scholarly journals Distinct dietary alpha-linolenic acid-dependent shifts in the fecal microbiome composition suppresses aging-associated inflammatory responses and thrombus formation

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S.S Saeedi Saravi ◽  
N.R Bonetti ◽  
G.G Camici ◽  
T.F Luscher ◽  
J.H Beer
Keyword(s):  
Platelet Aggregation ◽  
Shear Flow ◽  
Plasma Levels ◽  
Inflammatory Responses ◽  
Thrombus Formation ◽  
Funding Source ◽  
High Shear ◽  
Fecal Microbiome ◽  
Microbiome Composition

Abstract Background Aging is associated with alterations in the fecal microbiome composition. The microbiota-derived trimethylamine-N-oxide (TMAO) correlates with arterial thrombotic events, e.g. myocardial infarction and stroke, the leading causes of mortality worldwide. The omega-3 fatty acid (n-3 FA) α-linolenic acid (ALA) has been shown to be protective against thrombosis and associated pathologies. Therefore, we hypothesized that long-term dietary ALA supplementation protects against the aging-associated microbiome dysbiosis, and reduces inflammatory and thrombotic responses. Methods 24 week-old male C57BL/6 mice were fed either a high ALA (7.3g%) or low ALA (0.03g%) diet for 12 months. We examined the compositional changes of fecal microbiota of the animals treated with high vs. low ALA via 16S rRNA gene sequencing. The plasma levels of TMAO and its precursors choline and betaine, and LPS were measured by ELISA. Additionally, the platelet aggregation in response to thrombin, and thrombus formation on collagen under high-shear flow conditions of 3000/sec (to mimic blood flow in stenosed arteries) were investigated. Results Genomic analyses showed that the abundance of Phylum Proteobacteria and the family of desulfovibrio were reduced 71.72% and 51.73% in the aged high ALA-treated mice (p<0.01 and p<0.001, resp.) that may result in decrease in TAMO production and the subsequent inflammatory responses. However, microbial diversity of Bacteroidetes or Fermicutes and Bacteroidetes/Fermicutes ratio did not demonstrate a significant change between high vs. low ALA groups. Interestingly, the dietary intake of high ALA increased the abundance of Lachnospiraceae (p<0.01) that may exert anti-inflammatory effects. Importantly, high ALA significantly decreased the plasma levels of TMAO (p<0.01) and its precursor choline (P<0.05), but not betaine. The pro-inflammatory cytokine TNF-α showed a significant reduction (p<0.05), whereas plasma IL-1β did not change significantly following high ALA supplementation. An increased thrombus formation on collagen under high-shear flow (36.34%, p<0.01) and thrombin-induced platelet aggregation (31.31%, p<0.05) were found in the aged mice. Conclusion These studies demonstrate that an ALA-rich diet induces beneficial bacterial shifts in the aging-associated fecal microbiome that may lead to the suppression of inflammatory and thrombotic responses. Hence, long-term dietary ALA supplementation may be exploited as a nutritional antithrombotic strategy in the aging. Microbiome-Thrombosis-Aging Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Swiss National Science Foundation (SNSF)

scholarly journals Persistence of Supplemented Bifidobacterium longum subsp. infantis EVC001 in Breastfed Infants

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Steven A. Frese ◽  
Andra A. Hutton ◽  
Lindsey N. Contreras ◽  
Claire A. Shaw ◽  
Michelle C. Palumbo ◽  
...  
Keyword(s):  
Human Milk ◽  
Gut Microbiome ◽  
Bifidobacterium Longum ◽  
Specific Substrate ◽  
Fecal Microbiome ◽  
Microbiome Composition ◽  
Breastfed Infants ◽  
Gut Function ◽  
First Time

