Surface Expression Of P-selectin Research Articles

Abstract 40: Platelet Fragmentation During the Hemostatic Response and its Prevention by a P2Y12 Antagonist

Previous studies using intravital microscopy have shown that hemostatic plugs formed in the mouse microvasculature have a characteristic architecture in which the extent of platelet activation reflects gradients in the distribution of platelet agonists radiating outwards from the injury site. In that setting, we found minimal overlap of thrombin and ADP signaling, with thrombin primarily responsible for robust platelet activation close to the injury site and P2Y 12 -mediated ADP signaling resulting in accumulation of minimally activated platelets. Here we have taken these studies a major step forward by integrating fluorescence with scanning electron microscopy. Hemostatic plugs produced by needle injury in mouse jugular veins were imaged in situ 1 to 20 min after injury. The results show with unprecedented detail what could only be inferred previously, showing that platelet size, morphology and packing density vary remarkably depending on spatial localization within the hemostatic plug. The intraluminal and extravascular portions of the hemostatic mass presented distinct architectures. A large mass comprised almost exclusively of platelets was observed on the interior surface of the vein. Platelets closest to the injury edge had a highly activated morphology, including P-selectin surface expression, dense packing and platelet fragmentation, while those farther from the injury edge often remained discoid. In contrast, the extravascular portion of the hemostatic mass was rich in densely-packed, platelet-derived fragments intertwined with fibrin. Hemostatic plugs from mice treated with a P2Y 12 inhibitor were significantly smaller. The platelet activation gradient described above was less apparent and, notably, fragmentation of the platelets close to the injury edge was not observed with the inhibitor present. In conclusion, our findings indicate that 1) the development of a platelet activation gradient is a conserved feature of the hemostatic response across different vessels, 2) fragmentation of platelets closest to the injury site occurs very rapidly following injury, and 3) clinically relevant platelet signaling pathways play a role in regulating its formation.

An absence of platelet activation following thalidomide treatment in vitro or in vivo

Increased risk of thromboembolism and platelet hyperreactivity has been reported in patients receiving thalidomide therapy. Whether thalidomide induces platelet activation directly or through other factors remains unclear. The aim of this study was to evaluate the effect of thalidomide on platelet activation under resting conditions in vitro and in vivo. Isolated human or mouse platelets were treated with different concentrations of thalidomide (10, 50 and 100 μg/ml) for 60 min at 37°C followed by analysis of platelet surface expression of platelet receptors GPIbα, GPVI, αIIbβ3 and P-selectin, and PAC-1 or fibrinogen binding, by flow cytometry and collagen- or ADP-induced platelet aggregation. In addition, thalidomide (200 mg/kg) was intraperitoneally injected into mice for analysis of the effect of thalidomide on platelet activation in vivo. No increased expression of P-selectin, PAC-1 or fibrinogen binding was observed in either human and mouse platelets after thalidomide treatment in vitro for 60 min at 37°C. Thalidomide treatment also did not affect expression of GPIbα, GPVI or αIIbβ3, nor did it affect collagen- or ADP-induced platelet aggregation at threshold concentrations. However, while mice injected with thalidomide displayed no increased surface expression of platelet P-selectin or αIIbβ3, there was a significantly shortened tail bleeding time, thrombin time, prothrombin time together with higher levels of Factor IX and fibrinogen. In conclusion, thalidomide at therapeutic doses does not directly induce platelet activation under resting conditions in vitro or in vivo, but results in increased procoagulant activity, which could explain the thalidomide-dependent prothrombotic tendency in patients.

