Platelet CD36 Potentiates Thrombus Formation in Hyperlipidemic Conditions By Activating Redox Sensitive MAP Kinase ERK5

Abstract Atherosclerotic plaque instability is a pathological process that can lead to ischemic emergencies, such as myocardial infarction (MI) and stroke. Thrombosis in this context is promoted by circulating oxidized lipids present in LDL particles (oxLDL), which are generated during plaque formation. These particles are recognized by scavenger receptor CD36 present on platelets. CD36 binding to oxLDL lowers the threshold for platelet activation by recruitment and activation of Src kinases Fyn and Lyn, followed by signaling including generation of reactive oxygen species (ROS) through NADPH oxidase (Nox). Although these effectors are important for the prothrombotic properties of CD36, the downstream signaling that links CD36 to classic agonist-induced platelet activation pathways is incompletely defined. We hypothesize that platelet CD36 promotes thrombosis by generating specific ROS to modulate critical redox-sensitive signaling pathways in hyperlipidemia. Platelet MAP kinase ERK5 is a redox sensor that was shown to be required for optimal platelet activation in MI, a condition with greatly elevated ROS. We previously reported that oxLDL-CD36 signaling leads to the generation of the specific ROS superoxide radical anion (O2●-) and that O2●-/hydrogen peroxide (H2O2) are important mediators of platelet aggregation. Additionally, we reported that platelet ERK5 was activated by oxLDL in a CD36-dependent pathway requiring Src kinases, Nox and O2●-/H2O2 to promote platelet activation. We now report the function of platelet ERK5 in this context by performing an ex vivo microfluidic thrombosis assay in which mice whole blood is perfused over immobilized collagen. Platelet adhesion and accumulation were visualized in real time via mepacrine-tagged platelets. We found that stimulating whole blood from wild type C57Bl6 mice with oxLDL promoted platelet adhesion and accumulation by 12.9% compared to buffer stimulation (p=0.033). Whole blood from CD36 null mice showed indistinguishable platelet adhesion and accumulation when stimulated with oxLDL or buffer, suggesting CD36-dependency. We used the platelet ERK5 null mice, which was generated by crossing ERK5flox mice with PF4-cre+ mice (ERK5flox/PF4-cre+), and showed that platelet adhesion and accumulation by oxLDL was abrogated compared to control ERK5flox mice. Additionally, the in vivo relevance of platelet ERK5 activation by CD36 was determined by performing a novel collagen-mediated murine thrombosis assay. This assay is dependent on syngeneic transplantation of the epigastric artery into the carotid artery, where the collagen-rich outer adventitial layer is exposed to blood flow, thus generating a thrombus. Platelets and fibrin accumulation were visualized in real time by Rhodamine 6G and fluorophore-tagged anti-fibrin antibody, respectively. We transplanted bone marrows from donor ERK5flox mice or ERK5flox/PF4-cre+ mice into recipient atherogenic apoE null mice and generated hyperlipidemia by feeding the mice a high fat diet (HFD). ApoE null mice with ERK5flox bone marrows (apoE:ERK5floxBM) on HFD developed rapid platelet accumulation with subsequent thromboembolisms compared to apoE null mice with platelet ERK5-null bone marrows (apoE:ERK5flox/PF4-cre+BM) (p<0.01 at 10 min), demonstrating that this model is sensitive to detect a prothrombotic phenotype in diet-induced hyperlipidemia. Chow fed mice do not show potentiation of platelet accumulation in both apoE chimeras (p=0.56). Additionally, HFD-fed apoE:ERK5floxBM showed accelerated fibrin accumulation compared to apoE:ERK5flox/PF4-cre+BM, suggesting a role for CD36-ERK5 signaling in platelet procoagulant properties. Control diet-fed apoE null chimeras have indistinguishable fibrin accumulation kinetics. Subsequent studies to determine the mechanism for fibrin accumulation (via fluorophore-tagged Annexin V binding) showed that exposure of platelets to oxLDL induced dose-dependent phosphatidylserine (PS) exposure, a mechanism required for platelets to promote coagulation. Buffer or LDL controls had no effect. A CD36 blocking antibody, FA6, was able to abrogate oxLDL-mediated PS exposure compared to control antibody (p=0.041). These findings suggest that platelet CD36 potentiates thrombosis through a specific redox-regulated pathway requiring ERK5 and that ERK5 is a critical modulator of thrombosis in hyperlipidemic conditions. Disclosures No relevant conflicts of interest to declare.

