Platelet-leukocyte Aggregates Research Articles

Hyperglycemia enhances arsenic-induced platelet and megakaryocyte activation

ObjectiveLow to moderate inorganic arsenic (iAs) exposure is independently associated with cardiovascular disease (CVD), particularly for patients with diabetes mellitus (DM). The mechanism of increased CVD risk from iAs exposure in DM has not been adequately characterized. We evaluated whether increasing concentrations of glucose enhance the effects of iAs on platelet and megakaryocyte activity, key steps in atherothrombosis.MethodsHealthy donor whole blood was prepared in a standard fashion and incubated with sodium arsenite in a range from 0 to 10 µM. iAs-induced platelet activation was assessed by platelet receptor CD62P (P-selectin) expression and monocyte-platelet and leukocyte-platelet aggregation (MPA and LPA, respectively) in the presence of increasing sodium arsenite and glucose concentrations. Megakaryocyte (Meg-01) cell adhesion and gene expression was assessed after incubation with or without iAs and increasing concentrations of d-glucose.ResultsPlatelet activity markers increased significantly with 10 vs. 0 µM iAs (P < 0.05 for all) and with higher d-glucose concentrations. Platelet activity increased significantly following co incubation of 1 and 5 µM iAs concentrations with hyperglycemic d-glucose (P < 0.01 for both) but not after incubation with euglycemic d-glucose. Megakaryocyte adhesion was more pronounced after co incubation with iAs and hyperglycemic than euglycemic d-glucose, while gene expression increased significantly to iAs only after co incubation with hyperglycemic d-glucose.ConclusionWe demonstrate that glucose concentrations common in DM potentiate the effect of inorganic arsenic exposure on markers of platelet and megakaryocyte activity. Our results support recent observational cohort data that DM enhances the vasculotoxic effects of arsenic exposure, and suggest that activation of the platelet-megakaryocyte hemostatic axis is a pathway through which inorganic arsenic confers atherothrombotic risk, particularly for patients with DM.

Platelet inhibition and increased phosphorylated vasodilator-stimulated phosphoprotein following sodium nitrite inhalation

In the presence of red blood cells (RBCs), nitrite inhibits platelets through its conversion to nitric oxide (NO) by the reductase activity of partially deoxygenated hemoglobin. Inhaled sodium nitrite is being investigated as a therapy for pulmonary hypertension. Here, we measured platelet aggregation, P-selectin expression, platelet-leukocyte aggregates and phosphorylated vasodilator-stimulated phosphoprotein (P-VASPSer239) following sodium nitrite inhalation in healthy subjects. In vitro incubation of nitrite with deoxygenated whole blood showed an increase in P-VASPSer239, which was inhibited by ODQ, a soluble guanylyl cyclase (sGC) inhibitor. Immediately and 60 min after nitrite inhalation, P-VASPSer239 increased in platelets. Platelet aggregation, P-selectin expression, platelet-monocyte and platelet-lymphocyte aggregates decreased after inhalation. In conclusion, sodium nitrite administered to healthy subjects by inhalation can inhibit platelet activation and increase P-VASPSer239 in platelets. Platelet inhibition by nitrite administration may be useful in disorders associated with platelet hyperactivity.

Complement component 5 promotes lethal thrombosis

Extracellular histones promote platelet aggregation and thrombosis; this is followed by induction of coagulation disorder, which results in exhaustion of coagulation factors. Complement component 5 (C5) is known to be associated with platelet aggregation and coagulation system activation. To date, the pathological mechanism underlying liver injury has remained unclear. Here, we investigated whether C5 promotes liver injury associated with histone-induced lethal thrombosis. C5-sufficient and C5-deficient mice received single tail vein injections of purified, unfractionated histones obtained from calf thymus (45–75 μg/g). Subsequently, the mice were monitored for survival for up to 72 h. Based on the survival data, the 45 μg/g dose was used for analysis of blood cell count, liver function, blood coagulation ability, and promotion of platelet aggregation and platelet/leukocyte aggregate (PLA) production by extracellular histones. C5-deficient mice were protected from lethal thrombosis and had milder thrombocytopenia, consumptive coagulopathy, and liver injury with embolism and lower PLA production than C5-sufficient mice. These results indicate that C5 is associated with coagulation disorders, PLA production, and embolism-induced liver injury. In conclusion, C5 promotes liver injury associated with histone-induced lethal thrombosis.