Ethanol Induces Platelet Apoptosis

Alcohol abuse incurs severe medical conditions, such as thrombocytopenia and hemorrhage, but the pathogenesis is not totally understood. Alcohol has been reported to induce apoptosis in eukaryotic cells, such as hepatocyte, nerve cell, corneal fibroblasts. However, it is still unclear whether alcohol induces platelet apoptosis. Washed human platelets were pretreated with ethanol (EtOH), and apoptotic events and platelet function were detected. In invivo experiments, C57BL/6J mice were given EtOH by gavage. Platelet counts, tail bleeding time, and the stomach were examined. EtOH dose dependently induces depolarization of mitochondrial inner transmembrane potential, up-regulation of Bax, down-regulation of Bcl-2, and caspase-3 activation. EtOH does not induce surface expression of P-selectin or PAC-1 binding, whereas significantly reduces collagen-, thrombin-, and ADP-induced platelet aggregation. Moreover, EtOH induces c-Jun NH2-terminal kinase activation. In an invivo mouse model of the acute alcoholism, EtOH significantly reduces the number of circulating platelets, prolongs the tail bleeding time, and causes gastric mucosa hemorrhage. These data demonstrate that EtOH induces mitochondria-mediated intrinsic platelet apoptosis, results in the reduction of the number of circulating platelets, and impairs invivo hemostasis. These findings reveal the possible pathogenesis of hemorrhagic symptoms in patients experiencing acute alcohol intoxication.

Ascorbate inhibits platelet-endothelial adhesion in an in-vitro model of sepsis via reduced endothelial surface P-selectin expression.

Plugging of the capillary bed can lead to organ failure and mortality in sepsis. We have reported that intravenous ascorbate injection reduces platelet adhesion to the capillary wall and capillary plugging in septic mice. Both platelet adhesion and capillary plugging require P-selectin, a key adhesion molecule. To elucidate the beneficial effect of ascorbate, we hypothesized that ascorbate reduces platelet-endothelial adhesion by reducing P-selectin surface expression in endothelial cells. We used mouse platelets, and monolayers of cultured microvascular endothelial cells (mouse skeletal muscle origin) stimulated with lipopolysaccharide, to examine platelet-endothelial adhesion. P-selectin mRNA expression in endothelial cells was determined by real-time PCR and P-selectin protein expression at the surface of these cells by immunofluorescence. Secretion of von Willebrand factor from cells into the supernatant (a measure of P-selectin-containing granule exocytosis) was determined by ELISA. Lipopolysaccharide (10 μg/ml, 1 h) increased platelet-endothelial adhesion. P-selectin-blocking antibody inhibited this adhesion. Lipopolysaccharide also increased P-selectin mRNA in endothelial cells, P-selectin expression at the endothelial surface, and von Willebrand factor secretion. Ascorbate pretreatment (100 μmol/l, 4 h) inhibited the increased platelet adhesion, surface expression of P-selectin, and von Willebrand factor secretion, but not the increase in P-selectin mRNA. The lipopolysaccharide-induced increase in platelet-endothelial adhesion requires P-selectin presence at the endothelial surface. Ascorbate's ability to reduce this presence could be important in reducing both platelet adhesion to the capillary wall and capillary plugging in sepsis.

Decreased platelet reactivity in patients with cancer is associated with high risk of venous thromboembolism and poor prognosis.

Platelets are suggested to play a crucial role in cancer progression and the prothrombotic state of cancer patients. Here, we aimed to examine the activation status of platelets in cancer patients and investigate their association with risk of death and occurrence of venous thromboembolism (VTE) in a prospective observational cohort study. We measured platelet surface P-selectin, activated glycoprotein (GP) IIb/IIIa and monocyte-platelet aggregate (MPA) formation in vivo and platelet response to ex vivo stimulation with agonists of protease-activated receptor (PAR) -1, -4, and GPVI, by whole blood flow cytometry, before beginning of chemotherapy and repeatedly during the first six months thereafter (total number of samples analysed: 230). Endpoints of the study were occurrence of death or VTE during a two-year follow-up, respectively. Of 62 patients (median age [interquartile range, IQR]: 63 [54-70] years, 48 % female), 32 (51.6 %) died and nine (14.5 %) developed VTE. Association with a higher risk of death was found for lower platelet surface expression of P-selectin and activated GPIIb/IIIa in vivo and in response to PAR-1, -4 and GPVI activation, but not for MPA formation. Furthermore, reduced platelet responsiveness to PAR-1 and GPVI agonists was associated with higher risk of VTE (hazard ratio per decile increase of percentage P-selectin positive platelets: 0.73 [0.56-0.92, p=0.007] and 0.77 [0.59-0.98, p=0.034], respectively). In conclusion, cancer patients with a poor prognosis showed decreased platelet reactivity, presumably as a consequence of continuous activation. Our data suggest that decreased platelet reactivity is associated with increased mortality and VTE in cancer.