ABSTRACT The gut microbiome in early life plays an important role for long-term health and is shaped in large part by diet. Probiotics may contribute to improvements in health, but they have not been shown to alter the community composition of the gut microbiome. Here, we found that breastfed infants could be stably colonized at high levels by provision of B. infantis EVC001, with significant changes to the overall microbiome composition persisting more than a month later, whether the infants were born vaginally or by caesarean section. This observation is consistent with previous studies demonstrating the capacity of this subspecies to utilize human milk glycans as a nutrient and underscores the importance of pairing a probiotic organism with a specific substrate. Colonization by B. infantis EVC001 resulted in significant changes to fecal microbiome composition and was associated with improvements in fecal biochemistry. The combination of human milk and an infant-associated Bifidobacterium sp. shows, for the first time, that durable changes to the human gut microbiome are possible and are associated with improved gut function. Attempts to alter intestinal dysbiosis via administration of probiotics have consistently shown that colonization with the administered microbes is transient. This study sought to determine whether provision of an initial course of Bifidobacterium longum subsp. infantis (B. infantis) would lead to persistent colonization of the probiotic organism in breastfed infants. Mothers intending to breastfeed were recruited and provided with lactation support. One group of mothers fed B. infantis EVC001 to their infants from day 7 to day 28 of life (n = 34), and the second group did not administer any probiotic (n = 32). Fecal samples were collected during the first 60 postnatal days in both groups. Fecal samples were assessed by 16S rRNA gene sequencing, quantitative PCR, mass spectrometry, and endotoxin measurement. B. infantis-fed infants had significantly higher populations of fecal Bifidobacteriaceae, in particular B. infantis, while EVC001 was fed, and this difference persisted more than 30 days after EVC001 supplementation ceased. Fecal milk oligosaccharides were significantly lower in B. infantis EVC001-fed infants, demonstrating higher consumption of human milk oligosaccharides by B. infantis EVC001. Concentrations of acetate and lactate were significantly higher and fecal pH was significantly lower in infants fed EVC001, demonstrating alterations in intestinal fermentation. Infants colonized by Bifidobacteriaceae at high levels had 4-fold-lower fecal endotoxin levels, consistent with observed lower levels of Gram-negative Proteobacteria and Bacteroidetes. IMPORTANCE The gut microbiome in early life plays an important role for long-term health and is shaped in large part by diet. Probiotics may contribute to improvements in health, but they have not been shown to alter the community composition of the gut microbiome. Here, we found that breastfed infants could be stably colonized at high levels by provision of B. infantis EVC001, with significant changes to the overall microbiome composition persisting more than a month later, whether the infants were born vaginally or by caesarean section. This observation is consistent with previous studies demonstrating the capacity of this subspecies to utilize human milk glycans as a nutrient and underscores the importance of pairing a probiotic organism with a specific substrate. Colonization by B. infantis EVC001 resulted in significant changes to fecal microbiome composition and was associated with improvements in fecal biochemistry. The combination of human milk and an infant-associated Bifidobacterium sp. shows, for the first time, that durable changes to the human gut microbiome are possible and are associated with improved gut function.

Abstract 33: Platelet 12-lipoxygenase is a Key Regulator of Platelet Reactivity and Thrombus Formation in vivo

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Reheman Adili ◽  
Katherine Mast ◽  
Theodore R Holman ◽  
Michael Holinstat
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Platelet Reactivity ◽  
Thrombus Formation ◽  
High Shear ◽  
Vessel Occlusion ◽  
12 Lipoxygenase ◽  
Induced Aggregation