Reduced platelet hyper-reactivity and platelet-leukocyte aggregation after periodontal therapy

BackgroundPlatelets from untreated periodontitis patients are hyper-reactive and form more platelet-leukocyte complexes compared to cells from individuals without periodontitis. It is not known whether the improvement of the periodontal condition achievable by therapy has beneficial effects on the platelet function. We aimed to assess the effects of periodontal therapy on platelet reactivity.MethodsPatients with periodontitis (n = 25) but unaffected by any other medical condition or medication were included and donated blood before and after periodontal therapy. Reactivity to ADP or oral bacteria was assessed by flow cytometric analysis of membrane markers (binding of PAC-1, P-selectin, CD63) and platelet-leukocyte complex formation. Reactivity values were expressed as ratio between the stimulated and unstimulated sample. Plasma levels of soluble (s) P-selectin were determined by enzyme-linked immunosorbent assay (ELISA).ResultsBinding of PAC-1, the expression of P-selectin and CD63 in response to the oral bacterium P. gingivalis were lower at recall (1.4 ± 1.1, 1.5 ± 1.2, and 1.0 ± 0.1) than at baseline (2.7 ± 4.1, P = 0.026, 6.0 ± 12.5, P = 0.045, and 2.7 ± 6.7, P = 0.042, respectively). Formation of platelet-leukocyte complexes in response to P. gingivalis was also reduced at recall compared to baseline (1.2 ± 0.7 vs. 11.4 ± 50.5, P = 0.045). sP-selectin levels were significantly increased post-therapy.ConclusionsIn periodontitis patients, the improvement of the periodontal condition is paralleled by a reduction in platelet hyper-reactivity. We suggest that periodontal therapy, as an intervention for improved oral health, can facilitate the management of thrombotic risk, and on the long term can contribute to the prevention of cardiovascular events in patients at risk.Trial registrationCurrent Controlled Trials identifier ISRCTN36043780. Retrospectively registered 25 September 2013.

Activated Platelets Induce Endothelial Cell Activation via an Interleukin-1β Pathway in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is associated with the premature development of cardiovascular disease. The platelet-endothelium interaction is important in the pathogenesis of cardiovascular disease. In this study, we investigated the platelet phenotype from patients with SLE and matched controls, and their effect on endothelial cells. Platelet aggregability was measured in 54 SLE subjects off antiplatelet therapy (mean age 40.1±12.8 years; 82% female; 37% white) with age- and sex-matched controls. Platelets were coincubated with human umbilical vein endothelial cells (HUVECs) and changes to gene expression assessed by an RNA array and quantitative reverse transcription polymerase chain reaction. SLE disease activity index ranged from 0 to 22 (mean 5.1±3.9). Compared with controls, patients with SLE had significantly increased monocyte and leukocyte-platelet aggregation and platelet aggregation in response to submaximal agonist stimulation. An agnostic microarray of HUVECs cocultured with SLE platelets found a platelet-mediated effect on endothelial gene pathways involved in cell activation. Sera from SLE versus control subjects significantly increased (1) activation of control platelets; (2) platelet adhesion to HUVECs; (3) platelet-induced HUVEC gene expression of interleukin-8, and intercellular adhesion molecule 1; and (4) proinflammatory gene expression in HUVECs, mediated by interleukin-1β-dependent pathway. Incubation of SLE-activated platelets with an interleukin-1β-neutralizing antibody or HUVECs pretreated with interleukin-1 receptor antibodies attenuated the platelet-mediated activation of endothelial cells. Platelet activity measurements and subsequent interleukin-1β-dependent activation of the endothelium are increased in subjects with SLE. Platelet-endothelial interactions may play a role in the pathogenesis of cardiovascular disease in patients with SLE.

