Subsequent Platelet Aggregation Research Articles

A-154 Cost-effectiveness of On-site Vs Send-out Measurement of ADAMTS13 Activity in the Management of Acquired Thrombotic Thrombocytopenic Purpura

Abstract Background Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy (TMA) caused by congenital or acquired severe deficiency of disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS-13), an enzyme cleaving von Willebrand factor (VWF). ADAMTS-13 deficiency leads to the accumulation of ultra-large VWF multimers, and subsequent platelet aggregation and microthrombi formation. Aside clinical symptoms, the diagnosis of TTP is based thrombocytopenia, hemolytic anemia with the presence of schistocytes, and severely reduced activity of ADAMTS-13 (<10%). As TTP is a life-threatening condition, it is of great importance to shorten the time to the treatment, and according to the French recommendations, the triplet therapy including plasma exchange (PEX), rituximab, caplacizumab, plus corticosteroids, must be administered to patients with suspected TTP until the results of ADAMTS-13 was obtained, and be pursued only in the case of deficiency. Therefore, a rapid ADAMTS-13 testing is critical for early diagnosis and optimal management of acute TTP. Methods We compared the cost-effectiveness of a strategy based on on-site measurement of ADAMTS-13 activity using a rapid fully automated chemiluminescent assay (HemosIL AcuStar ADAMTS-13) vs its centralized measurement in a reference laboratory, as it is currently the case at our institution. For that purpose, we created two scenarios based on either its local measurement with a maximum time-to-result of 16 h (assay performed during daytime, 7/7) or its centralized measurement with a median time-to-result of 4 days (range: 2–8, as demonstrated from the 2018–2021 estimates). The triplet therapy was hypothesized to be started on admission and stopped if ADAMTS-13 activity was >10%. In case of ADAMTS-13 activity <10%, an inhibitor screening would be performed at the reference laboratory, as this assay was not expected to be locally implemented. For the calculation, we considered the costs of reagent, shipping/handling of samples to the reference laboratory, laboratory staff workload, reimbursement rates, and treatments administered in patients with suspected TTP until availability of the ADAMTS-13 result. Results Based on 2018 to 2021 estimates, there was a mean of 60 prescriptions of ADAMTS-13 activity per year. Most were for the follow-up of TTP patients and for patients with non-TTP TMA. Only seven were for the diagnosis of acute TTP, as suggested by the French score, which was confirmed in four cases (mean values). The local measurement of ADAMTS-13 activity was found to be cost-effective with a 16%-reduction of the costs compared to its centralized measurement in those seven patients with suspected TTP during the first 4 days. Conclusion Together with its short turnaround time (33 min) and its full automation, our results suggest that the HemosIL AcuStar ADAMTS-13 activity assay could be an assay of choice to rapidly measure ADAMTS-13 activity in the plasma and thus to establish the diagnosis of acute TTP in emergency settings. Considering the local costs, using the strategy based on the local evaluation of ADAMTS-13 activity led to a 16%-reduction of the overall costs during the first 4 days after admission of the seven patients with suspected acute TTP.

The role of CYP2C19 genotyping to guide antiplatelet therapy following ischemic stroke or transient ischemic attack

ABSTRACT Introduction Clopidogrel is an antiplatelet agent recommended for secondary prevention of ischemic stroke (IS) and transient ischemic attack (TIA). Conversion of clopidogrel to its active metabolite by hepatic cytochrome P450-2C19 (CYP2C19) is essential for the inhibition of the P2Y12 receptor and subsequent platelet aggregation to prevent thrombotic events. CYP2C19 is highly polymorphic, with over 30 loss of function (LoF) alleles. This review considers whether there is sufficient data to support genotype guided antiplatelet therapy after stroke. Areas covered A systematic literature review retrieved articles, which describe the interaction between CYP2C19 genotype and clinical outcomes following IS or TIA when treated with clopidogrel. The review documents efforts to identify optimal antiplatelet regimens and explores the value genotype guided antiplatelet therapy. The work outlines the contemporary understanding of clopidogrel metabolism and appraises evidence linking CYP2C19 LoF variants with attenuated platelet inhibition and poorer outcomes. Expert opinion There is good evidence that CYP2C19 LoF allele carriers of Han-Chinese ancestry have increased risk for further vascular events following TIA or IS when treated with clopidogrel. The evidence base is less certain in other populations. The expansion of pharmacogenetics into routine clinical practice will facilitate further research and help tailor other aspects of secondary prevention.