Remote Assessment of Platelet Function in Patients with Acute Stroke or Transient Ischaemic Attack

Background The TARDIS trial assessed the safety and efficacy of intensive versus guideline antiplatelet agents given for one month in patients with acute stroke or TIA. The aim of this substudy was to assess the effect of antiplatelet agents taken at baseline on platelet function reactivity and activation. Methods Platelet function, assessed by remotely measured surface expression of P-selectin, was assessed in patients at their time of randomisation. Data are median fluorescence values. Results The aspirin P-selectin test demonstrated that platelet expression was lower in 494 patients taking aspirin than in 162 patients not: mean 210 (SD 188) versus 570 (435), difference 360.3 (95% CI 312.2–408.4) (2p < 0.001). Aspirin did not suppress P-selectin levels below 500 units in 23 (4.7%) patients. The clopidogrel test showed that platelet reactivity was lower in 97 patients taking clopidogrel than in 585 patients not: 655 (296) versus 969 (315), difference 314.5 (95% CI 247.3–381.7) (2p < 0.001). Clopidogrel did not suppress P-selectin level below 860 units in 24 (24.7%) patients. Conclusions Aspirin and clopidogrel suppress stimulated platelet P-selectin, although one-quarter of patients on clopidogrel have high on-treatment platelet reactivity. Platelet function testing may be performed remotely in the context of a large multicentre trial. Trial registration ISRCTN47823388.

Developmental Stage-Specific Manifestations of Absent TPO/c-MPL Signalling in Newborn Mice.

Congenital amegakaryocytic thrombocytopaenia (CAMT) is a disorder caused by c-MPL mutations that impair thrombopoietin (TPO) signalling, resulting in a near absence of megakaryocytes (MKs). While this phenotype is consistent in adults, neonates with CAMT can present with severe thrombocytopaenia despite normal MK numbers. To investigate this, we characterized MKs and platelets in newborn c-MPL –/– mice. Liver MKs in c-MPL –/– neonates were reduced in number and size compared with wild-type (WT) age-matched MKs, and exhibited ultrastructural abnormalities not found in adult c-MPL –/– MKs. Platelet counts were lower in c-MPL –/– compared with WT mice at birth and did not increase over the first 2 weeks of life. In vivo biotinylation revealed a significant reduction in the platelet half-life of c-MPL –/– newborn mice (P2) compared with age-matched WT pups, which was not associated with ultrastructural abnormalities. Genetic deletion of the pro-apoptotic Bak did not rescue the severely reduced platelet half-life of c-MPL –/– newborn mice, suggesting that it was due to factors other than platelets entering apoptosis early. Indeed, adult GFP+ (green fluorescent protein transgenic) platelets transfused into thrombocytopenic c-MPL –/– P2 pups also had a shortened lifespan, indicating the importance of cell-extrinsic factors. In addition, neonatal platelets from WT and c-MPL –/– mice exhibited reduced P-selectin surface expression following stimulation compared with adult platelets of either genotype, and platelets from c-MPL –/– neonates exhibited reduced glycoprotein IIb/IIIa (GPIIb/IIIa) activation in response to thrombin compared with age-matched WT platelets. Taken together, our findings indicate that c-MPL deficiency is associated with abnormal maturation of neonatal MKs and developmental stage-specific defects in platelet function.

Patients with Splanchnic Vein Thrombosis Demonstrate Significantly Increased Platelet Activity

