MRP4 Expression and Platelet Activation in Children and Adolescents with Different Subtypes of Primary Thrombocythemia

Abstract Background and aims. Essential thrombocythemia (ET) rarely occurs in children and adolescents. In our experience, 40% of pediatric patients with primary thrombocythemia (PT) have JAK2 V617F or CALR mutations, 24% have a diagnosis of ET without any known molecular markers while 36% have hereditary thrombocytosis (HT) with a MPLS505A mutation. Thrombotic events, frequent in adult ET presenting high-risk factors and rare in pediatric PT, have been observed in pediatric HT patients with MPLS505A mutation in treatment with aspirin (ASA). The multidrug resistance protein-4 (MRP4) is an ATP-binding cassette transporter involved in the efflux of several pharmacological and physiological compounds. MRP4 has been identified as a modulator of ASA action in platelets; in addition, it also influences platelet activation. Recent studies have shown that MRP4 over-expression has a role in reducing the effect of ASA in patients who have undergone a bypass surgical procedure and that ASA induces platelet MRP4 upregulation. Aims of this study were to evaluate and correlate MRP4 expression and platelet function in children and adolescents aged <20 years at diagnosis with different subtypes of PT. Methods. MRP4 protein and mRNA expressions were evaluated in platelets obtained from healthy volunteers (HV) and from 41 PT patients: M/F ratio: 0.78; median age at diagnosis: 14.5 years; median platelet count at study: 671x109/L. Ten patients had MPLS505A mutation (HT), 10 were JAK2V617F-mutated (ET) and 6 harbored CALR mutations (ET) while 15 cases were JAK2, CALR and MPL wilde-type (ET). Expression of MRP4 protein and mRNA were analyzed by Western blot and RT-PCR, respectively, and the results were reported as ET/HV or HT/HV expression values. Platelet aggregation, using ADP at different concentration (0.8 to 2 μM) and collagen (1 μg/ml) as agonists, and platelet secretion, expressed as ATP release after U46619 + epinephrine (1 + 20 μM), using the luciferin-luciferase assay, were utilized as platelet function tests. This study was conducted in accordance with the Declaration of Helsinki. Results. Protein MRP4 expression was higher both in HT (4.23+/-1.88) and in ET patients with CALR mutations (4.27+/-2.60) compared to the values found in wild-type ET patients (3.55+/-1.52). In JAK2V617F-mutated ET patients, the MRP4 protein expression (2.03+/-1.46) was significantly lower compared to the values observed in all other ET and HT patients (p .05), whereas the MRP4 mRNA expression was significantly higher (ΔΔCt 0.021+0.003) compared both to HT patients (0.009+/-0.0039 ΔΔCt) (p .01) and CALR-mutated patients (ΔΔCt 0.014+/- 0.006). Patients with HT showed a significantly higher response to ADP 0.8 µM (83+/-29 Mx%) compared to all subgroups of ET patients who showed a similar response (60+/-34 Mx%; p .01). A significantly shorter lag-phase in response to collagen (1 µg/ml) was observed in HT compared to ET patients (33+/-3 sec vs 51+/-4 sec, p .005). Among the ET population, JAK2-mutated patients showed a significantly shorter lag-phase in response to collagen compared to wild-type patients (39+/-12.5 sec vs 50+/-15 sec), p .011. Platelet secretion was significantly higher in HT compared to ET patients, p .001. Conclusions. This study for the first time provides evidence that children and adolescents with MPLS505A-mutated HT show a higher platelet reactivity compared to age-matched patients with ET. Moreover, platelet reactivity correlates with MRP4 protein overexpression. These findings help to shed light into the thrombotic events observed in HT MPLS505A patients despite treatment with ASA. Disclosures No relevant conflicts of interest to declare.

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Primary Trombocythemia in Children and Adolescents Includes Different Subtypes Compared to Adult Essential Thrombocythemia

Blood ◽

10.1182/blood.v124.21.1865.1865 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1865-1865

Author(s):

Fiorina Giona ◽

Marica Laurino ◽

Luciana Teofili ◽

Sara Capodimonti ◽

Maurizio Martini ◽

...