Antibodies against Platelet Factor 4 and Their Associated Pathologies: From HIT/HITT to Spontaneous HIT-Like Syndrome, to COVID-19, to VITT/TTS.

Antibodies against platelet factor 4 (PF4), a protein released from alpha-granules of activated platelets, may cause a number of pathophysiological conditions. The most commonly known is heparin-induced thrombocytopenia (HIT), which develops in a small proportion of people treated with the anticoagulant drug heparin. Notably, PF4 binds with high affinity to heparin, and in HIT, complexes of PF4/H may, in a small proportion of susceptible patients, trigger the development of anti-PF4 antibodies and subsequent platelet activation and aggregation, ultimately leading to the development of pathological thrombosis at sites of vessel occlusion. Of more modern interest, antibodies against PF4 may also arise in patients with COVID-19 (Coronavirus Disease 2019) or in patients who have been vaccinated against COVID-19, especially in recipients of adenovirus-based vaccines. For this latter group of patients, the terms VITT (vaccine-induced [immune] thrombotic thrombocytopenia) and TTS (thrombotic thrombocytopenia syndrome) have been coined. Another category associated with this pathophysiology comprises those in whom a precipitating event is not clear; this category is referred to as ‘spontaneous HIT-like syndrome’. Despite its name, it arises as an HIT-mimicking disorder but without antecedent heparin exposure. In this narrative review, we describe the development of antibodies against PF4, and associated pathophysiology, in such conditions.

Effects of electronic cigarette flavorants on human platelet aggregation ex vivo

Because little is known about the effects of individual flavorants in electronic cigarette (e-cig) fluids on human platelet aggregation, we tested for the direct effects of 15 common e-cig flavorants on adenosine diphosphate (ADP)-induced human platelet aggregation ex vivo. To better understand a potential mechanism of action of flavorants, we quantified 2 phases of aggregation. Human platelet-rich plasma (PRP) was obtained from whole blood of healthy volunteers and used in a platelet aggregometry assay. PRP was incubated with 1 of 15 different flavorant compounds (e.g., benzyl alcohol, eugenol, citronellol, menthol, menthone, diacetyl, maltol, limonene, methylbutyric acid, isoamyl acetate, acetylpyridine, eucalyptol, 2,5-dimethylpyrazine, cinnamaldehyde, and vanillin) at 100 µM for 5 min at 37 °C prior to addition of ADP (10 µM). Subsequent ADP-induced platelet aggregation was tracked for 5 min using an aggregometer. Aggregation curves were analyzed for flavorant-induced effects on total (%) aggregation, Phase 1 and Phase 2 components, and compared with their ADP-only control via One-Way ANOVA. Notably, eugenol significantly inhibited total aggregation; an effect due solely to inhibition of Phase 2. No other flavor tested had any effect on total or phase-specific ADP-induced platelet aggregation. These results indicate that parent flavorant compounds commonly found in e-cig liquids neither activate nor inhibit ADP-induced human platelet aggregation. However, as flavorants are chemically altered during heating of e-cig, thermally-derived products of flavorants (e.g., flavor acetals) also will need to be tested for effects on platelet activation.

Resveratrol inhibits Ca2+ signals and aggregation of platelets

BackgroundResveratrol has been shown to inhibit platelet aggregation. However, the mechanism for this action of resveratrol remains to be clarified. The purpose of this study was to elucidate the Ca2+-related mechanism for the inhibitory action of resveratrol on platelet aggregation.MethodsCa2+ entry and subsequent aggregation of human platelets induced by different stimulants including thrombin, thapsigargin, and 1-oleoyl-2-acetylglycerol (OAG) were measured by the fluorescence method and light transmittance method, respectively. Each stimulant was added to a nominally Ca2+-free medium containing platelets, and then CaCl2 was added to the medium to induce Ca2+ influx into platelets.ResultsThapsigargin-induced Ca2+ entry into platelets and subsequent platelet aggregation were significantly inhibited in the presence of resveratrol at 6.25 μM or higher concentrations, while OAG-induced Ca2+ entry and subsequent platelet aggregation were not affected by resveratrol at concentrations up to 50 μM. In the nominally Ca2+-free medium, thrombin induced a small transient increase in intracellular Ca2+ concentrations, which was attenuated in the presence of resveratrol at 12.5 μM or higher concentrations. Thrombin-induced Ca2+ entry into platelets and subsequent platelet aggregation were significantly inhibited in the presence of resveratrol at 12.5 μM or higher concentrations.ConclusionsThe results suggest that resveratrol inhibits thrombin-induced platelet aggregation through decreasing Ca2+ release from its stores and inhibiting store-operated Ca2+ influx into platelets.

