Quantification Of Malondialdehyde Adducts In Platelet Activation As An Indicator Of Proinflammatory and Prothombotic State

Background The formation of malondialdehyde (MDA) has been previously described as a product of the thromboxane synthase. However, the reported approaches for its quantification have not been reliable, stymieing its use in research. As a reactive di-carbonyl, MDA reacts with primary amines, notably lysines on proteins, to form covalent adducts of several types. Three of the products of the reaction of MDA with lysine are an N-propenal adduct, a dihydropyridine ring adduct (N-lysyl-4-methyl-2, 6-dihydropyridine-3, 5-dicarbaldehyde), and a lysyl-MDA crosslink. Measurement of platelet protein modifications, such as MDA adducts, could provide a specific marker of in vivo activation of platelets, since these modifications accumulate over the lifespan of the platelet. Methods and Results To investigate thromboxane synthase-dependent formation of MDA adducts on platelet proteins, we developed an LC/MS/MS method for analysis of one of the MDA adducts, the lysyl-MDA crosslink, employing a [13C12] labeled internal standard. We demonstrated that levels of lysyl-MDA crosslink in human platelets are increased following its activation with arachidonic acid. This increase is inhibited by aspirin, the thromboxane synthase inhibitor, ozagrel and by γ-ketoaldehyde specific scavengers: 3-Methoxysalicylamine (3-MOSA) and Salicylamine (SA). To determine whether lysyl-MDA crosslinks reflect in vivo platelet activation, we analyzed samples from patients with medical conditions known to be associated with increased platelet activation. We employed traditional methods of measuring platelet activation: flow cytometry of p-selectin and reticulated platelets, and serum thromboxane, to measure platelet activation in patients with metabolic syndrome and sickle cell disease. These assays were compared with the levels of lysyl-MDA-crosslinks. In both populations, the levels of MDA-lysine-crosslink are increased by 2.5 fold compared to healthy volunteers and provide greater discrimination between groups than p-selectin expression and reticulated platelets. The inhibition of the lysyl-MDA crosslink adduct in patients taking NSAIDs further confirms the specificity for thromboxane synthase-dependent MDA modifications on platelet proteins. Discussion The results of this study provide compelling evidence that MDA-protein adducts in platelets may be a useful marker of in vivo platelet activation in humans and potentially helpful in predicting thrombotic risk and the benefit of antiplatelet therapy in patients with medical conditions associated with platelet hyperactivity. Disclosures: No relevant conflicts of interest to declare.

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Abstract 600: Novel Assays to Determine in vivo Platelet Activation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.600 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Iberia R Sosa ◽

Irene Zagol-Ikapitte ◽

Manju Bala ◽

Olivier Boutaud ◽

John Oates

Keyword(s):

Platelet Activation ◽

Ex Vivo ◽

Internal Standard ◽

Antiplatelet Agents ◽

High Rate ◽

Thrombotic Risk ◽

Thromboxane Synthase ◽

Coronary Syndrome ◽

Increased Risk

In vivo platelet activation is associated with several pathologic entities, including acute coronary syndrome, essential thrombocythemia, anti-phospholipid antibody syndrome, diabetes and metabolic syndrome. The need for more robust biomarkers with which to assess platelet activation in vivo in human diseases is well recognized. Our lab has developed two novel assays to study in vivo platelet activation and rate of turnover. During platelet activation, malondialdehyde (MDA) is produced by the thromboxane synthase in amounts equimolar to thromboxane A2 and also non-enzymatically by lipid peroxidation resulting from the oxidative processes that accompany activation. MDA is a reactive dicarbonyl that reacts with amines, notably lysines on proteins, yielding covalent modifications of the proteins that then accumulate over the lifetime of the platelet. We developed an LC/MS/MS method for quantification of the most stable of three MDA adducts, the dilysyl-MDA crosslink, employing a [13C12] labeled internal standard. We found that activation of platelets with arachidonic acid leads to an increase in the levels of dilysyl-MDA crosslinks in platelets, which is inhibited by the thromboxane synthase inhibitor, ozagrel, by the cyclooxygenase inhibitor, aspirin, and by scavengers of reactive carbonyls, 3- methoxysalicylamine and Salicylamine. High platelet turnover has been associated with increased risk for thrombosis and failure of antiplatelet agents. We propose a novel approach to studying platelet turnover by labeling platelets in vivo by oral administration of aspirin containing a deuterium labeled acetyl group (d3-aspirin). We can measure the clearance of d3-labeled platelets by using LC/MS/MS to measure the tryptic peptide (SLK) of COX-1 labeled with d3. This approach avoids exposure to radioactivity and the artifact resulting from manipulation of platelets labeled ex vivo. The results of this study provide two novel assays with the potential to serve as markers of in vivo platelet activation and turnover, which may be useful in predicting thrombotic risk and efficacy of antiplatelet therapy in patients with medical conditions associated with platelet hyperactivity and high rate of platelet turnover.