Keyword(s):

Children And Adolescents ◽

Essential Thrombocythemia ◽

Conflicts Of Interest ◽

Correct Diagnosis ◽

Young Patients ◽

Antiplatelet Agents ◽

Wild Type ◽

Primary Thrombocythemia ◽

Calr Mutations

Abstract The recent discovery of various mutations of the CALR gene that are mutually exclusive with JAK2 and MPL mutations has allowed a correct diagnosis in about 90% of adult cases of essential thrombocythemia (ET). Moreover, the mutation status of JAK2 and CARL defines subtypes of ET in adults with a substantially different clinical course and outcome. Based on our experience, we suggested that primary thrombocythemia (PT) in children is characterized by subtypes that differ from those found in adult ET. The present study was carried out in children and adolescents with PT in order to (a) characterize the various subtypes of the disease and (b) analyze their clinical and biologic features, treatment approach and outcome. PT patients aged <20 years (yrs) at diagnosis (dx) were evaluated for mutations of JAK2, thrombopoietin (TPO) and its receptor (MPL) and CALR genes, and for clonal hematopoiesis (females). The presence of MPLS505A (confirmed on DNA from buccal swabs) defined a hereditary thrombocytosis (HT). ET was diagnosed according to WHO 2008 criteria. For wild type patients, an additional inclusion criteria was a follow-up >24 months. Among 58 PT patients (males: 23; females: 35; median age at dx: 14.4 yrs), 21 (36%) had HT due to MPLS505A, 14 were JAK2V617F-mutated (24%), 9 (16%) harbored CALR mutations and 14 (24%) were wild type for JAK2, CALR and MPL (Fig 1). JAK2- and CALR-mutated were older than those with wild type ET or with HT (median age, 17.6 and 16.1 vs 10.4 and 13.7 yrs, p .028). As to the hematologic findings, HT patients showed both hematocrit values (median, 36.3%) and leukocytes counts (median, 9.53 x109/L) significantly lower than ET patients, whatever the subtypes (median, 41.2% and 11.2 x109/L, p .006 and p .029, respectively). No differences were found with regard to platelets both between HT and ET and among the different ET subtypes. JAK2-mutated patients exhibited more frequently symptoms (69%) compared to CALR-mutated (22%), wild-type ET (14%) and HT (14%) patients (p. 0057). Splenomegaly at diagnosis was recorded more frequently in JAK2-mutated than in CALR-mutated or wild type-ET or HT (50%, 33% 21% and 14% , respectively, p .122). Antiplatelet agents, mostly acetylsalicylic acid (ASA), were started less frequently in HT than in ET patients, irrespective of the subtypes (57% vs 81%, p .05). The use of ASA progressively decreased over the time; at the last follow-up, 2 patients with HT, 2 CALR-mutated and 1 JAK2-mutated patients were still receiving ASA, while no wild type ET patient was on treatment. Cytoreductive agents, hydroxyurea and/or interferon and/or anagrelide, were used in a minority of HT patients (19%) in comparison with ET patients (65%), p .001, mainly with those wild-type (78%, p <.001). At the last observation, one HT patient was still receiving cytoreductive agents compared to 30% of ET patients whatever the subtypes (p .024). After a median follow-up of 196 months (similar in the different subtypes), all patients are alive. On the whole, 5 thrombotic events were recorded in 3 patients with HT and in 2 ET patients (1 JAK2-mutated and 1 JAK2 and CALR wild-type), without any significant thrombophilic abnormalities during treatment with ASA and/or cytoreductive agents. A progressive splenomegaly was recorded in 9 (15%) patients (2 HT, 4 JAK2-mutated, 3 CALR-mutated) and it was combined with grade ≥2 medullar fibrosis in 2/4 JAK2-mutated and in 2/3 CALR-mutated patients. None of the JAK2 and CALR wild-type patients had spleen enlargement or reticulin fibrosis (p .022). Two untreated patients (1 HT and JAK2 and CALR wild-type) developed malignancies. On the whole, these data emphasize that in young patients with PT, hereditary forms can be frequently observed. Thrombotic events, recorded mainly in HT patients despite treatment with ASA, were probably due to a MRP4 protein overexpression that was found in our MPLS505A HT. Moreover, our observations highlight that, in contrast to adult ET, more than one third of young ET patients have no JAK2 or CALR mutations. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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microRNAs as Promising Biomarkers of Platelet Activity in Antiplatelet Therapy Monitoring