It's About Time: Transfusion effects on postinjury platelet aggregation over time.

Impaired postinjury platelet aggregation is common, but the effect of transfusion on this remains unclear. Data suggest that following injury platelet transfusion may not correct impaired platelet aggregation, and impaired platelet aggregation may not predict the need for platelet transfusion. We sought to further investigate platelet aggregation responses to transfusions, using regression statistics to isolate the independent effects of transfusions given in discrete time intervals from injury on both immediate and longitudinal platelet aggregation. We hypothesized that platelet aggregation response to platelet transfusion increases over time from injury. Serial (0-96 hours) blood samples were collected from 248 trauma patients. Platelet aggregation was assessed in vitro with impedance aggregometry stimulated by adenosine diphosphate, collagen, and thrombin receptor-activating peptide-6. Using regression, transfusion exposure was modeled against platelet aggregation at each subsequent timepoint and adjusted for confounders (Injury Severity Score, international normalized ratio (INR), base deficit, platelet count, and interval transfusions). The expected change in platelet aggregation at each timepoint under the intervention of transfusion exposure was calculated and compared with the observed platelet aggregation. The 248 patients analyzed were severely injured (Injury Severity Score, 21 ± 19), with normal platelet counts (mean, 268 × 10/L ± 90), and 62% were transfused in 24 hours. The independent effect of transfusions on subsequent platelet aggregation over time was modeled with observed platelet aggregation under hypothetical treatment of one unit transfusion of blood, plasma, or platelets. Platelet transfusions had increasing expected effects on subsequent platelet aggregation over time, with the maximal expected effect occurring late (4-5 days from injury). Controversy exists on whether transfusions improve impaired postinjury platelet aggregation. Using regression modeling, we identified that expected transfusion effects on subsequent platelet aggregation are maximal with platelet transfusion given late after injury. This is critical for tailored resuscitation, identifying a potential early period of resistance to platelet transfusion that resolves by 96 hours. Therapeutic, level V.

Two novel, putative mechanisms of action for citalopram-induced platelet inhibition

Citalopram, a selective serotonin reuptake inhibitor (SSRI), inhibits platelet function in vitro. We have previously shown that this action is independent of citalopram’s ability to block serotonin uptake by the serotonin transporter and must therefore be mediated via distinct pharmacological mechanisms. We now report evidence for two novel and putative mechanisms of citalopram-induced platelet inhibition. Firstly, in platelets, citalopram blocked U46619-induced Rap1 activation and subsequent platelet aggregation, but failed to inhibit U46619-induced increases in cytosolic Ca2+. Similarly, in neutrophils, citalopram inhibited Rap1 activation and downstream functions but failed to block PAF-induced Ca2+ mobilisation. In a cell-free system, citalopram also reduced CalDAG-GEFI-mediated nucleotide exchange on Rap1B. Secondly, the binding of anti-GPVI antibodies to resting platelets was inhibited by citalopram. Furthermore, citalopram-induced inhibition of GPVI-mediated platelet aggregation was instantaneous, reversible and displayed competitive characteristics, suggesting that these effects were not caused by a reduction in GPVI surface expression, but by simple competitive binding. In conclusion, we propose two novel, putative and distinct inhibitory mechanisms of action for citalopram: (1) inhibition of CalDAG-GEFI/Rap1 signalling, and (2) competitive antagonism of GPVI in platelets. These findings may aid in the development of novel inhibitors of CalDAG-GEFI/Rap1-dependent nucleotide exchange and novel GPVI antagonists.

Impact of shear stress on Src and focal adhesion kinase phosphorylation in fibrinogen-adherent platelets

: Shear stress alone can activate platelets resulting in a subsequent platelet aggregation, so-called 'shear-induced platelet aggregation'. In our work, we analyzed how differently elevated shear stress impacts the Src and focal adhesion kinase (FAK) activation in fibrinogen-adherent human platelets. We detected the extents of Src pY418 and FAK pY397 activations in platelets on immobilized fibrinogen and over BSA under shear conditions. Moreover, we analyzed the role of αIIbβ3 in the shear-induced platelet signaling by performing our experiments in the presence of the αIIbβ3-antagonist Abciximab. Abnormally high shear rates (5000 s) significantly increased the extent of phosphorylation of both tyrosine kinases after short (2 min) incubation time independently of the presence or absence of the integrin αIIbβ3 ligand, fibrinogen. We could see considerably greater Src activation on immobilized fibrinogen than on BSA, but the extent of FAK Y397 phosphorylation was independent on the matrix. Abciximab not only reduced the Src and FAK signaling in platelets exposed to 5000 s on immobilized fibrinogen, but in platelets exposed to 5000 s over BSA as well. Our data indicate that whereas Src activation under shear stress is dominantly ligand-dependent, FAK signaling seems to be mostly shear induced.