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Increased Thromboxane Biosynthesis in Essential Thrombocythemia

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649916 ◽

1995 ◽

Vol 74 (05) ◽

pp. 1225-1230 ◽

Cited By ~ 71

Author(s):

Bianca Rocca ◽

Giovanni Ciabattoni ◽

Raffaele Tartaglione ◽

Sergio Cortelazzo ◽

Tiziano Barbui ◽

...

Keyword(s):

Platelet Count ◽

Platelet Activation ◽

Essential Thrombocythemia ◽

Therapeutic Strategy ◽

Low Dose ◽

Thrombotic Risk ◽

Dose Aspirin ◽

Gender And Age ◽

Low Dose Aspirin

SummaryIn order to investigate the in vivo thromboxane (TX) biosynthesis in essential thromboeythemia (ET), we measured the urinary exeretion of the major enzymatic metabolites of TXB2, 11-dehydro-TXB2 and 2,3-dinor-TXB2 in 40 ET patients as well as in 26 gender- and age-matched controls. Urinary 11-dehydro-TXB2 was significantly higher (p <0.001) in thrombocythemic patients (4,063 ± 3,408 pg/mg creatinine; mean ± SD) than in controls (504 ± 267 pg/mg creatinine), with 34 patients (85%) having 11-dehydro-TXB2 >2 SD above the control mean. Patients with platelet number <1,000 × 109/1 (n = 25) had significantly higher (p <0.05) 11 -dehydro-TXB2 excretion than patients with higher platelet count (4,765 ± 3,870 pg/mg creatinine, n = 25, versus 2,279 ± 1,874 pg/mg creatinine, n = 15). Average excretion values of patients aging >55 was significantly higher than in the younger group (4,784 ± 3,948 pg/mg creatinine, n = 24, versus 2,405 ± 1,885 pg/mg creatinine, n = 16, p <0.05). Low-dose aspirin (50 mg/d for 7 days) largely suppressed 11-dehydro-TXB2 excretion in 7 thrombocythemic patients, thus suggesting that platelets were the main source of enhanced TXA2 biosynthesis. The platelet count-corrected 11-dehydro-TXB2 excretion was positively correlated with age (r = 0.325, n = 40, p <0.05) and inversely correlated with platelet count (r = -0.381, n = 40, p <0.05). In addition 11 out of 13 (85%) patients having increased count-corrected 11-dehydro-TXB2 excretion, belonged to the subgroup with age >55 and platelet count <1,000 × 1099/1. We conclude that in essential thrombocythemia: 1) enhanced 11-dehydro-TXB2 excretion largely reflects platelet activation in vivo;2) age as well as platelet count appear to influence the determinants of platelet activation in this setting, and can help in assessing the thrombotic risk and therapeutic strategy in individual patients.

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Thrombopoietin Stimulation Leads to Enhanced Polyploidization in p53-/- Bone Marrow Megakaryocytes: In Vivo and in Vitro Studies.

