Utility of Platelet Endothelial Cell Adhesion Molecule 1 in the Platelet Activity Assessment in Mouse and Human Blood.

In our previous study, we introduced the platelet endothelial cell adhesion molecule 1 (PECAM-1)/thrombus ratio, which is a parameter indicating the proportion of PECAM-1 in laser-induced thrombi in mice. Because PECAM-1 is an antithrombotic molecule, the higher the PECAM-1/thrombus ratio, the less activated the platelets. In this study, we used an extracorporeal model of thrombosis (flow chamber model) to verify its usefulness in the assessment of the PECAM-1/thrombus ratio in animal and human studies. Using the lipopolysaccharide (LPS)-induced inflammation model, we also evaluated whether the PECAM-1/thrombus ratio determined in the flow chamber (without endothelium) differed from that calculated in laser-induced thrombosis (with endothelium). We observed that acetylsalicylic acid (ASA) decreased the area of the thrombus while increasing the PECAM-1/thrombus ratio in healthy mice and humans in a dose-dependent manner. In LPS-treated mice, the PECAM-1/thrombus ratio decreased as the dose of ASA increased in both thrombosis models, but the direction of change in the thrombus area was inconsistent. Our study demonstrates that the PECAM-1/thrombus ratio can more accurately describe the platelet activation status than commonly used parameters such as the thrombus area, and, hence, it can be used in both human and animal studies.

Flow Cytometric Assessment of AKT Signaling in Platelet Activation: An Alternative Diagnostic Tool for Small Volumes of Blood.

INTRODUCTION: The diagnosis of platelet function disorder in children is challenging. Light transmission aggregometry is the gold standard for platelet function disorders. However, large blood volumes are required. Currently, there are no existing tools for the diagnosis of platelet function disorders that use small blood volumes. AKT signaling plays a central role in platelet activation during hemostasis and might be visualized by flow cytometry. METHODS: Platelet-rich plasma obtained by centrifugation of citrated blood from healthy volunteers was activated with arachidonic acid, thrombin receptor activating peptide-6 (TRAP-6), collagen, adenosine diphosphate ADP, collagen-related peptide (CRP), and epinephrine. After platelet activation, the phosphorylation of AKT was assessed by flow cytometer using a Navios cytometer. RESULTS: Healthy volunteers showed a reproducible phosphorylation of AKT upon activation. In comparison to nonactivated platelets, we documented an increase in pAKT expression with all agonists. Especially TRAP-6 and CRP caused considerable increase in percentage of pAKT expression throughout all the tested healthy volunteers. CONCLUSION: An activation of the AKT-signal pathway by different agonists can clearly be detected on the flow cytometer, indicating that the visualization of signaling in platelets by flow cytometry might be an efficient alternative for light transmission aggregometry to test platelet function in children.

Intravital Assessment of Blood Platelet Function. A Review of the Methodological Approaches with Examples of Studies of Selected Aspects of Blood Platelet Function.

Platelet biology owes to intravital studies not only a better understanding of platelets' role in primary hemostasis but also findings that platelets are important factors in inflammation and atherosclerosis. Researchers who enter the field of intravital platelet studies may be confused by the heterogeneity of experimental protocols utilized. On the one hand, there are a variety of stimuli used to activate platelet response, and on the other hand there are several approaches to measure the outcome of the activation. A number of possible combinations of activation factors with measurement approaches result in the aforementioned heterogeneity. The aim of this review is to present the most often used protocols in a systematic way depending on the stimulus used to activate platelets. By providing examples of studies performed with each of the protocols, we attempt to explain why a particular combination of stimuli and measurement method was applied to study a given aspect of platelet biology.

Assessment of Platelet Thrombus Formation under Flow Conditions in Patients with Acute Kawasaki Disease.