UEG Week 2016 Poster Presentations

Contact E-mail Address: nakuraokura0922@yahoo.co.jp.IntroductionSinusoidal obstruction syndrome (SOS) is a drug-induced liver injury caused by anticancer treatments, such as oxaliplatin-based chemotherapy. Injury of sinusoidal endothelial cells (SECs) and subsequent extravasated platelet aggregation (EPA) initiate development of SOS. Beraprost sodium (BPS) has a protective effect on SECs and antiplatelet effect. The aim of this study was to examine the protective effect of BPS against SECs injury resulting from monocrotaline (MCT)-induced SOS in mice.Aims & MethodsSOS was induced in Crl:CD1 (ICR) mice by intraperitoneal administration of MCT (270 mg/kg). To evaluate the effect of BPS on SOS, the mice were divided into two groups. In the BPS group, BPS (200 μl/kg) was injected by intraperitoneal administration at 1 hour before, 3 hours after, and 9 hours after MCT administration. The control group received intraperitoneal injections of the same volume of saline at the same time points. All mice were sacrificed at 48 hours after MCT administration and the protective effect on SECs was assessed by determining blood cell count, serum biochemical findings and immunohistochemical stains of sinusoidal endothelial cell antibody (SEC-1) and CD62P (P-selectin). Western blot analysis and real-time polymerase chain reaction (RT-PCR) were also used to examine plasminogen activator inhibitor-1 (PAI-1) and endothelial nitric oxide synthase (eNOS) expression.ResultsPlatelet count was maintained in the BPS group compared to that in the MCT group (48.2 vs. 4.8 ´104/μl, P < 0.001). Serum transaminase levels and hyaluronic acid (HA) in the BPS group were significantly reduced compared to the levels in the MCT group (AST 129 vs. 1013 IU/L, P < 0.05; ALT 90 vs. 923 IU/L, P < 0.01; HA 670 vs. 2243 ng/mL, P < 0.01). In the MCT group, liver histology showed endothelial damage of the central venule, congestion and obstruction of the sinusoids, and necrosis of hepatocytes in zone 3. In the BPS group, these findings were suppressed. Immunohistochemistry showed a protective effect of SECs on SEC-1 and a decrease in platelet infiltration in the space of Disse on CD62P in the BPS group compared to that in the MCT group. Expression of PAI-1 was significantly lower and expression of eNOS was significantly higher in the BPS group.ConclusionBPS preserved SECs and suppressed the progression of MCT-induced SOS in mice, suggesting that BPS is useful for the prevention of SOS.Disclosure of InterestAll authors have declared no conflicts of interest.

Development of a physiology-directed population pharmacokinetic and pharmacodynamic model for characterizing the impact of genetic and demographic factors on clopidogrel response in healthy adults

Clopidogrel (Plavix®), is a widely used antiplatelet agent, which shows high inter-individual variability in treatment response in patients following the standard dosing regimen. In this study, a physiology-directed population pharmacokinetic/pharmacodynamic (PK/PD) model was developed based on clopidogrel and clopidogrel active metabolite (clop-AM) data from the PAPI and the PGXB2B studies using a step-wise approach in NONMEM (version 7.2). The developed model characterized the in vivo disposition of clopidogrel, its bioactivation into clop-AM in the liver and subsequent platelet aggregation inhibition in the systemic circulation reasonably well. It further allowed the identification of covariates that significantly impact clopidogrel's dose-concentration-response relationship. In particular, CYP2C19 intermediate and poor metabolizers converted 26.2% and 39.5% less clopidogrel to clop-AM, respectively, compared to extensive metabolizers. In addition, CES1 G143E mutation carriers have a reduced CES1 activity (82.9%) compared to wild-type subjects, which results in a significant increase in clop-AM formation. An increase in BMI was found to significantly decrease clopidogrel's bioactivation, whereas increased age was associated with increased platelet reactivity. Our PK/PD model analysis suggests that, in order to optimize clopidogrel dosing on a patient-by-patient basis, all of these factors have to be considered simultaneously, e.g. by using quantitative clinical pharmacology tools.

