Platelet Activation Status Research Articles

Quantitative Optical Diffraction Tomography Imaging of Mouse Platelets.

Platelets are specialized anucleate cells that play a major role in hemostasis following vessel injury. More recently, platelets have also been implicated in innate immunity and inflammation by directly interacting with immune cells and releasing proinflammatory signals. It is likely therefore that in certain pathologies, such as chronic parasitic infections and myeloid malignancies, platelets can act as mediators for hemostatic and proinflammatory responses. Fortunately, murine platelet function ex vivo is highly analogous to human, providing a robust model for functional comparison. However, traditional methods of studying platelet phenotype, function and activation status often rely on using large numbers of whole isolated platelet populations, which severely limits the number and type of assays that can be performed with mouse blood. Here, using cutting edge 3D quantitative phase imaging, holotomography, that uses optical diffraction tomography (ODT), we were able to identify and quantify differences in single unlabeled, live platelets with minimal experimental interference. We analyzed platelets directly isolated from whole blood of mice with either a JAK2V617F-positive myeloproliferative neoplasm (MPN) or Leishmania donovani infection. Image analysis of the platelets indicates previously uncharacterized differences in platelet morphology, including altered cell volume and sphericity, as well as changes in biophysical parameters such as refractive index (RI) and dry mass. Together, these data indicate that, by using holotomography, we were able to identify clear disparities in activation status and potential functional ability in disease states compared to control at the level of single platelets.

MM-080: Platelet Hyperactivation in Multiple Myeloma Is Also Evident in Premalignant MGUS Patients and Persists During Treatment Initiation

Context: Thrombotic events are a common complication in multiple myeloma (MM) and smouldering myeloma (SM) patients, and are increasingly being recognized as a feature of MGUS. Platelet hyperactivation has been implicated in this prothrombotic state but remains to be characterized in MGUS patients and MM patients initiating treatment, when thrombotic risk is greatest. Objective: To address this, we evaluated platelet activation status with an extended panel of platelet activation markers (PAC-1, P-selectin, CD63, Annexin V) and platelet responsiveness to agonists (ADP, TRAP-6) in patients and healthy controls (HC). We also assessed additional parameters; reticulated platelet levels, platelet-leucocyte aggregates (PLA), and platelet-associated immunoglobulins (PAIg). Design, Setting & Patients: MGUS (n=19) and SM (n=4) patients who lacked anticoagulant use and underlying bleeding, thrombotic, and renal disorders were recruited from outpatient clinics. Newly diagnosed MM (n=12) patients were recruited using same criteria. A subset of MM patients (n=8) provided samples two months post-initiating treatment. HC (n=16) underwent paraprotein screening to exclude MGUS status. Interventions: No interventions to routine care. MM patients received Bortezomib (Velcade), dexamethasone and Lenalidomide (Revlimid) (VRD) (n=4) or cyclophosphamide (VCD) (n=4), and aspirin thromboprophylaxis (n=5). Main outcome measures: Platelets are hyperactivated and hyporesponsive to agonists in MGUS and SM/MM patients at diagnosis and in patients undergoing treatment. Results: Median platelet activation marker levels were elevated in patients versus HC, significantly for MGUS in terms of PAC-1 (0.72 v 1.7%, p=0.024), CD63 (0.55 v 1.29%, p=0.003), Annexin V (0.47 v 1.61%, p=0.004). In MM undergoing treatment, P-selectin (1.00 v 2.46%, p=0.010), CD63 (0.48 v 1.32%, p=0.020) and Annexin V (0.54 v 1.51%, p=0.038) were significantly elevated versus HC at diagnosis and remained elevated after treatment. MGUS patients had significantly reduced P-selectin levels in response to ADP (34.85 v 10.77%, p=0.005). In MM, hyporesponsiveness was evident versus HC at diagnosis and following treatment in terms of PAC-1 (58.5 v 34.91%, p=0.009) and P-selectin (42.21 v 23.49%, p=0.028). PLA (2.38 v 5.84%, p=0.023) and reticulated platelet levels (1.82 v 24.5%, p=0.031) increased significantly in MM after treatment. PAIg were evident in subsets of MGUS and SM/MM patients. Conclusions: This study reports changes in platelet parameters at various points in MM disease progression. Further investigation of the predictive and prognostic value of platelet dysregulation in such patients is warranted. Funding: Irish Research Council (GOIPG/2017/952) & TRAP Award, College of Medicine & Health, University College Cork.