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GPIb-dependent platelet activation is dependent on Src kinases but not MAP kinase or cGMP-dependent kinase

Blood ◽

10.1182/blood-2003-09-3319 ◽

2004 ◽

Vol 103 (7) ◽

pp. 2601-2609 ◽

Cited By ~ 67

Author(s):

Stuart J. Marshall ◽

Yotis A. Senis ◽

Jocelyn M. Auger ◽

Robert Feil ◽

Franz Hofmann ◽

...

Keyword(s):

Map Kinase ◽

Platelet Activation ◽

Map Kinases ◽

Thrombus Formation ◽

Critical Role ◽

Rabbit Model ◽

Guanosine Monophosphate ◽

Src Kinases ◽

Aggregate Formation ◽

No Donor

Abstract Glycoprotein Ib-IX-V (GPIb-IX-V) mediates platelet tethering to von Willebrand factor (VWF), recruiting platelets into the thrombus, and activates integrin αIIbβ3 through a pathway that is dependent on Src kinases. In addition, recent reports indicate that activation of αIIbβ3 by VWF is dependent on protein kinase G (PKG) and mitogen-activated protein (MAP) kinases. The present study compares the importance of these signaling pathways in the activation of αIIbβ3 by GPIb-IX-V. In contrast to a recent report, VWF did not promote an increase in cyclic guanosine monophosphate (cGMP), while agents that elevate cGMP, such as the nitrous oxide (NO) donor glyco–SNAP-1 (N-(β-D-glucopyranosyl)-N2-acetyl-S-nitroso-D,L-penicillaminamide) or the type 5 phosphosdiesterase inhibitor, sildenafil, inhibited rather than promoted activation of αIIbβ3 by GPIb-IX-V and blocked aggregate formation on collagen at an intermediate rate of shear (800 s-1). Additionally, sildenafil increased blood flow in a rabbit model of thrombus formation in vivo. A novel inhibitor of the MAP kinase pathway, which is active in plasma, PD184161, had no effect on aggregate formation on collagen under flow conditions, whereas a novel inhibitor of Src kinases, which is also active in plasma, PD173952, blocked this response. These results demonstrate a critical role for Src kinases but not MAP kinases in VWF-dependent platelet activation and demonstrate an inhibitory role for cGMP-elevating agents in regulating this process.

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Neutrophil Extracellular Traps Induce Platelet Adhesion and Thrombus Formation.

Blood ◽

10.1182/blood.v114.22.1345.1345 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1345-1345 ◽

Cited By ~ 4

Author(s):

Tobias Fuchs ◽

Alexander Brill ◽

Daniel Dürschmied ◽

Daphne Schatzberg ◽

John H. Hartwig ◽

...

Keyword(s):

Whole Blood ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Neutrophil Extracellular Traps ◽

Thrombin Inhibitor ◽

Human Neutrophils ◽

Dependent Manner ◽

Extracellular Traps ◽

Deep Vein

Abstract Abstract 1345 Introduction Thrombus stability is provided by very large polymers adhering to platelets and anchoring the thrombus to the vessel wall. The best described polymers are fibrin and von Willebrand Factor (VWF). Activated neutrophils and other leukocytes can form an extracellular fibrous network which is composed of DNA, histones, and granular proteins. These neutrophil extracellular traps (NETs) are present in various inflammatory diseases. In deep vein thrombosis (DVT) inflammation closely cooperates with thrombosis. Here we examine whether NETs provide a new means to support the adhesion and recruitment of platelets and whether NETs are present in DVT. Methods and Results: To study the interaction of platelets with NETs, we isolated human neutrophils, induced NET formation and perfused over the NETs human platelets in plasma or whole blood anticoagulated with the thrombin inhibitor PPACK. Microscopic analysis revealed that under flow platelets adhere avidly to NETs. Perfusion of whole blood at physiological shear resulted in formation of thrombi on NETs in a time dependent manner. Addition of DNase1 degraded NETs and removed all platelets and thrombi demonstrating their adhesion to NETs. Thrombus formation on NETs was absent if blood was supplemented with EDTA indicating the requirement for divalent cations. Perfusion of NETs with heparinized blood dismantled NETs and prevented thrombus formation. Incubation of NETs with heparin alone released histones from NETs, indicating that heparin destroys the chromatin backbone of NETs. Furthermore, immunocytochemistry revealed that NETs were able to bind platelet adhesion molecules VWF and fibronectin from human plasma. Immunohistochemical analysis of a baboon deep vein thrombus showed abundant extracellular chromatin which co-localized with fibronectin and VWF. Conclusions: We show that extracellular traps are able to promote thrombosis in vitro and are abundant in vivo in DVT. We propose that extracellular chromatin provides a new type of scaffold that promotes platelet adhesion, activation, and aggregation and may be important for thrombus initiation or stability. Disclosures No relevant conflicts of interest to declare.