Abstract 192: The Relationship of Platelet-leukocyte Aggregates and Early Brain Injury After Subarachnoid Hemorrhage

Introduction: Acute brain injury incurred after aneurysm rupture in subarachnoid hemorrhage (SAH) is a major predictor of poor functional outcome. We hypothesize that platelet-leukocyte aggregates (PLA) form early after SAH and contribute to acute brain injury. Methods: A prospective study of antiplatelet-naive SAH patients and controls (patients with unruptured aneurysms undergoing repair) was conducted from 3/2014-3/2016. Platelet-monocyte, platelet-lymphocyte and platelet-neutrophil aggregates in whole blood were measured with and without exposure to a platelet agonist (Thrombin receptor activating peptide [TRAP]) using flow cytometry. PLA within 24h and averaged over 72h from ictus (prior to the onset of delayed cerebral ischemia/vasospasm) were compared between patients with mild (admission Hunt-Hess [HH] 1-3) versus severe early brain injury (EBI; HH 4-5). Results: We enrolled 60 SAH patients and 13 controls. PLA were significantly lower in those with severe EBI compared to those with mild EBI (Platelet-monocyte-aggregates 36% versus 53%, P=0.011; Platelet-neutrophil-aggregates 15.2 versus 23.1%, P=0.002) within 24h of ictus and prior to aneurysm repair and remained significantly lower over 72h (both P&lt;0.05). Platelet-monocyte, platelet-neutrophil and platelet-lymphocyte aggregates were also significantly lower in those with severe EBI compared to controls (all P&lt;0.05). The ability of platelets to be stimulated/activated by TRAP to form PLA was also lower in severe EBI patients compared to mild EBI and control patients over 72h (platelet-neutrophil-aggregates 79.7, 88.2 and 92.7%, respectively, P=0.003; platelet-lymphocyte aggregates 9.2, 11.0 and 14.6%, respectively, P=0.022), consistent with prior platelet activation/degranulation. Conclusions: PLA are lower, and respond less to stimulation in patients with severe EBI after SAH compared to those with mild EBI and controls. These data suggest that in severe EBI: PLA may form earlier and are cleared, are adherent to endothelium and not shed in the blood, or have migrated into the parenchyma. These hypotheses bear further study.

Abstract WP447: Increased Platelet- and PMN-derived Extracellular Vesicles in Mice With Type 2 Diabetes

Introduction: Extracellular vesicles (EV) including microvesicles, microparticles and exosomes are now being recognized as key players in cardiovascular/metabolic diseases, hemostasis and thrombosis. Platelet-derived EVs are particularly important in mediating thrombotic events, whereas monocytes-associated EVs are known to increase inflammation, which can potentiate and exacerbate platelet activation and platelet-leukocyte aggregation. However, the signature of EVs in the db/db mice with metabolic syndrome (MetS) is yet to be established. Hypothesis: EV numbers or phenotype differ between the control db/+ mice and db/db mice that are procoagulant. Methods: Platelet poor plasma (PPP) was collected from adult db/+ and db/db mice subjected to experimental stroke via distal middle cerebral artery occlusion or sham surgery. PPP was tested by flow cytometry using panels of antibodies to detect EVs derived from leukocytes (CD11b), neutrophils (Ly6G), platelets (CD41), and RBCs (Ter119), as well as those expressing the oxidized LDL receptor CD36. The rate of clot formation and maximum clot firmness of whole blood was determined using thromboelastography for the intrinsic and extrinsic pathways. Results: No significant differences were seen in total EV counts or those bearing CD36, CD11b, or Ter119 between genotypes. However, PMN and platelet-derived EV counts were higher in db/db mice compared to db/+ mice. RBC-derived EVs were higher in mice post-stroke, likely reflecting fragmentation of RBCs during the clotting process. PPP from db/db mice accelerated the coagulation of blood from db/+ mice and increase their clot strength. Conclusions: The plasma of db/db mice is procoagulant and with higher levels of platelet-derived and proinflammatory EVs, which may underlie the impaired retrograde collateral flow and poor outcome after ischemic stroke. Ongoing studies will investigate the causal relationship between EVs and the thrombophilia tendency observed in those with type 2 diabetes.

Platelets protect lung from injury induced by systemic inflammatory response

Systemic inflammatory responses can severely injure lungs, prompting efforts to explore how to attenuate such injury. Here we explored whether platelets can help attenuate lung injury in mice resulting from extracorporeal circulation (ECC)-induced systemic inflammatory responses. Mice were subjected to ECC for 30 min, then treated with phosphate-buffered saline, platelets, the GPIIb/IIIa inhibitor Tirofiban, or the combination of platelets and Tirofiban. Blood and lung tissues were harvested 60 min later, and lung injury and inflammatory status were assessed. As expected, ECC caused systemic inflammation and pulmonary dysfunction, and platelet transfusion resulted in significantly milder lung injury and higher lung function. It also led to greater numbers of circulating platelet-leukocyte aggregates and greater platelet accumulation in the lung. Platelet transfusion was associated with higher production of transforming growth factor-β and as well as lower levels of tumour necrosis factor-α and neutrophil elastase in plasma and lung. None of these platelet effects was observed in the presence of Tirofiban. Our results suggest that, at least under certain conditions, platelets can protect lung from injury induced by systemic inflammatory responses.