International Journal of Molecular Sciences ◽

10.3390/ijms21103477 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3477

Author(s):

Teresa L. Krammer ◽

Manuel Mayr ◽

Matthias Hackl

Keyword(s):

Antiplatelet Therapy ◽

Platelet Activation ◽

Platelet Function ◽

Platelet Reactivity ◽

Therapy Monitoring ◽

Platelet Inhibition ◽

High Morbidity ◽

Platelet Function Tests ◽

Function Tests ◽

Plasma Mirna

Given the high morbidity and mortality of cardiovascular diseases (CVDs), novel biomarkers for platelet reactivity are urgently needed. Ischemic events in CVDs are causally linked to platelets, small anucleate cells important for hemostasis. The major side-effect of antiplatelet therapy are life-threatening bleeding events. Current platelet function tests are not sufficient in guiding treatment decisions. Platelets host a broad spectrum of microRNAs (miRNAs) and are a major source of cell-free miRNAs in the blood stream. Platelet-related miRNAs have been suggested as biomarkers of platelet activation and assessment of antiplatelet therapy responsiveness. Platelets release miRNAs upon activation, possibly leading to alterations of plasma miRNA levels in conjunction with CVD or inadequate platelet inhibition. Unlike current platelet function tests, which measure platelet activation ex vivo, signatures of platelet-related miRNAs potentially enable the assessment of in vivo platelet reactivity. Evidence suggests that some miRNAs are responsive to platelet inhibition, making them promising biomarker candidates. In this review, we explain the secretion of miRNAs upon platelet activation and discuss the potential use of platelet-related miRNAs as biomarkers for CVD and antiplatelet therapy monitoring, but also highlight remaining gaps in our knowledge and uncertainties regarding clinical utility. We also elaborate on technical issues and limitations concerning plasma miRNA quantification.

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In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation

Blood ◽

10.1182/blood-2011-11-393900 ◽

2012 ◽

Vol 119 (17) ◽

pp. 4066-4072 ◽

Cited By ~ 55

Author(s):

Bethan Psaila ◽

James B. Bussel ◽

Matthew D. Linden ◽

Bracken Babula ◽

Youfu Li ◽

...

Keyword(s):

Platelet Activation ◽

Platelet Function ◽

Whole Blood ◽

Immune Thrombocytopenia ◽

Ex Vivo ◽

Platelet Reactivity ◽

Adenosine Diphosphate ◽

High Dose ◽

Platelet Surface

Abstract The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

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Platelet reactivity in patients with aortic stenosis depends on LV-AO angle

European Heart Journal ◽

10.1093/ehjci/ehaa946.1866 ◽

2020 ◽

Vol 41 (Supplement_2) ◽

Author(s):

P Mourikis ◽

S Zako ◽

L Dannenberg ◽

R M'Pembele ◽

T Hohlfeld ◽

...

Keyword(s):