Amyloid precursor protein 770 is specifically expressed and released from platelets

Platelets not only play an essential role in hemostasis after vascular injury but are also involved in the development of coronary artery disease (CAD) and cerebrovascular lesions. Patients with CAD and cerebral ischemia are recommended to undergo antiplatelet therapy, but they have an increased incidence of major bleeding complications. Both assessment of the platelet activation status and response to antiplatelet therapy in each patient are highly desired. β-Amyloid precursor protein (APP) 770 is expressed in vascular endothelial cells, and its extracellular region, a soluble form of APP770 (sAPP770, also called nexin-2), is proteolytically cleaved for shedding. Abundant sAPP770 is also released from activated platelets. In this study, we used peripheral blood samples from patients with CAD and control subjects and evaluated sAPP770 as a specific biomarker for platelet activation. First, the plasma levels of sAPP770 correlated well with those of the soluble form CD40 ligand (CD40L), an established biomarker for platelet activation. Additionally, flow cytometry analysis using peripheral blood cells showed that CD40L expression is up-regulated in activated T cells, whereas APP770 expression is negligible in all blood cell types except platelets. Following stimulation with collagen or ADP, aggregating platelets immediately released sAPP770. Finally, patients with dual antiplatelet therapy showed significantly lower levels of plasma sAPP770 than those with no therapy. Taken together, our data show that plasma sAPP770 could be a promising biomarker for platelet activation.

An In Vitro Hemodynamic Loop Model to Investigate the Hemocytocompatibility and Host Cell Activation of Vascular Medical Devices

In this study, the hemocompatibility of tubes with an inner diameter of 5 mm made of polyvinyl chloride (PVC) and coated with different bioactive conjugates was compared to uncoated PVC tubes, latex tubes, and a stent for intravascular application that was placed inside the PVC tubes. Evaluation of hemocompatibility was done using an in vitro hemodynamic loop model that is recommended by the ISO standard 10993-4. The tubes were cut into segments of identical length and closed to form loops avoiding any gap at the splice, then filled with human blood and rotated in a water bath at 37 °C for 3 hours. Thereafter, the blood inside the tubes was collected for the analysis of whole blood cell count, hemolysis (free plasma hemoglobin), complement system (sC5b-9), coagulation system (fibrinopeptide A), and leukocyte activation (polymorphonuclear elastase, tumor necrosis factor and interleukin-6). Host cell activation was determined for platelet activation, leukocyte integrin status and monocyte platelet aggregates using flow cytometry. The effect of inaccurate loop closure was examined with x-ray microtomography and scanning electron microscopy, that showed thrombus formation at the splice. Latex tubes showed the strongest activation of both plasma and cellular components of the blood, indicating a poor hemocompatibility, followed by the stent group and uncoated PVC tubes. The coated PVC tubes did not show a significant decrease in platelet activation status, but showed an increased in complement and coagulation cascade compared to uncoated PVC tubes. The loop model itself did not lead to the activation of cells or soluble factors, and the hemolysis level was low. Therefore, the presented in vitro hemodynamic loop model avoids excessive activation of blood components by mechanical forces and serves as a method to investigate in vitro interactions between donor blood and vascular medical devices.

