Abstract

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   Collection date 2018 Jul.
   © 2018 The Authors. Research and Practice in Thrombosis and Haemostasis
   published by Wiley Periodicals, Inc on behalf of International Society
   on Thrombosis and Haemostasis.

   This is an open access article under the terms of the
   [19]http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which
   permits use and distribution in any medium, provided the original work
   is properly cited, the use is non‐commercial and no modifications or
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   [20]PMC Copyright notice
   PMCID: PMC6032109

PLATELETS

PB001: Platelet Chemokine Receptor CXCR7 Mediates an Anti‐thrombotic and
Anti‐thromboinflammatory Effect

   M. Chatterjee^1; M. Manke^1; M. Cebo^2; J. Rheinlaender^3; A. Witte^1;
   T. Schäffer^3; M. Lämmerhofer^2; O. Borst^1; M. Gawaz^1

   ^1University Clinic of Tübingen, Department of Cardiology and
   Cardiovascular Diseases, Tübingen, Germany, ^2University of Tübingen,
   Institute of Pharmaceutical Sciences, Tübingen, Germany, ^3University
   of Tübingen, Institute of Applied Physics, Tübingen, Germany

   Background: Elevated platelet surface expression of the chemokine
   CXCL12 and its receptors CXCR4‐CXCR7 in acute coronary syndrome
   influences functional recovery and prognosis.

   Aims: This study explores the anti‐thrombotic potential of CXCR7 and
   its effects on thromboinflammation.

   Methods: Flowcytometry, thrombinoscopy, thromboelastography, bleeding
   time, flow chamber assay, intravital microscopy, mass spectrometry,
   live imaging by scanning ion conductance microscopy.

   Results: Deletion of CXCR7 from platelets in Pf4‐Cre ^+ CXCR7
   ^flox/flox mice led to enhanced thrombotic disposition without
   affecting haemostasis. The pharmacological CXCR7‐agonist, counteracted
   GPVI‐PAR1‐P2Y12 induced degranulation, αIIbβIII‐integrin activation,
   aggregation, ex vivo thrombus formation in healthy subjects, STEMI
   patients and mice, reduced the rate and extent of platelet interaction
   with collagen, fibrinogen, ROS generation, the release of
   thromboinflammatory arachidonic acid, thromboxane A2, 12‐HETE, HHT.
   CXCR7‐agonist countered activation induced externalisation of
   thrombogenic phosphatidylserine, thus checked PRP but not plasma
   dependent (PPP) thrombin generation, or efficiency of clot formation,
   stabilisation and dissolution. In vivo CXCR7‐agonist administration
   decreased platelet activation, thrombus formation following carotid
   artery injury and subsequent inflammatory platelet interaction with
   leukocytes, without affecting tail bleeding time and coagulation
   parameters (prothrombin time, activated partial thromboplastin time).
   CXCR7 ligation inhibited agonist‐induced intracellular calcium
   mobilisation, phosphorylation of PLC‐γ‐Src‐PKC‐PI3K‐Akt‐p38MAPK. It
   induced inhibitory mediators cGMP, stabilised cAMP and downstream
   kinase PKG‐PKA mediated VASP phosphorylation to regulate αIIbβIII
   activation which was counteracted by guanylyl cyclase, PKG and PKA
   inhibitors.

   Conclusions: CXCR7 can be utilised as an anti‐thrombotic drug target
   without compromising physiological haemostasis.

PB002: Ticagrelor Inhibits Platelet‐induced Formation of Neutrophil
Extracellular Traps

   I.C. Moschonas^1; K.M. Hansson^2; D. Stakos^3; A.D. Tselepis^1

   ^1University of Ioannina, Atherothrombosis Research Centre/Laboratory
   of Biochemistry, Department of Chemistry, Ioannina, Greece,
   ^2AstraZeneca, Cardiovascular and Metabolic Diseases, Innovative
   Medicines and Early Development Biotech Unit, Mölndal, Sweden,
   ^3Democritus University of Thrace, Cardiology Department,
   Alexandroupolis, Greece

   Background: Neutrophil extracellular traps (NETs) are filamentous
   structures consisting of decondensed chromatin and granular proteins
   that have antimicrobial properties and are also involved in
   atherothrombosis. Ticagrelor is a potent reversible antagonist of the
   platelet ADP receptor P2Y12.

   Aims: We investigated the possible role of platelet activation and the
   effect of ticagrelor on NETs formation, in vitro.

   Methods: Platelet‐rich plasma (PRP) was prepared from whole blood of
   healthy volunteers and adjusted to 2.5×10^8 platelets/mL. Autologous
   neutrophils were isolated by double‐Ficoll gradient, placed on
   poly‐l‐lysine coverslips and incubated for 1 hour at 37°C, 5% CO2. PRP
   was incubated with/without 1.25 μM ticagrelor for 5 minutes in an
   aggregometer under stirring and then activated with 20 μM ADP for
   5 minutes. The platelet suspension was then transferred to neutrophils
   at a physiological number ratio of platelets/neutrophils and incubated
   for 3.5 hours at 37°C, 5% CO2. In other experiments PRP was premixed
   with neutrophils and incubated with/without 1.25 μM ticagrelor for
   5 minutes before activation in situ with 50 μM ADP for 3.5 hours at
   37°C, 5% CO2. Cells were then fixed, permeabilized and stained with
   anti‐myeloperoxidase antibody and DAPI. The % NETs formation was
   evaluated by fluorescence microscopy.

