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Platelets and Blood Cells
Anfibatide, a novel GPIb complex antagonist, inhibits platelet adhesion and thrombus formation in vitro and in vivo in murine models of thrombosis Xi Lei1*; Adili Reheman1*; Yan Hou1,2; Hui Zhou1; Yiming Wang1,3,4; Alexandra H. Marshall1; Chaofan Liang5; Xiangrong Dai6; Benjamin Xiaoyi Li5; Karen Vanhoorelbeke7; Heyu Ni1,3,4,8 Research Center, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, and Toronto Platelet Immunobiology Group, University of Toronto, Toronto, Ontario, Canada; 2Jilin Provincial Center for Disease Prevention and Control, Changchun, Jilil, China; 3Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 4Canadian Blood Services, Toronto, Ontario, Canada; 5Lee’s Pharmaceutical holdings limited, Shatin, Hong Kong, China; 6Zhaoke Pharmaceutical co. limited, Hefei, Anhui, China; 7Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Kulak, Kortrijk, Belgium; 8Department of Physiology and Department of Medicine, University of Toronto, Toronto, Ontario, Canada
Summary Platelet adhesion and aggregation at the sites of vascular injury are key events for thrombosis and haemostasis. It has been well demonstrated that interaction between glycoprotein (GP) Ib and von Willebrand factor (VWF) initiates platelet adhesion and contributes to platelet aggregation, particularly at high shear. GPIb has long been suggested as a desirable antithrombotic target, but anti-GPIb therapy has never been successfully developed. Here, we evaluated the antithrombotic potential of Anfibatide, a novel snake venom-derived GPIb antagonist. We found Anfibatide inhibited washed murine platelet aggregation induced by ristocetin and recombinant murine VWF. It also blocked botrocetin-induced binding of murine plasma VWF to recombinant human GPIb . Interestingly, Anfibatide did not inhibit botrocetin-induced aggregation of platelet-rich plasma, indicating that its binding site may differ from other snake venom-derived GPIb antagonists. Anfibatide strongly inhibited platelet adhesion, aggregation, and thrombus formation in perfusion chambers at high shear conditions Correspondence to: Dr. Heyu Ni, MD, PhD Canadian Blood Services and Department of Laboratory Medicine and Pathobiology St. Michael’s Hospital, University of Toronto Room 420, LKSKI – Keenan Research Center 209 Victoria Street, Toronto Ontario, M5B 1W8, Canada Tel.: +1 416 847 1738 E-mail: [email protected]
and efficiently dissolved preformed thrombi. Anfibatide also inhibited thrombus growth at low shear conditions, though less than at high shear. Using intravital microscopy, we found that Anfibatide markedly inhibited thrombosis in laser-injured cremaster vessels and prevented vessel occlusion in FeCl3-injured mesenteric vessels. Importantly, Anfibatide further inhibited residual thrombosis in VWF-deficient mice, suggesting that Anfibatide has additional antithrombotic effect beyond its inhibitory role in GPIb-VWF interaction. Anfibatide did not significantly cause platelet activation, prolong tail bleeding time, or cause bleeding diathesis in mice. Thus, consistent with the data from an ongoing clinical trial, the data from this study suggests that Anfibatide is a potent and safe antithrombotic agent.
Keywords Antithrombotic agent, snake venoms, platelet inhibitor, glycoprotein Ib-IX-V complex
Financial support: This work was supported partially by Canadian Institutes of Health Research and National Natural Science Foundation of China (China-Canada Joint Health Research Initiative Program), Lee’s Pharmaceutical Holdings limited, Canadian Institutes of Health Research (MOP 119540), Heart and Stroke Foundation of Canada, and Canadian Foundation for Innovation. Yan Hou is a recipient of State Scholarship Fund from China Scholarship Council (CSC) and Yiming Wang is a recipient of a Ph.D. Graduate Fellowship from Canadian Blood Services and the Meredith & Malcolm Silver Scholarship in Cardiovascular Studies from the Department of Laboratory Medicine and Pathobiology, University of Toronto. Received: June 17, 2013 Accepted after major revision: September 25, 2013 Prepublished online: October 31, 2013
* X. Lei and A. Reheman contributed equally to this work.
Introduction Platelet adhesion and subsequent aggregation at sites of vascular injury are required to maintain normal haemostasis; however, these processes can also contribute to pathological occlusive thrombosis (1-3). Interaction of the platelet surface glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) plays a key role in initiation of platelet adhesion, particularly at high shear © Schattauer 2014
doi:10.1160/TH13-06-0490 Thromb Haemost 2014; 111: 279–289
stress (e.g. shear rate >1,200 s-1) (1, 4). This interaction is also essential for complete vessel occlusion at sites of vascular stenosis (e.g. shear rate >10,000 s-1) (3, 4). Platelet surface integrin αIIbβ3 and its ligands (fibrinogen or other ligands) (5, 6) mediate platelet aggregation and contribute to platelet adhesion at lower shear stress (3). Thus, both the GPIb complex and integrin αIIbβ3 are important targets to consider for antithrombotic therapy. Interestingly, although several inhibitors of αIIbβ3 have been developed Thrombosis and Haemostasis 111.2/2014
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Lei, Reheman, et al. Anfibatide inhibits platelet adhesion and thrombus formation in mice
for antithrombotic therapies (7) and at least one anti-GPIb monoclonal antibody has been humanised (8), anti-GPIb therapy has not yet been developed. The GPIb complex is an adhesion receptor that consists of four membrane-spanning subunits including GPIbα, GPIbβ, GPIX, and GPV (9). Once activated by high shear stress, VWF undergoes a conformational change and binds to GPIbα (10, 11). In VWF knockout mice, arterial thrombosis still occurs but complete vessel occlusion is either prevented or postponed (4). ARC1779, an antiVWF aptamer, does reduce adenosine 5’-diphosphate (ADP)- and collagen-induced platelet aggregation but also prolongs cutaneous bleeding time in humans (12). In contrast to VWF knockout mice, thrombus formation is completely abolished in GPIb knockout mice (13), suggesting that GPIb receptor ligands, other than VWF, may play an important role in platelet aggregation and thrombus formation (14). Therefore, given that GPIb deficiency results in more severe impairment of thrombosis than VWF, GPIb is likely a more attractive candidate for antithrombotic therapy (15). Snaclecs are a subset of non-enzymatic proteins isolated from snake venom that include C-type lectins and their related proteins. Snaclecs have been isolated and tested for therapeutic potential for four decades; however, to our knowledge, none have successfully entered clinical practice. Agkisacucetin (trade name Anfibatide, following further purifications) is a snaclec purified from the venom of the Agkistrodon acutus snake. Anfibatide is a C-type lectin-like protein (α and β chains with interchain disulphide bonds, without Ca2+-/sugar-binding loops) derived from the protein complex agglucetin (16, 17). The structure of Anfibatide has high similarity to subunits of agglucetin (17); however, agglucetin is tetrameric protein. Earlier studies demonstrated that Agkisacucetin binds to GPIb to inhibit human VWF binding (17, 18). However, the effect of Anfibatide on thrombosis and haemostasis has not been studied. Here we report the first in vitro and in vivo assessment of the effects of Anfibatide on murine platelet function and thrombus formation. We found that Anfibatide is a potent antithrombotic agent that does not significantly affect haemostasis.
Material and methods Experimental animals C57BL/6J wild-type (WT) male mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). VWF deficient (-/-) mice were used as previously described (4, 19). C57BL/6J WT male and female mice were used to prepare washed murine platelets. All experimental protocols were reviewed and approved by the Animal Care Committee of St. Michael’s Hospital or the Institutional Animal Care and Use Committee of the KU Leuven, Belgium, as appropriate.
Purification of Anfibatide Anfibatide was purified from venom of the Agkistrodon acutus snake by anion-exchange and monoclonal antibody-based affinity Thrombosis and Haemostasis 111.2/2014
chromatography followed by Sephacryl S-100 column. After the purification, Anfibatide was analyzed by MALDI-TOF mass spectrometry and only one peak appeared indicating its purity.
VWF botrocetin cofactor activity assay The ELISA was performed as previously described (20). Briefly, a 96-well microtitre plate was coated with the anti-GPIbα monoclonal antibody 2D4 to capture 1 μg/ml recombinant fragment of human GPIbα (rfGPIbα) (21). The rfGPIbα was saturated with a constant amount of diluted murine plasma (1:16) containing 0.1 μg/ml of botrocetin (Sigma, St. Louis, MO, USA) in the presence or absence of a 1:2 dilution of Anfibatide (highest concentration was 6 μg/ml). Bound VWF was detected with anti-VWF immunoglobulins labelled with horse radish peroxidase (Dako, Glostrup, Denmark). Of note, ristocetin cannot be used to study the binding of plasma VWF to GPIbα in this assay.
Ristocetin-induced platelet aggregation of washed murine platelets As ristocetin-induced platelet aggregation can be performed with washed murine platelets (22) but not with murine platelet-rich plasma (PRP), the ristocetin-induced platelet aggregation assay was performed using washed murine platelets. Blood samples were collected via retro-orbital puncture on 80 μM PPACK (Calbiochem, Billerica, MA, USA) and 100 μl ACD-C (124 mM trisodium citrate, 130 mM citric acid, and 110 mM D-glucose, pH 6.5). Blood was centrifuged (100×g, 7 minutes [min]), then PRP was diluted in wash buffer (103 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5 mM D-Glucose, 36 mM citric acid, pH 6.5) containing 1 μM prostaglandin E1 (PGE1, Sigma) and 100 mU apyrase (Sigma). Platelets were centrifuged (800×g, 10 min), washed a second time, and finally resuspended in HEPES-Tyrode buffer. To induce aggregation, recombinant murine VWF (mVWF, 5 μg/ml, expressed in HEK293T cells [23]) and 1 mg/ml ristocetin (ABP, London, UK) were added to the washed platelets (250,000 platelets/μl). To study the effect of Anfibatide, 6 μg/ml was added. Platelet aggregation was evaluated by light transmission in a Chrono-Log dual channel aggregometer (Kordia BV, Leiden, The Netherlands).