OBJECTIVE: To assess platelet thrombus formation (PTF) under flow conditions in patients with Kawasaki disease. Previously available platelet activation data were limited for nonphysiological shear stress condition. The total thrombus-formation analysis system (T-TAS) was developed for quantitative PTF analysis. STUDY DESIGN: In total, 33 patients with acute Kawasaki disease were assessed. Whole blood samples, obtained immediately before treatment and 1 week and 1 month after treatment, were assessed using the T-TAS with a collagen-coated platelet chip under high shear values (1000 s-1 [PL12] and 2000 s-1 [PL24]). Measures, such as time to reach 5 kPa above the base pressure (T5+α) and area under the curve for flow pressure curve for 10 minutes (AUC10) were analyzed to quantify PTF. RESULTS: Immediately before treatment, the median PL12-T5+α and PL24-T5+α were 3.3 minutes (IQR 2.0-4.5) and 1.3 minutes (0.9-1.9), respectively, and both values were significantly lower in adult controls (3.5 minutes [2.9-6.4] and 2.8 minutes [1.8-4.8]; P = .015 and P < .001, respectively). In addition, the PL12-AUC10 (151.7 U [94.5-279.9]) significantly decreased in adult controls (234.1 U [110.5-306.5], P = .007). By contrast, at 1 week and 1 month after the start of treatment, the T5+α was longer, and the PL12-AUC10 and PL24-AUC10 decreased. CONCLUSIONS: In patients with acute Kawasaki disease, the PTF had an early onset and weak stability.

Influencing factors in quantitative measurement using activated platelet levels and platelet-activating capacity for the assessment of thrombosis in pre-metabolic syndrome and type 2 diabetes mellitus.

Activated platelet levels and platelet-activating capacity are well recognized as useful index parameters for the physiological and pharmacological prediction of thrombotic events. Recently, quantitative measurements for platelet functions using a flow cytometer have been increasing gradually. However, the relation of physiological factors, such as sex, aging, and laboratory tests, to platelet functions has not been well documented. We conducted a blood analysis of people with normal/pre-metabolic syndrome and patients with type 2 diabetes mellitus to clarify the pathological factors. The levels of basal (non-stimulated)-activated, platelet-expressed P-selectin and activated platelet stimulated by agonists were measured by a flow cytometer, and ratios of platelet-activating capacity were also calculated. Statistical analyses indicated significantly high basal-activated platelet in pre-metabolic syndrome, and basal-activated platelet was positively associated with hyperlipidemia and hepatic damage. Platelet-activating capacity was significantly low in aging and hyperlipidemia, but high in hyperglycemia, and was negatively associated with hyperlipidemia and hepatic damage. Aging and high nutrient condition impaired platelet functions. Quantitative measurements of basal-activated platelet and platelet-activating capacity are precise parameters for the prediction of thrombotic events.

Assessment of roles for the Rho-specific guanine nucleotide dissociation inhibitor Ly-GDI in platelet function: a spatial systems approach.

On activation at sites of vascular injury, platelets undergo morphological alterations essential to hemostasis via cytoskeletal reorganizations driven by the Rho GTPases Rac1, Cdc42, and RhoA. Here we investigate roles for Rho-specific guanine nucleotide dissociation inhibitor proteins (RhoGDIs) in platelet function. We find that platelets express two RhoGDI family members, RhoGDI and Ly-GDI. Whereas RhoGDI localizes throughout platelets in a granule-like manner, Ly-GDI shows an asymmetric, polarized localization that largely overlaps with Rac1 and Cdc42 as well as microtubules and protein kinase C (PKC) in platelets adherent to fibrinogen. Antibody interference and platelet spreading experiments suggest a specific role for Ly-GDI in platelet function. Intracellular signaling studies based on interactome and pathways analyses also support a regulatory role for Ly-GDI, which is phosphorylated at PKC substrate motifs in a PKC-dependent manner in response to the platelet collagen receptor glycoprotein (GP) VI-specific agonist collagen-related peptide. Additionally, PKC inhibition diffuses the polarized organization of Ly-GDI in spread platelets relative to its colocalization with Rac1 and Cdc42. Together, our results suggest a role for Ly-GDI in the localized regulation of Rho GTPases in platelets and hypothesize a link between the PKC and Rho GTPase signaling systems in platelet function.

Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia.

Platelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/l and group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogs in vitro varied depending on the platelet count and platelet activator.

Microfluidic approaches for the assessment of blood cell trauma: a focus on thrombotic risk in mechanical circulatory support devices.