   Results: PRP preactivated in the aggregometer increased NETosis by
   106±32% (P=0.03) whereas in situ platelet activation with ADP increased
   NETosis by 63±22% (P=0.011). Ticagrelor inhibited NETosis under both
   experimental conditions by 60±10% (P=0.028) and 77±15% (P=0.012),
   respectively. In the absence of platelets, neither ADP nor ticagrelor
   alone influenced NETosis.

   Conclusions: ADP‐activated platelets induce NETosis, which is
   significantly inhibited by ticagrelor. Given the antimicrobial
   properties of NETs and their implication in various pathophysiological
   conditions including inflammation and atherothrombosis, the above
   results may represent a novel effect of ticagrelor. The clinical
   significance of this effect remains to be established.

PB003: Differential Effects of PDE3 and ‐5 Inhibitors on Thrombus Formation
on Collagen versus Inflamed Endothelium

   D.M. Coenen^1; H.J. Albers^2,3; A.D. van derMeer^3; J.M.E.M. Cosemans^1

   ^1Maastricht University, Department of Biochemistry, Cardiovascular
   Research Institute Maastricht (CARIM), Maastricht, the Netherlands,
   ^2University of Twente, BIOS Lab‐on‐a‐Chip Group, MESA+ Institute for
   Nanotechnology, MIRA Institute for Biomedical Technology and Technical
   Medicine, Enschede, the Netherlands, ^3University of Twente, Applied
   Stem Cell Technologies Group, MIRA Institute for Biomedical Technology
   and Technical Medicine, Enschede, the Netherlands

   Background: Embolization of platelet‐thrombi formed after
   atherosclerotic plaque rupture may lead to acute myocardial infarcts
   and strokes. Despite antiplatelet treatment, the recurrence rate is ˜30
   percent, urging the need for optimized treatment. Our group showed that
   thrombi, formed ex vivo, remain active, secreting various mediators
   (Mastenbroek et al. ATVB 2015).

   Aims: This study aims to examine the effect of suppressing platelet
   secretion, by elevating cAMP and/or cGMP, on key functional thrombus
   activities.

   Methods: The effect of phosphodiesterase (PDE)3/5 inhibitors on
   platelet αIIbβ3 activation and α‐ and δ‐granule secretion was measured
   with flow cytometry. Microfluidics were used to study whole blood
   thrombus formation at 1000 s^−1 over a collagen type I + tissue factor
   surface or over a confluent monolayer of tumor necrosis factor‐α
   treated human umbilical vein endothelial cells.

   Results: Cilostazol (PDE3i, 5 μM) and tadalafil (PDE5i, 10 nM)
   significantly inhibited platelet αIIbβ3 activation of collagen‐related
   peptide‐treated washed platelets (P=0.005 and P=0.03), whereas platelet
   α‐ and δ‐granule secretion were not significantly affected (P>0.05). In
   whole blood, PDE3 (50 μM) or ‐5 (100 nM) inhibition resulted in smaller
   thrombi, although non‐significant. Cilostazol mainly affected the
   earlier phase of thrombus growth, while the effect of tadalafil was
   more pronounced at a later time point. Furthermore, PDE3 inhibition
   significantly increased time to fibrin (P=0.03 vs. P=0.17 with PDE5
   inhibition). Strikingly, a ten‐fold lower dose of cilostazol (5 μM)
   gave a strong decrease in size and compactness of platelet thrombi on
   inflamed endothelial cells.

   Conclusions: These data suggest that PDE3 and PDE5 are key enzymes in
   thrombus growth and stability, but not in initial platelet adhesion.
   The efficacy of PDE inhibition is enhanced by the vascular endothelium.
   Combination of PDE3/5 inhibition with current antiplatelet medication
   might be the key in the regulation of acute and prolonged thrombus
   activities.

PB005: Effects of Aspirin and Rivaroxaban Treatment on Murine Arterial Vessel
Wall Remodelling and Thrombus Activity

   M. Karel^1; T. Mastenbroek^1,2,3; M. Nagy^1; D. Coenen^1; W.
   Chaouyâ^1,4; A. Brouns^5; J. Debets^5; P. Leenders^5; H. van Essen^5;
   R. van Oerle^1; S. Heitmeier^6; H. Spronk^1; J. Heemskerk^1; M.
   Kuijpers^1; J. Cosemans^1

   ^1Maastricht University, Biochemistry, Maastricht, the Netherlands,
   ^2MERLN Institute for Technology‐Inspired Regenerative Medicine,
   Department of Complex Tissue Regeneration, Maastricht, the Netherlands,
   ^3Regenerative Medicine Crossing Borders (RegMed XB), Maastricht, the
   Netherlands, ^4Synapse BV, Maastricht, the Netherlands, ^5Maastricht
   University, Pharmacology & Toxicology, Maastricht, the Netherlands,
   ^6Bayer Pharma AG, Wuppertal, Germany

   Background: The COMPASS clinical study investigated the effect of
   combined therapy of ASA and 2.5 mg bid rivaroxaban on patients with
   coronary of peripheral artery disease. However little is known about
   the effects of this treatment on the vasculature.

   Aims: To study the effects of aspirin and/or rivaroxaban on thrombus
   activities and on injury‐induced vascular remodelling in mice.

   Methods: C57BL/6 mice (n=12/group) were either treated with vehicle,
   aspirin (5 mg aspirin/kg/d) and/or rivaroxaban (12 mg/g chow). Vascular
   injury was induced by temporary ligation of the carotid artery.
   Vascular stiffening was monitored for 2 weeks by non‐invasive
   ultrasound imaging. Two weeks after ligation, platelet‐leukocyte
   aggregates (PLA) and vascular changes were assessed. Whole blood
   thrombus formation and thrombus‐induced cleavage of dye‐quenched
   collagen was studied with microfluidics.