Background: Platelet reactivity is required to maintain hemostasis, however high platelet reactivity leads to thrombus formation, myocardial infarction, and stroke. Platelet 12-lipoxygenase (12-LOX) has been demonstrated by our lab and others to regulate agonist-mediated platelet reactivity suggesting a role for 12-LOX in regulation of in vivo thrombosis. The ability to target 12-LOX in vivo has not been established to date. Therefore, we sought to determine if 12-LOX regulates platelet reactivity and thrombus formation in vivo using the selective 12-LOX inhibitor ML355 to determine whether platelet 12-LOX is an effective target for anti-platelet therapeutics. Methods: ML355 effects on human platelet function was assessed in vitro by platelet aggregometry, ex vivo by perfusion chamber, and in vivo by thrombus formation and vessel occlusion in small and large vessels in 12-LOX -/- , WT mice, and mice treated with ML355 via intravital microscopy using the FeCl 3 and laser injury models. Results: In in vitro platelet aggregation, ML355 dose-dependently inhibited agonist-induced aggregation. In ex vivo flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX -/- mice were impaired in both laser and FeCl 3 -induced mesenteric, carotid artery and cremaster arteriole thrombosis models. Thrombi in 12-LOX -/- mice were unstable and frequently formed emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice. Conclusions: The 12-LOX inhibitor ML355 inhibits platelet aggregation induced by a number of platelet agonists. Ex vivo high shear conditions in both mice and human was attenuated in the presence of ML355. Thrombus formation and vessel occlusion were impaired in mice deficient in 12-LOX. Finally, ML355 attenuates thrombus formation and prevents vessel occlusion in vivo . Our data strongly indicates 12-LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics.

scholarly journals Distinct Localization of Coagulation Factor VIII, Von Willebrand Factor and Factor VIIIa-Mimetic Bispecific Antibody Contributing to Thrombus Formation Under Whole Blood Flow Conditions

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1483-1483
Author(s):  
Yasuaki Shida ◽  
Keiji Nogami ◽  
Hiroaki Minami ◽  
Hiroaki Yaoi ◽  
Tomoko Matsumoto ◽  
...  
Keyword(s):  
Shear Flow ◽  
Research Funding ◽  
Hemophilia A ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
High Shear ◽  
Flow Conditions ◽  
Von Willebrand ◽  
Low Shear

Abstract Background Factor VIII (FVIII) is an essential factor for coagulation system in the intrinsic pathway. Due to the short survival of FVIII in the plasma circulation, it requires von Willebrand factor (VWF) as a carrier protein to maintain the optimal level for hemostasis. VWF also plays an important role in primary hemostasis by bridging platelets to exposed subendothelial collagens, especially under high shear flow environment. Since VWF carries FVIII, it is conceivable that VWF takes FVIII to the sites of vascular injury. However, the role of FVIII at the local sites under flow conditions is not fully understood despite of the fact that increased level of FVIII is associated with the risk of venous thrombosis and the deficiency of FVIII is the pathology of the bleeding disorder, hemophilia A. The treatment of hemophilia A largely depends on the infusion of FVIII concentrates, which is often complicated by the development of the inhibitor. Recently, bispecific antibody（ACE910）that mimics the role of FVIIIa by recognizing FIXa and FX has been developed and is currently under clinical trial. This antibody theoretically works regardless of the presence of devastating inhibitors against FVIII. Furthermore, it could also improve the clinical outcome of the other bleeding disorders, such as von Willebrand disease (VWD). Aim To analyze the role of FVIII and VWF, and impact of ACE910 at the sites of vascular injury under various shear conditions, we have developed the flow-mediated thrombosis model using flow chamber system. Method Whole blood obtained from healthy donors, hemophilia A and VWD patients were perfused into the collagen coated flow chamber under high (2,500s-1) or low shear (50s-1) flow conditions with/without FVIII concentrate, FVIII/VWF concentrate and ACE910. Formed thrombus was fixed and immunostaining was performed with phalloidin (Platelet), anti-FVIII antibody (FVIII) and anti-thrombin antibody (Thrombin). For the detection of ACE910, anti-human IgG or anti-ACE antibody (rAQ8 or rAJ540) were used. Size of thrombi and distribution of platelet, FVIII, thrombin and ACE910 were analyzed. Result 1) Under high shear flow, thrombus formation of VWD blood was significantly impaired while blood from Hemophilia A demonstrated nearly normal thrombus formation. Addition of FVIII/VWF but not FVIII concentrate to the blood of these patients rescued the impaired thrombus formation. ACE910 enhanced the thrombus formation of blood from both VWD and hemophilia A. Under low shear flow, blood from both hemophilia A and VWD demonstrated decreased thrombus formation. FVIII, FVIII/VWF concentrates and ACE910 improved the size of thrombus. 2) Localization of FVIII was evaluated with thrombin as a marker for the activation of coagulation. Platelets and thrombin demonstrated complete co-localization and intensity of thrombin staining was associated with thrombus size. VWF localized mainly outer layer of thrombus and FVIII localized in and around thrombus. At high shear condition, FVIII and VWF mostly existed with platelets. By contrast, FVIII and VWF demonstrated less co-localization with platelets under low shear condition. ACE910 demonstrated similar tendency to FVIII localization although ACE910 did not appear around thrombus. Conclusion We have developed the flow chamber system to evaluate the extent of thrombogenesis under various shear environment. VWF showed dominant role under high shear conditions while FVIII plays a key role under low shear conditions. FVIII, VWF and ACE910 demonstrated distinct localization. Interestingly, the distribution of FVIII was broader than VWF and platelet. FVIII localized to platelets presumably prior to its activation and contributed for the subsequent thrombin generation at local sites. Finally, ACE910 demonstrated consistent enhancement of thrombus formation of blood from both hemophilia A and VWD and, therefore, is prompted for the treatment of these bleeding disorders. Disclosures Shida: Chugai Pharmaceutical Co., Ltd.: Research Funding. Nogami:Chugai Pharmaceutical Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Minami:Chugai Pharmaceutical Co., Ltd.: Research Funding. Yaoi:Chugai Pharmaceutical Co., Ltd.: Research Funding. Matsumoto:Chugai Pharmaceutical Co., Ltd.: Research Funding. Kitazawa:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Hattori:Chugai Pharmaceutical Co., Ltd.: Employment, Equity Ownership, Patents & Royalties. Shima:Chugai Pharmaceutical Co., Ltd.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Global Thrombosis Test: Occlusion is attributable to shear-induced platelet thrombus formation.