INTRODUCTION: Mechanical circulatory support devices (MCSDs) are emerging as a valuable therapeutic option for the management of end-stage heart failure. However, although recipients are routinely administered with anti-thrombotic (AT) drugs, thrombosis persists as a severe post-implant complication. Conventional clinical assays and coagulation markers demonstrate partial ability in preventing the onset of thrombosis. Through years, different laboratory techniques have been proposed as potential tools for the evaluation of platelets' hemostatic response in MCSD recipients. Most rely on platelet aggregation tests; they are performed in static or low shear conditions, neglecting the prominent contribution of MCSD shear-induced mechanical load in enhancing platelet activation (PA). On the other hand, those tests able to account for shear-induced PA have limited possibility of effective clinical translation. AIMS AND METHODS: Advances on this side have been addressed by microfluidic technology. Microfluidic devices have been developed for AT drug monitoring under flow, able to replicate physiological and/or constant shear flow conditions in vitro. In this paper, we present a newly developed microfluidic platform able to expose platelets to MCSD-specific dynamic shear stress patterns. We performed in vitro tests circulating human platelets in the microfluidic platform and quantifying the dynamics of PA by means of the Platelet Activity State (PAS) assay. RESULTS: Our results prove the feasibility of using microfluidics for the diagnosis of MCSD-related thrombotic risk. This study paves the way for the development of a miniaturized point-of-care device for monitoring AT drug regimen. Such a system may have significant impact on limiting the incidence of thrombosis in MCSD recipients.

Assessment of Platelet Activation and Immature Platelet Fraction as Predictors of Platelet Engraftment After Hematopoietic Stem Cell Transplantation.

Delayed platelet engraftment is a well-known complication of umbilical cord blood transplantation (CBT). Megakaryocytes derived from cord blood (CB) in vitro are smaller than megakaryocytes derived from bone marrow (BM) in adults. A small megakaryocyte size might contribute to delayed megakaryocytic maturation. This study included 37 patients undergoing hematopoietic stem cell transplantation (HSCT) at Chang Gung Children's Hospital between July 2011 and June 2013. Blood samples were obtained at different times: preconditioning and post-HSCT days 56 and 97. To test whether platelet activation persists posttransplantation, two commonly used platelet activation marker antibodies, CD62P (P-selectin) and CD42b, were evaluated using whole blood flow cytometry, combining thiazole orange and anti-CD41a staining, to assess reticulated platelets. Serial peripheral blood (PB) samples were obtained posttransplantation from patients undergoing CBT (CBT group; n = 15) and mobilized peripheral blood transplantation (PBT group; n = 22). Platelet activation in the postengraftment samples was considerably higher in the PBT group than the CBT group. Moreover, immature platelet fractions (IPF) were higher in the CBT group. Our results emphasize the role of IPF for dynamic prediction of platelet engraftment in CBT.

Assessment of mean platelet volume and soluble CD40 ligand levels in patients with non-dipper hypertension, dippers and normotensives.

UNLABELLED: Abstract Objective: Patients with a lack of nocturnal decline in blood pressure (BP) are at an increased risk for cardiovascular events. Mean platelet volume (MPV) and soluble CD40 ligand (sCD40L) are accepted biomarkers of platelet activation and considered as a risk factor for cardiovascular disease. The aim of this study was to determine whether MPV and sCD40L levels are higher in non-dipper hypertensive (NDHT) patients than in dipper hypertensive (DHT) patients and healthy controls. METHODS: 124 consecutive patients were included to this study. Patients were divided into three groups: NDHT patient group [n = 43; mean age 51.8 ± 6.6; 31 males (72.1%)]; DHT patient group [n = 41; mean age 50.2 ± 7.3; 22 males (53.7%)]; and normotensive group [n = 40; mean age 49.9 ± 6.7; 22 males (55%)]. Physical examination, laboratory work-up and 24-h ABPM were performed for all participants. RESULTS: The sCD40L and MPV levels were significantly higher in the NDHT group than in the DHT and normotensive groups (p < 0.05). In correlation analysis, MPV, 24-h systolic blood pressure (SBP), 24-h diastolic blood pressure (DBP), night-time SBP and night-time DBP were positively correlated with sCD40L. CONCLUSION: Our study demonstrated that MPV and sCD40L levels were significantly higher in NDHT patients compared to DHT and normotensive patients. sCD40L levels were positively correlated with MPV, 24-h SBP, 24-h DBP, night-time SBP and night-time DBP.

Assessment of hemostaseologic alterations induced by hyperbaric oxygen therapy using point-of-care analyzers.