In vitro platelet aggregation To investigate the specificity of Anfibatide and its effect on GPIbα-VWF-independent aggregation, PRP or gel-filtered platelet aggregation were tested as described (5, 24-26). Briefly, PRP was isolated from sodium-citrated murine whole blood by centrifugation. Gel-filtered platelets were isolated from PRP using a Sepharose 2B chromatography columns with PIPES buffer (5 mM PIPES, 137 mM NaCl, 4 mM KCl, 0.1% glucose, pH 7.0). Platelet count was adjusted to 3x108 platelets/ml. Platelet aggregation was assessed with or without adding Anfibatide (6 μg/ml) using a computerised Chrono-log aggregometer (Chrono-Log Corporation, Havertown, PA, USA). Platelet aggregation in PRP was induced by adenosine diphosphate (ADP, 1 to 20 μM, Sigma-Aldrich), col© Schattauer 2014
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Ex vivo perfusion chamber To study the effect of Anfibatide on thrombus formation under flow conditions, we used an ex vivo perfusion chamber system as described (6, 27, 28). Briefly, rectangular microcapillary glass tubes (0.1×1 mm) were coated with 100 μg/ml Horm collagen (Nycomed, Linz, Austria) overnight at 4°C. Whole blood was collected from anaesthetised mice, heparin-anticoagulated (15 U/ml), and fluorescently labelled by incubation with DiOC6 (1 μM, Sigma; 10 min, 37°C). Control or Anfibatide-treated (6 μg/ml) blood was perfused over the collagen-coated surface for 3 min using a syringe pump (from 100 s-1 to 5000 s-1; Harvard Apparatus, Holliston, MA, USA). To evaluate its therapeutic potential, we tested the effect of Anfibatide on preformed thrombi at high shear flow. Control whole blood was perfused over the collagen-coated surface (50 μg/ml Horm collagen) at a controlled shear rate of 1,800 s-1 for 3 min to form thrombi on collagen as described above. Then, without interruption, perfusion was continued either with untreated control blood or with Anfibatide-treated (6 μg/ml) whole blood for 3 min. Platelet adhesion, aggregation, and thrombus formation were recorded in real-time under a Zeiss Axiovert 135-inverted fluorescence microscope (60X-W objective). Quantitative dynamics of platelet fluorescence intensity were acquired by SlideBook software (Intelligent Imaging Innovations Inc., Denver, CO, USA).
Platelet activation To test whether Anfibatide causes platelet activation or granule release, resting gel-filtered WT platelets were assessed for platelet β3 integrin activation and P-selectin surface expression using flow cytometry, as described (6). Resting gel-filtered platelets were incubated with control buffer or Anfibatide (6 μg/ml) for 30 min, with the presence of phycoerythrin (PE)-conjugated rat anti-mouse αIIbβ3 antibodies (JON/A, EMFRET Analytics, Eibelstadt, Germany) and fluorescein isotheiocyanate (FITC)-conjugated rat anti-mouse CD62P (P-selectin) monoclonal antibodies (BD Biosciences Pharmingen, San Diego, CA, USA). Platelets were fixed with freshly prepared paraformaldehyde (1%) and antibodies were detected by a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA).
In vivo thrombosis studies To assess the effect of Anfibatide on platelet response to vascular injury in vivo, thrombus formation was studied by real-time in© Schattauer 2014
travital microscopy using two different in vivo thrombosis models that each represents a different vascular bed, size, and injury.
FeCl3-induced mesenteric arteriole thrombosis model The thrombotic response to ferric chloride (FeCl3)-induced vascular injury was assessed in WT control and Anfibatide-treated (6 μg/ml of blood) mice using an intravital microscopy thrombosis model, as previously described (24, 29-31). Briefly, gel-filtered platelets were fluorescently labelled with calcein acetoxymethyl ester (1 μg/ml; Molecular Probes) and injected via the tail vein of recipient mice (23-28 days old). The mesentery vascular bed was exteriorised through a midline abdominal incision. A single arteriole was chosen in each mouse based on vessel diameter (80-120 μm), shear rate (approximately 1,500 s-1), and visibility. Thrombus formation was induced in a 2-5 mm segment of the arteriole by topical application of 30 μl of 250 mM FeCl3 and was recorded using a Zeiss Axiovert 135-inverted fluorescent microscope (Zeiss, Oberkochen, Germany). We quantified 1) the number of fluorescent platelets deposited on the vessel wall during the 3-5 min interval following injury; 2) the time required for the formation of the first 20 μm thrombus; and 3) the vessel occlusion time (defined as complete cessation of blood flow for at least 10 seconds).
Laser-induced cremaster muscle arteriole thrombosis model The effect of Anfibatide on laser-induced cremaster arteriole thrombosis was studied in WT and VWF-/- mice, as previously described (24, 32). Briefly, the cremaster muscle of anaesthetised mice (8 weeks old) was prepared under a dissecting microscope and superfused with preheated bicarbonate-buffered saline throughout the experiment. Platelets were labelled by injection of rat anti-mouse CD41 antibody (BD Bioscience, 0.1 µg/g) secondarily-labelled with Alexa Flour 647 goat anti-rat IgG (Molecular Probes, 0.5 µg/g) via a jugular vein cannulus. Anfibatide (6 μg/ml) or control buffer was intravenously administered prior to injury. Multiple independent upstream injuries were then caused by a pulsed nitrogen dye laser. Images of thrombus formation were captured in real-time under an Olympus BX51WI microscope and analysed by Slidebook.
Tail bleeding time Tail bleeding time was assessed as previously described (33). WT mice (6-8 weeks old) were injected with Anfibatide (6 μg/ml via tail vein) or control buffer. Mice were anaesthetised 30 min postinjection with tribromoethanol (2.5 %, 0.015 ml/g body weight, intraperitoneal) and placed on a 37°C warming pad. A 3 mm terminal section of the tip of the tail was removed by a scalpel and the tail was immersed into isotonic saline (37°C) to bleed. Tail bleeding time was recorded until bleeding had ceased or for a maximum of 10 min, when further bleeding was prevented by cauterization.
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lagen (2.5 to 20 μg/ml, Nycomed Pharma, Ismaning, Germany), collagen-related peptide (CRP, 0.05 to 1 μg/ml; provided by Dr. Yotis Senis from the Institute of Biomedical Research, University of Birmingham, UK), or convulxin (0.5 to 5 μg/ml; also provided by Dr. Yotis Senis). Platelet aggregation in gel-filtered platelets was induced by thrombin receptor agonist peptide (TRAP; sequence AYPGKF-NH2; 100 to 500 μM; Peptides Int, Louisville, KY, USA).
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Statistical analysis Statistical analyses were performed using GraphPad Prism 6 and differences were evaluated by Student’s two-tailed t-test. Differences with a p-value less than 0.05 were considered statistically significant. Data are expressed as mean ± standard error of the mean (SEM). Sample size was >3 per group.
Results Anfibatide inhibited GPIb-VWF interaction and aggregation in vitro Ristocetin and botrocetin change the confirmation of VWF, allowing binding to GPIb. Interestingly, botrocetin-induced murine pla-
telet aggregation was not inhibited by Anfibatide (▶ Figure 1 A); however, ristocetin-induced aggregation of washed murine platelets and recombinant murine VWF was markedly inhibited (▶ Figure 1 B), suggesting that its binding site may differ from other snake venom-derived GPIb antagonists. In a separate study, we found that Anfibatide inhibits ristocetin-induced platelet aggregation using human PRP (unpublished data). To confirm these findings and to rule out a ristocetin-dependent effect of Anfibatide, the binding of murine plasma VWF to rfGPIbα was induced by botrocetin using an immunoassay (20). Complete inhibition of the VWF-GPIbα interaction was observed with the addition of Anfibatide (▶ Figure 1C). To examine whether the Anfibatide inhibition is specific to VWF-GPIb-dependent platelet aggregation, in vitro platelet aggregation was also induced by ADP, collagen, TRAP, convulxin, and
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G Figure 1: Anfibatide inhibits the VWF-/- GPIb interaction in vitro. Anfibatide did not inhibit botrocetin-induced aggregation of murine PRP (A). Anfibatide inhibited both ristocetin-induced aggregation of washed murine platelets supplemented with recombinant mVWF (B) and binding of plasma mVWF to rfGPIb in the presence of botrocetin in an immunoassay (n=3, C; ** represents p < 0.01 vs control). Anfibatide also did not inhibit platelet aggre-
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H gation of murine PRP in the presence of adenosine diphosphate (ADP; D), collagen (E), thrombin receptor activating peptide (TRAP; F), collagen related peptide (CRP; G), or convulxin (H). Representative tracings of three independent experiments are shown for all aggregation experiments. Black tracings: Control platelets, red tracings: Anfibatide-treated platelets.