Complementary roles of platelet αIIbβ3 integrin, phosphatidylserine exposure and cytoskeletal rearrangement in the release of extracellular vesicles

Background and aimsPlatelets can release extracellular vesicles (EVs) upon stimulation with various agonists. Interestingly, platelets from patients with Glanzmann thrombasthenia have reduced EV release. These platelets lack functional αIIbβ3 integrins, indicating that αIIbβ3 integrin is critical in vesicle release. Integrin activation is central in platelet function and is associated with e.g. adhesion, aggregation and cytoskeletal rearrangement. However, while platelet activation pathways are widely known, the mechanisms underlying EV release remain uncharacterized. We investigated the role of integrin αIIbβ3, phosphatidyl serine (PS) exposure, cytoskeletal rearrangement and their associated signalling pathways in EV release. MethodsEVs were isolated from activated platelets. Platelet activation status was measured by multicolour flow cytometry. A panel of pharmacologic inhibitors was used to interfere in specific signalling pathways. EV release was quantified enzymatically based on membrane PS content and nanoparticle tracking analysis. In addition, real-time visualization of EV shedding with confocal microscopy and EVs with Cryo-TEM imaging was performed. ResultsPlatelet activation with convulxin resulted in higher EV release than with activation by thrombin. Kinetic measurements indicated that EV release followed the pattern of αIIbβ3 integrin activation and subsequent closure paralleled by PS exposure. Prevention of αIIbβ3 activation with the inhibitor tirofiban dramatically suppressed EV release. Similar results were obtained using αIIbβ3-deficient platelets from patients with Glanzmann thrombasthenia. Inhibition of actin cytoskeleton rearrangement decreased EV release, whereas inhibition of individual signalling targets upstream of cytoskeletal rearrangement showed no such effects. ConclusionPlatelet EV release requires three main events: integrin activation and closure, PS exposure, and cytoskeletal rearrangement.

Safety of selective non-steroidal anti-inflammatory drugs: analysis of the last years data

Nonsteroidal anti-inflammatory drugs (NSAIDs) are the most commonly used pain relievers. However, their use often threatens with serious undesirable effects, associated mainly with damage to cardiovascular system (CVS), gastrointestinal tract, kidneys and liver. Contraindications to NSAIDs prescription are clearly regulated, algorithms for their personalized appointment are determined taking into account risk factors for cardiovascular and gastrointestinal adverse events. The severity of NSAIDs side effects is mainly due to the selectivity to cyclooxygenase-2 (COX-2), as well as the physicochemical properties of various drugs. Cardiovascular adverse events differ among various NSAIDs both within commonly used drugs and among COX-2 inhibitors. It is well known that NSAIDs selective for COX-2 are safer in terms of the effect on the gastrointestinal tract than non-selective drugs. A meta-analysis showed that relatively selective COX-2 inhibitors (meloxicam, etodolac) were associated with a comparable risk of developing symptomatic ulcers and ulcers identified by endoscopy, and safety and tolerability profiles of the drugs were similar.All NSAIDs are associated with cardiovascular toxicity, however, different drugs have significant risk differences. The mechanism of NSAIDs cardiovascular adverse effects is associated with an increase of blood pressure, sodium retention, vasoconstriction, platelet activation, and prothrombotic state. It has been shown that the risk of cardiovascular adverse events when taking COX-2 inhibitors (celecoxib, etoricoxib) significantly increases. According to a study of more than 8 million people, it was found that the risk of myocardial infarction was increased in patients taking ketorolac. Further, highest to lowest risk authors list indomethacin, etoricoxib, rofecoxib (not currently used), diclofenac, a fixed combination of diclofenac with misoprostol, piroxicam, ibuprofen, naproxen, celecoxib, meloxicam, nimesulide and ketoprofen. When taking NSAIDs, the risk of heart failure decompensation increases, and it turned out to be the greatest for ketorolac, etoricoxib, and indomethacin. Meloxicam, aceclofenac, ketoprofen almost did not increase heart failure risk. It should be noted that when using the drugs (except for indomethacin and meloxicam), there is a tendency to increase the total cardiovascular and renal risks with increasing doses. Thus, it is obvious that a very careful approach is required when choosing NSAIDs. If there is an increased risk of gastrointestinal complications associated with NSAIDs, selective NSAIDs are preferred, with both coxibs and traditional selective NSAIDs showing the best safety profile in the studies. To minimize cardiovascular side effects specialists should consider the risk level of cardiovascular complications, as well as results of large clinical studies where particular NSAIDs are compared.