Aortic Stenosis ◽

Platelet Activation ◽

Platelet Function ◽

Cox Regression ◽

Light Transmission ◽

Platelet Reactivity ◽

Funding Source ◽

Local Inflammation ◽

German Research Foundation ◽

Induced Aggregation

Abstract Background The pathogenesis of aortic stenosis (AS) is not fully understood. However, local inflammation of the valve appears to be a main player. Except haemostasis, platelets contribute to inflammatory processes in various ways. Furthermore, platelet function is altered in patients with AS. Moreover, a steep angle between the left ventricle and the aorta (LV-AO-angle) leads to turbulent blood flow. However, it is not known if platelet reactivity is associated with steep LV_AO angle in patients with AS. Methods We included 289 patients with severe AS and performed cardiac computertomography to assess the LV-AO-angle. Platelet function was evaluated by light transmission aggregometry by using collagen and adenosine-diphosphate to induce platelet activation Results ADP- and collagen induced aggregation showed a significant negative correlation with LV-AO-angle (ADP: r=−0.19, p=0.0009, R2=0.022; collagen: r=−0.21, p=0.0004, R2=0.027). ADP-induced MoA was significant higher in patients with a LV-AO-angle <160° in comparison to patients with an angle ≥160° (<160°: 66.99±20.72% vs. ≥160°: 60.66±19.85%, p=0.009). Collagen-induced platelet reactivity was significant higher in patients with a LV-AO-angle <160° in comparison to patients with an angle ≥160° (<160°: 78.67±13.19% vs. ≥160°: 73.85±14.44%, p=0.003). Multivariate cox-regression revealed that LV-AO angle <160 was a robust predictor of ADP- and collagen-induced platelet aggregation Conclusion A steep LV-AO-angle is associated with enhanced platelet reactivity in patients with AS. Platelet activation is known to lead to local inflammation. Therefore, enhanced platelet reactivity could play crucial in the progression of AS. The clinical significance of a steep LV-AO-angle needs be evaluated in further trials. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): German Research Foundation

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Platelet activation and multidrug resistance protein-4 expression in children and adolescents with different subtypes of primary thrombocythemia

Haematologica ◽

10.3324/haematol.2019.226266 ◽

2019 ◽

Vol 105 (2) ◽

pp. e65-e67

Author(s):

Fiorina Giona ◽

Isabella Massimi ◽

Maria Luisa Guarino ◽

Michelina Santopietro ◽

Marica Laurino ◽

...

Keyword(s):

Multidrug Resistance ◽

Children And Adolescents ◽

Platelet Activation ◽

Multidrug Resistance Protein ◽

Primary Thrombocythemia ◽

Resistance Protein

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Aronia juice consumption prior to half-marathon race can acutely affect platelet activation in recreational runners

Applied Physiology Nutrition and Metabolism ◽

10.1139/apnm-2019-0267 ◽

2020 ◽

Vol 45 (4) ◽

pp. 393-400

Author(s):

Vuk Stevanović ◽

Ana Pantović ◽

Irena Krga ◽

Milica Zeković ◽

Ivana Šarac ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Repeated Measures ◽

Platelet Reactivity ◽

Activation Parameters ◽

Long Distance ◽

Activation Markers ◽

Marathon Race ◽

Recreational Runners

Long-distance running, especially in non-professional runners, can increase cardiac arrest risk by enhancing platelet activation and aggregation. Polyphenols can exert cardioprotective effects by positively influencing platelet function. This study aimed to examine the acute effects of polyphenol-rich aronia juice consumption, before simulation of a half-marathon race, on platelet activation and aggregation with leukocytes in recreational runners. In this acute crossover study,10 healthy male runners (age 30.8 ± 2.3 years) consumed breakfast with 200 mL of aronia juice or 200 mL of placebo. They warmed-up and ran a simulated half-marathon race (21.1 km). Blood was collected at baseline, and at 15 min, 1 h, and 24 h after the run. All variables were analyzed with 4 (time) × 2 (group) ANOVA with repeated measures on both factors. Results revealed a significant effect of group on platelet activation parameters: P-selectin and GPIIb-IIIa expressions significantly decreased in the aronia group compared with the placebo group (F[1,9] = 10.282, p = 0.011 and F[1,9] = 7.860, p = 0.021, respectively). The effect of time was significant on both platelet aggregation markers: platelet-monocyte and platelet-neutrophil aggregates were significantly lower after the race (F[3,7] = 4.227, p = 0.014 and F[3,7] = 70.065, p = 0.000, respectively), with changes more pronounced in the later. All effects remained when platelets were exposed to an agonist. These results suggest that aronia consumption could counteract the half-marathon race-induced changes in platelet function. Novelty Aronia juice consumption significantly decreased the expression of platelet activation markers but did not affect platelet aggregation. The race itself did significantly reduce platelet-neutrophil aggregation. Aronia juice may serve as a supplement beverage for recreational runners to alleviate enhanced platelet reactivity caused by prolonged running.