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Anfibatide strongly inhibited platelet adhesion and aggregation, and dissolved preformed thrombi in an ex vivo thrombosis model The effect of Anfibatide on thrombosis was first assessed by the ex vivo perfusion chamber model at both high (1,800 s-1) and low (300 s-1) shear rates under a real-time fluorescence microscope. As shown in ▶ Figure 2, immediately following the perfusion of control whole blood over a collagen-coated surface, fluorescent pla-
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E Figure 2: Anfibatide inhibited platelet function and thrombosis in whole blood at high and low shear rate, and dissolved preformed thrombi in ex vivo perfusion chamber. The representative images (A) and quantification (B) of platelet adhesion and aggregation at 1,800 s-1 shear rate for 3 min. The means were significantly different, p < 0.05. The representative images (C) and quantification (D) of platelet adhesion and aggregation at 300 s-1 for 3 min. The representative images (E) and quantifi-
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CRP, agonists that lead to platelet and αIIbβ3 activation independent of the GPIb-VWF interaction. As hypothesised, ADP-, collagen-, TRAP-, CRP-, and convulxin-induced aggregation were not inhibited by Anfibatide pretreatment (▶ Figure 1D-H). There was also no significant effect on platelet aggregation induced by low doses of ADP, collagen, CRP, and TRAP (Suppl. Figure 1, available online at www.thrombosis-online.com). These results suggest that Anfibatide itself did not significantly affect signalling pathways downstream of GPIb.
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F cation (F) of platelet adhesion and aggregation during 6 min thrombolysis experiment. Control blood was perfused until 3 min in both groups, then either control or Anfibatide-treated blood was perfused at 1,800 s-1 until 6 min. B, D, F) The kinetic curves represent platelet mean fluorescence intensity (MFI) and the shaded regions represent SEM. Green: fluorescent platelets. Black: Untreated control whole blood. Red: Anfibatide-treated whole blood. The means were significantly different, p < 0.05.
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telets firmly adhered to the collagen, formed stable platelet aggregates, and gradually formed large thrombi with collagen fibrils after 3 min at high shear conditions. In sharp contrast to controls, Anfibatide pretreatment strongly inhibited platelet adhesion, aggregation, and thrombus formation at high shear (p < 0.01, ▶ Figure 2A and B); only a few single platelets adhered to the collagen surface during the entire 3 min perfusion. Platelet adhesion and subsequent aggregate formation was also notably decreased at low shear (p < 0.05, ▶ Figure 2C and D); only some adhered platelets and small aggregates were observed after 3 min of perfusion.
We tested the effect of Anfibatide under a range of shear stress conditions in perfusion chambers, including 100 s-1, 200 s-1, 500 s-1, 3,000 s-1, and 5,000 s-1. As shear stress increased, the inhibitory effect of Anfibatide became stronger (data not shown). The inhibition was approximately 100 times stronger at high shear (1,800 s-1) as compared to that at low shear (300 s-1). At 5,000 s-1, Anfibatide-treated platelet adhesion and aggregation on collagen was undetectable. To mimic a pathological scenario where the thrombus has already formed, we tested the effect of Anfibatide on preformed
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Figure 3: Anfibatide markedly inhibited thrombosis in wild-type (WT) mice in arteriole thrombosis models. Representative images (A) and quantification (B-D) of thrombus formation induced by ferric chloride in Control WT mice and Anfibatide-treated WT mice in mesenteric arteriole thrombosis model are shown from 4 to 18 min. B) The number of fluorescent platelets deposited on the vessel wall per min during the 3–5 min interval following injury. C) The time required for the formation of the first 20 μm thrombus. * Means were significantly different, p < 0.01. D) The time to com-
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plete vessel occlusion. *Means were significantly different, p < 0.01. Representative images (E) and quantification (F) of thrombus formation induced by laser injury in cremaster arteriole thrombosis model in Control WT mice and Anfibatide-treated WT mice. There was a significant difference between the means, p < 0.01. The kinetic curves represent platelet mean fluorescence intensity (MFI), and the shaded regions represent SEM. Green or white: fluorescent platelets. Black: Untreated control whole blood. Red: Anfibatidetreated whole blood.
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Anfibatide inhibited thrombosis and prevented vessel occlusion in vivo in two complimentary intravital microscopy thrombosis models We used the FeCl3-induced mesenteric arteriole thrombosis model to evaluate the effects of Anfibatide on platelet adhesion, aggregation, and occlusive thrombus formation in vivo in real-time 30 min after treatment. As shown in ▶ Figure 3A-D, in control mice, platelets interacted with and deposited on the injured vessel wall shortly after FeCl3 application. Within a few minutes of injury, visible platelet aggregates formed and grew into thrombi that eventually resulted in stable vessel occlusion and complete disruption of blood flow. Consistent with our ex vivo findings, Anfibatide treatment significantly inhibited the initial platelet interaction and adhesion response to vascular injury in mice (▶ Figure 3A-D). The number of interacted platelets per minute at 3-5 min after injury was significantly less in Anfibatide-treated mice compared with WT controls (p < 0.05, ▶ Figure 3B). Formation of visible platelet aggregates was delayed and small aggregates were easily dissolved, rarely growing into large thrombi. The time required for the formation of first thrombus (>20 μm in diameter) at the site of vessel injury was significantly longer in Anfibatide-treated mice compared to WT controls (p < 0.01, ▶ Figure 3C). Moreover, An-
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Anfibatide has anti-thrombotic functions beyond GPIb-VWF interaction blockade To investigate whether Anfibatide inhibits GPIb interaction with other ligands or agonists in addition to VWF, we tested thrombus formation in VWF-/- mice with or without Anfibatide. Interestingly, in VWF-/- mice where laser-induced thrombosis is impaired compared to WT mice, Anfibatide inhibited thrombus formation even further than knockout alone (p < 0.05, ▶ Figure 4). Furthermore, Anfibatide inhibition was more potent in VWF-/- mice (▶ Figure 4B) than that in WT mice (▶ Figure 3F). These results suggest that Anfibatide not only inhibits GPIb-VWF interaction
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Figure 4: Anfibatide further inhibited thrombosis in von Willebrand factor-deficient (VWF-/-) mice in the cremaster muscle arteriole thrombosis model. Representative images (A) and quantification (B) of thrombus formation in VWF-/- mice and VWF-/- mice treated with Anfibatide (6
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fibatide treatment delayed complete vessel occlusion in all mice (recorded up to 40 min post-injury, p < 0.01, ▶ Figure 3D). We used the laser-induced cremaster arteriole thrombosis model to determine the effect of Anfibatide treatment on platelet accumulation within a growing thrombus. In WT control mice, platelets adhered and quickly accumulated at the site of injury immediately after laser-induced arteriole injury, as shown in ▶ Figure 3 E-F. The thrombus rapidly increased in size and reached a maximal size approximately 1 min after injury. Then the thrombus decreased in size and stabilised on the site of injury. By contrast, Anfibatide pretreatment significantly inhibited thrombus growth in injured arterioles (p < 0.01, ▶ Figure 3 F). Platelet thrombus formation at the site of injury was limited and quickly decreased in size or stabilised without the rapid platelet accumulation observed in WT control mice. In summary, thrombus formation in Anfibatide-treated mice was delayed, unstable, easily dissolved, and failed to form an occlusive thrombus.
g/ml) at 1 min. The kinetic curves represent platelet mean fluorescence intensity (MFI), and the shaded regions represent SEM. The means were significantly different, p < 0.05. Green: fluorescent platelets. Black: Untreated VWF-/- mice. Red: Anfibatide-treated VWF-/- mice.
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thrombi in perfusion chambers at high shear stress (1,800 s-1). At 3 min, large and stable thrombi were already formed in the perfusion chambers. Continued perfusion of control whole blood increased the size of the preformed thrombi, and more platelets adhered and formed new thrombi. Continued perfusion of Anfibatide-treated blood dissolved the preformed thrombi in the perfusion chambers (▶ Figure 2E and F).
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but also attenuates other thrombotic pathways mediated by the GPIbα.
Anfibatide did not significantly activate platelets in vitro or prolong bleeding time in vivo To test the possible side effects, it is important to assess the effects of Anfibatide on platelet activation and haemostasis. By flow cytometry, we found no significant difference in P-selectin expression (CD62P; ▶ Figure 5 A) or integrin αIIbβ3 activation (JON/A; ▶Figure 5B) between in vitro Anfibatide-treated and control platelets. Moreover, in vivo, no significant difference was observed in tail vein bleeding time in mice treated with Anfibatide compare to
control mice (▶ Figure 5C). There was also no spontaneous bleeding or bleeding from sites of vascular access during surgeries (data not shown).
Discussion This study is the first in vitro and in vivo assessment of the effects of Anfibatide on murine platelet function and thrombosis. We demonstrated that Anfibatide strongly inhibited platelet adhesion, aggregation, and thrombus formation at both high and low shear conditions, dissolved preformed thrombi ex vivo, and prevented vessel occlusion in in vivo thrombosis models. Interestingly, we
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C Figure 5: Anfibatide did not significantly affect platelet activation in vitro or tail vein bleeding time in vivo. A) P-selectin (CD62P) expression on the platelet surface. B) 3 integrin activation tested by JON/A antibody. A, B) Left panel shows the representative histogram. Grey: Control platelets.
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Red: Anfibatide-treated platelets. Right panel shows the mean fluorescence intensity (MFI). Means were not significantly different. C) Tail bleeding time in untreated WT mice and Anfibatide-treated WT mice. Black: WT mice. Red: Anfibatide-treated WT mice. No significant differences detected.