Influence of Different Antithrombotic Regimens on Platelet-Mediated Thrombin Generation in Patients with Left Ventricular Assist Devices.

We characterized the biologic background of prothrombotic platelet function in the setting of durable left ventricular assist devices (LVADs) evaluating the role of different antithrombotic regimens. Platelet-mediated thrombin generation was quantified using the Platelet Activity State (PAS) Assay and the Thrombin Generation Test (TGT) in 78 patients implanted with the HeartMate II (n = 10, 13%), the HeartMate 3 (HM3) (n = 30, 38%), or the HVAD (n = 38, 49%) and managed with oral anticoagulation plus aspirin (n = 46, 59%) or anticoagulation alone (n = 32, 41%). Coagulation parameters (platelet count, International Normalized Ratio (INR), activated Partial Thromboplastin Time, Fibrinogen and D-Dimer levels) and hemolysis (lactate dehydrogenase levels [LDH]) were also recorded to comprehensively characterize the hemostatic profile in the two groups. In patients without aspirin, the PAS assay revealed low-intensity increase in platelet prothrombinase activity (1.11-fold, p = 0.03). Similarly the TGT revealed moderate higher platelet reactivity when compared with patients receiving aspirin, consistent with reduction in lag time (0.87-fold, p < 0.001), increase in peak of thrombin generation (1.5-fold, p = 0.002) and thrombin generation rate (2-fold, p = 0.02), but comparable endogenous thrombin potential (p = 0.50). Coagulation parameters and LDH were comparable in the two groups (p > 0.05). Moreover, no differences were noted in platelet prothrombinase activity of patients implanted with the HM3 or HVAD. Our results suggest that, in the setting of durable LVADs, aspirin minimally modulates the biochemical pathway of platelet-mediated thrombin generation. Accordingly, re-evaluation of current antithrombotic management criteria in patients stratified according to bleeding/thromboembolic risk might be safe and beneficial to prevent adverse events.

Intestinal microbiota dysbiosis play a role in pathogenesis of patients with primary immune thrombocytopenia

BackgroundThe intestinal microbiota is essential for the maintenance of the physiology of immune homeostasis. Dysbiosis has been described in some autoimmune diseases, however its role is still elusive in primary immune thrombocytopenia (ITP), which is one kind of autoimmune diseases. This study aimed to characterize the phylogenetic diversity of the fecal microbiota and its relationship with the platelet activation status in patients with ITP. MethodsThe platelet activation status was assessed by 2 platelet markers, PAC-1 (antibody that recognizes the activated GPIIb/IIIa complex) and CD62p (Platelet surface P-selectin) by flow cytometry. Total DNA was extracted from fecal samples of ITP patients and healthy controls (HC). Sequencing the V4 hypervariable region of bacterial 16S rRNA genes was used to identify the changes in phylogenetic diversity and composition of the intestinal flora. The obtained sequencing reads were assigned to operational taxonomic units (OTUs, 97% sequence identity) and taxonomically classified to assess composition and diversity. ResultsThe percentage of PAC-1+ platelets in ITP patients was higher than that in control group (p < 0.001), The percentage of CD62p+ and PAC-1+CD62p+ platelets in ITP patients both higher than those in control group (p < 0.001). At the phylum level, eight different phyla were identified in ITP individuals, with a majority of Bacteroidetes (45.96%) and Firmicutes (38.59%), followed by Proteobacteria (11.43%), Fusobacteria(1.29%), and Actinobacteria (1.22%). While in the Healthy volunteers, ten phyla were detected, with a predominance of Firmicutes (50.92%) and Bacteroidetes (34.26%), came before Proteobacteria (13.60%), and Actinobacteria (0.90%). The gut microbiota was skewed in ITP, with an increased proportion of Proteobacteria, Bacteroidetes and Bacteroidetes/Firmicutes ratio, a decreased proportion of Firmicutes compared with HC. Disease specific alterations in diversity was also identified, especially the potential markers (Anaerorhabdus, sutterella, Peptostreptococcaceae, Clostridium_XI and carnobacteriaceae, p < 0.05) for ITP. ConclusionsThe results suggested that the distinct microbiota dysbiosis in ITP characterized by alterations in biodiversity and composition, which could provide insights for diet therapy and fecal microbiota transplantation treatment to cure ITP. There might be somehow compensatory enhancement of platelet activation in ITP patients. And there is associate between platelet activation and intestinal microbiota in patients with ITP.