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Development Of a Novel Method To Assess Neonatal Platelet Function

Blood ◽

10.1182/blood.v122.21.4740.4740 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4740-4740

Author(s):

Kristina M. Haley ◽

Michael Recht ◽

Owen J.T. McCarty

Keyword(s):

Cord Blood ◽

Platelet Function ◽

Whole Blood ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Platelet Response ◽

Primary Hemostasis ◽

Platelet Aggregometry ◽

Age Dependent ◽

Static Conditions

Background The hemostatic system is developmentally regulated, resulting in qualitative and quantitative differences in the mediators of primary and secondary hemostasis as well as fibrinolysis. Age-dependent values of pro- and anti-coagulant proteins have been determined. However, the task of defining age-dependent normal values of neonatal platelet function has been met with challenges owing to difficulties in obtaining adequate blood volumes for functional assays and inconsistent results amongst varying testing methods. In order to overcome many of these challenges, cord blood is often used as a source of neonatal platelets. Platelet aggregometry comparing adult and cord blood derived platelets has demonstrated a near lack of platelet response to epinephrine, collagen, and thromboxane in cord blood samples. In contrast, other studies of platelet function, such as flow cytometry, have failed to demonstrate this phenotypic difference. Assays of primary hemostasis reveal that neonatal blood mediates primary hemostasis as effectively as adult blood. In order to overcome the challenges associated with studying neonatal platelets, we have developed a novel platelet function assay employing small volumes of blood obtained directly from the neonate in order to assess platelet adhesion, activation, and aggregation simultaneously. Methods Eight-well slide chambers were coated with either fibrillar collagen or fibrinogen and allowed to adsorb at room temperature for one hour. Blood was obtained from healthy adult controls via venipuncture and neonatal samples via heelstick into sodium citrate. The blood was separated into two 200 µl aliquots, and TRAP (Thrombin Receptor Activating Peptide: 30 mM) was added to one aliquot. 100 µl of plain whole blood was added to both a collagen and a fibrinogen coated well and 100 µ of whole blood plus TRAP was added to a fibrinogen coated well. The samples were then incubated at 37°C for 30 minutes. Non-adherent cells were washed three times with modified HEPES-Tyrode buffer. FITC-P-selectin was then added (10 µg/ml), and the samples were incubated at 37 oC for 10 minutes and subsequently washed. Samples were imaged with differential interference contrast (DIC) and fluorescence microscopy on a Zeiss Axiovert 200 M microscope. Results Platelet adhesion, activation, and aggregation were assessed for 3 neonatal samples and 3 adult control samples. Both adult and neonatal platelets adhered to fibrinogen and collagen equally. Exposure to collagen and fibrinogen (+/- TRAP) resulted in alpha granule release and P-selectin expression in both neonatal and adult platelets. In addition, both adult and neonatal platelets were observed to undergo the characteristic cytoskeletal changes that result in platelet spreading on fibrinogen (+/- TRAP) and collagen surfaces. Both neonatal and adult platelets were observed to form platelet aggregates on both surfaces under static conditions. (Figure 1) Conclusions We have successfully developed a novel platelet function assay using small volumes of whole blood to assess three key platelet functions: adhesion, activation, and aggregation. This is the first study to demonstrate that neonatal platelets spread on adhesive and extracellular matrix proteins and suggests that neonatal platelets contain the cytoskeletal machinery necessary to undergo this change in platelet formation. This assay fills a critical need in clinical pediatric hematology where efforts to diagnose and treat neonatal platelet dysfunction are often met with technical challenges related to conventional platelet function assays. Disclosures: No relevant conflicts of interest to declare.