Blood ◽

10.1182/blood.v114.22.2531.2531 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2531-2531

Author(s):

Pani A. Apostolidis ◽

Stephan Lindsey ◽

William M. Miller ◽

Eleftherios T. Papoutsakis

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Platelet Surface ◽

Platelet Counts ◽

Reticulated Platelets ◽

High Ploidy ◽

And Control ◽

Platelet Formation

Abstract Abstract 2531 Poster Board II-508 BACKGROUND AND HYPOTHESIS. We have previously shown that tumor suppressor p53 is activated in differentiating megakaryocytic (Mk) cells and its knock-down (KD) leads to increased polyploidization and delayed apoptosis in CHRF, a human Mk cell line. Furthermore, bone marrow (BM)-derived Mks from p53−/− mice reach higher ploidy classes in culture. Accordingly, we hypothesized that the role of p53 during megakaryopoiesis is to delimit polyploidization and control the transition from endomitosis by inhibiting DNA synthesis and promoting apoptosis. Here, we test this hypothesis by examining the differential effect of mouse thrombopoietin (rmTpo) on the ploidy of p53−/− and p53+/+ mouse Mk cells. METHODS. 8–10 week-old, male p53−/− mice and p53+/+ littermates were injected once with 1.2 μg rmTpo or saline. On days 2 and 5 after Tpo/saline treatment, tail-bleeding assays were performed to measure bleeding times/volumes, mice were bled for platelet counts and sacrificed to harvest BM. We employed flow cytometry to examine baseline ploidy in BM-resident Mks in p53−/− and p53+/+ mice as well as Mk cells generated from BM progenitors after 4 and 6 days of culture with rmTpo. RESULTS. At steady state, ploidy in BM-resident CD41+ Mk cells was similar in p53−/− and p53+/+ mice: 11.8±2.3% and 10.7±1.3% of p53−/− and p53+/+ Mks, respectively, reaching a ploidy of ≥32N (n=3-4). Platelet counts were 1.3×106±1×105/μl (12.5±1.0% reticulated) and 1.1×106±5×104/μl (12.4±1.3% reticulated) in p53−/− and p53+/+ mice, respectively (n=8). Two days following Tpo treatment of the mice, we did not observe significantly increased platelet levels, while ploidy was marginally affected. However, 5 days following Tpo treatment, we found greater ploidy in the BM in the absence of p53: 22±1.6% 16N and 10.1±0.8% ≥32N Mks in the p53−/− versus 18.6±3.3% 16N and 7.1±1.4% ≥32N Mks in the p53+/+ (n=2). This was accompanied by increased platelet formation: 23.6±8.3% reticulated platelets in the p53−/− versus 17.8±2.6% in the p53+/+ (n=2). Culture of BM cells from non-Tpo treated mice with 50ng/ml rmTpo resulted in a 50% increase in total Mks and increased polyploidy by day 6 of culture: 38.6±4.6% of p53−/− versus 19.2±2.3% of p53+/+ Mks reached ploidy classes of ≥32N (n=3-4, p < 0.01). Lack of p53 led to hyperploid Mk cells; by day 6 of culture 10.3±2.2% of p53−/− Mks were in ploidy classes of 128N and higher, while only 0.6±0.1% p53+/+ Mks achieved such high ploidy (n=3-4). In addition, a 6 day culture with Tpo of BM cells derived from p53−/− and p53+/+ mice pre-treated with Tpo 5 days prior to sacrifice led to more profound polyploidization compared to Mks generated from the non-Tpo treated mice but only in the p53−/− Mks: 48.8±1.1% of p53−/− versus only 17.6±0.2% of p53+/+ Mks reached ploidy ≥32N (n=2). Microarray analysis comparing p53KD to control CHRF cells undergoing Mk differentiation revealed down-regulation of genes coding for platelet surface complex CD41/CD61 and CD62P in the p53KD cells. To examine the possibility of altered functionality of platelets in p53−/− mice, we performed tail-bleeding assays on the mice that did not receive Tpo. Bleeding times and volumes were generally prolonged in the absence of p53 (all p53−/− mice exceeded the 10 min duration of the assay; mean p53−/− and p53+/+ blood loss was 17μl and 10μl, respectively, n=3-4). CONCLUSIONS. Our data indicate that in vivo polyploidization and platelet formation from Mks is increased in the p53−/− relative to p53+/+ mice after Tpo administration. These data are in line with our hypothesis that p53 activation decreases the ability of Mks to respond to Tpo and undergo polyploidization. Additionally, our preliminary data on platelet functionality suggest that p53 may have a role in hemostasis. Disclosures: No relevant conflicts of interest to declare.