Background:Splanchnic vein thrombosis (SVT) includes extrahepatic portal vein obstruction (EHPVO), Budd-Chiari syndrome (BCS) and mesenteric vein thrombosis. Myeloproliferative neoplasms (MPN) account for the majority of non-cirrhotic SVT and are diagnosed in 30% of patients with EHPVO and 40% with BCS. Recurrent thrombosis is a common and significant complication in patients with PVT and MPN. Causes of thrombosis in this patient group are unknown; it is likely multifactorial, with proposed risk factors including increased platelet mass and platelet hyper-reactivity. There is evidence that in general, patients with MPN have increased platelet reactivity when compared to healthy controls, but, there is no data looking specifically at patients with SVT. However patients with SVT clearly demonstrate a significantly more aggressive phenotype in terms of systemic thrombosis, with risk of recurrent thrombotic episodes. MASCOT is a multicenter observational study assessing morbidity and portal circulation in patients with SVT, in a subset of whom we have assessed platelet activity.Methods:Whole blood flow cytometry was used to assess platelet activity by surface expression of P-selectin (CD62P) an established marker of platelet activity. We measured CD62P at baseline in unstimulated platelets and following stimulation with increasing concentrations of thrombin receptor activator protein (TRAP), range 10-50μmol. The guidelines from the European consensus on platelet flow cytometry were used for this assay (Schmitz G et al 1998). Demographic and clinical data were collected. Results were analysed in GraphPad Prism using t-tests; all values displayed as mean (95% confidence interval).Results:We assessed platelet activity in 14 patients with SVT using healthy controls (n=6) for comparison. In our patient cohort 8/14 (57%) were male and 6/14 (42%) female. The average age of our patients was 49 years. 12/14 (85%) were positive for the JAK2 mutation and 2/14 (15%) had a positive calreticulin mutation (CALR+). 2/14 (14%) patients had myelofibrosis (MF), 6/14 (42%) polycythaemia vera (PV), 4/14 (28%) essential thrombocytosis (ET) and 2/14 (14%) were positive for the JAK2 mutation but did not show evidence of MPN in their bone marrow and had normal blood counts. 10/14 (71%) were on warfarin alone, 2/14 (14%) on warfarin and aspirin, 1/14 (7%) on rivaroxaban and 1/14 (7%) on aspirin alone.Platelet activity as measured by CD62P expression was increased in patients with SVT at baseline compared with controls 13.1% (5.5-20.7) vs 0.5% (0.2-0.7) (p=0.003). Platelet activation and reactivity was significantly greater in the patient group compared with controls at all concentrations of TRAP (figure 1). Control platelets were relatively unreactive to trap stimulation and a significant increase from baseline in CD62P at the highest concentration of TRAP used (50 μmol) 0.5% (0.2-0.7) vs 7.31% (1.17-13.4) (p=0.03), however this is a modest increase, the biological significance of which is uncertain.In the patient population, the platelets were hyper-reactive to TRAP induced up-regulation of CD62P. This was significant when comparing unstimulated platelets at the 25 and 50 μmol concentrations 13.1% (5.5-20.7) vs 22.6% (13.8-39.4) (p=0.0008) and 13.1% (5.5-20.7) vs 34.9% (22.6-47.1) (p<0.0001) respectively. Although not significant at the lower concentration of 10 μmol TRAP there was a trend towards significance 13.1% (5.5-20.7) vs 22% (12.1-38.9) (p=0.07).Discussion:These results demonstrate that patients with SVT have both significantly increased baseline markers of platelet activation and increased reactivity to stimulation. As part of this longitudinal study, we propose to monitor changes in platelet activity in this patient cohort over time to assess whether or not changes in treatment modality influence platelet activity and whether or not changes in platelet activity can predict further thrombotic episodes; this data will be available in the coming year. Our data provides a platform for further investigation; in due course we will control this data using patients with MPN without thrombosis, as further patients within our study group are analysed. [Display omitted] DisclosuresChen:Novartis: Other: Advisory Board. Sekhar:Novartis: Research Funding.

Platelet Alpha-Storage Pool Defect Combined with Non-Classic Delta-Storage Pool Deficiency - with a Severe Bleeding Tendency