TH Open ◽  
2021 ◽  
Author(s):  
Diana Adrienne Gorog ◽  
J. Yamamoto
Keyword(s):  
Platelet Aggregation ◽  
Experimental Approach ◽  
Thrombus Formation ◽  
Published Data ◽  
High Shear ◽  
Coagulation Time ◽  
P2y12 Inhibitors ◽  
Large Platelet ◽  
Platelet Thrombus ◽  
Platelet Aggregates

Herein, we set out a rebuttal to the publication by Claveria and co-workers published in TH Open this month entitled “Global Thrombosis Test: Occlusion by Coagulation or SIPA?” We strongly believe that the conclusions of their paper, suggesting that occlusion (OT) in the Global Thrombosis Test (GTT) is due to coagulation, rather than shear-induced platelet thrombus formation, is incorrect and the evidence and arguments they present are fundamentally flawed, with major errors both in the experimental approach and in the interpretations of the results. The evidence which they demonstrate, shows that occlusion in the GTT is, in fact, caused by high shear induced platelet thrombus formations. We set out herein the evidence for that, based on histology of the thrombus from the GTT in earlier work using electron microscopy showing large platelet aggregates, the very brief timescale of OT in the GTT compared to coagulation time and the sensitivity of the OT in the GTT to the effects of heparin, t-PA and P2Y12 inhibitors. In addition, we revisit the known pathomechanism of high shear-mediated platelet aggregation to underpin our rationale and show that the modifications to the instrument proposed by Claveria and co-authors would render the technique unphysiological. We highlight several methodological concerns and apparent misinterpreted of the data obtained. We present evidence predominantly from the authors’ own data, together with our earlier published data and evidence from the literature, showing that occlusion in the GTT occurs do to shear-induced platelet aggregation.

scholarly journals Aquaporin-1: a major modulator of aging-mediated platelet/endothelial dysfunction in atherothrombosis