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STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Blood ◽

10.1182/blood.v116.21.485.485 ◽

2010 ◽

Vol 116 (21) ◽

pp. 485-485

Author(s):

Firdos Ahmad ◽

Lucia Stefanini ◽

Timothy Daniel Ouellette ◽

Teshell K Greene ◽

Stefan Feske ◽

...

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Critical Role ◽

Phosphatidyl Serine ◽

Flow Chamber ◽

Procoagulant Activity ◽

Static Conditions

Abstract Abstract 485 Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca2+/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca2+ concentration of platelets is regulated by Ca2+ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca2+ sensor that couples Ca2+ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca2+-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1fl/fl). STIM1fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin. Disclosures: No relevant conflicts of interest to declare.

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Inflammation Drives Coagulopathies in Sars-Cov-2 Patients

Blood ◽

10.1182/blood-2020-142848 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 34-35

Author(s):

Martha MS Sim ◽

Meenakshi Banerjee ◽

Melissa Hollifield ◽

Hammodah Alfar ◽

Xian Li ◽

...

Keyword(s):

Endothelial Cells ◽

Platelet Activation ◽

Von Willebrand Factor ◽

Conflicts Of Interest ◽

Similar Process ◽

Aids Patients ◽

Thrombotic Risk ◽

S Protein ◽

Von Willebrand ◽

Willebrand Factor

Background:A hypercoagulable state has been consistently reported in patients with severe Coronavirus Disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), characterized by elevated D-dimer, prolonged PT, and mild thrombocytopenia, though the mechanism is unclear. We have previously shown that human immunodeficiency virus (HIV) infection causes depletion of the anticoagulant protein S and virus-mediated platelet activation. Based on early reports, we hypothesized that a similar process contributed to COVID-19-associated thrombosis. Aim:To probe platelet activation and coagulation factor activity in SARS-CoV-2-infected patients. Methods:Blood was collected from consenting patients with differing COVID-19 severity: outpatients (15), hospitalized inpatients (15), and healthy controls (8). Platelet-leukocyte aggregate (PLA) formation and monocyte profiling were measured by flow cytometry. Coagulation factors were assessed by enzymatic assays. PS, von Willebrand Factor (vWF), PC, cytokines, and anti-S-Protein (viral spike protein) IgG were measured by ELISAs. Results:Ninety percent of SARS-CoV-2+ out-patients and in-patients had circulating anti-S-Protein IgG, but plasma IL-6 and TNFα were only elevated in three in-patients, consistent with reports that systemic inflammation is relatively rare in this population. Immune response did not correlate with disease severity. Unlike in HIV1+/AIDS patients, total PS was not reduced in SARS-CoV-2+ patients. However, the anticoagulant pool of PS ("free PS") was reduced in plasma samples from in-patients compared to controls (47.2%±23.3% vs. 100.8±42.6%, p=0057), while out-patients had an intermediate concentration (73.1%±28.9%). Specific loss of free PS is likely mediated by an increase in C4-binding protein (C4bp), which binds PS. In-patients also had a trend toward elevated plasma tissue factor (TF) compared to controls (79.5±121.4 fM vs. 37.8±39.7 fM, p = 0.32). Endothelial cells and monocytes can express TF under inflammatory conditions. We evaluated endothelial damage and dysfunction by measuring E-Selectin, which was unchanged in either in-patients or out-patients, and von Willebrand Factor (vWF), which was elevated in in-patients compared to controls (143±29.8 ng/mL vs. 56.2±41.9 ng/mL, p=0.0023). Plasma from in-patients also had elevated myeloperoxidase (524±187 ng/mL vs. 127±35 ng/mL, p=0.0026) and had a trend toward increased platelet-leukocyte aggregates (14.6±11.7% vs. 5.2±3.7%, p=0.24), indicating platelet and leukocyte stimulation. Unlike in the HIV1+/AIDS patients, no virus was detectable in any of the SARS-CoV-2+ patient plasmas. Consistent with a lack of direct platelet-virus interaction, plasma PF4 and platelet Akt phosphorylation were unchanged in the patient samples. We also observed a trend toward increased TF on TF+/CD64+/CD11b+ monocytes from in-patients compared to controls (MFI = 3244±2340 vs. 1741±382, p=0.18). Two inpatients were followed until they were SARS-CoV-2-negative. In both, PLAs, IL-6, vWF, and plasma TF remained elevated out to 28 days and PS remained reduced, suggesting that hemostatic dysregulation persists after SARS-CoV-2 is undetectable. Conclusions:We propose that localized inflammation in SARS-CoV-2+ patients results in a decrease in anticoagulant PS, through a shift of the free and C4bp-bound forms. At the same time, this inflammation causes stimulation of endothelial cells, which secrete procoagulant vWF, monocytes, which express TF and release it into plasma on microvesicles, and platelets, which form platelet-leukocyte aggregates. These changes may not return to baseline post-infection, suggesting that long-term monitoring of thrombotic risk may be necessary for SARS-CoV-2+ patients. Disclosures No relevant conflicts of interest to declare.