A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting.

Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbβ3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbβ3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability.

Variants in COX-2, PTGIS, and TBXAS1 Are Associated with Carotid Artery or Intracranial Arterial Stenosis and Neurologic Deterioration in Ischemic Stroke Patients

Eicosanoids may play a role in ischemic stroke (IS). However, the association of variants in eicosanoid genes with symptomatic carotid artery or intracranial arterial stenosis and neurologic deterioration (ND) is not fully understood. The aim of the present study was to investigate the association of 11 variants in eicosanoid genes with symptomatic carotid artery or intracranial arterial stenosis and ND. Eleven variants in eicosanoid genes were examined using mass spectrometry method in 297 IS patients. The symptomatic carotid artery or intracranial arterial stenosis was assessed by computed tomographic angiography. Platelet aggregation and platelet-leukocyte aggregates were measured. The primary outcome was ND within 10 days of admission. ND was defined as an increase of 2 or more points in National Institutes of Health Stroke Scale score. Among 297 IS patients, 182 (61.3%) cases had symptomatic carotid artery or intracranial arterial stenosis, and 88 (29.6%) patients experienced ND within 10 days after admission. Symptomatic carotid artery or intracranial arterial stenosis was significantly associated with higher ND (P < .001). Rs20417CC, rs41708TT, and rs5629CC were independent risk factors for symptomatic carotid artery or intracranial arterial stenosis and ND, and associated with higher platelet aggregation and platelet-leukocyte aggregates. Symptomatic carotid artery or intracranial arterial stenosis was associated with higher ND. Rs20417CC, rs41708TT, and rs5629CC were not only independent risk factors for symptomatic carotid artery or intracranial arterial stenosis, but also independent risk predictors for ND.

Platelet, monocyte and neutrophil activation and glucose tolerance in South African Mixed Ancestry individuals

Platelet activation has been described in patients with chronic inflammation, however in type 2 diabetes mellitus it remains controversial. We compared levels of platelet leucocyte aggregates, monocyte and granulocyte activation across glucose tolerance statuses in mixed ancestry South Africans. Individuals (206) were recruited from Bellville-South, Cape Town, and included 66% with normal glucose tolerance, 18.7% pre-diabetes, 8.7% screen-detected diabetes and 6.3% known diabetes. Monocyte and neutrophil activation were measured by calculating the percentage of cells expressing CD142 and CD69 while platelet monocyte aggregates were defined as CD14++ CD42b+ events and platelet neutrophil aggregates as CD16++ CD42b+ events. The percentage of monocytes and neutrophils expressing CD69 and CD142 was significantly higher in known diabetes and prediabetes, but, lowest in screen-detected diabetes (both p ≤ 0.016). The pattern was similar for platelet monocyte and neutrophil aggregates (both p ≤ 0.003). In robust linear regressions adjusted for age and gender, known diabetes was significantly and positively associated with the percentage of monocytes expressing CD69 [beta 11.06 (p = 0.016)] and CD42b (PMAs) [19.51 (0.003)] as well as the percentage of neutrophils expressing CD69 [14.19 (<0.0001)] and CD42b [17.7 (0.001)]. We conclude that monitoring platelet activation in diagnosed diabetic patients may have a role in the management and risk stratification.