BACKGROUND: Exposure to hyperbaric conditions influences the coagulation system. Thromboembolic events and disseminated intravascular coagulation were observed. OBJECTIVES: To detect the effects of a hyperbaric environment on the human coagulation system using the point-of-care coagulation analyzers Multiplate and ROTEM. PATIENTS/METHODS: 20 patients were included. Each received 90 minutes of oxygen intermittently at 2.4 atmospheres absolute, as per the TS 240-90 wound-healing protocol. Blood samples were taken before and after hyperbaric exposure and ROTEM, Multiplate and standard laboratory assays were subsequently performed. RESULTS: ROTEM showed a significant increase of the maximum clot firmness (EXTEM MCF; p < 0.05) and the thromboelastometric platelet component of the clot firmness (MCF(EXTEM) - MCF(FIBTEM); p < 0.01). Multiplate showed a platelet activation mediated by thrombin (AU TRAP-test; p < 0.05) and by arachidonic acid (AUC ASPI-test; p < 0.01). Standard laboratory assays revealed a lower activated partial thromboplastin time (p < 0.05) and a higher leukocyte count (p < 0.05). No further changes were detected. A t-test was performed after testing if data followed normal distribution. CONCLUSIONS: ROTEM and Multiplate were able to detect an activation of platelets after HBO2 therapy via thrombin and arachidonic acid pathways. Previously reported fibrinolysis could not be confirmed.

Biocompatibility Assessment of the CentriMag-Novalung Adult ECMO Circuit in a Model of Acute Pulmonary Hypertension.

Extracorporeal membrane oxygenation (ECMO) is rarely used in patients with severe pulmonary hypertension (PH) as a bridge to lung transplantation. In this study, we assess the blood biocompatibility of the integrated CentriMag-Novalung ECMO system (venoarterial) in an acute model of PH. Severe PH (≥2/3 systemic) was induced in eight sheep through progressive ligation of the main pulmonary artery. System performance, platelet activation, thromboelastography (TEG) parameters, fibrinogen, plasma-free hemoglobin, and total plasma protein were measured at initiation, 3, and 6 hr of support in the ECMO (N = 4) and sham (N = 4) groups. A stable ECMO flow (2.2 ± 0.1 L/min), low transmembrane pressure gradient, and steady blood O2 and CO2 levels were maintained. Platelet activation was low (<4%) in both the groups, whereas platelet responsiveness to agonist (platelet activating factor) was reduced in the sham group when compared with the ECMO group. There were no differences in the TEG parameters, fibrinogen concentration, plasma-free hemoglobin (<10 mg/dl), and plasma total protein between the two groups. The findings of low levels of platelet activation and plfHb suggest adequate blood biocompatibility of the integrated CentriMag-Novalung circuit use for short-term support in a model of PH.

Assessment of spontaneous platelet aggregation using laser light scattering in healthy subjects: an attempt to standardize.

INTRODUCTION: When measuring platelet aggregation using laser light scattering, small aggregates forming without the addition of agonists may be observed. This event is called 'spontaneous platelet aggregation (SPA)'. The platelet hyperactivity observed in arterial thrombotic diseases can be detected with relative ease by measuring SPA. Standardization is urgently needed because of differences between measurement conditions among various laboratories. METHODS: We conducted a systematic study of factors that affect SPA measurement, compared SPA results to flow cytometry detection of surface antigens expressed on activated platelet membranes (P-selectin, activated glycoprotein IIb/IIIa), and determined conditions that yield stable measurements. RESULTS AND CONCLUSIONS: We evaluated results from 125 healthy volunteers and established conditions for a stable measurement of SPA. As the occurrence of SPA tended to increase with age, we determined conditions valid for subjects aged 20-60 years. Blood should be collected using a syringe, and the sample should be prepared after allowing the whole blood to rest for 30 min after collection. To isolate platelet-rich plasma, a 2-mL tube should be used and centrifuged at 150 g. The sample should be stored at room temperature, the platelet count of the sample should be (250 ± 10) × 10(9) /L, and the measurement should be completed within 90 min of blood collection.

Automated assessment of the neutrophil and platelet activation status in patients with essential thrombocythemia.

Neutrophil and platelet activation are consistently found in essential thrombocythemia (ET), but the techniques employed to demonstrate such abnormalities are complex. To ascertain whether the ADVIA 120 analyzer can be employed to assess neutrophil and platelet activation status in ET, 55 such patients and the same number of matched healthy individuals were studied and the results correlated with neutrophil CD11b and platelet P-selectin expressions measured by flow cytometry. Compared with controls, ET patients had significantly higher values of neutrophil myeloperoxidase index (MPXI), mean platelet volume (MPV), platelet distribution width (PDW), and platelet component distribution width, and significantly lower values of neutrophil lobularity index and mean platelet component (MPC). Patients with the JAK2 mutation had significantly lower values of MPC and higher values of MPV and PDW than those with wild-type allele. A positive correlation was observed between MPXI and neutrophil CD11b expression and a negative correlation between MPC and platelet P-selectin expression. The intensity of the agreement between the variables obtained by the two methods was moderate. These results support the possible value of MPC as surrogate parameter of platelet activation in ET.