Platelets in Amyloidogenic Mice Are Activated and Invade the Brain.

Alzheimer’s disease (AD) is a neurodegenerative disease with a complex and not fully understood pathogenesis. Besides brain-intrinsic hallmarks such as abnormal deposition of harmful proteins, i.e., amyloid beta in plaques and hyperphosphorylated Tau in neurofibrillary tangles, blood-derived elements, in particular, platelets have been discussed to be involved in AD pathogenesis. The underlying mechanisms, however, are rather unexplored. Here, we investigate a potential role of platelets in an AD transgenic animal model with severe amyloid plaque formation, the APP-PS1 transgenic mice, and analyzed the presence, spatial location and activation status of platelets within the brain. In APP-PS1 mice, a higher number of platelets were located within the brain parenchyma, i.e., outside the cerebral blood vessels compared to WT controls. Such platelets were activated according to the expression of the platelet activation marker CD62P and to morphological hallmarks such as membrane protrusions. In the brain, platelets were in close contact exclusively with astrocytes suggesting an interaction between these two cell types. In the bloodstream, although the percentage of activated platelets did not differ between transgenic and age-matched control animals, APP-PS1 blood-derived platelets showed remarkable ultrastructural peculiarities in platelet-specific organelles such as the open canalicular system (OCS). This work urges for further investigations on platelets and their yet unknown functional roles in the brain, which might go beyond AD pathogenesis and be relevant for various age-related neurodegenerative diseases.

Platelet activation during chronic hepatitis B infection exacerbates liver inflammation and promotes fibrosis.

Recurrent hepatitis activity during chronic hepatitis B virus infection results in fibrosis and even hepatocellular carcinoma. It is still unclear what causes acute exacerbation. As platelets have recently been identified as a significant role in inflammation, we here investigated the role of platelets in mediating liver damage in patients with chronic hepatitis B virus infection. Platelet aggregation testing and flow cytometry were carried out to evaluate platelet activation status in 121 patients chronically infected with hepatitis B across different phases of the condition. The correlation between platelet aggregation rate and liver inflammation or liver fibrosis index was evaluated. To investigate the genesis of platelet activation, several serum cytokines were also assessed by MILLIPLEX microsphere-based multiplex cytokine assay. Active hepatitis patients showed a higher aggregation rate than others. Levels of CD62p, a marker of platelet activation, were also increased in this group of patients. Positive correlations between platelet aggregation rate and liver inflammation or liver fibrosis were also noted, indicating a significant role of platelet in the progression of liver disease. The level of tumor necrosis factor-alpha, which is known to trigger platelet activation, was markedly higher in the active hepatitis group (P < .005). Based on the findings in our study, platelet activation plays a vital role in the progression of chronic hepatitis B virus infection. Antiplatelet therapy may provide a new means of hepatitis B infection treatment.

A Comparative Study of The Effects of Anticoagulants on Pure Platelet-Rich Plasma Quality and Potency.