   Results: Treatment with aspirin with(out) rivaroxaban decreased the
   number of adhered platelets per PLA (P<0.05) in unstimulated blood and
   blood stimulated with stable ADP analogue or PAR4 peptide (flow
   cytometry). Importantly, treatment with aspirin with(out) rivaroxaban
   protected the arteries against ligation‐induced stiffening (P<0.05),
   while rivaroxaban alone showed a trend herein (P=0.08). Histological
   analysis indicated that ligation of the carotid artery resulted in
   local intima‐media thickening, which was reduced by treatment with
   aspirin or rivaroxaban (P<0.01). Treatment of mouse blood with aspirin
   and/or rivaroxaban did not alter the size of thrombi formed ex vivo.
   Yet, interestingly, thrombus‐induced collagenolytic activity ex vivo
   was reduced (P<0.05) upon treatment with aspirin or rivaroxaban.

   Conclusions: This study provides new insight into the vascular‐directed
   effects of aspirin and/or rivaroxaban treatment after thrombotic
   injury. Treatment with aspirin significantly antagonised, and
   rivaroxaban showed potential to inhibit, vascular stiffening,
   intima‐media thickening, PLA formation and thrombus‐collagenolytic
   activity.

PB006: Association of PEAR1 Gene Variant with Platelet Reactivity and
Clinical Outcomes in Patients Undergoing Stenting and Treated with Aspirin
and Clopidogrel

   K. Xu^1; N. Chan^2; F. Wang^1; L. Yang^1; H. Zhu^1,3; J. Li^1,4; J.
   Zhang^1; Y. Fan^1; J. Chen^5; L. Xu^6; L. Ying^1,7; X. Hu^1; S. Ye^1;
   C. Li^1

   ^1The First Affiliated Hospital of Nanjing Medical University,
   Department of Cardiology, Nanjing, China, ^2McMaster University,
   Department of Medicine, Hamilton, Canada, ^3The Second People's
   Hospital of Changzhou Affiliated to Nanjing Medical University,
   Changzhou, China, ^4Fuyang Fifth People's Hospital, Fuyang, China,
   ^5Maanshan People's Hospital, Department of Cardiology, Maanshan,
   China, ^6Sir Run Run Shaw Hospital, Nanjing Medical University,
   Nanjing, China, ^7The Affiliated Huai'an Hospital of Xuzhou Medical
   University and Huai'an Second People's Hospital, Department of
   Cardiology, Huai'an, China

   Background: Dual antiplatelet therapy (DAPT) with aspirin and
   clopidogrel is the standard therapy for patients undergoing coronary
   stenting. Genetic determinants of variable response to aspirin and
   clopidogrel are incompletely characterized. Polymorphisms in platelet
   endothelial aggregation receptor‐1 (PEAR1, rs12041331) were initially
   described as being important for platelet aggregation. The prevalence
   of PEAR1 A/A homozygotes, its associations with laboratory and clinical
   outcome in a Chinese population undergoing stenting and DAPT, and the
   mechanism explaining any association are unclear.

   Aims: To explore the prevalence of PEAR1 rs12041331 A/A homozygotes and
   its association with platelet aggregation and clinical outcome.

   Methods: We recruited 2007 consecutive patients undergoing stenting and
   DAPT. Arachidonic acid‐ and ADP‐induced platelet aggregation (PLAA and
   PLADP) were performed on the 5th day after the procedure. Genotyping
   was performed using an improved multiplex ligation detection reaction
   technique. All patients underwent a 30‐day follow‐up and data for MACE
   (cardiovascular death, nonfatal myocardial infarction or ischemic
   stroke) were collected. This study was approved by the local ethics
   committee and registered. Informed consent was obtained.

   Results: PLADP was significantly lower in AA‐ than non‐AA‐ genotypes
   (P<0.001) but not PLAA. Independent of CYP2C19*2 polymorphism, 30‐day
   MACEs occurred significantly more in AA‐ than in GG‐homozygotes or
   heterozygotes (adjusted HR, 3.13; 95% CI, 1.05‐9.31; P=0.041).

   FIGURE 1 Genotypes of PEAR1 (rs12041331) and platelet reactivity. ADP‐
   and AA‐induced platelet aggregation were showed in (A) and (B),
   respectively

   graphic file with name RTH2-2-1-g001.jpg

   FIGURE 2 Cumulative incidence of 30‐day MACEs in patients undergoing
   stenting and DAPT stratified by PEAR1 rs12041331 genotype

   graphic file with name RTH2-2-1-g002.jpg

   Conclusions: In the largest study to date, PEAR1 A/A homozygosity
   occurs in 14.8% of Chinese patients and is associated with a 3‐fold
   increase in MACE risk after stenting. PLAA was unaffected and PLADP was
   lowest in AA‐homozygotes. The discordant laboratory findings raise the
   possibility that PEAR1 polymorphism might impact clinical outcome
   through mechanisms independent or distal to the inhibition by aspirin
   or clopidogrel.