HMGB1: a novel protein that induced platelets active and aggregation via Toll-like receptor-4, NF-κB and cGMP dependent mechanisms.

BackgroundThrombotic diseases are a group of prevalent and life-threatening diseases. Selective inhibition of pathological thrombosis holds the key to treat variety of thrombotic diseases. The pathological thrombosis can be induced by either tissue necrosis and deregulated inflammation. HMGB1, as an important proinflammatory cytokine and a late mediator, also involves on thrombosis disease. However, the underlying mechanisms are not fully understood.MethodsImmunofluorescence, ELISA assay, Platelet Aggregation, Thromboelastogram (TEG) analyzes. Flow cytometric analysis and Western blot analysis were used to investigated the role of HMGB1 in platelet aggregation and obtained following observations.ResultsBy doing so, we obtained the following observations: i) Highly purified HMGB1 recombinant protein induces platelet aggregation and secretion in a dose-dependent manner in the presence of serum. ii) Low concentration of extracellular HMGB1 could synergistically promote subthreshold concentration of collagen or thrombin induced platelet aggregation. iii) Extracellular HMGB1 promoted platelet aggregation in a platelet-expressed GPIIb/IIIa-dependent manner. iv) We proposed that extracellular HMGB1 seems to promote the phosphorylation of GPIIb/IIIa and subsequent platelet aggregation via TLR4/NF-κB and cGMP pathway.ConclusionsIn this study, we provide evidence for the hypothesis that HMGB1 interact with platelet might play an important role in the haemostasis and thrombotic diseases. Our research might be provide an interesting avenue for the treatment of thrombotic diseases in the future.

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Photothrombotic Ischemia: A Minimally Invasive and Reproducible Photochemical Cortical Lesion Model for Mouse Stroke Studies

The photothrombotic stroke model aims to induce an ischemic damage within a given cortical area by means of photo-activation of a previously injected light-sensitive dye. Following illumination, the dye is activated and produces singlet oxygen that damages components of endothelial cell membranes, with subsequent platelet aggregation and thrombi formation, which eventually determines the interruption of local blood flow. This approach, initially proposed by Rosenblum and El-Sabban in 1977, was later improved by Watson in 1985 in rat brain and set the basis of the current model. Also, the increased availability of transgenic mouse lines further contributed to raise the interest on the photothrombosis model. Briefly, a photosensitive dye (Rose Bengal) is injected intraperitoneally and enters the blood stream. When illuminated by a cold light source, the dye becomes activated and induces endothelial damage with platelet activation and thrombosis, resulting in local blood flow interruption. The light source can be applied on the intact skull with no need of craniotomy, which allows targeting of any cortical area of interest in a reproducible and non-invasive way. The mouse is then sutured and allowed to wake up. The evaluation of ischemic damage can be quickly accomplished by triphenyl-tetrazolium chloride or cresyl violet staining. This technique produces infarction of small size and well-delimited boundaries, which is highly advantageous for precise cell characterization or functional studies. Furthermore, it is particularly suitable for studying cellular and molecular responses underlying brain plasticity in transgenic mice.

Abstract 27: Dok-1 Negatively Regulates Integrin aIIbß3 Outside-in Signaling and Inhibits Thrombosis

Background Platelet agonists activate integrin αIIbβ3 to allow the binding of soluble fibrinogen (a process known as inside-out signaling). Subsequent platelet aggregation leads to αIIbβ3 outside-in signaling, which results in tyrosine phosphorylation of β3 and other proteins, cytoskeletal reorganization, and platelet spreading. It has been reported that the adapter protein Dok-1 binds to the cytoplasmic tail of β3 and inhibits αIIbβ3 activation, but the specific role of Dok-1 in regulating inside-out or outside-in platelet signaling remains undefined. Methods We assessed the effects of Dok-1 on platelet signaling and thrombosis in Dok-1 null (Dok-1-/-) mice. Inside-out signaling was assessed by measuring αIIbβ3 activation (using the JON/A antibody) and fibrinogen binding by flow cytometry after stimulation of platelets with thrombin, ADP, and/or the thromboxane A2 receptor agonist U46619. Outside-in signaling was examined by measuring platelet spreading and clot retraction. Tail-transection bleeding time and susceptibility to thrombotic occlusion of the carotid artery in response to photochemical injury (rose bengal) were also measured. Results No significant differences in JON/A or fibrinogen binding were detected between wild-type (Dok-1+/+) and Dok-1-/- platelets, suggesting that Dok-1 does not regulate inside-out signaling. In contrast, Dok-1-/- platelets exhibited increased clot retraction and enhanced spreading on fibrinogen upon thrombin stimulation compared to Dok-1+/+ platelets (P&lt;0.05), suggesting that Dok-1 negatively regulates outside-in signaling. Compared with Dok-1+/+ mice, Dok-1-/- mice had shorter bleeding times (181±168 vs. 379±193 seconds; P&lt;0.001) and Dok-1-/- mice had shorter times to stable occlusion times of the carotid artery after photochemical injury compared with Dok-1+/+ mice (16.4±6.1 vs. 25.0±8.1 minutes; P&lt;0.05). Conclusions The adaptor protein Dok-1 functions to negatively regulate integrin αIIbβ3 outside-in signaling. Deficiency of Dok-1 results in a prothrombotic phenotype, with shorten bleeding times and enhanced arterial thrombosis.