It is generally accepted that citrate or the A-form of acid-citrate-dextrose (ACD-A) are suitable for preparing platelet-rich plasma (PRP) for regenerative therapy. However, this is based on evidence from blood transfusions and not from regenerative medicine. Thus, we examined the effects of anticoagulants, such as ACD-A, ethylenediaminetetraacetic acid (EDTA), and heparin, on the regenerative quality of PRP to address this gap. The blood samples were collected in the presence of anticoagulants and were processed to prepare pure-PRP. Platelet size, activation status, and intra-platelet free Ca2+ concentration were determined while using a hematology analyzer and flow cytometer. Platelet-derived growth factor-BB (PDGF-BB) was quantified while using an ELISA. In pure-PRP samples, EDTA caused platelet swelling and activation, but yielded the highest number of platelets. Heparin aggregated platelets and disturbed the overall counting of blood cells. However, no significant differences in PDGF-BB levels were observed among the anticoagulants tested. Moreover, when considering the easy preparation of platelet suspensions, without the need for high-level pipetting skills, these findings suggest the comparable potency of EDTA-derived pure-PRP in tissue regeneration and support the use of EDTA in the preparation of pure-PRP. Further in vivo studies are required in animal models to exclude the possible negative effects of including EDTA in pure-PRP preparations.

Reduced miR-26b Expression in Megakaryocytes and Platelets Contributes to Elevated Level of Platelet Activation Status in Sepsis.

In sepsis, platelets may become activated via toll-like receptors (TLRs), causing microvascular thrombosis. Megakaryocytes (MKs) also express these receptors; thus, severe infection may modulate thrombopoiesis. To explore the relevance of altered miRNAs in platelet activation upon sepsis, we first investigated sepsis-induced miRNA expression in platelets of septic patients. The effect of abnormal Dicer level on miRNA expression was also evaluated. miRNAs were profiled in septic vs. normal platelets using TaqMan Open Array. We validated platelet miR-26b with its target SELP (P-selectin) mRNA levels and correlated them with clinical outcomes. The impact of sepsis on MK transcriptome was analyzed in MEG-01 cells after lipopolysaccharide (LPS) treatment by RNA-seq. Sepsis-reduced miR-26b was further studied using Dicer1 siRNA and calpain inhibition in MEG-01 cells. Out of 390 platelet miRNAs detected, there were 121 significantly decreased, and 61 upregulated in sepsis vs. controls. Septic platelets showed attenuated miR-26b, which were associated with disease severity and mortality. SELP mRNA level was elevated in sepsis, especially in platelets with increased mean platelet volume, causing higher P-selectin expression. Downregulation of Dicer1 generated lower miR-26b with higher SELP mRNA, while calpeptin restored miR-26b in MEG-01 cells. In conclusion, decreased miR-26b in MKs and platelets contributes to an increased level of platelet activation status in sepsis.

Short-Term Exposure to Waterpipe/Hookah Smoke Triggers a Hyperactive Platelet Activation State and Increases the Risk of Thrombogenesis.

Cardiovascular disease is a major public health problem. Among cardiovascular disease's risk factors, tobacco smoking is considered the single most preventable cause of death, with thrombosis being the main mechanism of cardiovascular disease mortality in smokers. While tobacco smoking has been on the decline, the use of waterpipes/hookah has been rising, mainly due to the perception that they are less harmful than regular cigarettes. Strikingly, there are few studies on the negative effects of waterpipes on the cardiovascular system, and none regarding their direct contribution to thrombus formation. Approach and Results: We used a waterpipe whole-body exposure protocol that mimics real-life human exposure scenarios and investigated its effects, relative to clean air, on platelet function, hemostasis, and thrombogenesis. We found that waterpipe smoke (WPS)-exposed mice exhibited both shortened thrombus occlusion and bleeding times. Further, our results show that platelets from WPS-exposed mice are hyperactive, with enhanced agonist-induced aggregation, dense and α-granule secretion, αIIbβ3 integrin activation, phosphatidylserine expression, and platelet spreading, when compared with clean air-exposed platelets. Finally, at the molecular level, it was found that Akt (protein kinase B) and ERK (extracellular signal-regulated kinases) phosphorylation are enhanced in the WPS and in nicotine-treated platelets. Our findings demonstrate that WPS exposure directly modulates hemostasis and increases the risk of thrombosis and that this is mediated, in part, via a state of platelet hyperactivity. The negative health impact of WPS/hookah, therefore, should not be underestimated. Moreover, this study should also help in raising public awareness of the toxic effects of waterpipe/hookah.