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
S.S Saeedi Saravi ◽  
G.G Camici ◽  
T.F Luscher ◽  
J.H Beer
Keyword(s):  
Endothelial Cells ◽  
Platelet Aggregation ◽  
Endothelial Dysfunction ◽  
Shear Flow ◽  
Platelet Adhesion ◽  
Funding Source ◽  
Rolling Velocity ◽  
Caveolin 1 ◽  
Aquaporin 1 ◽  
Aqp1 Expression

Abstract Background Aging is associated with development of cardiovascular diseases, including atherothrombosis. Aquaporin-1 (AQP1) is a water channel that also transports hydrogen peroxide (H2O2). Regarding to the role of oxidative stress in atherothrombosis, we hypothesized that AQP1 modulates aging-associated platelet/endothelial dysfunction. Methods Human aortic endothelial cells (HAEC) from passages 5 (young) to 15 (senescent/old) were subjected to fluorescent immunocytochemistry to detect AQP1 protein expression and subcellular localization. The cells were also probed for AQP1 and the phospho/total proteins (AMPK, acetyl-coA-carboxylase (ACC), caveolin-1 and eNOS) for studying the signal transduction by immunoblotting. The endothelial cells were transfected with constructs containing H2O2 biosensor HyPer targeted to cell nucleus or cytosol followed by fluorescence imaging. The transcriptional levels of pro-inflammatory/pro-atherogenic vs. anti-inflammatory/atheroprotective genes in the cells were assessed by qRT-PCR. Human blood samples were taken and treated with or without AQP1 inhibitor (Bacopaside II, 10 μM) to examine the platelet adhesion and rolling velocity on vWF under high shear flow (100 dyn/cm2). Also, platelet aggregation in response to collagen (2 μM), ADP (1 μM) and TRAP (1 μM) were recorded. Results First, the senescence of HAEC was adjusted by a significant increase in β-galactosidase activity from passage 5 to 15. AQP1 immunofluorescence showed a remarkable increase in the young (P.5) compared to senescent (P.15) cells. Immunoblot analyses showed that aging leads to significant increases in AQP1 intensity and phosphorylation of caveolin-1 (Tyr14) and ACC (Ser79), along with decreases in phosphorylation of eNOS (Ser1177) and AMPK (Thr172) (p<0.01, n>6). Fluorescence imaging documented a robust H2O2 production in the senescent endothelial cell cytosol, but not nucleus, and activated TNF-α gene, whereas the transcription of hemoxygenase-1 gene enhanced in the young cells (p<0.01). AQP1 inhibition reduced platelet adhesion and thrombus formation, and elevated platelet rolling velocity on vWF under shear flow (p<0.01). Also, a decrease was found in platelet aggregation in response to AQP1 inhibition (p<0.05). Conclusion These studies, for the first time, demonstrate that aging induces AQP1 expression in endothelial cells and platelets, and modulates the dephosphorylation of AMPK/eNOS. These may lead to platelet/endothelial dysfunction and production of pro-coagulant/pro-inflammatory factors via ACC activation. Therefore, AQP1 inhibition could potentially be exploited as a therapeutic strategy for improving age-related atherothrombosis. Figure 1. Aging increases AQP1 expression in HAEC that leads to dephosphorylation of AMPK and eNOS. AQP inhibition also improves platelet function. *p<0.05, **p<0.01 compared to control. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF)

ALX-0081 Nanobody™, an Engineered Bivalent Anti-Thrombotic Drug Candidate with Improved Efficacy and Safety as Compared to the Marketed Drugs.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 896-896
Author(s):  
Karen Silence ◽  
Heidi Jonckheere ◽  
Peter Casteels ◽  
Jan Roodt ◽  
Muriel Meiring ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Half Life ◽  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Bleeding Complications ◽  
High Shear ◽  
Single Chain ◽  
A1 Domain ◽  
Low Shear