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ASK1, a Novel Regulator of Thrombosis, Is Activated Downstream of G-Protein Coupledreceptors in Human Platelets

Blood ◽

10.1182/blood.v124.21.4163.4163 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4163-4163

Author(s):

Randall Derstine ◽

Meghna Ulhas Naik ◽

Ramya Turaga ◽

Ulhas P Naik

Keyword(s):

Map Kinase ◽

G Protein ◽

Platelet Activation ◽

Molecular Mechanisms ◽

Conflicts Of Interest ◽

Human Platelets ◽

P2y12 Receptor ◽

Kinase Signaling ◽

Dense Granules ◽

Map Kinase Signaling

Abstract In the event of vascular injury, platelets rapidly adhere to sub-endothelial matrix proteins such as collagen and Von Willebrand factor and activate to form a stable hemostatic platelet plug. Defects in the molecular mechanisms dictating platelet plug formation are responsible for numerous thrombotic disorders. Elucidating the signaling pathways and molecular mechanisms of platelet activation is paramount to the development of safer and more effective anti-thrombotic drugs. While it is known that MAP-Kinase signaling participates in platelet activation, it is unknown how MAP-Kinase signaling specifically mediates platelet activation. Our laboratory has identified the presence and activation of a MAP-Kinase Kinase Kinase known as Apoptosis Signal Regulating Kinase 1 (ASK1). We have demonstrated using an ASK1 knockout mouse model that ablation of ASK1 leads to a significantly increased (p = .0003) time of vessel occlusion associated with unstable thrombus formation following a carotid artery injury induced by 10% FeCl3. Furthermore, ASK1 knockout mice display protection from pulmonary thromboembolism induced by an intravenous injection of collagen and epinephrine. In order to determine the kinetics of ASK1 activation by physiological agonists, washed human platelets (4 x 108 platelets/mL) were treated with 0.1 U/mL of thrombin for 30”, 1’, 3’, 5’, and 8’. Robust activation of ASK1 by thrombin occurred as early as 30 seconds up until 5 min, after which ASK1 activation decreased sharply. Platelets treated with 100 µM of PAR1 (SFLLRN) or PAR4 (AYPGKF) peptides resulted in strong ASK1 activation, suggesting that both the PAR1 and PAR4 receptors lead to ASK1 activation. Inhibition of Src family kinases by PP2 or PI3K by wortmannin or Rho kinase by Y-27632 had no effect on thrombin-induced ASK1 activation. However, inhibition of PLC-β2, a mediator of platelet activation downstream of the PAR1/4 receptors, strongly inhibited ASK1 activation by thrombin. We next determined whether TxA2 generation was responsible for ASK1 activation by thrombin. Washed platelets were pre-treated with 1 mM aspirin to block TxA2 generation, followed by treatment with 0.1 U/mL of thrombin. It was found that blocking TxA2 generation eliminated ASK1 activation by thrombin at 30” and 1’, but not at a later time point, suggesting there may be an additional pathway contributing to ASK1 activation. The observation that TxA2 generation contributes to ASK1 activation by thrombin seemed to correlate with the finding that treatment of platelets with 1 µM of the TxA2 mimetic U46619, which activates the TP-α receptor, could also activate ASK1. We also determined whether ADP released from dense granules, which would activate the P2Y1 and P2Y12 receptors, leads to ASK1 activation. To test this, washed platelets were pre-treated with 1 U/mL of apyrase to hydrolyze secreted ADP. It was found that apyrase treatment completely eliminates ASK1 activation by thrombin, suggesting a strong dependency of thrombin-induced ASK1 activation on ADP release from dense granules. To further investigate this possibility, washed platelets were pre-treated with 50 µM of the P2Y1 antagonist MRS2179 or P2Y12 antagonist 2-MeSAMP, followed by treatment with 0.1 U/mL of thrombin. Antagonism of the P2Y12 receptor and not P2Y1 receptor severely diminished ASK1 activation by thrombin. This indicates that ASK1 activation by thrombin is also dependent on ADP released from dense granules and subsequent activation of the P2Y12 receptor. Surprisingly, collagen, a strong activator of platelets, was unable to activate ASK1 in washed platelets at a concentration of 2 µg/mL. Similarly, 2 µM epinephrine treatment also had no effect. However, when washed platelets were treated with 2 µg/mL collagen and 2 µM epinephrine together, a strong ASK1 activation was observed (p=.0012). This suggests the existence of a novel mechanism for ASK1 activation by simultaneous stimulation of the collagen receptors GPVI/α2β1 and epinephrine receptor α2A. The finding that ASK1 activation occurs downstream of TP-α, P2Y12, and possibly α2A receptors highlights the importance of ASK1 in regulation of these G-Protein Coupled Receptors in platelet activation. In conclusion, our data indicates ASK1 to be a key mediator in platelet activation and represents a novel target for anti-thrombotic drug therapy. Disclosures No relevant conflicts of interest to declare.