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switch the thrombus growth toward thrombus dissolution under flow conditions. It is also very likely that GPIb-VWF interaction may deliver outside-in signaling via the GPIb complex that activate newly recruited platelets in the thrombi and maintain their αIIbβ3 integrins in active conformation, which is required for fibrinogen- and other integrin ligand-mediated platelet aggregation. In addition, GPIb-P-selectin and GPIb-other haemostatic/thrombotic factor interactions may provide additional bridges between adjacent activated platelets to enhance thrombus stability. Furthermore, we also will not exclude the potential interaction of Anfibatide with other plasma proteins that may also favor thrombus dissolution and thrombolysis. Thus, Anfibatide may induce thrombus dissolution, inhibit thrombus growth, and prevent vessel occlusion in diseased arteries, which would prevent myocardial infarction and ischemic stroke and save lives. Deep vein thrombosis (DVT) is another major cause of mortality worldwide. Recent studies have clearly demonstrated that both
What is known about this topic?
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Platelet adhesion and aggregation at the sites of vascular injury are key events for thrombosis and haemostasis. Interaction between the glycoprotein (GP)Ib complex and von Willebrand factor (VWF) initiates platelet adhesion and contributes to platelet aggregation, particularly at high shear. This interaction is also essential for complete vessel occlusion at sites of vascular stenosis. GPIb has long been suggested as a desirable target for antithrombotic therapy, but an anti-GPIb agent for therapy has never been developed. Anfibatide (trade name for Agkisacucetin) is a GPIb antagonist derived from snake venom. Its structure has been described but its effect on thrombosis and haemostasis has not been adequately studied.
What does this paper add?
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We evaluated the antithrombotic potential of Anfibatide, a novel snake venom-derived GPIb antagonist, in vitro, ex vivo, and in vivo in murine thrombosis models. Anfibatide strongly inhibited platelet adhesion, aggregation, and thrombus formation, dissolved preformed thrombi on collagen in ex vivo perfusion chambers at high shear, and prevented vessel occlusion in in vivo thrombosis models. Thus, Anfibatide is a strong antithrombotic agent for arterial thrombosis. Importantly, Anfibatide further inhibited residual thrombosis in VWF-deficient mice and inhibited thrombosis at low shear. These results suggest that Anfibatide has additional antithrombotic effect beyond inhibition of GPIb-VWF interaction and may have therapeutic potential for both arterial and deep vein thrombosis. No significant platelet activation was detected after Anfibatidetreatment, and bleeding time was not significantly increased in Anfibatide-treated mice. Therefore, Anfibatide may represent a novel and potent antithrombotic agent that will not cause severe bleeding complications in patients.
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found that Anfibatide not only inhibits the GPIb-VWF interaction but also attenuates other thrombotic pathways mediated by the GPIb complex. Importantly, no platelet activation was detected after Anfibatide-treatment and bleeding time was not increased in Anfibatide-treated mice. Therefore, Anfibatide may represent a novel and potent antithrombotic agent that can control both arterial and deep vein thrombosis without significantly causing bleeding complications. Earlier research demonstrated that purified Agkisacutacin (trade name Anfibatide) selectively binds to GPIb (18). Flavocetin-A and echicetin are two snaclecs that also inhibit VWF access to GPIb with similar structures to Anfibatide. Surprisingly, though they both bind to GPIb to inhibit platelet aggregation, they are also able to increase platelet agglutination by forming tetramers or by binding to immunoglobulin Mκ (IgMκ), respectively (17, 34). In contrast, each Anfibatide αβ-heterodimer molecule binds to one GPIb molecule and inhibits GPIb-VWF interaction without causing GPIb clustering (17). Akitonin and jararaca GPIb-BP are GPIb antagonists that decrease platelet aggregation in vitro but we have not found any information regarding their efficacy and bleeding diathesis in vivo (35, 36). According to our in vitro and in vivo results, Anfibatide is a promising therapeutic agent given our evidence that it does not significantly increase bleeding time (▶ Figure 5). Ristocetin and botrocetin both bind to the A1 domain of VWF to induce a conformational change of VWF, which allows for GPIb binding and leads to platelet aggregation in vitro (37, 38). Interestingly, we found that Anfibatide did not inhibit botrocetin-induced murine platelet aggregation in PRP (▶ Figure 1 A) but did block GPIb-VWF binding induced by botrocetin in ELISA and inhibited washed murine platelet aggregation induced by ristocetin and recombinant murine VWF (▶ Figure 1B and C). In an ongoing clinical trial, Anfibatide inhibited ristocetin-induced human platelet aggregation in PRP (unpublished data). These results suggest that the tertiary interactions between Anfibatide and human and mouse GPIb may be different and its binding site may differ from other reported snake venom-derived GPIb antagonists. Our comprehensive ex vivo and in vivo studies revealed that Anfibatide inhibited platelet adhesion, aggregation, and thrombus formation in murine models of thrombosis (▶ Figure 2 and ▶ Figure 3). The GPIb-IX-V complex plays a pivotal role in initiating and propagating both haemostasis and thrombosis when bound to VWF (39). Initiation of platelet adhesion and aggregation is absolutely dependent on GPIb and its ligand VWF above a limiting shear rate (~10,000 s-1) (40). In diseased arteries, shear stress is elevated when lumen restriction increases blood flow velocity (41). The VWF and GPIb interaction therefore precipitates the complete thrombotic occlusion of the vessel causing obstruction of blood flow and subsequent tissue damage (1). In this study we demonstrated that GPIb antagonism by Anfibatide almost completely inhibited platelet adhesion, aggregation, and thrombus formation and dissolved preformed thrombi ex vivo at high shear (▶ Figure 2). It is notable that thrombus growth and thrombolysis are dynamic processes and likely occur simultaneously, blocking GPIb-VWF interaction may prevent platelet recruitment and
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VWF and platelets are important in this process (42), which is consistent with an earlier study that demonstrated GPIb-VWF interaction also plays a role in platelet adhesion at low shear in veins (43). In our perfusion chambers experiments, we found that Anfibatide inhibited platelet adhesion, aggregation, and thrombus formation at venous shear stress (▶ Figure 2), suggesting that Anfibatide may be able to inhibit DVT. It is also notable that shear rate escalates in DVT as luminal stenosis progresses. Therefore, the inhibitory effect of Anfibatide at both low and high shear indicates that it may also have therapeutic potential in DVT. Interestingly, we found that Anfibatide inhibited thrombus formation even further in VWF-/- mice (▶ Figure 4). Therefore, Anfibatide not only selectively inhibits GPIb-VWF interaction but also attenuates other thrombotic pathways mediated by the GPIb complex. Previous research demonstrated that the role of GPIb far exceeds its main ligand VWF and that a GPIb ligand other than VWF may exist in the injured vessel (13). Recent studies showed that GPIb interacts with thrombin, P-selectin, Mac-1, and other molecules (39, 44). It will be of interest to further elucidate whether Anfibatide can disrupt binding of thrombin or other thrombotic/haemostatic factors to the GPIb complex. If Anfibatide indeed inhibits additional interactions, this may broaden its role in vascular biology. Currently the most common anti-thrombotic agents are fibrinogen receptor antagonists, aspirin, and P2Y12 antagonists, which all inhibit β3 integrin-mediated platelet aggregation. Recent clinical data with intravenous fibrinogen receptor antagonists (45) and novel anti-platelet drugs such as Prasugrel (46) demonstrated platelet inhibition and prevention of ischaemic complications, but these agents were also associated with increased bleeding complications. αIIbβ3 inhibitors represent effective antiplatelet/antithrombotic agents, but they often lead to thrombocytopenia and/ or life-threatening bleeding complications, such as cerebral, alveolar, and gastrointestinal system haemorrhages (47-50). Until now, no anti-GPIb drug has been successfully introduced into clinical practice, although several potential candidates are under development (15). We demonstrate that Anfibatide, a GPIb antagonist, had no significant effect on platelet activation or bleeding time (▶ Figure 5). There was also no spontaneous bleeding or bleeding from sites of vascular access during surgeries. There are a couple of possible explanations for this exciting phenomenon. First, VWF-GPIb interaction may mainly be involved in thrombosis and haemostasis at high shear stress, and blocking this interaction may have less effect on haemostasis occurring at low shear stresses. Second, Anfibatide may have no effect on VWF-mediated haemostatic pathways that are independent of GPIb (e.g. VWF-integrin interaction). Therefore, Anfibatide may offer a more favorable safety profile than currently available antithrombotic agents. In summary, we report that Anfibatide, a novel GPIb antagonist, effectively inhibited platelet adhesion, aggregation, and thrombus formation ex vivo, prevented vessel occlusion in in vivo thrombosis models, and has a favorable safety profile. Anfibatide may represent the first successful GPIb antagonist to offer potent antithrombotic treatment without affecting haemostasis. Thrombosis and Haemostasis 111.2/2014
Acknowledgements
This work was partially supported by Lee’s Pharmaceutical Holdings limited. The authors would like to thank Dr. Yotis Senis for generously providing the CRP and convulxin, Inge Pareyn and Aline Vandenbulcke for their technical assistance, and Dr. Guangheng Zhu and Dr. Pingguo Chen for their insightful and valuable comments during the manuscript preparation. Conflicts of interest
This work was partially supported by Lee's Pharmaceutical Holdings limited. C. Liang and B. X. Li are employees of Lee's Pharmaceutical Holdings limited, the company which holds the patent for Anfibatide. X. Dai is an employee of Zhooke Pharmaceutical Co. Ltd. None of the other authors reports any conflicts of interest.
