation. Final occlusion time and time to first thrombus (>20 μm and stable for >2 min) were analyzed, n = 7. (c) The distal 5 mm of the tail was transected and immediately immersed in 10 mL of saline maintained at 37°C, and then, the bleeding time was measured. Data are expressed as mean ± standard error of mean

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The bleeding time on tail snip was increased significantly by clopidogrel (1260 ± 83 s vs. 534 ± 62 s in the vehicle control, P < 0.01) but not by AOS pretreatment (581 ± 60 s vs. 534 ± 62 s, P = 0.59) [Figure 5]c.

Alginate oligosaccharide decreases phosphorylated mitogen-activated protein kinasess

AOS concentration dependently decreased phosphorylated p38 (Thr180/Tyr182, Cell Signal Inc., monoclonal, cat# 4511, Beverly, MA), Erk1/2 (Thr202/Tyr204, Cell Signal Inc., polyclonal, cat# 9101), and JNK (Thr183/Tyr185, Cell Signal Inc., monoclonal, cat# 4668) in platelets stimulated with thrombin and collagen [Figure 6].
Figure 6: The effects of alginate oligosaccharide on the phosphorylation of mitogen-activated protein kinases in human washed platelets. (a) Alginate oligosaccharide concentration dependently inhibited the phosphorylation of mitogen-activated protein kinases in human washed platelets stimulated with thrombin and collagen. (b) Quantification data of the phosphorylation of p38, Erk 1/2, and JNK in (a). The results shown are representative of three experiments using platelets from different donors. *P < 0.05 versus the control group; **P < 0.01 versus the control group

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Alginate oligosaccharide attenuates lipopolysaccharide-induced production of proinflammatory cytokines

We examined the potential effects of AOS on the pro-inflammatory activity of platelets. Meg-01 cells were exposed to LPS in the absence/presence AOS of varying concentrations. The results showed a concentration-dependent reduction of pro-inflammatory cytokine production, including interleukin (IL)-1 β, tumor necrosis factor (TNF)-α, and IL-6 (R&D Systems, Minneapolis, MN) [Figure 7]a,[Figure 7]b,[Figure 7]c. AOS also concentration dependently decreased phosphorylated IκB (Santa Cruz Biotechnology, monoclonal, cat# sc–8404, Santa Cruz, CA) but not p65 (Santa Cruz Biotechnology, monoclonal, cat# sc–8008) in the cell lysate [Figure 7]d.
Figure 7: Effects of alginate oligosaccharide on pro-inflammatory cytokine productions in lipopolysaccharide-induced Meg-01 cells. Cells preincubated with different concentrations of alginate oligosaccharide for 2 h were treated with 1 μg/mL lipopolysaccharide for 24 h. Interleukin-1β (a), tumor necrosis factor-α (b), and interleukin-6 (c) in the supernatant were detected by an enzyme-linked immunosorbent assay kit. Values are the mean ± standard error of mean of three independent experiments. *P < 0.05 versus the lipopolysaccharide-treated group; **P < 0.01 versus the lipopolysaccharide-treated group. (d) Alginate oligosaccharide concentration dependently inhibited lipopolysaccharide-induced IκB phosphorylation and degradation in Meg-01 cells

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  Discussion Top


Platelet adhesion and aggregation at sites of vascular injury are two critical steps in hemostasis and thrombosis.[28] Despite the wide use of antiplatelet drugs, such as aspirin, clopidogrel, and ticagrelor, as well as cilostazol, recurrent ischemic events are common. Furthermore, antiplatelet drugs are associated with the risk of bleeding. Novel antiplatelet agents with improved efficacy and safety profile are needed. In this study, we showed that AOS could inhibit platelet aggregation and ATP release induced by agonists, as well as platelet clot retraction and spreading. We also conformed the antithrombotic effects of AOSin vivo using the model of FeCl3-induced thrombus formation in mouse mesenteric arterioles. AOS exhibited weaker antithrombotic efficacyin vivo than clopidogrel; however, it caused dramatically less bleeding, indicating the low bleeding risk of AOS. The underlying mechanisms may include inhibition of MAPKs phosphorylation. AOS also inhibited the pro-inflammatory cytokine productions in platelets by the inactivation of NF-κB. All of these data underlie the antiplatelet, antithrombotic, and anti-inflammatory effects of AOS.

Platelet activation is induced by a variety of agonists, such as thrombin and collagen, as well as adenosine diphosphate and thromboxane A2, which are released from the granules of activated platelets, further promoting platelet activation, forming a feed-forward loop.[29],[30] Although different agonists induce platelet activation through different mechanisms by binding with their specific receptors, they all resulted in the phosphorylation of MAPKs, which is critical for platelet activation, including integrin activation, thromboxane A2 synthesis, and granule secretion.[29] MAPKs' activation is also a major intracellular event in response to environmental stress. Thus, it is not surprising that MAPKs play vital roles in platelet activation. In the current study, AOS concentration dependently decreased phosphorylated p38, Erk1/2, and JNK in platelets. We speculate that decreased MAPKs phosphorylation may be the underlying mechanism that underlies the observation of inhibition of platelet activation by AOS.

There has been a growing appreciation of the important inflammatory roles of platelets. Previous studies have implicated platelets in inflammatory processes under a variety of disease conditions, including atherosclerosis and infectious diseases.[31],[32] The anti-inflammatory action of natural oligosaccharides has been widely studied in recent years. Chitosan oligosaccharides were found to be effective in inhibiting the release of pro-inflammatory cytokines from RAW 264.7 cells through the inhibition of NF-κB signaling.[33],[34] AOS reduces the secretion of pro-inflammatory cytokines in LPS-induced RAW 264.7 cells.[12] In the present study, we demonstrated that AOS could inhibit the release of pro-inflammatory cytokines (IL-1 β, TNF-α, and IL-6) from LPS-induced Meg-01 cells in a concentration-dependent manner. This finding encourages future efforts in developing AOS as a treatment for platelets-related inflammatory diseases.

AOS has a very favorable safety profile.[35] We speculate that adding AOS to dual antiplatelet therapy as secondary prevention could possibly enhance efficacy without increasing the risk of bleeding, particularly in carriers of the loss-of-function hepatic cytochrome 2C19 allele and thus hyporesponsive to clopidogrel.[36]

The present study has several limitations. First, a mechanistic investigation was based on observed changes in molecular pathways after AOS treatment and not intervention at the likely pathways. Second, only the phosphorylation of MAPKs was examined. A previous study showed that at an oral dosage of 10 mg/day, AOS is effective and safe in repressing aneurysm recurrence.[35] The doses at which AOS could produce antiplatelet and antithrombotic activity in humans remain unknown.


  Conclusions Top


AOS has antiplatelet and antithrombotic activity with minimal impact on bleeding time.

Acknowledgment

The authors would like to thank Qingdao BZ Oligo Biotech Co., Ltd (Qingdao, China) for the preparation of AOS.

Financial support and sponsorship

This work was supported by the National Natural Science Foundation of China, Innovation Research Group Science Foundation (Grant No. 81521001).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]



 

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