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Human Apolipoprotein a-I Decreases Platelet Activation Responses and Protects Mice from Arterial Thrombosis

Blood ◽

10.1182/blood.v124.21.4159.4159 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4159-4159 ◽

Cited By ~ 1

Author(s):

Brian R. Branchford ◽

Christopher Ramos ◽

Wilbert Jones ◽

Christine Brzezinski ◽

Luke Law ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Function ◽

Arterial Thrombosis ◽

Aggregate Formation ◽

Wild Type ◽

Platelet Aggregate ◽

Microfluidic Analysis ◽

Induced Aggregation ◽

Platelet Function Assays

Abstract Introduction: High-density lipoprotein (HDL) protects against thromboembolic coronary disease via reverse cholesterol transport mediated by HDL's primary lipoprotein subunit, apolipoprotein A-I (apoA-I), binding to scavenger receptor BI (SR-BI). Absence of SR-BI increases platelet aggregation and venous/arterial thrombosis in mice, consistent with an antithrombotic role of this signaling axis. To date, the effects of the isolated apoA-I subunit on platelet activation remain unknown. We hypothesize that the antithrombotic effect is mediated by apoA-I signaling through platelet-specific SR-BI. Methods: Platelet function assays were performed on samples from healthy human volunteers (n=3). ApoA-I levels were determined using an ELISA kit. Microfluidic analysis of platelet aggregate formation on collagen under physiologic flow conditions (650 sec-1) was carried out in whole blood samples. Light transmission platelet aggregation in response to collagen and ristocetin was conducted using both platelet rich plasma (PRP) and washed platelets (WP). Venous and arterial thromboses were induced in wild type C57Bl/6 mice in a collagen (0.3 mg/kg)/epinephrine (0.03 mg/kg)-induced pulmonary embolism model and a 6% ferric chloride (FeCl3)-induced carotid artery thrombosis model, respectively. Recombinant human apoA-I was used with a final concentration of 300 µg/mL. Results/Discussion: ApoA-I plasma levels in the subjects studied fell within previously published ranges. Microfluidic analysis of platelet aggregate formation on collagen under shear stress did not demonstrate significant difference in total surface area coverage (n=3 in quadruplicate), but apoA1-treated samples demonstrated a significantly greater proportion of small aggregates (3-10 platelets/aggregate, 40.2% vs 31.8%, p<0.0001), compared to controls which had a significantly greater proportion of large aggregates (51-100 plts/agg, 6.3 vs 2.3%, p<0.001 and >100 plts/agg, 2.3% vs 0.7%, p<0.05). Platelet aggregation studies (Fig. 1) revealed significantly decreased collagen-induced aggregation in platelets treated with apoA-I compared to vehicle-treated controls in both PRP and WP samples, suggesting direct action of apoA-I on platelets. The difference was less pronounced in ristocetin-induced aggregation, though still significant, consistent with decreased activation of VWF, which has been recently shown to bind apoA-I. Apoa-I-treated PRP samples also had significantly decreased dense granule (ATP) release compared to non-treated samples. Pre-treatment with native HDL had no significant effect, consistent with studies demonstrating that only oxidized HDL inhibits aggregation. Recombinant human SR-BI (rhSR-BI) alone had little effect in PRP but inhibited collagen-induced aggregation in WP. Addition of rhSR-BI and apoA-I together inhibited aggregation in both PRP and WP to a greater degree than either component alone. Rabbit anti-human SR-BI alone had little effect in PRP but inhibited collagen-induced aggregation in WP. FeCl3 applicationinduced initial arterial occlusion within 8.8 +/-0.8 minutes in apoA-I-treated mice treated (n=3) compared to 6.1 +/- 0.3 minutes in vehicle-treated controls (n=3, p<0.01, t-test). Systemic thrombosis resulting in pulmonary embolism was induced by injection of collagen/epinephrine. Median time of survival after injection in mice pre-treated with apoA-I (n=5) was 30 +/- 0 minutes, compared to 3.3 +/- 0.4 minutes in vehicle-treated controls (n=5, p<0.001, t-test). Experiments were stopped after 30 minutes, at which time all apoA-1-treated mice were still alive, compared to zero controls. Conclusion: Though the mechanism is not yet completely understood, the data show an inhibitory effect of isolated human apoA-I on human platelet activation and murine arterial/venous thrombosis, likely through platelet inhibition. The increased inhibition seen with rhSR-BI and apoA-I together is consistent with previous studies demonstrating SR-BI's regulatory role in platelet function. Ongoing studies include increasing sample size for human platelet function assays and murine venous/arterial thrombosis models in wild type mice with and without infusion of apoA-I compared to SR-BI-/- and apoA-I-/- mice with and without infusion of apoA-I. Figure 1: Maximum aggregation values (mean +/- SEM) following addition of collagen or ristocetin Figure 1:. Maximum aggregation values (mean +/- SEM) following addition of collagen or ristocetin Disclosures No relevant conflicts of interest to declare.