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Biphasic Roles for the Soluble Guanylyl Cyclase (sGC) In Platelet Activation In Mice

Blood ◽

10.1182/blood.v116.21.486.486 ◽

2010 ◽

Vol 116 (21) ◽

pp. 486-486

Author(s):

Guoying Zhang ◽

Binggang Xiang ◽

Radek C. Skoda ◽

Susan S. Smyth ◽

Xiaoping Du ◽

...

Keyword(s):

Platelet Activation ◽

Guanylyl Cyclase ◽

Conflicts Of Interest ◽

Soluble Guanylyl Cyclase ◽

Wild Type ◽

No Donor ◽

Cgmp Production ◽

Deficient Mice

Abstract Abstract 486 The role of intracellular secondary messenger cGMP in platelet activation has been controversial, with both stimulatory and inhibitory roles reported. The platelet cGMP is believed to be predominantly synthesized by soluble guanylyl cyclase (sGC), which is activated by nitric oxide (NO). To specifically determine the role of sGC-dependent cGMP synthesis in platelet function and in vivo thrombosis and hemostasis, we produced mice harboring a “floxed” sGC beta1 allele. In the “floxed” sGC beta1 mice (sGC beta1fl/fl), the exons 7 and 8 of sGC beta1 gene and an inserted Neo cassette were flanked with three LoxP sites. Platelet-specific deletion of sGC beta1fl/fl allele was accomplished through breeding of the sGC beta1fl/fl mice with pf4-Cre recombinase transgenic mice. Immunoblotting showed the complete absence of this protein in sGC beta1fl/fl/Cre platelets. Mice lacking sGC beta1 in platelets appeared to develop normally and had normal blood counts, including platelets. Blood pressure of platelet-specific sGC deficient mice was comparable to that of wild-type littermates. Inactivating the sGC beta1 gene in platelets abolished cGMP production induced by either NO donors or platelet agonists that are known to activate endogenous NO synthesis, confirming that both the platelet agonist-induced and NO donor-induced platelet cGMP production are predominantly mediated by sGC. Platelets lacking sGC exhibit a marked defect in aggregation and secretion in response to low doses of platelet agonists, collagen and thrombin. Importantly, tail-bleeding times were significantly prolonged in the platelet-specific sGC deficient mice compared with the wild-type littermates. In a FeCl3-induced carotid artery thrombosis model, time to occlusive thrombosis was prolonged in the platelet-specific sGC deficient mice, compared to wild type littermates. Thus, the agonist-stimulated sGC activation is important in promoting platelet granule secretion and aggregation. On the other hand, NO donor SNP-induced inhibition of platelet activation was abolished in sGC-deficient platelets. However, at high concentrations (>100μM), SNP inhibited platelet activation in both wild type and sGC deficient mice, indicating that both cGMP-dependent and -independent mechanisms are involved in NO donor-induced inhibition of platelet activation. Together, our data demonstrate that sGC contributes to both agonist-induced platelet activation and NO donor-induced platelet inhibition. Disclosures: No relevant conflicts of interest to declare.