Aims:We report about a 35 year old male patient with severe and frequent epistaxis and hematoma since infancy. He presented with mild thrombocytopenia and increased mean platelet volume. Von Willebrand's disease and subhemophilia had been excluded. Previously, he was diagnosed with immune thrombocytopenic purpura. He never underwent elective surgery. His parents were asymptomatic. However, his young daughter also suffers from severe bleeding symptoms and was referred to our coagulation outpatient clinic to investigate the bleeding disorder.Methods:Platelet function was assessed by light transmission-, lumi-aggregometry and flow cytometry. Lysates of gel-filtered platelets were analyzed for total granule P-selectin, CD63 and von Willebrand factor (VWF) content by Western blotting and for serotonin levels by ELISA, respectively. Molecular genetic analysis was performed using whole exome sequencing and pyrosequencing.Results: Platelet aggregation in response to ADP was not inducible. Platelet aggregation in response to epinephrine resulted in monophasic curves. Platelet aggregation in response to TRAP-6 or collagen was inducible but impaired and accompanied by disaggregation. In contrast, arachidonic acid- and U46619-induced platelet aggregation as well as ristocetin-induced platelet agglutination/aggregation was within the normal range. Also platelet surface expression of GPIIb/IIIa, GPIb/V/IX, GPVI and CD29 was normal. Activation of GPIIb/IIIa in response to ADP, convulxin or TRAP-6 was inducible but diminished and in response to epinephrine or arachidonic acid normal. P-selectin (alpha-granule marker) and CD63 (dense body/lysosome marker) surface expression was not inducible by thrombin or convulxin and markedly reduced in response to TRAP-6 and arachidonic acid. Immunoblot analysis of isolated platelets demonstrated pronouncedly decreased content of total P-selectin, VWF and CD63. Fluorescent staining of adenine nucleotides by mepacrine in patient’s platelet dense bodies and secretion of ATP induced by arachidonic acid (normal aggregation) were absent. However, patient’s platelet serotonin levels were within the normal range. The molecular genetic diagnostic showed an undescribed mutation in the GATA1 gene (c.886A>C hemizygot, p.T296P). The bioinformatic check confirmed a pathogenetic importance of this mutation located in the domain I of the C-terminal zinc finger.Conclusion: We describe the first case of a combined platelet alpha- and partial delta-storage pool disorder where the dense granules lack adenine nucleotides but contain normal levels of serotonin - causing a severe bleeding disorder. DisclosuresNo relevant conflicts of interest to declare.

Platelets Can Phagocytose Influenza Virus Which May Contribute to the Occurrence of Thrombocytopenia during Influenza Infection

▪Thrombocytopenia is a common and sometimes life-threatening symptom in patients with influenza, which indicates that platelets may play a significant role during virus infection. However, the mechanisms underlying influenza associated thrombocytopenia are still unclear to date. In this study, the relationship between platelets and influenza infection was studied in vivo and in vitro.In the period 2009-2012 we randomly measured viral load and platelet count in laboratory-confirmed influenza patients (A/H1N1 subtype; n=85) presented at the emergency department of Erasmus MC. In a cross-sectional study we found, after excluding patients with HIV infection, chemotherapy treatment and immediate referral to the ICU, that a high viral load was significantly correlated with a low platelet count at presentation (n=36; Spearman correlation coefficient 0.341; P=0.042). To study this relationship further we inoculated ferrets (n=120) with an H3N2 (n=30), pandemic H1N1 (n=30), or H5N1 influenza A virus (n=30) and measured platelet counts in six ferrets per time point at day 0, 1, 2, 3 and 4 after infection. Thrombocytopenia was seen at day 3 in all ferrets compared to the control animals (n=30). The decrease in platelet counts was significant in the H5N1 infected ferrets (p<0.05). For the sialidase and influenza virus inhibitor oseltamivir, we have previously shown that it functions as an inhibitor of platelet degradation. Interestingly, thrombocytopenia in ferrets could also be inhibited by oral administration of oseltamivir. However, these results were not statistically significant.Finally, we studied the interaction between platelets and influenza viruses by electron microscopy. In a platelet suspension incubated for 1 minute with influenza virus we observed platelets with virus containing vacuoles, suggesting that the platelets had rapidly phagocytosed the viruses. Characteristics of these platelets incubated with influenza virus were measured including membrane expression of the GPIb-IX-V complex, CD62P, and surface glycans sialic acid and GlcNac using flowcytometry and functional capacity employing aggregometry using collagen, thrombin and ADP as agonists. However, no effect was seen in expression of GPIba and GPIX, P-selectin expression and surface expression of sialic acid and GlcNAc after incubation with influenza virus. Also no effect was seen on functional capacity of these platelets.Our study shows that influenza virus is significantly correlated with a lower platelet count. Phagocytosis of influenza virus by platelets may play an important role in the occurrence of thrombocytopenia during influenza infection and may be a mechanism of virus clearance during infection. DisclosuresOsterhaus:Viroclinics Biosciences: Other: chief scientific officer and hold certificates of shares.

Intracoronary platelet and monocyte activation status within platelet-monocyte complexes are determinants of inflammation in ST elevation myocardial infarction1.