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12-HETrE, An Endogenous Inhibitor of Platelet Activation,

Blood ◽

10.1182/blood.v118.21.3254.3254 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3254-3254

Author(s):

Jennifer Yeung ◽

Kenneth Ikei ◽

Joanne Vesci ◽

Alex Arnouk ◽

Theodore R Holman ◽

...

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Platelet Activation ◽

Platelet Function ◽

Conflicts Of Interest ◽

Platelet Reactivity ◽

Fatty Acid Content ◽

Pharmacological Intervention ◽

Direct Role ◽

Granule Secretion

Abstract Abstract 3254 Platelet activation plays a pivotal role in thrombosis and hemostasis. Understanding the signaling events mediating this process is essential in preventing unwanted clot formation, which can lead to the development of heart attack, stroke, and venous thromboembolism. Fatty acids may play an important role in determining the level of platelet reactivity, however the mechanism(s) by which this occurs are not entirely clear. 12-lipoxygenase (12-LOX) has been shown to oxidize the fatty acid, arachidonic acid (AA), in order to produce the bioactive eicosanoid 12-HETE, which has recently been shown to play a role in tissue factor activation and subsequent thrombin generation in the platelet (Thomas et al., 2010, J Biol Chem; 285:6891–903). 12-HETE has also been shown to signal to cells, in part, through the G protein-coupled receptor GPR31 (Guo et al, 2011, J Biol Chem; epub). While research on 12-LOX-mediated eicosanoid regulation of platelets has primarily focused on 12-HETE, the potential for regulation of platelets by eicosanoids derived from other fatty acids has been overlooked. As the fatty acid content on the platelet membrane is extremely dynamic and fatty acid supplementation is correlated with a reduced risk for cardiovascular disease, we hypothesized that 12-LOX oxidation of another fatty acid may play a direct role in regulating platelet function. To test this hypothesis, the Ω-6 fatty acid dihomo-γ-linolenic acid (DGLA) as well as its eicosanoid derived from 12-LOX oxidation, 12-hydroxyeicosatrienoic acid (12-HETrE), were applied to washed platelets followed by stimulation with thrombin, PAR1-AP, or PAR4-AP. Platelets treated with either DGLA or 12-HETrE showed significant attenuation in platelet aggregation following stimulation with thrombin or PAR-AP. A number of biochemical intermediates were also tested in the presence of 12-HETrE or DGLA including Rap1 activation, αIIbβ3 integrin activation, α-granule secretion, and dense granule secretion. All endpoints tested were attenuated in the presence of DGLA or 12-HETrE relative to control. To confirm the regulation was unique to DGLA and its metabolite, the same endpoints were measured in the presence of AA or 12-HETE. Pre-treatment with either AA or 12-HETE did not attenuate any of the agonist-mediated platelet activation endpoints. Thus, our data supports a unique role for 12-HETE and 12-HETrE and that fatty acid regulation of platelet function may be highly dependent on the lipid content of the platelet. Shifting the ratios of fatty acids in the platelet through dietary supplementation or pharmacological intervention may be sufficient to induce a cardio-protective state, in part, through increasing 12-HETrE formation and subsequently inhibiting platelet activation. Disclosures: No relevant conflicts of interest to declare.