Regulation of integrin αV subunit expression by sulfatide in hepatocellular carcinoma cells

Integrin is important in migration and metastasis of tumor cells. Changes of integrin expression and distribution will cause an alteration of cellular adhesion and migration behaviors. In this study, we investigated sulfatide regulation of the integrin αV subunit expression in hepatoma cells and observed that either exogenous or endogenous sulfatide elicited a robust upregulation of integrin αV subunit mRNA and protein expression in hepatoma cells. This regulatory effect occurred with a corresponding phosphorylation (T739) of the transcription factor Sp1. Based on the electrophoretic mobility shift assay, sulfatide enhanced the integrin αV promoter activity and strengthened the Sp1 complex super-shift. The results of chromatin immunoprecipitation analysis also indicated that sulfatide enhanced Sp1 binding to the integrin αV promoter in vivo. Silence of Sp1 diminished the stimulation of integrin αV expression by sulfatide. In the early stage of sulfatide stimulation, phosphorylation of Erk as well as c-Src was noted, and inhibition of Erk activation with either U0126 or PD98059 significantly suppressed Sp1 phosphorylation and integrin αV expression. We demonstrated that sulfatide regulated integrin αV expression and cell adhesion, which was associated with Erk activation.

Pharmacogenomics of Oral P2Y12 Receptor Blockers

Dual antiplatelet therapy of aspirin and a P2Y12 receptor antagonist is the cornerstone of therapy for patients. Thienopyridines (ticlopidine, clopidogrel and prasugrel) are prodrugs that need cytochrome–mediated conversion to active metabolites to block P2Y12 receptors and subsequent ADP-induced platelet aggregation. Clopidogrel response variability has been attributed to a variable generation of its active metabolite which is in turn influenced by single nucleotide polymorphisms of genes associated with intestinal absorption protein, ABCB1 and hepatic cytochrome isoenzymes particularly CYP2C19. Moreover, presence of loss-of-function allele of CYP2C19 gene has been associated with poor active metabolite generation, poor antiplatelet response and increased risk for cardiovascular event occurrences particularly stent thrombosis in patients treated with clopidogrel. Recent studies suggest that genetic variations in CYP2C19 and CYP2B6 may affect response to the drug; however its clinical significance is unknown at this time. Although, ticagrelor (a cyclopentyl-triazolo-pyrimidine), is a prodrug, it is metabolized by CYP3A4 and its active metabolite is as potent as parent drug. To date there are no reports of significant influence of genotype variations on ticagrelor metabolism, its antiplatelet response or clinical outcome. The optimal strategy to overcome the influence of LoF carriage in patients treated with clopidogrel is probably to switch therapy to either prasugrel or ticagrelor, although large-scale studies assessing this approach have not been performed.