PB007: Beta‐1,4‐galactosyltransferase 2 c.909C>T Gene Variant Is Predictive
of On‐clopidogrel Platelet Reactivity

   T. Belleville‐Rolland^1,2; N. Pallet^3,4; A. Savalle^3; M. Lejeune^3;
   L. Mauge^1,5; S. Bertil^1; M.‐A. Loriot^3,4; P. Gaussem^1,2

   ^1AP‐HP, Hôpital Européen Georges Pompidou, Hematology, Paris, France,
   ^2Paris Descartes University, Inserm UMR‐S1140, Paris, France, ^3AP‐HP,
   Hôpital Européen Georges Pompidou, Clinical Chemistry, Paris, France,
   ^4Paris Descartes University, Inserm UMR‐S1147, Paris, France, ^5Paris
   Descartes University, Inserm UMR‐S970, Paris, France

   Background: CYP2C19*2 loss‐of function variant influences clopidogrel
   response but only accounts for a small part of the variability of
   platelet reactivity, suggesting the involvement of other gene variants.
   Recently, exome sequencing of coronary patients under dual antiplatelet
   therapy identified a variant of the gene encoding
   beta‐1,4‐galactosyltransferase 2 (B4GALT2), a platelet‐expressed
   galactosyltransferase. Carriers of the B4GALT2 c.909C>T variant had
   lower platelet reactivity, as assessed by the VerifyNow P2Y12 assay and
   light‐transmission aggregation, compared with non‐carriers, indicating
   that B4GALT2 could influence clopidogrel sensitivity.

   Aims: To determine if B4GALT2 c.909C>T influences clopidogrel
   pharmacodynamics as assessed by vasodilator‐stimulated phosphoprotein
   (VASP) assay and ADP‐induced platelet aggregation in a non‐selected
   cohort of patients.

   Methods: We retrospectively analyzed 353 patients under clopidogrel
   (mono or bi‐antiplatelet therapy) addressed to our center between
   January 2010 and January 2017 to undergo genetic and pharmacodynamic
   testing (ADP‐induced light‐transmission aggregation and VASP assay).
   All patients gave written informed consent.

   Results: Among the 353 patients screened, DNA was available for 174 of
   them: 28% were under clopidogrel monotherapy, and 72% under
   clopidogrel+aspirin. In individuals under dual antiplatelet therapy,
   B4GALT2 c.909C>T was associated with a significant lower ADP‐induced
   platelet aggregation. Surprisingly, it did not influence VASP assay.
   Otherwise, B4GALT2 c.909C>T and CYP2C9*2 were independent genetic
   predictors of on‐treatment platelet reactivity.

   Conclusions: Our findings confirm that B4GALT2 c.909C>T influences
   on‐treatment platelet reactivity in a cohort of non‐selected patients
   under clopidogrel‐based antiplatelet therapy, independently of the
   P2Y12 signaling pathway. B4GALT2 could influence platelet adhesion
   through posttranslational modification of integrins and/or other cell
   surface glycoproteins.

PB008: High On‐treatment Platelet Reactivity (HTPR) on Clopidogrel but Not on
Novel P2Y12 Antagonists in Patients with ACS Undergoing PCI – Single Centre
Study

   J. Staško^1; R. Šimonová^1; M. Samoš^1; J. Sokol^1; L. Stančiaková^1;
   I. Škorňová^1; T. Simurda^2; P. Kubisz^1

   ^1Comenius University in Bratislava, Jessenius Faculty of Medicine in
   Martin, University Hospital Martin, National Centre of Hemostasis and
   Thrombosis, Martin, Slovakia, ^2Comenius University in Bratislava,
   Jessenius Faculty of Medicine in Martin, National Centre of Hemostasis
   and Thrombosis, Martin, Slovakia

   Background: P2Y12 receptor antagonists together with acetylsalicylic
   acid (ASA) are used in treatment of patients with acute coronary
   syndrome (ACS) and secondary prophylaxis of thrombotic complications
   after percutaneous coronary intervention (PCI). High on‐treatment
   platelet reactivity (HTPR) during dual antiplatelet treatment (DAPT)
   represents a risk of thrombosis. On the other side low on‐treatment
   platelet reactivity (LTPR) can lead to bleeding.

   Aims: To monitor the on‐treatment platelet reactivity (PR) in patients
   with ST segment elevation myocardial infarction (STEMI), to find out
   prevalence of HTPR and LTPR, to reveal the prevalence of ischemic and
   bleeding episodes during DAPT and their relation to laboratory
   findings.

   Methods: 73 patients with acute STEMI after PCI and on DAPT were
   included in the prospective monocentric study. PR was assessed using
   both the VASP phosphorylation analysis and light transmittance
   aggregometry (LTA) in 3 intervals: T1 (time of PCI), T2 (1 day after
   PCI) and T3 (30 days after PCI). Patients were observed during a period
   of 1 month with the aim to reveal thrombotic and bleeding episodes.

   Results: High variability in PR was found in clopidogrel treated
   patients in T2 and T3. Significant difference in PR was found between
   clopidogrel and novel P2Y12 antagonists in T2 and T3, while no
   significant difference between PR in prasugrel and ticagrelor group was
   found. Prevalence of HTPR in clopidogrel treated group in T2 and T3
   exceeded 45%. HTPR in prasugrel/ticagrelor treated group was rare (5%).
   Stent thrombosis was diagnosed in 12% of clopidogrel treated patients
   (all with HTPR). In spite of high prevalence of LTPR in novel P2Y12
   antagonist treatment no bleeding episodes were found.

   Conclusions: Results confirm a high effectiveness of PR monitoring in
   patients with acute STEMI undergoing PCI on DAPT. VASP analysis has a
   high specificity to signal route of P2Y12 receptor of platelets thus it
   seems to be suitable for monitoring of response to its antagonists.