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Neutrophil but not Monocyte Activation Inhibits P-Selectin-Mediated Platelet Adhesion

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648953 ◽

1994 ◽

Vol 72 (05) ◽

pp. 750-756 ◽

Cited By ~ 15

Author(s):

Henry M Rinder ◽

Jayne L Tracey ◽

Christine S Rinder ◽

David Leitenberg ◽

Brian R Smith

Keyword(s):

Platelet Activation ◽

Whole Blood ◽

Platelet Adhesion ◽

Platelet Activating Factor ◽

Leukocyte Activation ◽

Monocyte Activation ◽

Activated Platelets ◽

Simultaneous Activation ◽

Cell Fractions

SummarySelectins are Ca2+-dependent glycoprotein receptors that mediate the adhesion of activated platelets or endothelial cells to unstimulated leukocytes. Using purified cell fractions, we examined activated neutrophil adhesion to P-selectin-expressing platelets and found that phorbol 12-myristate 13-acetate (PMA), platelet activating factor C16 (PAF), and n-formyl-met-leu-phe (fMLP) pretreatment of neutrophils inhibited activated platelet adhesion. Furthermore, PMA and PAF were capable of dissociating established resting neutrophil-activated platelet conjugates. Since L-selectin is downregulated after leukocyte activation and has been postulated as a ligand for P-selectin, we preincubated resting neutrophils with Dreg-2 and Dreg-56, blocking monoclonal antibodies (MoAb) to L-selectin; these MoAb failed to inhibit activated platelet adhesion. To more closely approximate in vivo conditions of leukocyte and platelet activation, we also employed a whole blood (WB) model of leukocyte-platelet adhesion. We found that simultaneous activation of both platelets and leukocytes by PMA caused an immediate rise in the % of P-selectin-positive platelets accompanied by a rapid increase in monocyte-platelet and neutrophil-platelet conjugates; however, the % of neutrophil-platelet conjugates subsequently declined over 30-60 min to baseline levels while monocyte-platelet adhesion remained elevated over 90 min. By contrast, selective platelet activation in WB by thrombin resulted in an increase in platelet P-selectin expression accompanied by a sustained (90 min) elevation in both monocyte- and neutrophil-platelet conjugates. This increase in leukocyte-platelet conjugates after thrombin was not inhibited by preincubation of WB with Dreg-2 or Dreg-56. We conclude that neutrophil activation decreases the expression of the ligand for platelet P-selectin within 30-60 min resulting in inhibition of neutrophil-platelet adhesion and dissociation of existing neutrophil-platelet conjugates. By contrast, monocyte activation over 90 min does not affect monocyte adhesion to activated platelets in whole blood.