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Phosphatidylserine-Mediated Platelet Clearance By Endothelium Decreases Platelet Aggregates and Procoagulant Activity in Sepsis

Blood ◽

10.1182/blood.v128.22.2538.2538 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2538-2538

Author(s):

Ruishuang Ma ◽

Xiaoming Wu ◽

Lixiu Wang ◽

Lu Zhao ◽

Yan Zhang ◽

...

Keyword(s):

Endothelial Cells ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Disease Process ◽

Procoagulant Activity ◽

Activation Markers ◽

Fibrin Formation ◽

Activated Platelets

Abstract Introduction: Disorders of coagulation are common in sepsis, with disseminated intravascular coagulation (DIC) occurring in approximately 35 % of severe cases, contributing to microvascular dysfunction and death. Intensive platelet activation in sepsis facilitates platelet aggregation, leading to the formation of microthrombi and platelet depletion. This results in the development of DIC and sepsis-associated thrombocytopenia. Therefore, platelets must be cleared locally and quickly in the early phase of activation. Previous studies mainly focused on the clearance of activated cold-stored and aging platelets as well as platelets in immune-mediated thrombocytopenia. However, platelet activation and their clearance in sepsis are poorly understood. Platelets can form aggregates with leukocytes resulting in leukocyte death, the release of extracellular traps (ETs), increased endothelial permeability, and aggravated thrombosis. This study explored an alternate pathway for platelet disposal mediated by endothelial cells (ECs) through phosphatidylserine (PS) and examined the effect of platelet clearance on procoagulant activity (PCA) in sepsis. Methods: The subjects were septic patients (n=48) and healthy controls (n=48). Platelet engulfment by ECs was observed by electron microscopy, immunofluorescence, or immunochemistry both in vitro and in animal models. The PCA of platelets was measured by clotting time, purified coagulation complex assays, and fibrin formation. Results: Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered to and were ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or integrin on ECs attenuated platelet clearance, resulting in increased platelet count in a mouse model of sepsis (p<0.05). Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets (p<0.01). Pretreatment with lactadherin increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. A large decline in PS exposure on platelets, associated platelet PCA, and PLA formation is seen in patients in remission, which could be attributed to the elimination of abnormal platelets. Conclusions: Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contributes to maintaining platelet homeostasis during acute inflammation. Antiplatelet treatment has been suggested as a novel strategy in sepsis, and we speculate that promoting efficient removal of activated and apoptotic platelets could further improve patient outcomes. Therefore, clearance of activated platelets earlier in the disease process could hasten recovery of homeostasis in circulation by eliminating catalytic platforms for the coagulation pathway, protecting blood cells from excessive activation, and restoring their normal function. Endothelium, at least in part, contributes to platelet disposal and may further improve the hypercoagulable status in inflammation. It is noteworthy that PS-mediated and lactadherin-strengthened platelet engulfment may modify coagulopathy, and thus provide a new modality for treatment of septic clotting disorders. Figure 1 Phagocytosis of platelets by endothelial cells in vitro. Figure 1. Phagocytosis of platelets by endothelial cells in vitro. Figure 1 Effect of lactadherin-mediated phagocytosis on procoagulant activity and fibrin formation. Figure 1. Effect of lactadherin-mediated phagocytosis on procoagulant activity and fibrin formation. Disclosures No relevant conflicts of interest to declare.