Abstract In patients with plaque rupture, platelets adhere, aggregate and form a thrombus. Current strategies to prevent thrombus formation consist of the use of Aspirin®, Plavix® and integrin αIIbβ3 blockers (e.g. Reopro®) in combination with Heparin®. These drugs are associated with high bleeding risk. Several in vivo experiments have shown that neutralizing the collagen von Willebrand Factor (vWF) platelet glycoprotein (GP)Ib-IX-V axis strongly inhibits arterial thrombosis without bleeding complications, therefore, these targets are of high interest to develop new anti-thrombotic drugs. Nanobodies are antibody-derived therapeutic proteins with the structural and functional properties of naturally occurring single-chain antibodies derived from camelids. ALX-0081 is a bivalent humanized Nanobody targeting the GPIb-IX-V binding site at the A1 domain of vWF. The precursor molecule was isolated from a llama immunized with the recombinant A1 domain of vWF and then humanized and engineered into a bivalent format to maximally benefit from the avid binding to vWF. In vitro, ALX-0081 can completely inhibit platelet adhesion to collagen at nanomolar concentrations. This inhibition is specific for the high shear rates relevant for coronary and carotid arteries whilst platelet adhesion and aggregation under low shear conditions is unaltered. The Nanobody also inhibits platelet adhesion to ultra large vWF (ULvWF) whilst it does not inhibit cleavage of ULvWF by ADAMTS-13. In a modified Folt’s model in baboons ALX-0081 inhibits thrombus formation more efficiently than a combination of Aspirin, Heparin and Plavix. Inhibition of thrombus formation is sustained in the presence of epinephrine and upon a new injury confirming the strong anti-thrombotic effect of ALX-0081. The Nanobody is effective at doses approximately 10–20 times lower than the dose required for Reopro. Ex vivo analysis of plasma samples after ALX-0081 administration in baboons in the ristocetin induced platelet aggregation (RIPA) assay reflects the efficacy seen in the Folt’s model. Therefore, this assay seems to be suited to predict effective ALX-0081 concentrations in vitro. In comparison to Reopro and Plavix, ALX-0081 is associated with less bleeding complications, even at doses exceeding the effective dose by a factor of 10 probably because of its selective inhibition of platelet aggregation under high shear but not under low shear conditions. After treatment with ALX-0081 no effect on other hematological parameters such as PT, aPTT, platelet count, VWF concentration and FVIII levels is seen and no immunogenicity is detected in baboons after repeated administration of ALX-0081. The terminal half-life of ALX-0081 in baboons is 8 hours, indicating that the molecule adopts the half-life of vWF. This high efficacy combined with an improved safety compared to the currently marketed drugs suggests that Ablynx’ drug development candidate ALX-0081 can become a powerful drug to treat acute thrombotic events in indications such as ACS, stroke, and TTP.