Targeting factor VIII expression to platelets for hemophilia A gene therapy does not induce an apparent thrombotic risk in mice

Background Targeting factor (F) VIII expression to platelets is a promising gene therapy approach for hemophilia A, and is successful even in the presence of inhibitors. It is well known that platelets play important roles not only in hemostasis, but also in thrombosis and inflammation. Objective To evaluate whether platelet-FVIII expression might increase thrombotic risk and thereby compromise the safety of this approach. Methods In this study, platelet-FVIII-expressing transgenic mice were examined either in steady-state conditions or under prothrombotic conditions induced by inflammation or the FV Leiden mutation. Native whole blood thrombin generation assay, rotational thromboelastometry analysis and ferric chloride-induced vessel injury were used to evaluate the hemostatic properties. Various parameters associated with thrombosis risk, including D-dimer, thrombin-antithrombin complexes, fibrinogen, tissue fibrin deposition, platelet activation status and activatability, and platelet-leukocyte aggregates, were assessed. Results We generated a new line of transgenic mice that expressed 30-fold higher levels of platelet-expressed FVIII than are therapeutically required to restore hemostasis in hemophilic mice. Under both steady-state conditions and prothrombotic conditions induced by lipopolysaccharide-mediated inflammation or the FV Leiden mutation, supratherapeutic levels of platelet-expressed FVIII did not appear to be thrombogenic. Furthermore, FVIII-expressing platelets were neither hyperactivated nor hyperactivatable upon agonist activation. Conclusion We conclude that, in mice, more than 30-fold higher levels of platelet-expressed FVIII than are required for therapeutic efficacy in hemophilia A are not associated with a thrombotic predilection.

Flow Cytometry Protocols for Assessment of Platelet Function in Whole Blood.

Flow cytometry is a powerful tool for rapid evaluation of multiple functional properties of large numbers of platelets in whole blood. In the following chapter, we provide a number of flow cytometry-based protocols broadly aimed at (1) assessment of constitutively expressed platelet membrane receptors to diagnose inherited platelet bleeding disorders and (2) investigation of basal and agonist-induced platelet functional responses including generation of platelet-leukocyte aggregates, alpha and dense granule release, calcium flux, and phosphatidylserine exposure.

Platelet toll-like receptors in thromboinflammation.

Besides their undiscussed role in hemostasis and thrombosis, platelets are also key effector cells capable of assisting and modulating inflammatory reactions and immune responses. Platelets play a sentinel role in immune surveillance by recognizing danger signals from pathogens and cell damage through the expression of toll-like receptors (TLRs) on its surface and internal compartments. Platelets express all 10 TLRs transcripts and its signalosome, including adaptor proteins and transcription factors. The activation of these receptors in platelets triggers hemostatic and inflammatory responses which participate in the host's response to bacterial and viral infections, linking thrombosis with infection and immunity. Among the responses elicited by the activation of platelet TLRs are platelet adhesion and aggregation, formation of mixed platelet-leukocyte aggregates, expression and secretion of cytokines and quemokines, and thrombin generation. TLRs also are expressed in megakaryocytes, and their activation regulates not only platelet biogenesis, but also pro-inflammatory and antiviral responses. This review will focus on work that has shown the role of platelet TLRs, mainly in thromboinflammatory responses elicited by platelets and megakaryocytes.

Platelets from Fresh and Cold-Stored Whole Blood Contribute to Clot Formation after Transfusion in Rats with Acute Traumatic Coagulopathy