PB009: Detection of Anthocyanins Effects to Alleviating Thrombotic Risks in
Comparison to Aspirin

   A. Gaiz^1; A. Kundur^2; S. Mosawy^2; N. Colson^2; I. Singh^2

   ^1Almustansiriah University, National Center of Hematology, Baghdad,
   Iraq, ^2Griffith University/Menzies Health Institute of Queensland,
   Southport, Australia

   Background: Platelet hyperactivity has a significant role in initiating
   vascular thrombosis and subsequent cardiovascular disease (CVD).
   several studies demonstrate resistance to currently used antiplatelet
   therapies such as aspirin (AS). The antioxidant activity of
   polyphenolic compounds including anthocyanin (AC) has been shown to
   reduce platelet activity and reduce CVD morbidity.

   Aims: Comparing the antiplatelet effect of pure AC compounds with
   Aspirin.

   Methods: Healthy human subject (n=50) were recruited in this study and
   divided into two groups. Each participant consumed either 80 mg
   Aspirin/day or 320 mg of AC/day in the form of capsules for 28 days.
   Fasting blood samples were collected at baseline (before) and after the
   treatment period. Flow cytometry was used to assess platelet activation
   by measuring platelet surface marker expression of CD41a and
   P‐selectin. Platelet aggregation studies were performed by stimulating
   platelets with ADP, collagen or arachidonic acid. Haematological
   indices, lipid profile, glucose, uric acid, and high sensitivity CRP
   were assessed.

   Results: There was a significant suppressive effect of AC on the
   expression of P‐selectin. There was a substantial reduction of
   ADP‐stimulated platelet aggregation. On the other side, Aspirin
   significantly reduced collagen‐stimulated and arachidonic
   acid‐stimulated platelet aggregation assays, but it has an
   insignificant inhibitory effect on ADP‐stimulated platelet aggregation
   and expression of P‐selectin. There were trends of lowering impact of
   AC on total cholesterol and triglycerides, but they were not
   significant.

   Conclusions: The results show that AC may reduce platelet aggregation
   and activation as demonstrated by inhibition of ADP‐stimulated platelet
   aggregation and P‐selectin expression. These results provide greater
   insight into the effect of AC and the possible mechanism by which AC
   can reduce platelet function. Hence, AC may act as a complement to
   other anti‐platelet agents to reduce the occurrence of thrombotic
   events.

PB011: Assessment of Clopidogrel Response in Patients with Prior
Atherothrombotic Events

   Z. Kaya^1; P. Özen^1; A.P. Nas^1; H. Batur Çağlayan^2; G. Aydoğdu
   Taçoy^3; B. Nazlıel^2

   ^1Gazi University Medical Faculty, Pediatric Hematology Unit of the
   Department of Pediatrics, Ankara, Turkey, ^2Gazi University Medical
   Faculty, Department of Neurology, Ankara, Turkey, ^3Gazi University
   Medical Faculty, Department of Cardiology, Ankara, Turkey

   Background: Clopidogrel is a prophylactic antiplatelet drug that has
   been successful at preventing recurrences in patients with
   atherothrombotic events. Abnormal platelet count, high MPV, and PDW
   indicating platelet activation, are additional risk factors for
   thrombosis in patients with prior cardiac events.

   Aims: We retrospectively evaluated platelet tests and clopidogrel
   response for 80 patients with prior cardiovascular or cerebrovascular
   events.

   Methods: All patients had received 75 mg/day clopidogrel for at least
   7 days prior to platelet aggregation testing. Clopidogrel response was
   assessed by light transmission aggregometry (Chrono‐log Model 700)
   using ADP agonist. According to the manufacturer's description, high
   responders to the drug were defined as having platelet reactivity below
   49%, low responders between 50% and 61%, and non‐responders between 62%
   and 70%. Platelet count, MPV and PDW were measured as a part of each
   patient's complete blood count in a Sysmex^®, XN‐3000 automated
   hematology analyzer.

   Results: There were 29 patients with prior cardiovascular events
   (median age: 70 years) and 51 patients with prior cerebrovascular
   events (median age: 62 years). Of the cardiovascular patients, there
   were 12 (42%) clopidogrel high responders with ADP, 10 (34%) low
   responders and 7 (24%) non‐responders. Of the cerebrovascular patients,
   there were 28 (54%) clopidogrel high responders with ADP, 15 (30%) low
   responders and 8 (16%) non‐responders. Mean platelet count was
   significantly lower in the cardiovascular group than in the
   cerebrovascular group (210.680±73.830 μL vs. 280.530±65.860 μL,
   respectively) (P<0.05). Mean MPV and PDW were significantly higher in
   the cardiovascular group than in the cerebrovascular group (MPV
   10.9±0.8 /fL vs. 10.1±0.9 /fL; PDW 13.9±1.9 fL vs. 11.6±2.1 fL) (both
   P<0.05).

   Conclusions: Our findings suggest that clopidogrel dose adjustment is
   necessary to prevent recurrences in patients with previous
   cardiovascular events who are low responders clopidogrel.

PB013: Assessment of Platelet Reactivity with Multiple Electrode Impedance
Aggregometry (MEA) in Patients Reseiving Ticargelor

   D. Dineva; I. Paskaleva

   National Heart Hospital, Sofia, Bulgaria

   Background: Monitoring platelet reactivity during P2Y12‐inhibitors
   helps prevent stent thrombosis (ST) and/ or bleeding. High platelet
   reactivity (HPR) is associated with an increased risk for ischaemic
   complications, while low platelet reactivity (LPR) is associated with
   greater bleeding events.

   Aims: The purpose of this study was to evaluate the effect of
   ticagrelor on residual platelet reactivity.