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Lei, Reheman, et al. Anfibatide inhibits platelet adhesion and thrombus formation in mice
Platelets and Blood Cells
Anfibatide, a novel GPIb complex antagonist, inhibits platelet adhesion and thrombus formation in vitro and in vivo in murine models of thrombosis Xi Lei1*; Adili Reheman1*; Yan Hou1,2; Hui Zhou1; Yiming Wang1,3,4; Alexandra H. Marshall1; Chaofan Liang5; Xiangrong Dai6; Benjamin Xiaoyi Li5; Karen Vanhoorelbeke7; Heyu Ni1,3,4,8 Research Center, Li Ka Shing Knowledge Institute, St. Michael’s Hospital, and Toronto Platelet Immunobiology Group, University of Toronto, Toronto, Ontario, Canada; 2Jilin Provincial Center for Disease Prevention and Control, Changchun, Jilil, China; 3Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada; 4Canadian Blood Services, Toronto, Ontario, Canada; 5Lee’s Pharmaceutical holdings limited, Shatin, Hong Kong, China; 6Zhaoke Pharmaceutical co. limited, Hefei, Anhui, China; 7Laboratory for Thrombosis Research, IRF Life Sciences, KU Leuven Kulak, Kortrijk, Belgium; 8Department of Physiology and Department of Medicine, University of Toronto, Toronto, Ontario, Canada
Summary Platelet adhesion and aggregation at the sites of vascular injury are key events for thrombosis and haemostasis. It has been well demonstrated that interaction between glycoprotein (GP) Ib and von Willebrand factor (VWF) initiates platelet adhesion and contributes to platelet aggregation, particularly at high shear. GPIb has long been suggested as a desirable antithrombotic target, but anti-GPIb therapy has never been successfully developed. Here, we evaluated the antithrombotic potential of Anfibatide, a novel snake venom-derived GPIb antagonist. We found Anfibatide inhibited washed murine platelet aggregation induced by ristocetin and recombinant murine VWF. It also blocked botrocetin-induced binding of murine plasma VWF to recombinant human GPIb . Interestingly, Anfibatide did not inhibit botrocetin-induced aggregation of platelet-rich plasma, indicating that its binding site may differ from other snake venom-derived GPIb antagonists. Anfibatide strongly inhibited platelet adhesion, aggregation, and thrombus formation in perfusion chambers at high shear conditions Correspondence to: Dr. Heyu Ni, MD, PhD Canadian Blood Services and Department of Laboratory Medicine and Pathobiology St. Michael’s Hospital, University of Toronto Room 420, LKSKI – Keenan Research Center 209 Victoria Street, Toronto Ontario, M5B 1W8, Canada Tel.: +1 416 847 1738 E-mail: [email protected]
and efficiently dissolved preformed thrombi. Anfibatide also inhibited thrombus growth at low shear conditions, though less than at high shear. Using intravital microscopy, we found that Anfibatide markedly inhibited thrombosis in laser-injured cremaster vessels and prevented vessel occlusion in FeCl3-injured mesenteric vessels. Importantly, Anfibatide further inhibited residual thrombosis in VWF-deficient mice, suggesting that Anfibatide has additional antithrombotic effect beyond its inhibitory role in GPIb-VWF interaction. Anfibatide did not significantly cause platelet activation, prolong tail bleeding time, or cause bleeding diathesis in mice. Thus, consistent with the data from an ongoing clinical trial, the data from this study suggests that Anfibatide is a potent and safe antithrombotic agent.
Keywords Antithrombotic agent, snake venoms, platelet inhibitor, glycoprotein Ib-IX-V complex
Financial support: This work was supported partially by Canadian Institutes of Health Research and National Natural Science Foundation of China (China-Canada Joint Health Research Initiative Program), Lee’s Pharmaceutical Holdings limited, Canadian Institutes of Health Research (MOP 119540), Heart and Stroke Foundation of Canada, and Canadian Foundation for Innovation. Yan Hou is a recipient of State Scholarship Fund from China Scholarship Council (CSC) and Yiming Wang is a recipient of a Ph.D. Graduate Fellowship from Canadian Blood Services and the Meredith & Malcolm Silver Scholarship in Cardiovascular Studies from the Department of Laboratory Medicine and Pathobiology, University of Toronto. Received: June 17, 2013 Accepted after major revision: September 25, 2013 Prepublished online: October 31, 2013
* X. Lei and A. Reheman contributed equally to this work.
Introduction Platelet adhesion and subsequent aggregation at sites of vascular injury are required to maintain normal haemostasis; however, these processes can also contribute to pathological occlusive thrombosis (1-3). Interaction of the platelet surface glycoprotein (GP) Ib-IX-V complex with von Willebrand factor (VWF) plays a key role in initiation of platelet adhesion, particularly at high shear © Schattauer 2014
doi:10.1160/TH13-06-0490 Thromb Haemost 2014; 111: 279–289
stress (e.g. shear rate >1,200 s-1) (1, 4). This interaction is also essential for complete vessel occlusion at sites of vascular stenosis (e.g. shear rate >10,000 s-1) (3, 4). Platelet surface integrin αIIbβ3 and its ligands (fibrinogen or other ligands) (5, 6) mediate platelet aggregation and contribute to platelet adhesion at lower shear stress (3). Thus, both the GPIb complex and integrin αIIbβ3 are important targets to consider for antithrombotic therapy. Interestingly, although several inhibitors of αIIbβ3 have been developed Thrombosis and Haemostasis 111.2/2014
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Lei, Reheman, et al. Anfibatide inhibits platelet adhesion and thrombus formation in mice
for antithrombotic therapies (7) and at least one anti-GPIb monoclonal antibody has been humanised (8), anti-GPIb therapy has not yet been developed. The GPIb complex is an adhesion receptor that consists of four membrane-spanning subunits including GPIbα, GPIbβ, GPIX, and GPV (9). Once activated by high shear stress, VWF undergoes a conformational change and binds to GPIbα (10, 11). In VWF knockout mice, arterial thrombosis still occurs but complete vessel occlusion is either prevented or postponed (4). ARC1779, an antiVWF aptamer, does reduce adenosine 5’-diphosphate (ADP)- and collagen-induced platelet aggregation but also prolongs cutaneous bleeding time in humans (12). In contrast to VWF knockout mice, thrombus formation is completely abolished in GPIb knockout mice (13), suggesting that GPIb receptor ligands, other than VWF, may play an important role in platelet aggregation and thrombus formation (14). Therefore, given that GPIb deficiency results in more severe impairment of thrombosis than VWF, GPIb is likely a more attractive candidate for antithrombotic therapy (15). Snaclecs are a subset of non-enzymatic proteins isolated from snake venom that include C-type lectins and their related proteins. Snaclecs have been isolated and tested for therapeutic potential for four decades; however, to our knowledge, none have successfully entered clinical practice. Agkisacucetin (trade name Anfibatide, following further purifications) is a snaclec purified from the venom of the Agkistrodon acutus snake. Anfibatide is a C-type lectin-like protein (α and β chains with interchain disulphide bonds, without Ca2+-/sugar-binding loops) derived from the protein complex agglucetin (16, 17). The structure of Anfibatide has high similarity to subunits of agglucetin (17); however, agglucetin is tetrameric protein. Earlier studies demonstrated that Agkisacucetin binds to GPIb to inhibit human VWF binding (17, 18). However, the effect of Anfibatide on thrombosis and haemostasis has not been studied. Here we report the first in vitro and in vivo assessment of the effects of Anfibatide on murine platelet function and thrombus formation. We found that Anfibatide is a potent antithrombotic agent that does not significantly affect haemostasis.
Material and methods Experimental animals C57BL/6J wild-type (WT) male mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). VWF deficient (-/-) mice were used as previously described (4, 19). C57BL/6J WT male and female mice were used to prepare washed murine platelets. All experimental protocols were reviewed and approved by the Animal Care Committee of St. Michael’s Hospital or the Institutional Animal Care and Use Committee of the KU Leuven, Belgium, as appropriate.
Purification of Anfibatide Anfibatide was purified from venom of the Agkistrodon acutus snake by anion-exchange and monoclonal antibody-based affinity Thrombosis and Haemostasis 111.2/2014
chromatography followed by Sephacryl S-100 column. After the purification, Anfibatide was analyzed by MALDI-TOF mass spectrometry and only one peak appeared indicating its purity.
VWF botrocetin cofactor activity assay The ELISA was performed as previously described (20). Briefly, a 96-well microtitre plate was coated with the anti-GPIbα monoclonal antibody 2D4 to capture 1 μg/ml recombinant fragment of human GPIbα (rfGPIbα) (21). The rfGPIbα was saturated with a constant amount of diluted murine plasma (1:16) containing 0.1 μg/ml of botrocetin (Sigma, St. Louis, MO, USA) in the presence or absence of a 1:2 dilution of Anfibatide (highest concentration was 6 μg/ml). Bound VWF was detected with anti-VWF immunoglobulins labelled with horse radish peroxidase (Dako, Glostrup, Denmark). Of note, ristocetin cannot be used to study the binding of plasma VWF to GPIbα in this assay.
Ristocetin-induced platelet aggregation of washed murine platelets As ristocetin-induced platelet aggregation can be performed with washed murine platelets (22) but not with murine platelet-rich plasma (PRP), the ristocetin-induced platelet aggregation assay was performed using washed murine platelets. Blood samples were collected via retro-orbital puncture on 80 μM PPACK (Calbiochem, Billerica, MA, USA) and 100 μl ACD-C (124 mM trisodium citrate, 130 mM citric acid, and 110 mM D-glucose, pH 6.5). Blood was centrifuged (100×g, 7 minutes [min]), then PRP was diluted in wash buffer (103 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5 mM D-Glucose, 36 mM citric acid, pH 6.5) containing 1 μM prostaglandin E1 (PGE1, Sigma) and 100 mU apyrase (Sigma). Platelets were centrifuged (800×g, 10 min), washed a second time, and finally resuspended in HEPES-Tyrode buffer. To induce aggregation, recombinant murine VWF (mVWF, 5 μg/ml, expressed in HEK293T cells [23]) and 1 mg/ml ristocetin (ABP, London, UK) were added to the washed platelets (250,000 platelets/μl). To study the effect of Anfibatide, 6 μg/ml was added. Platelet aggregation was evaluated by light transmission in a Chrono-Log dual channel aggregometer (Kordia BV, Leiden, The Netherlands).