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12-lipoxygenase activity plays an important role in PAR4 and GPVI-mediated platelet reactivity

Thrombosis and Haemostasis ◽

10.1160/th13-01-0014 ◽

2013 ◽

Vol 110 (09) ◽

pp. 569-581 ◽

Cited By ~ 29

Author(s):

Jennifer Yeung ◽

Patrick L. Apopa ◽

Joanne Vesci ◽

Moritz Stolla ◽

Ganesha Rai ◽

...

Keyword(s):

Arachidonic Acid ◽

Platelet Activation ◽

Platelet Function ◽

Platelet Reactivity ◽

Selective Inhibition ◽

Platelet Activity ◽

12 Lipoxygenase ◽

Deficient Mice ◽

Cox 1

SummaryFollowing initial platelet activation, arachidonic acid is metabolised by cyclooxygenase-1 and 12-lipoxygenase (12-LOX). While the role of 12-LOX in the platelet is not well defined, recent evidence suggests that it may be important for regulation of platelet activity and is agonist- specific in the manner in which it regulates platelet function. Using small molecule inhibitors selective for 12-LOX and 12-LOX-deficient mice, the role of 12-LOX in regulation of human platelet activation and thrombosis was investigated. Pharmacologically inhibiting 12-LOX resulted in attenuation of platelet aggregation, selective inhibition of dense versus alpha granule secretion, and inhibition of platelet adhesion under flow for PAR4 and collagen. Additionally, 12-LOX-deficient mice showed attenuated integrin activity to PAR4-AP and convulxin compared to wild-type mice. Finally, platelet activation by PARs was shown to be differentially dependent on COX-1 and 12-LOX with PAR1 relying on COX-1 oxidation of arachidonic acid while PAR4 being more dependent on 12-LOX for normal platelet function. These studies demonstrate an important role for 12-LOX in regulating platelet activation and thrombosis. Furthermore, the data presented here provide a basis for potentially targeting 12-LOX as a means to attenuate unwanted platelet activation and clot formation.

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Human Gamma-Thrombin: Lack of Correlation Between a Platelet Functional Response and Glycoprotein V Hydrolysis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646015 ◽

1987 ◽

Vol 58 (03) ◽

pp. 915-920 ◽

Cited By ~ 18

Author(s):

Martine Jandrot-Perrus ◽

Marie-Claude Guillin ◽

Alan T Nurden

Keyword(s):

Platelet Aggregation ◽

Protein Binding ◽

Functional Response ◽

Platelet Activation ◽

Platelet Function ◽

Catalytic Site ◽

Lag Phase ◽

Induce Platelet Aggregation ◽

Threshold Levels ◽

Dense Bodies

SummaryThe ability of purified human gamma-thrombin to stimulate platelet function was related to its capacity to degrade GP V. Compared to alpha-thrombin, much greater amounts of gamma- thrombin were required to induce platelet aggregation; and this also applied to secretion from dense bodies, alpha-granules and lysosomal granules. Platelet stimulation by gamma-thrombin was additionally characterized by the presence of a lag-phase. Platelets with 3H-labelled surface glycoproteins showed the same functional response to both alpha- and gamma-thrombin as unlabelled platelets. But while threshold levels of alpha-thrombin induced little GP V hydrolysis confirming McGowan et al. (1), amounts of gamma-thrombin which induced substantial degradation (e. g. 8.3 nM degraded 36% of platelet GP V in 3 min) were unable to sustain either platelet aggregation or secretion. These results suggest that protein-binding regions remote from the catalytic site of alpha-thrombin are more important for platelet activation than GP V hydrolysis. They also provide further support to the argument that GP V hydrolysis may not be the essential trigger of platelet activation by thrombin.

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