Background: The hemostatic and platelet functions of fresh whole blood (FWB) are well preserved when stored at 4°C for 14 days (human) or 7 days (rats). Resuscitation of either FWB or whole blood stored for 7-days at 4°C (SWB) in rats with acute traumatic coagulopathy (ATC) induced by polytrauma and hemorrhage (T/H), shows identical correction of hemodynamic and metabolic function, as well as the equivalent impact on platelet aggregation in vitro. Platelet hemostatic function is influenced by platelet number, aggregation, and platelet-leukocyte interactions. Therefore, the objective of this study is to identify the in-vivo fate and activity of platelets from FWB and SWB, and determine whether transfused platelets contribute to clot formation in tissue injury after transfusion.Method: Isoflurane anesthetized Sprague-Dawley rats underwent polytrauma, followed by 40% hemorrhage. The rats were resuscitated (20% of bled volume) 1hr after trauma using either FWB or SWB (n=5 each) collected from green fluorescence (GFP) donor rats. SWB was kept in mini-blood-bag and refrigerated at 40C for 7 days, and warmed up to 370C prior to transfusion. Sham rats had no trauma, but underwent 20% blood replacement by FWB or SWB (n=4 each). Immediately after transfusion, a liver incision was made, followed by the measurement of kidney bleeding time (both standardized incisions was made by a Surgicutt adult template device). The incisional liver sample was taken 10min after injury for immunohistochemistry stained for CD61 (platelets) and GFP. Blood samples taken from donor rats (FWB and SWB) and recipient rats at baseline, immediately before resuscitation, and 0min and 10min after transfusion were analyzed by flow cytometry using CD42d, CD45, and CD62P antibodies for defining platelets, leukocytes and activated platelets respectively. Lactadherin was used to detect phosphatidylserine (PS). Platelet-leukocyte aggregates were defined as CD42d+/CD45+. The transfused and native platelet or leukocyte was differentiated by GFP signal.Results: GFP signal intensity was equally expressed in platelets derived from either FWB or SWB. Storage at 4°C led to a significant increase in number of platelets expressing CD62P (76±2% vs. 11±4%) and PS (8.6±0.7% vs. 1.1±0.4%) as compared to FWB. SWB also showed a significant rise in intensity, but not percentage, of platelet-leukocyte aggregates (45781±6235 vs. 10978±2345 MFI). After transfusion, recipient rats showed a significant elevation in the percentage of GFP+ platelets after transfusion of FWB as compared to SWB (Sham:13.0±1.0% vs. 4.9±0.4%; T/H: 17.0±0.8% vs. 6.8±0.%). Similarly, GFP+ leukocytes aggregates from FWB were three times higher than SWB (Sham: 6.15±0.5% vs. 1.9±0.5%; T/H: 4.4±0.4% vs. 1.2±0.2%). There was greater percentage of GFP+ platelet-leukocyte aggregates in both T/H and sham rats transfused with SWB than FWB (Sham: 11.7±3.3% vs. 33.8±2.3%; T/H: 9.8±1.6% vs. 47.2±5.1%). Transfusion with SWB led to a significant increase in percentage of activated GFP+ platelets in T/H rats as compared to FWB (30.0±2.7% vs. 2.5±0.5%). However, the activity of native platelets was not significantly different between SWB and FWB in T/H rats after transfusion (3.9±1.2% vs. 3.7±0.3%). The kidney bleeding time was not significantly different in T/H rats receiving FWB and SWB (131±4 vs.127±7 seconds) under equivalent mean arterial blood pressure (82±7 vs 85±9 mmHg). The clot that formed at the site of liver incision was identified by platelet aggregates stained by the CD61 antibody. Using co-localization of CD61 and GFP, we found that the platelets from both FWB and SWB equivalently incorporated into the clot at incisional site.Conclusion: The platelets from SWB are higher in platelet activation state, clearance rate and platelet-leukocyte aggregates than FWB after transfusion in both sham rats and rats with polytrauma and hemorrhage. However, platelets from both fresh and cold-stored whole blood contribute to hemostasis of tissue injury after transfusion. This study suggests that cold-stored whole blood is an alternative resource to treat trauma patients for restoration of hemostatic function. Future study is necessary to optimize the storage of whole blood to prolong the platelet survival rate and optimize hemostatic function. This project was funded by US Army Medical Research and Material Command. DisclosuresNo relevant conflicts of interest to declare.

Chronic Administration of the PDE9 Inhibitor PF-04447943 Reduces Leukocyte-Platelet Aggregates and Markers of Endothelial Activation in a Mouse Model of Sickle Cell Disease