   Methods: The platelet function was assessed with Multiplate impedance
   aggregometry by ADP‐test in 91 patients on 2×90 mg/d ticagrelor therapy
   who underwent PCI. Blood was collected at the first month after stent
   implantation. Adequate response to ADP receptor blocking was defined as
   ADP‐test <450 AU, on the base of cut‐off value, determined by ROC
   analysis (AUC 0.864 with 0.84 specificity and 0.78 sensitivity). Low
   platelet reactivity was defined as ADP‐test <150 AU.

   Results: A total of 91 patients were enrolled, 88 of them showed
   adequate response on ticagrelor treatment (ADP 203±89 AU). We observed
   residual HPR in three patients (ADP 635±78 AU) and they were switched
   on prasugrel 10 mg/d. Additional platelet inhibition of 52.8% was
   observed when switching them from ticagrelor to prasugrel (663 AU vs.
   313 AU).

   Minor bleeding complications like cutaneous hematoma, epistaxis,
   haemorrhoidal bleeding and hemoptoe were found in patients with lower
   values of ADP test (107±64 AU). Antiplatelet therapy in patients with
   bleeding was switched to clopidogrel 75 mg/d. Reduction of platelet
   inhibition and decreasing of the bleeding was seen when ticagrelor was
   switched to clopidogrel.

   Conclusions: We found impaired response to ticagrelor 2×90 mg in 3 of
   91 patients (3.29%). ADP‐test <150 AU was associated with increasing
   risk of minor bleeding. Tailored treatment with different P2Y12
   inhibitors leads to optimal level of ADP inhibition.

PB014: Results of Platelet Aggregation Testing by Light Transmission
Aggregometry using Drug Specific Agonists in Patients Taking Antiplatelet
Therapy

   S. Margetić^1; N. Vrkić^1,2; B. Getaldic^1; D. Ruzic Ferenec^1; I.
   Vuga^1

   ^1Sestre Milosrdnice University Hospital Center, Department of Clinical
   Chemistry, Zagreb, Croatia, ^2Faculty of Pharmacy and Biochemistry
   University of Zagreb, Zagreb, Croatia

   Background: In the recent years platelet aggregation testing has
   expanded in the assessment of the effectiveness of antiplatelet
   therapy.

   Aims: To evaluate results of platelet aggregation by light transmission
   aggregometry (LTA) using drug specific agonists in patients on
   antiplatelet therapy.

   Methods: Platelet aggregation testing was performed in 107 patients on
   antiplatelet therapy: ASA, ADP receptor inhibitor (clopidogrel or
   ticagrelor) or dual therapy (ASA plus ADP receptor inhibitor) as well
   as in 27 patients in whom antiplatelet therapy was discontinued from 2
   to 8 days due to surgery. Inhibition of platelet aggregation was
   assessed by ADP (2 μmol/L) agonist for ADP receptor inhibitor and by
   arachidonic acid (AA, 1 mmol/L) as agonist for acetylsalicylic acid
   (ASA) drug on Sysmex CS2500 analyzer using Hyphen BioMed (France)
   reagents.

   Results: Platelet aggregation with drug specific agonists was
   significantly inhibited in all patients, as shown in Table 1. Platelet
   inhibition with ADP was statistically different (P=0.003) between
   patients treated with clopidogrel and ticagrelor. However, there were
   no differences in AA or ADP inhibitions between patients treated with
   one or both drugs (Table 1). Among all patients on ASA and ADP receptor
   inhibitor alone and among those on dual therapy, only one patient was
   poor responsive to AA inhibition and no one was poor responsive to ADP
   inhibition. Among 27 patients with discontinued antiplatelet therapy,
   platelet function was recovered to the values above the limit of the
   reference range (60%) 6th day for AA and 7th day for ADP agonist after
   drug withdrawal.

   TABLE 1 Results of platelet aggregation testing using specific agonists
   by light transmission aggregometry (LTA) in patients on antiplatelet
   therapy
   Antiplatelet drug acetylsalicylic acid (ASA) 100 mg/day ADP inhibitor
   ASA + ADP inhibitor P ADP inhibitor clopidogrel 75 mg/day ADP inhibitor
   ticagrelor 90 mg/day P
   Agonist / N N=35 N=24 N=48 N=50 N=22
   Arachidonic acid (AA) % aggregation
    Median 12 — 10 0.342 — — —
    95% CI for median 6‐18 — 6‐14 — — —
    Interquartile range (IQR) 6‐19 — 11‐20 — — —
   Adenosine‐diphosphate (ADP) % aggregation
    Median — 29 30 0.539 32 22 0.003
    95% CI for median — 11‐40 24‐35 28‐40 9‐26 —
    Interquartile range (IQR) — 13‐39 22‐40 24‐41 12‐26 —
   [21]Open in a new tab

   Conclusions: Use of specific agonists for certain antiplatelet drugs
   contribute to the better understanding of platelet aggregation
   inhibition in patients taking antiplatelet therapy. Recovery time of
   platelet function of 6‐7 days after drug withdrawal could be explained
   by the fact that the function of inhibited enzyme or receptor recovers
   for about 10% a day as a result of the entry of new platelets in
   circulation.