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Premature Platelet Activation and Resistance to P2Y12 Inhibitors in Rasa3 Mutant Mice

Blood ◽

10.1182/blood.v124.21.91.91 ◽

2014 ◽

Vol 124 (21) ◽

pp. 91-91

Author(s):

Wolfgang Bergmeier ◽

David S Paul ◽

Lucia Stefanini ◽

Raymond F. Robledo ◽

E. Ricky Chan ◽

...

Keyword(s):

Platelet Activation ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Small Gtpase ◽

Nucleotide Exchange ◽

P2y12 Inhibitors ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

Abstract The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the guanine nucleotide exchange factor CalDAG-GEFI and an unknown regulator operating downstream of the ADP receptor, P2Y12, the target of antithrombotic therapy. Here we provide evidence that the GTPase-activating protein, RASA3, is a critical inhibitor of platelet activation and the missing link in the P2Y12/RAP1 signaling pathway. Genetic inactivation of Rasa3 led to premature activation and markedly reduced lifespan of circulating platelets in mice (t1/2=14 hrs vs. 55 hrs in controls). The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Importantly, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Thus, constitutively active RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling. At sites of vascular injury, P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation. Our findings provide critical mechanistic insights for the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders. Disclosures No relevant conflicts of interest to declare.

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Phosphatidylinositol-3,4-Bisphosphate-Akt Signaling Pathway Promotes Platelet Activation

Blood ◽

10.1182/blood-2018-99-115066 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1131-1131

Author(s):

Jasna Marjanovic ◽

Brad Rumancik ◽

Luke Weber ◽

Felix Wangmang ◽

Dane Fickes ◽

...

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Conflicts Of Interest ◽

Glycogen Synthase ◽

Thrombus Formation ◽

Type I ◽

Dependent Manner ◽

Wild Type ◽

Phosphorylated Akt

Abstract Phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2) is a messenger that accumulates in platelets in a phosphoinositide 3-kinase and platelet aggregation-dependent manner. PtdIns(3,4)P2 is broken down by inositol polyphosphate 4-phosphatases, type I (INPP4A) and type II (INPP4B). These enzymes do not catalyze hydrolysis of phosphoinositides other than PtdIns(3,4)P2, and therefore provide unique means for studying the role of this lipid in platelet activation. We have found that the dominant isoform of 4-phosphatases expressed in platelets is INPP4A and we have generated radiation chimera mice with the deficiency in INPP4A restricted to hematopoietic cell lineage. Compared to wild type platelets, agonist-stimulated INPP4A-deficient platelets accumulated higher levels of PtdIns(3,4)P2. An increase in platelet aggregation in INPP4A-deficient platelets was observed with all tested agonists. To study platelet function in vivo, we performed carotid artery injury mouse thrombosis model experiments. Time to occlusion was dramatically reduced in mice with INPP4A deficiency. These data support the hypothesis that by regulating PtdIns(3,4)P2 levels, INPP4A downregulates platelet aggregation and thrombus formation. To investigate mechanisms mediating INPP4A-dependent signals, we compared levels of phosphorylated Akt and phosphorylated glycogen synthase kinase (GSK) in wild type and INPP4A-deficient platelets in response to agonist stimulation. An increase in phospho-Akt levels was observed in INPP4A-deficient platelets, suggesting that in addition to its well-characterized regulator, PtdIns(3,4,5)P3, PtdIns(3,4)P2 can promote Akt activation. Interestingly, this was not accompanied by a significant increase in phospho-GSK levels, suggesting a possible novel mechanism involved in platelet aggregation. Disclosures No relevant conflicts of interest to declare.