▪Sickle cell disease (SCD), caused by a single amino change in the β-globin gene, exhibits a complex pathophysiology. Hypoxia in the microvascular venous bed leads to inflammation of the endothelium, adhesion of neutrophils, and a decrease in neutrophil rolling and flow velocity. The cell aggregates become trapped in the vasculature through interactions with endothelial cells. The adhesive interactions of the sickle erythrocytes, leukocytes, and endothelial cells obstruct the vasculature leading to vaso-occlusion. The vaso-occlusion can result in endothelial cell dysfunction and inflammation. Soluble adhesion molecules, including soluble E-Selectin, are associated with disease severity (Kato et al, Br.J.Hematol, 2005). PF-04447943, a selective inhibitor of the cyclic guanosine monophosphate-specific phosphodiesterase-9A, is currently in clinical development for the prophylactic treatment of SCD. The Townes mouse model of SCD exhibits many of the pathological symptoms observed in patients. Previously, we demonstrated vaso-occlusion in the Townes model by intravital microscopy (IVM), and that a single dose of PF-04447943 reduced leukocyte-platelet aggregates and the adhesion of neutrophils to the endothelium. Here we examined the pharmacodynamic effects of chronic repeat dosing with PF-04447943 in male Townes sickle cell mice.Mice were dosed orally with PF-04447943 twice daily for 4 weeks in the presence and absence of hydroxyurea (HU). Treatment effects were evaluated in vivo using IVM after 4 weeks. Surgical exposition of the cremaster muscle elicits inflammation-induced vaso-occlusion in the mouse vasculature and neutrophil rolling, adhesion and neutrophil-platelet aggregates were analyzed using IVM. Administration of PF-04447943 (10 mg/kg) in combination with HU (25 mg/kg) showed a reduction in neutrophil adhesion, an increase in neutrophil rolling on the microvasculature, and a reduction in neutrophil-platelet aggregates compared to vehicle alone. A 50% reduction in neutrophil platelet aggregates was observed when 10 mg/kg of PF-04447943 was administered as a single agent (50%), but this effect was more pronounced with co-administration of PF-04447943 and HU (73%). Significant reductions (11.1%) were observed in soluble plasma E-selectin levels in mice treated with a combination of PF-04447943 and HU for 28 days compared to vehicle treated mice. Analysis of hematological parameters showed a 42% reduction in total peripheral white blood cell count in mice that received a co-administration of PF-04447943 and HU. A small but significant increase was seen in the mean corpuscular hemoglobin (MCH) as well as mean corpuscular volume (MCV).In conclusion, the phosphodiesterase-9A inhibitor PF-04447943 results in reductions in leukocyte-platelet aggregates and soluble E-selectin in a chronic treatment study in SCD mice. Long term treatment with PF-04447943 may be beneficial in improving pharmacodynamic parameters, as well as markers of inflammation that may mediate vaso-occlusion associated with SCD.All experiments were within guidelines and were reviewed and approved by Pfizer Institutional Animal Care and use Committee. DisclosuresJasuja:Pfizer: Employment. Murphy:Pfizer: Employment. Pittman:Pfizer: Employment.

Deletion of mPGES-1 affects platelet functions in mice.

Microsomal prostaglandin E2 synthase-1 (mPGES-1) constitutes an essential player in inflammation and is involved in the pathogenesis of rheumatoid arthritis. Platelets participate in the regulation of inflammatory processes by the release of proinflammatory mediators and platelet-derived microparticles (PMPs). However, the role of the inducible mPGES-1/PGE2 pathway in platelet functions has not been investigated. In the present study we report a significant impact of mPGES-1 on platelet functions during inflammation. Wild-type (WT) and mPGES-1-/- knockout (KO) mice were stimulated with lipopolysaccharide (LPS) for 24h. Platelet counts and activation were assessed by flow cytometry analysing CD62P-CD154 expression, PMP numbers, platelet-leukocyte aggregates and platelet aggregation. The accumulation of platelets and fibrinogen in the liver was analysed by immunofluorescent staining. In native platelets from WT and mPGES-1 KO mice, there were no differences among the investigated functions. After LPS treatment, the number of platelets was significantly decreased in WT, but not in KO mice. Platelet activation, platelet-leukocyte aggregates and PMP numbers were all significantly lower in KO mice compared with WT mice after LPS treatment. In addition, KO mice displayed a significant reduction in platelet aggregation ex vivo In the liver of LPS-stimulated WT and KO mice, there were no differences in platelet accumulation, although the percentage of total vessel area in the KO liver was significantly lower compared with the WT one. Our results demonstrate that systemic inhibition of mPGES-1 prevents platelet activation, which should have important implications with regard to the cardiovascular safety of mPGES-1 inhibitors.

The effects of hydrogen sulfide on platelet–leukocyte aggregation and microvascular thrombolysis