Study on Mechanism for Preventing Thrombus Formation by Nano-Biological Catheter Pump

Based on prediction model for thrombus proposed by Grigioni and Danny Bluestein, the mechanism for preventing thrombus formation by a Nano-biological catheter pump was studied herein by applying computational fluid dynamics (CFD). The shearing force and exposure time for platelets during the movement of nano-biologic catheter pump were calculated, and the platelet activation state (PAS) was analyzed, so as to simulate thrombus formation of Nano-biologic catheter pump, predict the thrombus formation and prevent the occurrence of thrombus. Through calculation and analysis, it was shown that the pump can meet the requirements for left ventricle pump when the flow rate of the Nano-biological catheter pump is 3.8 L/min, the rotation speed is 9800 r/min and the head is 14.798 kPa. PAS in the prediction model by Grigioni and Danny Bluestein were 6.39 × 10–6 and 7.68 × 10–4, respectively. Since the values were small, the thrombus almost never formed. To explore the mechanism for preventing thrombus formation, and prediction of hydraulic performance of the pump can provide theoretical basis for optimizing the structure of the pump.

New approaches for the assessment of platelet activation status in thrombus under flow condition using confocal microscopy.

The goal of the study was the assessment of heterogeneous platelet activation status in thrombus. In a ferric(III) chloride (FeCl3) thrombosis (intravital) model of C57BL/6J mice, the area of irreversibly activated (phosphatidylserine (PS)-positive) platelets was assessed after 1-s exposure of a vessel to FeCl3. In a laser-induced thrombosis (intravital) model of GFP mice, the area of the thrombus composed of PS-negative platelets was evaluated. The ratio of the area of PECAM-1 to the area of the thrombus was used as a marker to assess the activity of PS-negative platelets. In the in vitro flow chamber model, the thrombus area (PS-negative and PS-positive platelets) and the platelet activation index (ratio of the area of PS-positive platelets to the area of thrombus) were determined. To assess platelet activation status with these models, acetylsalicylic acid (ASA) and iloprost (Ilo) were used. In the FeCl3 thrombosis, ASA (10mg/kg, 100mg/kg) decreased the area of PS-positive platelets. In the laser thrombosis, ASA (10mg/kg) decreased the thrombus area, but the decrease in platelet activity was evident even at 3mg/kg by an increased PECAM-1/thrombus ratio. In the flow chamber, ASA (0.02mg/ml, 0.2mg/ml) equally decreased the platelet activation index, whereas only at 0.2mg/ml, it decreased the thrombus area. Ilo (3.6ng/ml, 36ng/ml) decreased the thrombus area but at 36ng/ml increased the platelet activation index. We showed that intravital models and flow chamber provide a detailed assessment of platelet activation status and the mechanism of drug action.

Comparative proteomics reveals unexpected quantitative phosphorylation differences linked to platelet activation state

There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbβ3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after in-vitro -activation and –inhibition with their respective untreated controls using unbiased fluorescence two-dimensional differential in-gel electrophoresis. The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen’s d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.

Platelet Activation State Intermixing in a Venous Puncture Model Indicates Novel Patterns of Thrombus Formation

No relevant conflicts of interest to declare.

Repurposing pyridoxamine for therapeutic intervention of intravascular cell-cell interactions in mouse models of sickle cell disease.