scholarly journals Inhibitory Effect of a Novel DNA Aptamer with Artificial Nucleic Bases for Von Willebrand Factor A1 Domain on Platelet Thrombosis Formation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2522-2522
Author(s):  
Kazuya Sakai ◽  
Hideo Yagi ◽  
Masaki Hayakawa ◽  
Tatsuhiko Someya ◽  
Kaori Harada ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Inhibitory Effect ◽  
Final Concentration ◽  
Dna Aptamer ◽  
High Shear ◽  
Inhibitory Effects ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract <Introduction> von Willebrand factor (VWF) is a blood glycoprotein that plays an important role in platelet thrombus formation through interaction with its A1 domain and platelet glycoprotein 1b. Thus, VWF A1 domain was thought to be a good therapeutic target candidate for VWF mediated thrombosis. In this study, we analyzed the inhibitory effects of a novel DNA aptamer (TAGX-0004) on platelet aggregation compared with another VWF A1 domain binding aptamer (ARC1779) which had entered to Phase II clinical trial for acquired thrombotic thrombocytopenic purpura (TTP) in 2011. <Methods> TAGX-0004 was generated by SELEX (systematic evolution of ligands by exponential enrichment) and consisted of artificial nucleic acid base, 7-(2-thienyl) imidazo [4,5-b] pyridine (Ds) as well as natural bases (Adenine, Thymine, Cytosine and Guanine). The dissociation constant (Kd) of the aptamers was analyzed by electrophoretic mobility shift assay (EMSA). 100 nM of each DNA aptamer was mixed with A1 domain of VWF protein (final concentration was 0 to 800 nM) in binding buffer (1x PBS, 0.005% NP-40) and incubated for 30 min at 37 °C. The samples were subjected to EMSA with 8% native PAGE, then stained by SYBR Gold. Kd value was determined with scatchard plot analysis. To characterize the binding sites of VWF A1 to these DNA aptamers, various mutants of VWF A1 domain were generated with an alanine substitution technique. Platelet aggregation was induced with ristocetin (RIPA), botrocetin (BIPA), collagen, epinephrine and adenosine diphosphate (ADP). The change of light transmission rate of platelet rich plasma (PRP) compared to platelet poor plasma (PPP) at 37 °C was recorded for 6 min, then the inhibition ratio of platelet aggregation was determined. Total thrombus-formation analysis system (T-TAS) (Fujimori Kogyo Co. Ltd., Tokyo, Japan), which is a novel micro-chip flow-chamber system, was employed to analyze thrombus formation visually and quantitatively in whole blood samples. The micro-chip coated with type 1 collagen was used for this analysis. Anti-VWF A1 inhibitory effects of these aptamers at high shear stress (initial rate of 2000 s-1) were calculated by continuous pressure levels within its narrow capillary. The course of thrombus formation was also optically recorded with a video-microscope located under the microchip. <Results> Biophysical interaction analysis using EMSA showed that TAGX-0004 had approximately 15-times higher binding activity to VWF A1 domain than ARC1779. Based on an alanine scan analysis, it was revealed that a couple of residues we tested were critical for binding of ARC1779 but not for TAGX-0004, which indicating that TAGX-0004 interacts with VWF A1 domain via different amino acid residue(s). Both aptamers did not inhibit the platelet aggregation induced by collagen, epinephrine or ADP. In RIPA, the 80% inhibition was observed by TAGX-0004 and ARC1779 at a final concentration of 10 nM and 750 nM, respectively. In BIPA, those were seen at those of 50 nM and 1000 nM, respectively. In T-TAS analysis, TAGX-0004 inhibited the thrombus formation completely at a final concentration of 100 nM, whereas ARC1779 exhibited partial inhibition of thrombosis formation even at 1000 nM. These results indicated that TAGX-0004 had stronger inhibitory effect on the platelet aggregation compared with ARC1779 under both static and high-shear conditions. <Conclusions> In the present study, we showed the affinity to VWF-A1 domain of TAGX-0004 was stronger than that of ARC1779. In addition, TAGX-0004 was more effective in suppressing platelet aggregation under both static and high-shear stress condition than ARC1779. In the published results of clinical study of ARC1779, there were no hemorrhagic complications in the small clinical trial of patients (7 patients of ARC1779 group) despite the almost complete suppression of VWF function in severely thrombocytopenic patients. Therefore, TAGX-0004 could be developed as a promising therapeutic agent for VWF mediated thrombotic disorders, such as acute coronary syndrome and TTP. Disclosures No relevant conflicts of interest to declare.

Anfibatide, a novel GPIb complex antagonist, inhibits platelet adhesion and thrombus formation in vitro and in vivo in murine models of thrombosis

2014 ◽  
Vol 111 (02) ◽  
pp. 279-289 ◽  
Author(s):  
Yan Hou ◽  
Hui Zhou ◽  
Yiming Wang ◽  
Alexandra Marshall ◽  
Chaofan Liang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Snake Venom ◽  
Platelet Adhesion ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
High Shear ◽  
Antithrombotic Agent ◽  
Vessel Occlusion ◽  
Induced Aggregation

SummaryPlatelet 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 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.

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