The volatile transmitter hydrogen sulfide (H2S) is known for its various functions in vascular biology. This study evaluates the effect of the H2S-donor GYY4137 (GYY) on thrombus stability and microvascular thrombolysis. Human whole blood served for all in vitro studies and was analyzed in a resting state, after stimulation with thrombin-receptor activating peptide (TRAP) and after incubation with 10 or 30 mM GYY or its vehicle DMSO following TRAP-activation, respectively. As a marker for thrombus stability, platelet–leukocyte aggregation was assessed using flow cytometry after staining of human whole blood against CD62P and CD45, respectively. Furthermore, morphology and quantity of platelet–leukocyte aggregation were studied by means of scanning electron microscopy (scanning EM). Therefore, platelets were stained for CD62P followed by immuno gold labeling. In vivo, the dorsal skinfold chamber preparation was performed for light/dye induction of thrombi in arterioles and venules using intravital fluorescence microscopy. Thrombolysis was assessed 10 and 22 h after thrombus induction and treatment with the vehicle, GYY, or recombinant tissue plasminogen activator (rtPA).Flow cytometry revealed an increase of CD62P/CD45 positive aggregates after TRAP stimulation of human whole blood, which was significantly reduced by preincubation with 30 mM GYY. Scanning EM additionally showed a reduced platelet–leukocyte aggregation and a decreased leukocyte count within the aggregates after preincubation with GYY compared to TRAP stimulation alone. Further on, morphological signs of platelet activation were found markedly reduced upon treatment with GYY. In mice, both GYY and rtPA significantly accelerated arteriolar and venular thrombolysis compared to the vehicle control. In conclusion, GYY impairs thrombus stability by reducing platelet–leukocyte aggregation and thereby facilitates endogenous thrombolysis.

Statin and Aspirin Pretreatment Are Associated with Lower Neurological Deterioration and Platelet Activity in Patients with Acute Ischemic Stroke

Aspirin and statin are recommended for the treatment of acute ischemic stroke. However, whether aspirin and statin pretreatment is associated with clinical outcomes has not been well addressed. This study aimed to evaluate the effect of pre-existing statin and aspirin use on platelet activation and clinical outcome in acute ischemic stroke patients. We conducted a prospective, multicenter observational study in patients with acute ischemic stroke. Platelet aggregation and platelet-leukocyte aggregates were measured on admission and during 7-10 days after admission. The primary outcome of the study was neurological deterioration (ND) within 10 days after admission. The secondary outcome was a composite of recurrent ischemic stroke, myocardial infarction, and death during the first 3 months after admission. Physical disability was evaluated using the modified Rankin Scale score at 3 months after admission. Among 1124 enrolled patients, 270 (24%) experienced ND. Higher platelet aggregation and platelet-leukocyte aggregates on admission and during 7-10 days were associated with ND. Platelet aggregation and platelet-leukocyte aggregates on admission were significantly lower in the patients with pre-existing statin or aspirin use than those without treatment. Patients with prestroke concomitant statin and aspirin treatment had significantly lower incidence of ND than those without treatment. Diabetes mellitus, fasting glucose, platelet-leukocyte aggregates, and prestroke concomitant statin and aspirin use were independently associated with ND. Prestroke concomitant statin and aspirin use is associated with lower neurological deterioration and platelet activity in patients with acute ischemic stroke.

Imaging flow cytometry in the assessment of leukocyte-platelet aggregates

Platelets are subcellular blood elements with a well-established role in haemostasis. Upon activation platelets undergo granule exocytosis, resulting in α-granule P-Selectin being expressed on the cell membrane. This allows binding of activated platelets to P-Selectin glycoprotein ligand 1 (PSGL-1) expressing leukocytes, forming leukocyte-platelet aggregates (LPAs). Whole blood flow cytometry (FCM) has demonstrated that elevated circulating LPAs (especially monocyte LPAs) are linked to atherothrombosis in high risk patients, and that activated platelet binding influences monocytes towards a pro-adhesive and pro-atherogenic phenotype. However, a limitation of conventional FCM is the potential for coincident events to resemble LPAs despite no tethering. Imaging cytometry can be used to characterize LPA formation and distinguish circulating MPAs from coincidental events. Platelets and leukocyte subsets are identified by expression of surface markers (e.g. the lipopolysachharide receptor CD14 on monocytes, glycoprotein Ib CD42b on platelets). In conventional FCM, all events with both leukocyte and platelet characteristics are designated as LPAs. However, by using an ‘internal’ mask based on the brightfield image and the fluorescent platelet identifier, imaging flow cytometry is able to distinguish leukocytes with tethered platelets (genuine LPAs) from leukocyte with coincidental, untethered platelets nearby. Mechanisms (e.g. adhesion molecules) or consequences (e.g. signal transduction) can then be separately analysed in platelet tethered and untethered leukocytes. Imaging flow cytometry therefore provides a more accurate approach for both enumeration and analysis of LPAs than conventional FCM.