Adherent neutrophils on vascular endothelium positively contribute to cell-cell aggregation and vaso-occlusion in sickle cell disease (SCD). In the present study, we demonstrated that pyridoxamine, a derivative of vitamin B6, might be a therapeutic agent to alleviate intravascular cell-cell aggregation in SCD. Using real-time intravital microscopy, we found that one oral administration of pyridoxamine, in a dose-dependent manner, increased the rolling influx of neutrophils and reduced neutrophil adhesion to endothelial cells in cremaster microvessels of SCD mice challenged with hypoxia-reoxygenation. Short-term treatment also mitigated neutrophil-endothelial cell and neutrophil-platelet interactions in the microvessels and improved the survival of SCD mice challenged with tumor necrosis factor-α. The inhibitory effects of pyridoxamine on intravascular cell-cell interactions were enhanced by co-treatment with hydroxyurea. We observed that long-term (5.5 months) oral treatment with pyridoxamine significantly reduced the adhesive function of neutrophils and platelets, and down-regulated the expression of E-selectin and intercellular adhesion molecule-1 on the vascular endothelium in tumor necrosis factor-α-challenged SCD mice. Ex vivo studies revealed that the surface amount of αMβ2 integrin was significantly decreased in stimulated neutrophils isolated from SCD mice treated with pyridoxamine-containing water. Studies using platelets and neutrophils from SCD mice and patients suggested that treatment with pyridoxamine reduced the activation state of platelets and neutrophils. These results suggest that pyridoxamine may be a novel therapeutic and a supplement to hydroxyurea to prevent and treat vaco-occlusion events in SCD.

Modeling sensitivity and uncertainties in platelet activation models applied on centrifugal pumps for extracorporeal life support

Two platelet activation models were studied with respect to uncertainties of model parameters and variables. The sensitivity was assessed using two direct/deterministic approaches as well as the statistical Monte Carlo method. The first two, are linear in character whereas the latter is non-linear. The platelet activation models were applied on platelets moving within an extracorporeal centrifugal blood pump. The phenomenological, Lagrangian stress- and time-based power law-based models under consideration, have experimentally calibrated parameters and the stress expressed in a scalar form. The sensitivity of the model with respect to model parameters and the expression of the scalar stress was examined focusing on a smaller group of platelets associated with an elevated risk of activation. The results showed a high disparity between the models in terms of platelet activation state, found to depend on the platelets’ trajectory in the pump and the expression used for the scalar stress. Monte Carlo statistics was applied to the platelets at risk for activation and not to the entire platelet population. The method reveals the non-linear sensitivity of the activation models. The results imply that power-law based models have a restricted range of validity. The conclusions of this study apply to both platelet activation and hemolysis models.

Identification of low level monoclonality using quantitative immunoprecipitation mass spectrometry (QIP-MS) as a first line monoclonal gammopathy screening strategy

The venous thromboembolism, which may be caused by increased platelet activation, is a risk factor for tumor prognosis. We determined the platelet activation status for diagnosis and predicting postoperative prognosis of hepatocellular carcinoma.We conducted a prospective study of 191 patients diagnosed with HCC at Zhongshan Hospital from April 2016 to July 2016 as well as 99 healthy people. The platelet activation status was assessed by 2 platelet markers, PAC-1 and CD62p, using flow cytometry. The patients were treated with TACE or resection and monitored for ≥6 months. The diagnostic value of marker-positive platelets was determined by the receiver operating characteristic curve and the postoperative value were analyzed using the Kaplan-Meier method and COX regression model.All the 3 groups with high levels of marker-positive platelets were likely to be diagnosed with HCC and the PAC-1+ percentage had the best efficacy. The univariate analysis showed that the levels of PAC-1+ and CD62p+ platelets was risker factors for poor postoperative prognosis after both TACE and resection. Moreover, the multivariate analysis revealed that the level of PAC-1+ platelets was an independent risk factor for poor prognosis.The PAC-1+ percentage of platelets is a new indicator for diagnosis and predicting postoperative prognosis.