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Platelet Glycoprotein VI Dimerisation Is An Active Process and Enables the Receptor to Be Competent

Blood ◽

10.1182/blood.v116.21.3194.3194 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3194-3194 ◽

Cited By ~ 1

Author(s):

Stéphane Loyau ◽

Bénédicte Dumont ◽

Nadine Ajzenberg ◽

Martine Jandrot-Perrus

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Platelet Adhesion ◽

Transmembrane Domain ◽

Conflicts Of Interest ◽

Blood Platelets ◽

Matrix Proteins ◽

Platelet Glycoprotein ◽

Active Process ◽

Platelet Surface

Abstract Abstract 3194 In the blood, platelets are normally prevented from activation by endothelial inhibitors (i.e. prostacycline, ectonucleotidase). Dysfunctional endothelial cells loose their protective properties and favor platelet adhesion to matrix proteins, platelet aggregation and thrombus growth. Collagen fibers are highly thrombogenic and the platelet Glycoprotein (GP)VI predominantly mediates collagen-induced platelet responses. GPVI is a platelet specific receptor of the immunoglobulin (Ig) superfamily containing two extracellular Ig domains, a single transmembrane domain and a short cytoplasmic tail. GPVI signals through the immunoreceptor tyrosine-based activation motifs (ITAM) of the non-covalently associated immune receptor adaptor FcRg dimer. There is growing evidence that optimal binding of GPVI to collagen depends on the formation of GPVI dimers at the platelet surface: only dimeric GPVI binds to collagen and inhibits collagen-induced platelet aggregation and not monomeric GPVI. Moreover, crystallographic data showed dimerization of GPVI ectodomains. However, the valence of GPVI on resting and activated platelets is still debated. We have obtained an anti-human GPVI monoclonal antibody (9E18), that binds to dimeric GPVI with a 200 fold higher affinity than to monomeric GPVI. In flow cytometry on whole blood, while the 3J24 antibody labels >95% platelets, 9E18 hardly binds to resting platelets with less than 3% positive platelets. The level of 9E18-positive platelets moderately increased (10-15%) after platelet isolation suggesting it could reflect platelet activation. Binding of 9E18 was indeed significantly increased on ADP- or TRAP-activated washed platelets (25±1.9 % and 36±7% positive platelets respectively). Additionally, increased binding of 9E18 was triggered by the GPVI agonists, collagen, convulxin or the activating 9O12 IgG. At sites of vascular lesion, platelet adhesion is initiated by the shear-dependent interaction of GPIb with vWF, assumed to favor GPVI-collagen interaction. When a platelet rich plasma was submitted to a shear of 4000 s-1 for 5 min, 9E18-positive platelets increased from 3.6±1.6% to 7±2% in the whole platelet population and to 26±7.7% on small aggregates (p<0.05).When a2b1 and aIIbb3 were blocked, the relation between the 9E18 binding to stimulated platelets and platelet binding to collagen was linear (r2 = 0.847, p=0.0012, n=8). Interestingly, the cAMP elevating agent PGE1 further lowered the level of 9E18-binding to resting platelets and dropped it to basal values on ADP- or TRAP-treated platelets. Apyrase reduced by 50% TRAP-induced binding of 9E18 whereas indomethacin had no effect. PMA triggered binding of 9E18 on platelets (p<0.001) while the Tyr-phosphatase inhibitor PAO, strongly inhibited PMA-induced 9E18 binding to platelets (p<0.0019) and GPVI-dependent platelet adhesion to collagen. Altogether, these data indicate that 9E18 permit to quantify GPVI dimers on platelets. They show that (i) GPVI is mainly monomeric on resting platelets, (ii) dimerisation is an active process triggered by shear, soluble agonists and matrix proteins, (iii) the level of GPVI dimers is related to the capacity of platelets to adhere to collagen, (iv) GPVI dimerisation is completely prevented in the presence of agents increasing cAMP or by PAO. These data suggested that the formation of GPVI dimer is strictly controlled on resting platelets and that GPVI dimers could thus represent a new marker of platelet activation and susceptibility to collagen. Indeed, in a population of hospitalized patient, a positive correlation was observed between 9E18 binding and P-selectin exposure on platelets. Disclosures: No relevant conflicts of interest to declare.

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Microfluidic and Flow Cytometric Studies Support a Role for Monocytes and Coated Platelets in the Prothrombotic State in Heparin-Induced Thrombocytopenia (HIT)

Blood ◽

10.1182/blood.v118.21.539.539 ◽

2011 ◽

Vol 118 (21) ◽

pp. 539-539

Author(s):

Valerie Tutwiler ◽

Hyun Sook Ahn ◽

Douglas B. Cines ◽

Rodney M. Camire ◽

Mortimer Poncz ◽

...