In vitro platelet aggregation To investigate the specificity of Anfibatide and its effect on GPIbα-VWF-independent aggregation, PRP or gel-filtered platelet aggregation were tested as described (5, 24-26). Briefly, PRP was isolated from sodium-citrated murine whole blood by centrifugation. Gel-filtered platelets were isolated from PRP using a Sepharose 2B chromatography columns with PIPES buffer (5 mM PIPES, 137 mM NaCl, 4 mM KCl, 0.1% glucose, pH 7.0). Platelet count was adjusted to 3x108 platelets/ml. Platelet aggregation was assessed with or without adding Anfibatide (6 μg/ml) using a computerised Chrono-log aggregometer (Chrono-Log Corporation, Havertown, PA, USA). Platelet aggregation in PRP was induced by adenosine diphosphate (ADP, 1 to 20 μM, Sigma-Aldrich), col© Schattauer 2014
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Ex vivo perfusion chamber To study the effect of Anfibatide on thrombus formation under flow conditions, we used an ex vivo perfusion chamber system as described (6, 27, 28). Briefly, rectangular microcapillary glass tubes (0.1×1 mm) were coated with 100 μg/ml Horm collagen (Nycomed, Linz, Austria) overnight at 4°C. Whole blood was collected from anaesthetised mice, heparin-anticoagulated (15 U/ml), and fluorescently labelled by incubation with DiOC6 (1 μM, Sigma; 10 min, 37°C). Control or Anfibatide-treated (6 μg/ml) blood was perfused over the collagen-coated surface for 3 min using a syringe pump (from 100 s-1 to 5000 s-1; Harvard Apparatus, Holliston, MA, USA). To evaluate its therapeutic potential, we tested the effect of Anfibatide on preformed thrombi at high shear flow. Control whole blood was perfused over the collagen-coated surface (50 μg/ml Horm collagen) at a controlled shear rate of 1,800 s-1 for 3 min to form thrombi on collagen as described above. Then, without interruption, perfusion was continued either with untreated control blood or with Anfibatide-treated (6 μg/ml) whole blood for 3 min. Platelet adhesion, aggregation, and thrombus formation were recorded in real-time under a Zeiss Axiovert 135-inverted fluorescence microscope (60X-W objective). Quantitative dynamics of platelet fluorescence intensity were acquired by SlideBook software (Intelligent Imaging Innovations Inc., Denver, CO, USA).
Platelet activation To test whether Anfibatide causes platelet activation or granule release, resting gel-filtered WT platelets were assessed for platelet β3 integrin activation and P-selectin surface expression using flow cytometry, as described (6). Resting gel-filtered platelets were incubated with control buffer or Anfibatide (6 μg/ml) for 30 min, with the presence of phycoerythrin (PE)-conjugated rat anti-mouse αIIbβ3 antibodies (JON/A, EMFRET Analytics, Eibelstadt, Germany) and fluorescein isotheiocyanate (FITC)-conjugated rat anti-mouse CD62P (P-selectin) monoclonal antibodies (BD Biosciences Pharmingen, San Diego, CA, USA). Platelets were fixed with freshly prepared paraformaldehyde (1%) and antibodies were detected by a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA).
In vivo thrombosis studies To assess the effect of Anfibatide on platelet response to vascular injury in vivo, thrombus formation was studied by real-time in© Schattauer 2014
travital microscopy using two different in vivo thrombosis models that each represents a different vascular bed, size, and injury.
FeCl3-induced mesenteric arteriole thrombosis model The thrombotic response to ferric chloride (FeCl3)-induced vascular injury was assessed in WT control and Anfibatide-treated (6 μg/ml of blood) mice using an intravital microscopy thrombosis model, as previously described (24, 29-31). Briefly, gel-filtered platelets were fluorescently labelled with calcein acetoxymethyl ester (1 μg/ml; Molecular Probes) and injected via the tail vein of recipient mice (23-28 days old). The mesentery vascular bed was exteriorised through a midline abdominal incision. A single arteriole was chosen in each mouse based on vessel diameter (80-120 μm), shear rate (approximately 1,500 s-1), and visibility. Thrombus formation was induced in a 2-5 mm segment of the arteriole by topical application of 30 μl of 250 mM FeCl3 and was recorded using a Zeiss Axiovert 135-inverted fluorescent microscope (Zeiss, Oberkochen, Germany). We quantified 1) the number of fluorescent platelets deposited on the vessel wall during the 3-5 min interval following injury; 2) the time required for the formation of the first 20 μm thrombus; and 3) the vessel occlusion time (defined as complete cessation of blood flow for at least 10 seconds).
Laser-induced cremaster muscle arteriole thrombosis model The effect of Anfibatide on laser-induced cremaster arteriole thrombosis was studied in WT and VWF-/- mice, as previously described (24, 32). Briefly, the cremaster muscle of anaesthetised mice (8 weeks old) was prepared under a dissecting microscope and superfused with preheated bicarbonate-buffered saline throughout the experiment. Platelets were labelled by injection of rat anti-mouse CD41 antibody (BD Bioscience, 0.1 µg/g) secondarily-labelled with Alexa Flour 647 goat anti-rat IgG (Molecular Probes, 0.5 µg/g) via a jugular vein cannulus. Anfibatide (6 μg/ml) or control buffer was intravenously administered prior to injury. Multiple independent upstream injuries were then caused by a pulsed nitrogen dye laser. Images of thrombus formation were captured in real-time under an Olympus BX51WI microscope and analysed by Slidebook.
Tail bleeding time Tail bleeding time was assessed as previously described (33). WT mice (6-8 weeks old) were injected with Anfibatide (6 μg/ml via tail vein) or control buffer. Mice were anaesthetised 30 min postinjection with tribromoethanol (2.5 %, 0.015 ml/g body weight, intraperitoneal) and placed on a 37°C warming pad. A 3 mm terminal section of the tip of the tail was removed by a scalpel and the tail was immersed into isotonic saline (37°C) to bleed. Tail bleeding time was recorded until bleeding had ceased or for a maximum of 10 min, when further bleeding was prevented by cauterization.
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lagen (2.5 to 20 μg/ml, Nycomed Pharma, Ismaning, Germany), collagen-related peptide (CRP, 0.05 to 1 μg/ml; provided by Dr. Yotis Senis from the Institute of Biomedical Research, University of Birmingham, UK), or convulxin (0.5 to 5 μg/ml; also provided by Dr. Yotis Senis). Platelet aggregation in gel-filtered platelets was induced by thrombin receptor agonist peptide (TRAP; sequence AYPGKF-NH2; 100 to 500 μM; Peptides Int, Louisville, KY, USA).
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Statistical analysis Statistical analyses were performed using GraphPad Prism 6 and differences were evaluated by Student’s two-tailed t-test. Differences with a p-value less than 0.05 were considered statistically significant. Data are expressed as mean ± standard error of the mean (SEM). Sample size was >3 per group.
Results Anfibatide inhibited GPIb-VWF interaction and aggregation in vitro Ristocetin and botrocetin change the confirmation of VWF, allowing binding to GPIb. Interestingly, botrocetin-induced murine pla-
telet aggregation was not inhibited by Anfibatide (▶ Figure 1 A); however, ristocetin-induced aggregation of washed murine platelets and recombinant murine VWF was markedly inhibited (▶ Figure 1 B), suggesting that its binding site may differ from other snake venom-derived GPIb antagonists. In a separate study, we found that Anfibatide inhibits ristocetin-induced platelet aggregation using human PRP (unpublished data). To confirm these findings and to rule out a ristocetin-dependent effect of Anfibatide, the binding of murine plasma VWF to rfGPIbα was induced by botrocetin using an immunoassay (20). Complete inhibition of the VWF-GPIbα interaction was observed with the addition of Anfibatide (▶ Figure 1C). To examine whether the Anfibatide inhibition is specific to VWF-GPIb-dependent platelet aggregation, in vitro platelet aggregation was also induced by ADP, collagen, TRAP, convulxin, and
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G Figure 1: Anfibatide inhibits the VWF-/- GPIb interaction in vitro. Anfibatide did not inhibit botrocetin-induced aggregation of murine PRP (A). Anfibatide inhibited both ristocetin-induced aggregation of washed murine platelets supplemented with recombinant mVWF (B) and binding of plasma mVWF to rfGPIb in the presence of botrocetin in an immunoassay (n=3, C; ** represents p < 0.01 vs control). Anfibatide also did not inhibit platelet aggre-
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H gation of murine PRP in the presence of adenosine diphosphate (ADP; D), collagen (E), thrombin receptor activating peptide (TRAP; F), collagen related peptide (CRP; G), or convulxin (H). Representative tracings of three independent experiments are shown for all aggregation experiments. Black tracings: Control platelets, red tracings: Anfibatide-treated platelets.