Insufficient Efficiency of Prasugrel in Diabetic Patients with Acute STEMI Undergoing Ppci of Culprit Coronary Lesion – Review of Current Knowledge

The activation and subsequent platelet aggregation plays a key role in the formation of arterial thrombosis and therefore is the key therapeutic target in the treatment of acute coronary syndromes. Dual antiplatelet therapy containing aspirin and P2Y12 ADP receptor antagonist forms currently the basis in acute ST – elevation myocardial infarction (STEMI) pharmacological treatment. Nevertheless, there is a wide variability in pharmacodynamic response to administration of clopidogrel, the most frequently used P2Y12 ADP receptor antagonist. High platelet reactivity after clopidogrel administration is associated with increased risk of in - stent thrombosis and points to the suitability of laboratory monitoring of antiplatelet therapy effectivity in clinical practice. Laboratory monitoring of antiplatelet therapy by ex vivo platelet function tests may help to identify individuals with poor antiplatlet response. Recently, there is growing number of data reporting a failure in antiplatelet response following clopidogrel administration, which is specifically associated with insulin resistance and diabetes mellitus. Prasugrel, a new, potent P2Y12 ADP receptor antagonist, provides faster and more consistent inhibition of platelet function compared with clopidogrel. Prasugrel therapy was repeatedly described as an effective method to overcome clopidogrel resistance and prasugrel resistance has not yet been reliably described. Article gives a review about prasugrel resistance and its role in acute coronary syndrome patients.

The Impact of Shear Stress On the Interaction of Human Platelet Integrin αIIbβ3 with Fibrinogen.

AbstractAbstract 2167 Objectives:Shear stress can activate platelets resulting in subsequent platelet aggregation. The so-called “shear-induced platelet aggregation” (SIPA) contributes to various vascular diseases (Speich et al., Am J Physiol Cell Physiol 2008). Several signaling pathways were proposed to be involved in this process, e.g., αIIbβ3-mediated signaling (Feng et al., Am J Physiol Cell Physiol 2006). We investigated the impact of shear stress on the αIIbβ3–ligand interaction in human platelets adherent onto fibrinogen. Platelets on immobilized fibrinogen were exposed to various shear rates and signaling of Src and FAK tyrosine kinases, both essential in the integrin downstream signaling pathways, were examined. Specifically, we analyzed the role of αIIbβ3 in shear-induced platelet signaling (i) by comparing the Src Y418 and FAK Y397 phosphorylation activities between platelets on immobilized fibrinogen and platelets on BSA matrix in response to shear stress, and (ii) by performing experiments in the presence of the αIIbβ3 antagonist abciximab. Methods:Human washed platelets were incubated on immobilized fibrinogen 100 μg/ml or 1% BSA either under static conditions or exposed to shear rates of 500 s−1 or 5000 s−1, respectively. Specific phosphorylation of Src (pY418) and FAK (pY397) was determined by Western blot and quantified densitometrically. Experiments under flow conditions were performed in a cone-plate viscometer. Results:Both Src and FAK exhibited phosphorylation under static conditions on immobilized fibrinogen after 2 min of adhesion. A shear rates of 500 s−1 did not increase the phosphorylation activities. By contrast, high shear rates (5000 s−1) significantly enhanced both Src and FAK phosphorylations in fibrinogen-adherent platelets (3-fold increase each, p<0.05). In the absence of immobilized fibrinogen, platelets incubated with BSA matrix did not show any Src activation under static conditions and only a very low Y418 phosphorylation activity in response to a shear rate of 500 s−1. A shear rate of 5000 s−1 considerably induced Src pY418 activity compared to platelets exposed to physiological shear stress (10-fold increase, p< 0.01). In response to shear rates of 500 s−1 or 5000 s−1, we detected a significantly higher Src activation in platelets adherent onto fibrinogen (500 s−1: 10-fold higher, p<0.01; 5000 s−1: 2-fold higher, p<0.05) than in platelets incubated over a BSA matrix indicating a ligand-dependent signaling. When platelets over BSA were exposed to a shear rate of 5000 s−1, FAK also exhibited a significant elevation of pY397 activity (9-fold increase, p<0.05). By contrast to Src, in platelets exposed to a shear rate of 500 s−1 or 5000 s−1, we observed approximately equal FAK pY397 activation, independent of the presence or absence of immobilized fibrinogen. In platelets incubated for 10 min on a fibrinogen matrix under static conditions, we did not detect any change in the Src activation compared to 2 min incubation. The activity of FAK pY397, however, was time-dependent and showed a 3-fold higher phosphorylation extent after 10 min than after 2 min adhesion (p<0.05). In response to a shear rate of 500 s−1 both Src Y418 and FAK Y397 phosphorylations exhibited a considerable time-dependent enhancement (comparing the phosphorylation activities after incubation for 2 or 10 min). This enhancement could be seen both in platelets adherent onto fibrinogen and in platelets over BSA (3 to 6-fold increase, p<0.05). In platelets exposed to a shear rate of 5000 s−1 for 10 min, the Src and FAK phosphorylation activities were similar to platelets after 2 min. Abciximab inhibited the Src and FAK signaling in platelets exposed to 5000 s−1 on immobilized fibrinogen. The same inhibition was seen in platelets exposed to 5000 s−1 over BSA (p<0.05). Conclusions:Exposure of platelets to high shear rates induces a significant increase of both Src and FAK signaling compared to platelets under static conditions. Whereas Src activation remains predominantly ligand-dependent in fibrinogen-adherent platelets even under shear stress, FAK signaling appears to be shear-induced. The finding that, abciximab inhibits the activation of both Src and FAK in the absence of fibrinogen, emphasizes the role of integrin αIIβ3 in the shear-induced platelet signaling. Disclosures:No relevant conflicts of interest to declare.