PB015: Optimal Antiplatelet Therapy in Moderate to Severe Asymptomatic and
Symptomatic Atherosclerotic Carotid Stenosis: A Systematic Review of the
Literature

   S. Murphy^1; R. Naylor^2; J.‐B. Ricco^3; H. Sillesen^4; S. Kakkos^5; A.
   Halliday^6; G. de Borst^7; M. Vega de Ceniga^8; G. Hamilton^9; D.
   McCabe^10

   ^1Adelaide and Meath Hospital, Department of Neurology, Dublin,
   Ireland, ^2Glenfield Hospital, Department of Vascular Surgery,
   Leicester, United Kingdom, ^3University of Strasbourg, Department of
   Vascular Surgery, Strasbourg, France, ^4Rigshospitalet, University of
   Copenhagen, Department of Vascular Surgery, Copenhagen, Denmark,
   ^5University of Patras Medical School, Department of Vascular Surgery,
   Patras, Greece, ^6University of Oxford, Nuffield Department of Surgical
   Sciences, Oxford, United Kingdom, ^7University Medical Center Utrecht,
   Department of Vascular Surgery, Utrecht, the Netherlands, ^8Hospital de
   Galdakao‐Usansolo, Department of Angiology and Vascular Surgery,
   Bizkaia, Spain, ^9University College London Medical School, Department
   of Vascular Surgery, London, United Kingdom, ^10School of Medicine,
   Trinity College Dublin, Academic Unit of Neurology, Dublin, Ireland

   Background: Carotid stenosis patients are at risk of cerebrovascular
   events despite antiplatelet therapy. Data on prescribed antiplatelet
   regimens have not been comprehensively‐collated from trials to guide
   optimal therapy in this population.

   Aims: To perform a comprehensive systematic review of the literature on
   randomised trials of patients with extracranial moderate to severe
   carotid stenosis treated with antiplatelet therapy.

   Methods: We searched Medline, Ovid, Embase, Web of Science and Google
   Scholar from 01/1988 to 03/2016 for randomised trials in this patient
   population on any form of antiplatelet therapy in which cerebrovascular
   outcome events were reported.

   Results: 22 studies were deemed eligible for inclusion. Data from one
   RCT showed a non‐significant benefit from aspirin vs. placebo in
   asymptomatic carotid stenosis, but it is still reasonable to recommend
   aspirin (81‐325 mg daily) for prevention of vascular events in these
   patients. Low‐medium dose aspirin (81‐325 mg daily) is superior to
   higher doses (>650 mg daily) at preventing recurrent vascular events in
   patients undergoing endarterectomy. Data from endovascular treatment
   (EVT) trials support peri‐procedural treatment of asymptomatic and
   symptomatic patients with 81‐325 mg of aspirin daily. The use of
   peri‐procedural aspirin‐clopidogrel in patients undergoing EVT is based
   on one pilot trial, but appears safe. Short‐term treatment with
   aspirin‐dipyridamole or aspirin‐clopidogrel are equally effective at
   reducing micro‐embolic signals on transcranial Doppler ultrasound in
   patients with ≥50% symptomatic carotid stenosis. There is insufficient
   evidence to recommend routine aspirin‐clopidogrel combination therapy
   to reduce the risk of ‘recurrent clinical ischaemic events’ in patients
   with symptomatic moderate‐severe carotid stenosis.

   Conclusions: This systematic review facilitates an evidence‐based
   approach to ‘optimal antiplatelet therapy’ in carotid stenosis
   patients. Future trials should randomise such patients to receive
   different antiplatelet regimens.

PB016: The Original Algorithm for Platelet Functional and Molecular Genetic
Analysis to Optimize Antiplatelet Therapy in Outpatients with Peripheral
Artery Diseases

   O. Sirotkina^1,2; N. Surint^3; A. Topanova^1; T. Vavilova^1,3

   ^1Almazov National Medical Research Centre, Saint‐Petersburg, Russian
   Federation, ^2B.P. Konstantinov Petersburg Nuclear Physics Institute of
   National Research Centre «Kurchatov Institute», Saint‐Petersburg,
   Russian Federation, ^3First City Consultative and Diagnostic Center,
   Saint‐Petersburg, Russian Federation

   Background: Inhibition of platelet aggregation is the main approaches
   to treatment and prevention of CVD.

   Aims: The development of original algorithm for laboratory and
   molecular genetic analysis to optimize antiplatelet therapy in
   outpatients with peripheral artery disease (PAD) underwented
   reconstructive surgery.

   Methods: 105 patients (age 68±1) with PAD were included into the study.
   We analyzed the Leu33Pro GPIIIa, C807T GPIa, G36T P2Y12, CYP2C19*2,
   CYP2C19*3 and CYP3A5*3 polymorphism by PCR, ADP/collagen induced
   platelet aggregation by impedance method.

   Results: The distribution of appraised genotypes was not significantly
   different from previously explored healthy donors. The presence of 36T
   P2Y12 allele in homo or heterozygous state was a risk factor for high
   platelet reactivity in patients on standard antiplatelet therapy: 19
   (6‐32) in carriers GG and 83 (66‐91) in carriers GT/TT genotype
   (p=0.0006) for ADP induced aggregation and 34 (29‐48) and 63 (55‐74),
   respectively (P=0.003) for collagen induced aggregation. Moreover 16%
   of patients have polymorphism of CYP3A5 and CYP2C19 genes, associated
   with poor clopidogrel metabolism. Individual selection of antiplatelet
   therapy was carried out considering the results of complex of clinical
   data, platelet functional and molecular genetic tests. According to
   this approach all patients were divided into 5 groups with own regimen
   of antiplatelet therapy: ASA 100 mg or 150 mg, clopidogrel 75 mg,
   clopidogrel 75 mg+ASA 100 mg or 150 mg. After 3 months of individual
   antiplatelet therapy all patients showed a significant decrease in
   platelet reactivity from 58 (43‐75) to 28 (6‐58) (p=0.01) and from 54
   (41‐68) to 40 (25‐55) (P=0.05) for ADP and collagen induced aggregation
   respectively. We have achieved a good clinical result. No limb
   amputation was performed within 5 years of follow‐up.