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Anfibatide strongly inhibited platelet adhesion and aggregation, and dissolved preformed thrombi in an ex vivo thrombosis model The effect of Anfibatide on thrombosis was first assessed by the ex vivo perfusion chamber model at both high (1,800 s-1) and low (300 s-1) shear rates under a real-time fluorescence microscope. As shown in ▶ Figure 2, immediately following the perfusion of control whole blood over a collagen-coated surface, fluorescent pla-
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E Figure 2: Anfibatide inhibited platelet function and thrombosis in whole blood at high and low shear rate, and dissolved preformed thrombi in ex vivo perfusion chamber. The representative images (A) and quantification (B) of platelet adhesion and aggregation at 1,800 s-1 shear rate for 3 min. The means were significantly different, p < 0.05. The representative images (C) and quantification (D) of platelet adhesion and aggregation at 300 s-1 for 3 min. The representative images (E) and quantifi-
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CRP, agonists that lead to platelet and αIIbβ3 activation independent of the GPIb-VWF interaction. As hypothesised, ADP-, collagen-, TRAP-, CRP-, and convulxin-induced aggregation were not inhibited by Anfibatide pretreatment (▶ Figure 1D-H). There was also no significant effect on platelet aggregation induced by low doses of ADP, collagen, CRP, and TRAP (Suppl. Figure 1, available online at www.thrombosis-online.com). These results suggest that Anfibatide itself did not significantly affect signalling pathways downstream of GPIb.
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F cation (F) of platelet adhesion and aggregation during 6 min thrombolysis experiment. Control blood was perfused until 3 min in both groups, then either control or Anfibatide-treated blood was perfused at 1,800 s-1 until 6 min. B, D, F) The kinetic curves represent platelet mean fluorescence intensity (MFI) and the shaded regions represent SEM. Green: fluorescent platelets. Black: Untreated control whole blood. Red: Anfibatide-treated whole blood. The means were significantly different, p < 0.05.
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telets firmly adhered to the collagen, formed stable platelet aggregates, and gradually formed large thrombi with collagen fibrils after 3 min at high shear conditions. In sharp contrast to controls, Anfibatide pretreatment strongly inhibited platelet adhesion, aggregation, and thrombus formation at high shear (p < 0.01, ▶ Figure 2A and B); only a few single platelets adhered to the collagen surface during the entire 3 min perfusion. Platelet adhesion and subsequent aggregate formation was also notably decreased at low shear (p < 0.05, ▶ Figure 2C and D); only some adhered platelets and small aggregates were observed after 3 min of perfusion.
We tested the effect of Anfibatide under a range of shear stress conditions in perfusion chambers, including 100 s-1, 200 s-1, 500 s-1, 3,000 s-1, and 5,000 s-1. As shear stress increased, the inhibitory effect of Anfibatide became stronger (data not shown). The inhibition was approximately 100 times stronger at high shear (1,800 s-1) as compared to that at low shear (300 s-1). At 5,000 s-1, Anfibatide-treated platelet adhesion and aggregation on collagen was undetectable. To mimic a pathological scenario where the thrombus has already formed, we tested the effect of Anfibatide on preformed
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Figure 3: Anfibatide markedly inhibited thrombosis in wild-type (WT) mice in arteriole thrombosis models. Representative images (A) and quantification (B-D) of thrombus formation induced by ferric chloride in Control WT mice and Anfibatide-treated WT mice in mesenteric arteriole thrombosis model are shown from 4 to 18 min. B) The number of fluorescent platelets deposited on the vessel wall per min during the 3–5 min interval following injury. C) The time required for the formation of the first 20 μm thrombus. * Means were significantly different, p < 0.01. D) The time to com-
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plete vessel occlusion. *Means were significantly different, p < 0.01. Representative images (E) and quantification (F) of thrombus formation induced by laser injury in cremaster arteriole thrombosis model in Control WT mice and Anfibatide-treated WT mice. There was a significant difference between the means, p < 0.01. The kinetic curves represent platelet mean fluorescence intensity (MFI), and the shaded regions represent SEM. Green or white: fluorescent platelets. Black: Untreated control whole blood. Red: Anfibatidetreated whole blood.
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Anfibatide inhibited thrombosis and prevented vessel occlusion in vivo in two complimentary intravital microscopy thrombosis models We used the FeCl3-induced mesenteric arteriole thrombosis model to evaluate the effects of Anfibatide on platelet adhesion, aggregation, and occlusive thrombus formation in vivo in real-time 30 min after treatment. As shown in ▶ Figure 3A-D, in control mice, platelets interacted with and deposited on the injured vessel wall shortly after FeCl3 application. Within a few minutes of injury, visible platelet aggregates formed and grew into thrombi that eventually resulted in stable vessel occlusion and complete disruption of blood flow. Consistent with our ex vivo findings, Anfibatide treatment significantly inhibited the initial platelet interaction and adhesion response to vascular injury in mice (▶ Figure 3A-D). The number of interacted platelets per minute at 3-5 min after injury was significantly less in Anfibatide-treated mice compared with WT controls (p < 0.05, ▶ Figure 3B). Formation of visible platelet aggregates was delayed and small aggregates were easily dissolved, rarely growing into large thrombi. The time required for the formation of first thrombus (>20 μm in diameter) at the site of vessel injury was significantly longer in Anfibatide-treated mice compared to WT controls (p < 0.01, ▶ Figure 3C). Moreover, An-
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Anfibatide has anti-thrombotic functions beyond GPIb-VWF interaction blockade To investigate whether Anfibatide inhibits GPIb interaction with other ligands or agonists in addition to VWF, we tested thrombus formation in VWF-/- mice with or without Anfibatide. Interestingly, in VWF-/- mice where laser-induced thrombosis is impaired compared to WT mice, Anfibatide inhibited thrombus formation even further than knockout alone (p < 0.05, ▶ Figure 4). Furthermore, Anfibatide inhibition was more potent in VWF-/- mice (▶ Figure 4B) than that in WT mice (▶ Figure 3F). These results suggest that Anfibatide not only inhibits GPIb-VWF interaction
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Figure 4: Anfibatide further inhibited thrombosis in von Willebrand factor-deficient (VWF-/-) mice in the cremaster muscle arteriole thrombosis model. Representative images (A) and quantification (B) of thrombus formation in VWF-/- mice and VWF-/- mice treated with Anfibatide (6
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fibatide treatment delayed complete vessel occlusion in all mice (recorded up to 40 min post-injury, p < 0.01, ▶ Figure 3D). We used the laser-induced cremaster arteriole thrombosis model to determine the effect of Anfibatide treatment on platelet accumulation within a growing thrombus. In WT control mice, platelets adhered and quickly accumulated at the site of injury immediately after laser-induced arteriole injury, as shown in ▶ Figure 3 E-F. The thrombus rapidly increased in size and reached a maximal size approximately 1 min after injury. Then the thrombus decreased in size and stabilised on the site of injury. By contrast, Anfibatide pretreatment significantly inhibited thrombus growth in injured arterioles (p < 0.01, ▶ Figure 3 F). Platelet thrombus formation at the site of injury was limited and quickly decreased in size or stabilised without the rapid platelet accumulation observed in WT control mice. In summary, thrombus formation in Anfibatide-treated mice was delayed, unstable, easily dissolved, and failed to form an occlusive thrombus.
g/ml) at 1 min. The kinetic curves represent platelet mean fluorescence intensity (MFI), and the shaded regions represent SEM. The means were significantly different, p < 0.05. Green: fluorescent platelets. Black: Untreated VWF-/- mice. Red: Anfibatide-treated VWF-/- mice.
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thrombi in perfusion chambers at high shear stress (1,800 s-1). At 3 min, large and stable thrombi were already formed in the perfusion chambers. Continued perfusion of control whole blood increased the size of the preformed thrombi, and more platelets adhered and formed new thrombi. Continued perfusion of Anfibatide-treated blood dissolved the preformed thrombi in the perfusion chambers (▶ Figure 2E and F).
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but also attenuates other thrombotic pathways mediated by the GPIbα.
Anfibatide did not significantly activate platelets in vitro or prolong bleeding time in vivo To test the possible side effects, it is important to assess the effects of Anfibatide on platelet activation and haemostasis. By flow cytometry, we found no significant difference in P-selectin expression (CD62P; ▶ Figure 5 A) or integrin αIIbβ3 activation (JON/A; ▶Figure 5B) between in vitro Anfibatide-treated and control platelets. Moreover, in vivo, no significant difference was observed in tail vein bleeding time in mice treated with Anfibatide compare to
control mice (▶ Figure 5C). There was also no spontaneous bleeding or bleeding from sites of vascular access during surgeries (data not shown).
Discussion This study is the first in vitro and in vivo assessment of the effects of Anfibatide on murine platelet function and thrombosis. We demonstrated that Anfibatide strongly inhibited platelet adhesion, aggregation, and thrombus formation at both high and low shear conditions, dissolved preformed thrombi ex vivo, and prevented vessel occlusion in in vivo thrombosis models. Interestingly, we
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C Figure 5: Anfibatide did not significantly affect platelet activation in vitro or tail vein bleeding time in vivo. A) P-selectin (CD62P) expression on the platelet surface. B) 3 integrin activation tested by JON/A antibody. A, B) Left panel shows the representative histogram. Grey: Control platelets.
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Red: Anfibatide-treated platelets. Right panel shows the mean fluorescence intensity (MFI). Means were not significantly different. C) Tail bleeding time in untreated WT mice and Anfibatide-treated WT mice. Black: WT mice. Red: Anfibatide-treated WT mice. No significant differences detected.