Migraine and patent foramen ovale: state of the science.

Migraine is a prominent cause of recurrent pain, affecting 12% of the population. In several case series, approximately 50% of migraineurs with aura were found to have patent foramen ovale (PFO). The pathophysiological mechanism is speculated to be passage of microemboli and vasoactive chemicals through the PFO, thereby evading pulmonary filtration and triggering migraine symptoms. This article presents the results of retrospective and prospective research studies documenting the effects of PFO closure on migraine symptoms and presents emerging theories on possible pathologic mechanisms that may partially explain the increased risk of ischemic stroke in the migraine population. Finally, evidence-based recommendations are presented for health care providers for managing patients who have migraine and PFO.

Assessment of thrombin generation: useful or hype?

Thrombin plays an important role in hemostasis through its multiple functions across blood coagulation, platelet activation, and fibrinolysis. The measurement of thrombin generation is therefore viewed as a potentially useful test that could be applied to the screening, monitoring, and/or diagnosis of hemostatic abnormalities. Indeed, advances in thrombin generation assays have created significant interest and debate as to whether they may provide a more physiologically relevant testing system than do traditional coagulation tests. A variety of thrombin generation assays, including commercially available systems, have been investigated for their correlation with hypocoagulable and hypercoagulable states. Although there is an extensive body of literature that has investigated the application of thrombin generation assays, some limitations remain. These include poor standardization of reagents and methods and a lack of large prospective studies that demonstrate clear relationships between thrombin generation with bleeding and thrombosis phenotypes, as well as with monitoring anticoagulation. Whether thrombin generation assays become more "useful" than "hype" will require well-designed, large, prospective multicenter trials using standardized methods.

Assessment of platelet activation indices using the ADVIATM 120 amongst 'high-risk' patients with hypertension.

BACKGROUND: Platelet activation is involved in the pathogenesis of the thrombotic complications of hypertension. Novel surrogate markers of platelet activation (mean platelet volume (MPV), mean platelet component (MPC, measure of platelet density), platelet component distribution width (PCDW, a marker of platelet shape change) and the number of platelet clumps) have been related to cardiovascular risk. We hypothesized a stepwise increase in these platelet activation indices between healthy controls (HC, n = 60), 'high-risk' essential hypertensive subjects (HBP, n = 45) and treated, previously diagnosed patients with malignant phase hypertension (MHT, n = 45). METHODS: In a cross-sectional study, we measured comparative platelet counts and indices of platelet activation (MPV, MPC, PCDW and the number of platelet clumps) using the Bayer ADVIATM haematology system, in our three study groups. RESULTS: There was a stepwise increase in MPV (P = 0.0002) and MPM (P = 0.03), and a stepwise decrease in the MPC (P = 0.03) and PCDW (P = 0.001) across the three study groups, despite similarities in platelet count. These differences were only significantly different (on post-hoc analysis) between the healthy controls and the MHT group. On multivariate analysis, there was a significant relationship (R2 = 66.5%; P<0.0001) between the MPV and the PCDW (P<0.0001), systolic blood pressure (P = 0.008) and platelet count (P<0.0001). CONCLUSION: There is a stepwise increase in platelet activation indices, despite similar platelet counts, with increasing severity of hypertensive disease. This may contribute to the pathogenesis of thrombosis-related complications in hypertension.

[Value of coagulation function and fibrinolytic system assessment in patients with gestational diabetes mellitus].

OBJECTIVE: To study the clinical implications of changes of coagulation function and fibrinolytic system in patients with gestational diabetes mellitus (GDM). METHODS: Twenty non-pregnant women, 20 with normal pregnancy and 46 with GDM were enrolled in this study for examinations of platelet alpha-granule membrane protein (GMP-140), Von Willebrand factor (vWF), antithrombin III activity (AT-III), plasminogen activity (PLG), activity of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI). RESULTS: vWF, GMP-140, PLG, D-dimer, PAI were obviously elevated while t-PA was lower in GDM patients as compared with the measurement in non-pregnant women and women with normal pregnancy (P<0.01). AT-III and ProC measurement showed no significant differences between GDM patients and women of the other two groups. CONCLUSION: GDM patients may have elevated platelet activation and fibrinolyic activity as well as vascular endothelial injuries, and antenatal assessment of the coagulation function can be of value for prevention and treatment of GDM.