Keyword(s):

Whole Blood ◽

Platelet Adhesion ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Passive Immunization ◽

Annexin V ◽

Thrombin Inhibitor ◽

Prothrombotic State ◽

Flow Cytometric ◽

Activated State

Abstract Abstract 539 HIT is an immune thrombocytopenia associated with a high risk of developing thrombosis. A passive immunization murine model of this disorder has provided important insights into the underlying pathogenesis of this disease, but is limited by its inability to study human cells and limited ability to define the contribution of various hematopoeitic and vascular cells to the prothrombotic state. We used a microfluidic system in conjunction with flow cytometry to further our understanding of the prothrombotic nature of HIT. Platelet adhesion and aggregation was studied in whole blood labeled with Calcein AM, perfused through a microfluidic channel (BioFlux 200 system, Fluxion) coated with von Willebrand factor (vWf) at shear stress of 20 dyne/cm2 at 37°C. A 40–60% increase in platelet adhesion (relative area covered by platelets) with up to a 4 fold increase in average aggregate size was seen in the presence of the pathogenic HIT-like monoclonal antibody (moAb) KKO (50 μg/ml) in conjunction with PF4 (10 μg/ml) when compared to control samples with PF4 only or with PF4 plus a non-pathogenic anti-PF4 moAb RTO (p <0.01). Monocyte-depletion decreased platelet aggregation by 20 – 40% relative to whole blood or after monocyte-repletion (P<0.0001). In HIT, thrombin plays a key role in the formation of platelet aggregates. Addition of thrombin inhibitor PPACK to the whole blood stimulated by KKO and PF4 decreased thrombus formation in the microfluidic chamber by 40% (p<0.001). Coated platelets are prothrombotic and characterized by phosphatidylserine (PS) exposure and binding of FVa and FXa. This activated state requires dual stimulation via thrombin and ITAM receptors. Flow cytometric studies of annexin V and FXa binding showed extensive induction of coated platelets in whole blood by KKO plus PF4 in contrast to PF4 or PF4 plus RTO (annexin V: p<0.0001; Factor Xa p<0.01). These new studies, focused on human blood, support our finding in the passive murine HIT model as to the importance of monocytes to thrombus formation and suggest that the prothrombotic nature of HIT may also be promoted by the generation of coated platelets. Identification of coated platelets may also lead to new diagnostic tests and new therapeutic interventions in HIT. Disclosures: No relevant conflicts of interest to declare.

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A Humanized Anti FcγRIIa Scfv Inhibits Platelet Activation, Aggregation and Thrombus Formation in Heparin-Induced Thrombocytopenia (HIT)

Blood ◽

10.1182/blood.v126.23.10.10 ◽

2015 ◽

Vol 126 (23) ◽

pp. 10-10

Author(s):

Jose Perdomo ◽

Jaa Yien New ◽

Zohra Ahmadi ◽

Xing-Mai Jiang ◽

Beng H Chong

Keyword(s):