   Conclusions: One of the ways to improve the long‐term results of
   reconstructive interventions in PAD might be the development of the
   algorithm for an integrated approach to individual antiplatelet
   therapy.

PB017: Impact of Time‐to‐treatment on Freedom‐from‐relapse for
Steroid‐Refractory Immune Thrombocytopenia

   P. Vishnu^1; W.A. Hammond^2; E.M. Rodriguez^3; Z. Li^1; C.E. Rivera^1

   ^1Mayo Clinic Florida, Jacksonville, United States, ^2Baptist MD
   Anderson Cancer Center, Jacksonville, United States, ^3University of
   Puerto Rico at Mayagüez, Puerto Rico, United States

   Background: Splenectomy (S) or rituximab (R) are potentially curative
   options for patients with steroid‐refractory ITP (sr‐ITP) with good
   responses, but the long‐term outcome is not known and the treatment
   order is uncertain.

   Aims: To evaluate freedom‐from‐relapse (FFR) after therapy with S or R
   for sr‐ITP and the impact of time from diagnosis to 2nd line treatment
   (TT) based on sequence of therapy, and the difference in efficacy of R
   in patients with or without a prior S.

   Methods: Patients age ≥18 years with sr‐ITP treated with S or R between
   Jan 2002 and Dec 2015 at Mayo Clinic who underwent S or R as 2nd line
   therapy. TT, FFR and response following 2nd‐ and 3rd‐line treatment
   with S or R were measured.

   Results: We identified 237 patients; 122 had S and 115 received R for
   sr‐ITP. Patients in S group were younger at diagnosis compare to R
   (median 49 v 60 years, P=0.001) and at time of 2nd‐line treatment
   (median, 53 v 61 years, P=0.007). Most patients required 2nd line
   therapy within 12 months of steroid refractoriness (65.4%). There were
   more patients with TT of ≥12 months in S group compared to R group
   (42.6 v. 26%; P=0.0075). Patients in S group had a higher response
   (87.7% v 43.5%, P<0.001) and a higher 5‐year FFR (53.25% v 18.9%,
   P<0.001) compared to R. Patients treated with R after failure of S
   achieved similar response (88.5% vs. 82.8%, P=0.39) and 2‐y FFR (67.31%
   vs. 58.13%, P=0.68) compared to those who had S for relapsed ITP after
   R. The 5‐year FFR after R was significantly longer in patients with a
   prior S than in those with an intact spleen (43.51% vs. 18.70%,
   P<0.001). Multivariate model predicting FFR following 2nd line therapy
   was in favor of splenectomy (HR 0.33, 95% CI 0.22, 0.50; P<0.001).

   Conclusions: Sequence of S before R has a clinical benefit for
   treatment of sr‐ITP. Use of R following failure of S appears to be as
   effective as S following failure to R. Although the impact of time to
   2nd line treatment was significant, it is likely that the treatment
   type was the impetus for the different outcomes.

PB018: Guidance on the Diagnosis and Management of PT‐VWD: A Project from the
Platelet Physiology Subcommittee of the ISTH

   M. Othman^1,2; P. Gresele^3

   ^1Queen's University, Biomedical and Molecular Sciences, Kingston,
   Canada, ^2St Lawrence College, School of Baccalaureate Nursing,
   Kingston, Canada, ^3University of Perugia, Department of Medicine ‐
   Section of Internal and Cardiovascular Medicine, Perugia, Italy

   Background: Only 55 patients with PT‐VWD have been reported worldwide.
   Literature is scarce in providing information required to establish
   standards for diagnosis and management of the disease. Currently there
   are no clear guidelines/consensus and many patients remain undiagnosed
   or undertreated.

   Aims: To generate guidance for a standard diagnostic and management
   approach for PT‐VWD based on consensus expert views.

   Methods: RAND based approach to obtain a formal consensus among
   experts. A panel formed of 11 experts (9 countries, 5 continents) was
   formulated. A series of 46 survey statements on history and clinical
   presentation (14), diagnosis (21) and management (11) of PT‐VWD were
   prepared for the experts to review. Statements were rated by the
   experts from 1‐9 where 1 is completely inappropriate and 9 is fully
   appropriate. Statements were classified as inappropriate (scores of
   1‐3), uncertain (scores of 4‐6), or appropriate (scores of 7‐9).

   Results: Experts have agreed largely on most statements related to
   basic history, clinical features and methodology for diagnosis and
   management approaches. Of these are: common bleeding symptoms and
   severity, family history, GP1BA gene mutation and using RIPA mixing
   studies as essential lab tests with some disagreement on ristocetin
   cutoff. Few statements were judged uncertain with respect to common
   bleeding symptoms in children, the association with cancer,
   inflammation and bone pathologies indicating research is required in
   these areas. Some uncertainty/ambiguity remained around the use of
   DDAVP, rFVIIa, or appropriate perinatal monitoring. These ambiguous
   statements will be resolved in a second survey. Large agreement was
   made towards a proposed algorithm for diagnosis and management of the
   disease.

   Conclusions: A consensus around a standard diagnostic and management
   approach for PT‐VWD can be established and would be critical to
   improving the diagnosis and treatment of this bleeding disorder.

   TABLE 1
   Acknowledgement of RAND Survey Participants‐ Alphabetical Order