Identification of Hic‐5 as a novel regulatory factor for integrin αIIbβ3 activation and platelet aggregation in mice

Integrin αIIbβ3 plays key roles in platelet aggregation and subsequent thrombus formation. Hydrogen peroxide-inducible clone-5 (Hic-5), a member of the paxillin family, serves as a focal adhesion adaptor protein associated with αIIbβ3 at its cytoplasmic strand. Hic-5 function in αIIbβ3 activation and subsequent platelet aggregation remains unknown. To address this question, platelets from Hic-5(-/-) mice were analyzed. Hic-5(-/-) mice displayed a significant hemostatic defect and resistance to thromboembolism, which were explained in part by weaker thrombin-induced aggregation in Hic-5(-/-) platelets. Mechanistically, Hic-5(-/-) platelets showed limited activation of αIIbβ3 upon thrombin treatment. Morphological alteration in Hic-5(-/-) platelets after thrombin stimulation on fibrinogen plates was also limited. As a direct consequence, the quantity of actin co-immunoprecipitating with the activated αIIbβ3 was smaller in Hic-5(-/-) platelets than in wild-type platelets. We identified Hic-5 as a novel and specific regulatory factor for thrombin-induced αIIbβ3 activation and subsequent platelet aggregation in mice.

Disaggregation Signals Initiated by Ligand Binding and Platelet Aggregation

Abstract 2196Accumulating evidence suggests that activation of the major platelet integrin, αIIbβ3, and subsequent platelet aggregation are intrinsically dynamic and reversible processes, especially when platelets are activated by low-dose agonists like ADP. When aggregation is not robust enough to activate feed-forward autocrine amplification pathways, platelet integrins revert to their inactivated state and fibrinogen dissociates, resulting in platelet disaggregation. Failure to disaggregate following low-dose agonist stimulation has been known for many years to correlate with occlusive arterial disease and diabetes mellitus, however the mechanisms by which integrins become inactivated leading to dissociation of fibrinogen is not well understood. Because phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling is known to be required to maintain integrins in an activated state and to support the formation of stable platelet aggregates, we examined whether the PI3K/Akt signaling might be actively reversed when platelets aggregate in response to ADP. We found that Akt becomes completely dephosphorylated in an aggregation-dependent manner. SHIP-1, a negative regulator of PI3K signaling, also becomes phosphorylated and activated during the early stages of platelet aggregation. These results suggest that fibrinogen binding and platelet aggregation initiate negative feedback through the PI3K/Akt signaling pathway to control unnecessary platelet aggregation responses. To gain further insight into the mechanism of platelet disaggregation, we examined the dynamic interaction of αIIbβ3 with talin – a cytosolic protein whose association with the integrin β3 cytoplasmic domain is required for integrin activation. We found that talin translocates to αIIbβ3 upon ADP stimulation, but dissociates from it during the process of platelet disaggregation. Blocking ADP-induced platelet aggregation with RGDW inhibited dissociation of talin from the integrin, again supporting the notion that that platelet aggregation initiates negative, as well as positive, signals to regulate the extent of agonist-induced platelet aggregation. Finally, we found that platelets missing PLCγ2 or FcγRIIa, each of which plays a positive role downstream of αIIbβ3-mediated outside-in signaling, are less prone to disaggregate following low-dose agonist stimulation than are their wild-type counterparts, although neither of them plays a role in ADP-induced platelet aggregation. Taken together, these data demonstrate that platelet aggregation and integrin αIIbβ3-mediated outside-in signaling not only initiates signals that amplify integrin activation and platelet aggregation, but also guards against inadvertent activation by shifting activated integrin αIIbβ3 back to its resting state, triggering platelet contraction and leading to dissociation of platelet aggregates. Disclosures:No relevant conflicts of interest to declare.