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Apoptosis Signal-Regulating Kinase (ASK1) Regulates Thrombosis in Part By Regulating cPLA2 phosphorylation-Dependent TxA2 Generation

Blood ◽

10.1182/blood.v128.22.3719.3719 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3719-3719

Author(s):

Pravin Patel ◽

Meghna U. Naik ◽

Ulhas Naik

Keyword(s):

Map Kinase ◽

Platelet Activation ◽

Platelet Function ◽

Map Kinases ◽

Conflicts Of Interest ◽

Cellular Stress ◽

Substantial Reduction ◽

Genetic Ablation ◽

Independent Mechanism

Abstract When vascular endothelium is injured, circulating platelets are activated by primary agonists. Activation causes platelets to change shape, aggregate, and release secondary agonists which reinforce initial platelet activation as well as help recruit additional platelets to the site of vascular injury. MAP kinases have been shown to be important regulators of platelet function and secondary agonist production. One important secondary agonist released by activated platelets is TxA2. TxA2 is generated by metabolism of Arachidonic acid (AA). AA is released from platelet membrane phospholipids via the activity of PLAs. In platelets cPLA2 activity has been shown to be regulated by MAP kinases, however, the mechanisms which regulate platelet MAP kinase activity are not well understood. Our laboratory has identified that ASK1 (a Ser/Thr kinase of the MAP3K family) is present in both human and murine platelets and is activated by physiological agonists. ASK1 is known to be activated by a number of cellular stress response pathways. When challenged by cellular stress, ASK1 auto phosphorylates Thr845 on its activation loop, which is required for its ability to phosphorylate its substrates. Here we show that ASK1 regulates platelet function in part by regulating agonist-induced TxA2 generation. To determine the role of Ask1 in hemostasis and thrombosis, we evaluated in vivo thrombosis using carotid artery injury induced by 10% FeCl3 or pulmonary thromboembolism induced by injecting mixture of collagen/epinephrine. We found that genetic ablation of Ask1 renders mice significant protection from thrombosis. To determine the mechanism by which Ask1 regulates platelet activation leading to thrombosis, we evaluated the MAP kinase cascade using Ask1 null platelets. We found that genetic ablation of Ask1 blocked agonist-induced activation of the MAP2Ks (MKK3 and MKK4) in murine platelets. Since MKK3 can activate p38 and MKK4 can activate both p38 and JNK, we assessed MAPKs activation in murine platelets. When stimulated by various agonists, activation of p38 was entirely lost in Ask1 null platelets while activation of ERK1/2 and JNK remained unaffected indicating that Ask1 solely regulates p38 activity in platelets. Activity of p38 has been linked to agonist-induced generation of TxA2, an important contributing factor to thrombosis. We therefore evaluated agonist-induced production of TxA2 by measuring TxB2 (a stable metabolite of TxA2). We saw a substantial reduction (~50% in thrombin- and ~70% in convulxin-induced) production of TxA2 in Ask1 null platelets suggesting a separate Ask1 independent mechanism for TxA2 generation. Since TxA2 is a metabolite of AA, whose production in platelets is caused by cPLA2 enzymatic activity and cPLA2 activity is regulated by phosphorylation of its Ser505 residue by p38, we evaluated phosphorylation of cPLA2 (p-Ser505). We found that agonist-induced phosphorylation of cPLA2 (Ser505) was completely lost in Ask1 null platelets. Although in Ask1 null platelets cPLA2 phosphorylation (Ser505) is completely abolished, substantial amount (~50%) of TxA2 was generated in response to thrombin suggesting that there exists an Ask1 independent mechanism of activation of cPLA2. To rule out the possibility that an alternative PLA2 is responsible for the residual TxA2 production found in Ask1 null platelets, we evaluated agonist-induced TxA2 production in the presence of pyrrophenone, a cPLA2 specific inhibitor. Pretreatment with pyrrophenone completely abolished agonist-induced TxA2 production in murine as well as human platelets, suggesting that cPLA2 is solely responsible for the majority of agonist induced AA/TxA2 in platelets. In addition to its phosphorylation, it is documented that cPLA2 activity is also dependent on intracellular Ca2+, which facilitates translocation of cPLA2 to AA containing membranes. It is therefore possible that the remainder of TxA2 formed is dependent on Ca2+-dependent activity of cPLA2. Taken together these in vivo and in vitro results strongly suggest that ASK1 plays a key role in regulating thrombosis, in part, by regulating the signaling mechanisms involved in agonist-induced production of TxA2. Disclosures No relevant conflicts of interest to declare.

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