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© Schattauer 2014
switch the thrombus growth toward thrombus dissolution under flow conditions. It is also very likely that GPIb-VWF interaction may deliver outside-in signaling via the GPIb complex that activate newly recruited platelets in the thrombi and maintain their αIIbβ3 integrins in active conformation, which is required for fibrinogen- and other integrin ligand-mediated platelet aggregation. In addition, GPIb-P-selectin and GPIb-other haemostatic/thrombotic factor interactions may provide additional bridges between adjacent activated platelets to enhance thrombus stability. Furthermore, we also will not exclude the potential interaction of Anfibatide with other plasma proteins that may also favor thrombus dissolution and thrombolysis. Thus, Anfibatide may induce thrombus dissolution, inhibit thrombus growth, and prevent vessel occlusion in diseased arteries, which would prevent myocardial infarction and ischemic stroke and save lives. Deep vein thrombosis (DVT) is another major cause of mortality worldwide. Recent studies have clearly demonstrated that both
What is known about this topic?
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Platelet adhesion and aggregation at the sites of vascular injury are key events for thrombosis and haemostasis. Interaction between the glycoprotein (GP)Ib complex and von Willebrand factor (VWF) initiates platelet adhesion and contributes to platelet aggregation, particularly at high shear. This interaction is also essential for complete vessel occlusion at sites of vascular stenosis. GPIb has long been suggested as a desirable target for antithrombotic therapy, but an anti-GPIb agent for therapy has never been developed. Anfibatide (trade name for Agkisacucetin) is a GPIb antagonist derived from snake venom. Its structure has been described but its effect on thrombosis and haemostasis has not been adequately studied.
What does this paper add?
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We evaluated the antithrombotic potential of Anfibatide, a novel snake venom-derived GPIb antagonist, in vitro, ex vivo, and in vivo in murine thrombosis models. Anfibatide strongly inhibited platelet adhesion, aggregation, and thrombus formation, dissolved preformed thrombi on collagen in ex vivo perfusion chambers at high shear, and prevented vessel occlusion in in vivo thrombosis models. Thus, Anfibatide is a strong antithrombotic agent for arterial thrombosis. Importantly, Anfibatide further inhibited residual thrombosis in VWF-deficient mice and inhibited thrombosis at low shear. These results suggest that Anfibatide has additional antithrombotic effect beyond inhibition of GPIb-VWF interaction and may have therapeutic potential for both arterial and deep vein thrombosis. No significant platelet activation was detected after Anfibatidetreatment, and bleeding time was not significantly increased in Anfibatide-treated mice. Therefore, Anfibatide may represent a novel and potent antithrombotic agent that will not cause severe bleeding complications in patients.
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found that Anfibatide not only inhibits the GPIb-VWF interaction but also attenuates other thrombotic pathways mediated by the GPIb complex. Importantly, no platelet activation was detected after Anfibatide-treatment and bleeding time was not increased in Anfibatide-treated mice. Therefore, Anfibatide may represent a novel and potent antithrombotic agent that can control both arterial and deep vein thrombosis without significantly causing bleeding complications. Earlier research demonstrated that purified Agkisacutacin (trade name Anfibatide) selectively binds to GPIb (18). Flavocetin-A and echicetin are two snaclecs that also inhibit VWF access to GPIb with similar structures to Anfibatide. Surprisingly, though they both bind to GPIb to inhibit platelet aggregation, they are also able to increase platelet agglutination by forming tetramers or by binding to immunoglobulin Mκ (IgMκ), respectively (17, 34). In contrast, each Anfibatide αβ-heterodimer molecule binds to one GPIb molecule and inhibits GPIb-VWF interaction without causing GPIb clustering (17). Akitonin and jararaca GPIb-BP are GPIb antagonists that decrease platelet aggregation in vitro but we have not found any information regarding their efficacy and bleeding diathesis in vivo (35, 36). According to our in vitro and in vivo results, Anfibatide is a promising therapeutic agent given our evidence that it does not significantly increase bleeding time (▶ Figure 5). Ristocetin and botrocetin both bind to the A1 domain of VWF to induce a conformational change of VWF, which allows for GPIb binding and leads to platelet aggregation in vitro (37, 38). Interestingly, we found that Anfibatide did not inhibit botrocetin-induced murine platelet aggregation in PRP (▶ Figure 1 A) but did block GPIb-VWF binding induced by botrocetin in ELISA and inhibited washed murine platelet aggregation induced by ristocetin and recombinant murine VWF (▶ Figure 1B and C). In an ongoing clinical trial, Anfibatide inhibited ristocetin-induced human platelet aggregation in PRP (unpublished data). These results suggest that the tertiary interactions between Anfibatide and human and mouse GPIb may be different and its binding site may differ from other reported snake venom-derived GPIb antagonists. Our comprehensive ex vivo and in vivo studies revealed that Anfibatide inhibited platelet adhesion, aggregation, and thrombus formation in murine models of thrombosis (▶ Figure 2 and ▶ Figure 3). The GPIb-IX-V complex plays a pivotal role in initiating and propagating both haemostasis and thrombosis when bound to VWF (39). Initiation of platelet adhesion and aggregation is absolutely dependent on GPIb and its ligand VWF above a limiting shear rate (~10,000 s-1) (40). In diseased arteries, shear stress is elevated when lumen restriction increases blood flow velocity (41). The VWF and GPIb interaction therefore precipitates the complete thrombotic occlusion of the vessel causing obstruction of blood flow and subsequent tissue damage (1). In this study we demonstrated that GPIb antagonism by Anfibatide almost completely inhibited platelet adhesion, aggregation, and thrombus formation and dissolved preformed thrombi ex vivo at high shear (▶ Figure 2). It is notable that thrombus growth and thrombolysis are dynamic processes and likely occur simultaneously, blocking GPIb-VWF interaction may prevent platelet recruitment and
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VWF and platelets are important in this process (42), which is consistent with an earlier study that demonstrated GPIb-VWF interaction also plays a role in platelet adhesion at low shear in veins (43). In our perfusion chambers experiments, we found that Anfibatide inhibited platelet adhesion, aggregation, and thrombus formation at venous shear stress (▶ Figure 2), suggesting that Anfibatide may be able to inhibit DVT. It is also notable that shear rate escalates in DVT as luminal stenosis progresses. Therefore, the inhibitory effect of Anfibatide at both low and high shear indicates that it may also have therapeutic potential in DVT. Interestingly, we found that Anfibatide inhibited thrombus formation even further in VWF-/- mice (▶ Figure 4). Therefore, Anfibatide not only selectively inhibits GPIb-VWF interaction but also attenuates other thrombotic pathways mediated by the GPIb complex. Previous research demonstrated that the role of GPIb far exceeds its main ligand VWF and that a GPIb ligand other than VWF may exist in the injured vessel (13). Recent studies showed that GPIb interacts with thrombin, P-selectin, Mac-1, and other molecules (39, 44). It will be of interest to further elucidate whether Anfibatide can disrupt binding of thrombin or other thrombotic/haemostatic factors to the GPIb complex. If Anfibatide indeed inhibits additional interactions, this may broaden its role in vascular biology. Currently the most common anti-thrombotic agents are fibrinogen receptor antagonists, aspirin, and P2Y12 antagonists, which all inhibit β3 integrin-mediated platelet aggregation. Recent clinical data with intravenous fibrinogen receptor antagonists (45) and novel anti-platelet drugs such as Prasugrel (46) demonstrated platelet inhibition and prevention of ischaemic complications, but these agents were also associated with increased bleeding complications. αIIbβ3 inhibitors represent effective antiplatelet/antithrombotic agents, but they often lead to thrombocytopenia and/ or life-threatening bleeding complications, such as cerebral, alveolar, and gastrointestinal system haemorrhages (47-50). Until now, no anti-GPIb drug has been successfully introduced into clinical practice, although several potential candidates are under development (15). We demonstrate that Anfibatide, a GPIb antagonist, had no significant effect on platelet activation or bleeding time (▶ Figure 5). There was also no spontaneous bleeding or bleeding from sites of vascular access during surgeries. There are a couple of possible explanations for this exciting phenomenon. First, VWF-GPIb interaction may mainly be involved in thrombosis and haemostasis at high shear stress, and blocking this interaction may have less effect on haemostasis occurring at low shear stresses. Second, Anfibatide may have no effect on VWF-mediated haemostatic pathways that are independent of GPIb (e.g. VWF-integrin interaction). Therefore, Anfibatide may offer a more favorable safety profile than currently available antithrombotic agents. In summary, we report that Anfibatide, a novel GPIb antagonist, effectively inhibited platelet adhesion, aggregation, and thrombus formation ex vivo, prevented vessel occlusion in in vivo thrombosis models, and has a favorable safety profile. Anfibatide may represent the first successful GPIb antagonist to offer potent antithrombotic treatment without affecting haemostasis. Thrombosis and Haemostasis 111.2/2014
Acknowledgements
This work was partially supported by Lee’s Pharmaceutical Holdings limited. The authors would like to thank Dr. Yotis Senis for generously providing the CRP and convulxin, Inge Pareyn and Aline Vandenbulcke for their technical assistance, and Dr. Guangheng Zhu and Dr. Pingguo Chen for their insightful and valuable comments during the manuscript preparation. Conflicts of interest
This work was partially supported by Lee's Pharmaceutical Holdings limited. C. Liang and B. X. Li are employees of Lee's Pharmaceutical Holdings limited, the company which holds the patent for Anfibatide. X. Dai is an employee of Zhooke Pharmaceutical Co. Ltd. None of the other authors reports any conflicts of interest.
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Lei, Reheman, et al. Anfibatide inhibits platelet adhesion and thrombus formation in mice