Platelet Aggregation ◽

Platelet Activation ◽

Immune Complexes ◽

Whole Blood ◽

Conflicts Of Interest ◽

Dependent Manner ◽

Single Chain ◽

Platelet Factor ◽

Heparin Induced Thrombocytopenia ◽

Micro Fluidics

Abstract Introduction. Heparin is widely used as an anticoagulant to prevent thrombosis and to treat venous thromboembolism and myocardial infarction. A complication of heparin use is the development of heparin-induced thrombocytopenia (HIT), which is a limb- and life-threatening disorder due to associated thrombotic events. HIT arises through the formation of immune complexes between heparin, platelet factor 4 and HIT autoantibodies. These immune complexes engage with FcγRIIa receptors on platelets, leading to platelet activation and aggregation and subsequent initiation of the coagulation pathway. Current HIT treatment consists of cessation of heparin administration and substitution with parenteral anticoagulants such as argatroban and danaparoid. While these anticoagulants are generally beneficial in reducing thrombocytopenia, they are only partially effective since the risk of thrombosis continues due to the underlying FcγRIIa-mediated platelet activation. Thus, alternative anticoagulants do not reduce morbidity and mortality rates, highlighting the need for more effective HIT interventions. Methods. IV.3 is a monoclonal antibody that recognizes and blocks the FcγRIIa receptor and is used in assays to confirm the presence of HIT antibodies. We derived the VH and VL sequences of IV.3 and constructed a single-chain variable fragment (scFv) antibody in the form of VH-linker-VL. Using a complementarity determining region grafting and point mutation approach the scFv was humanized with the aim of reducing potential immunogenicity for future clinical applications. The molecule was expressed in E. coli and purified by FPLC. We reconstituted the HIT condition in a micro-fluidics device on a Vena8 Fluoro+ biochip coated with vWf using whole blood flowing at 20 dyne/cm2 at 37oC. Whole blood was stained with DiOC6 and the formation of platelet aggregates was monitored by fluorescence microscopy. Video images were acquired at 1 frame every 2 sec for 460 sec. Results. The purified scFv interacts with FcγRIIa on platelets. Platelet aggregation and serotonin release assays show that the scFv effectively prevents aggregation and activation induced by HIT immune complexes. We demonstrate that in the HIT condition reconstituted in a micro-fluidics system the scFv precludes thrombus deposition in a dose-dependent manner as determined by thrombus coverage area and mean thrombus diameter (Figure 1). Conclusions. These data provide evidence that a humanized scFv binds and neutralizes FcγRIIa on platelets. This interaction prevents HIT immune complex-induced platelet aggregation and activation in vitro and stops thrombus deposition ex vivo. This molecule, therefore, inhibits a critical initiating event in HIT and may serve as a potential treatment for this condition. 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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Sub-Optimal Inhibition of Thrombus Formation by Aspirin (as measured by RTTP analysis) In Primary Thrombocythemia

Blood ◽

10.1182/blood.v116.21.4218.4218 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4218-4218

Author(s):

Gillian Stephens ◽

Jan Tauscher ◽

Fabian Siegel ◽

David R. Phillips ◽

Patrick Andre ◽

...

Keyword(s):

Healthy Volunteers ◽

Real Time ◽

Whole Blood ◽

Conflicts Of Interest ◽

Thrombus Formation ◽

Myeloproliferative Disease ◽

Fluorescent Intensity ◽

Shear Rates ◽

Primary Thrombocythemia ◽

Platelet Deposition

Abstract Abstract 4218 Primary thrombocythemia (PT) is a myeloproliferative disease with a high incidence of thrombotic complications. First-line therapy is the inhibition of platelet aggregation with aspirin (via COX-1 inhibition) for the prevention of thromboembolic complications. However, this does not account for the potential for COX-2 expression in newly synthesized platelets. The purpose of the present study was 1) to determine whether aspirin reduces the thrombotic potential of PT patients who did not undergo cytoreductive therapy to the levels of those achieved in healthy volunteers (HV), and 2) to determine whether the presence of the JAK2V617F mutation is responsible for the elevated thrombotic potential in PT patients. We determined the thrombotic potential of a cohort of 16 PT patients and compared these values to those of 10 healthy volunteers all treated with 100mg aspirin QD. Thrombotic potential was determined using a custom built Real Time Thrombosis Profiler (RTTP). Whole blood anticoagulated with a Factor Xa inhibitor (to preserve physiological Ca++ concentration) with Rhodamine 6G-labeled platelets was perfused over a fibrillar collagen surface at shear rates approximating those in veins (100s-1), arteries (600 s-1) and moderately stenosed arteries (1600s-1). The deposition of fluorescently labeled platelets on the collagen surface was monitored in real time by fluorescence microscopy in the RTTP. Endpoint thrombosis (size of thrombi at t=300sec expressed as Mean Fluorescent Intensity/Area of coverage) and rate of thrombus growth (initial rate of platelet deposition) were recorded for each individual as measures of thrombotic potential and data are expressed as mean ± SEM (statistics performed using GraphPad Prism v4.03 using unpaired, 2-tailed Students t-test). Despite aspirin therapy, PT patients were characterized by significant increases in thrombotic potential at venous and arterial shear rates (Table 1) with a greater than 2-fold increase in the rate of platelet deposition. Interestingly only the size of thrombi formed but not the rate of formation was elevated in PT patients at moderately stenosed shear, likely a reflection of the increasing contribution of shear and decreasing dependence on absolute platelet count and other individual cellular factors in whole blood. The Disclosures: No relevant conflicts of interest to declare.

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