Platelet Monocyte Complexes (PMCs) are commonly expressed in coronary artery disease but their pathologic significance in ST elevation myocardial infarction (STEMI) is unclear. This study evaluates the relationship between locally activated PMCs and intracoronary inflammation in stable and unstable coronary disease. Micro catheter aspirated blood samples of 15 STEMI and 7 stable angina patients are collected from the coronary artery (CA), aorta (AO) and right atrium (RA). Samples are labelled with monoclonal antibodies and prepared for flow cytometry. CD 14 and CD 61 double positive cells are identified as PMC. P-selectin expression is identified by additional CD62P positivity and TF expression by additional CD142 positivity. Plasma TNF-alpha and IL-6 are measured using ELISA and CRP is measured in plasma using a high sensitivity automated microparticle enhanced latex turbidimetric immunoassay. No site-specific difference is seen in overall PMC expression in STEMI or stable angina. Surface P-selectin expression in STEMI [median (IQR)] is significantly higher in CA [35.01 (23.15-56.99)] compared with AO [15.99 (10.3-18.85)] or RA [14.02 (10.42-26.08)] (p = 0.003). Intracoronary PMC correlates significantly with intracoronary TNF-alpha (r = 0.87, p = 0.001) and intracoronary IL-6 (r = 0.76, p = 0.03). Bound monocytes within P-selectin positive and tissue factor positive complexes correlate positively with intracoronary TNF-alpha (r = 0.81, p = 0.008 & r = 0.80, p = 0.009 respectively) and IL-6 (r = 0.54, p = 0.16 & r = 0.71, p = 0.05 respectively). No such correlation is observed in the peripheral circulation of STEMI and stable angina patients. Inflammation is not attributable to PMC formation per se. However, increased intracoronary P-selectin expression by activated platelets and tissue factor expression by activated monocytes within the complexes are determinants of local intracoronary inflammatory burden in STEMI.

Humanizing the Protease-Activated Receptor (PAR) Expression Profile in Mouse Platelets by Knocking PAR1 into the Par3 Locus Reveals PAR1 Expression Is Not Tolerated in Mouse Platelets

Anti-platelet drugs are the mainstay of pharmacotherapy for heart attack and stroke prevention, yet improvements are continually sought. Thrombin is the most potent activator of platelets and targeting platelet thrombin receptors (protease-activated receptors; PARs) is an emerging anti-thrombotic approach. Humans express two PARs on their platelets–PAR1 and PAR4. The first PAR1 antagonist was recently approved for clinical use and PAR4 antagonists are in early clinical development. However, pre-clinical studies examining platelet PAR function are challenging because the platelets of non-primates do not accurately reflect the PAR expression profile of human platelets. Mice, for example, express Par3 and Par4. To address this limitation, we aimed to develop a genetically modified mouse that would express the same repertoire of platelet PARs as humans. Here, human PAR1 preceded by a lox-stop-lox was knocked into the mouse Par3 locus, and then expressed in a platelet-specific manner (hPAR1-KI mice). Despite correct targeting and the predicted loss of Par3 expression and function in platelets from hPAR1-KI mice, no PAR1 expression or function was detected. Specifically, PAR1 was not detected on the platelet surface nor internally by flow cytometry nor in whole cell lysates by Western blot, while a PAR1-activating peptide failed to induce platelet activation assessed by either aggregation or surface P-selectin expression. Platelets from hPAR1-KI mice did display significantly diminished responsiveness to thrombin stimulation in both assays, consistent with a Par3-/- phenotype. In contrast to the observations in hPAR1-KI mouse platelets, the PAR1 construct used here was successfully expressed in HEK293T cells. Together, these data suggest ectopic PAR1 expression is not tolerated in mouse platelets and indicate a different approach is required to develop a small animal model for the purpose of any future preclinical testing of PAR antagonists as anti-platelet drugs.

Whole Blood Analysis of Leukocyte-Platelet Aggregates.

In inflammatory and thrombotic syndromes, platelets aggregate with circulating leukocytes, especially monocytes and neutrophils. This leukocyte-platelet aggregate formation is initiated primarily through platelet surface expression of P-selectin (CD62P), following activation-dependent degranulation of α-granules, binding to its constitutively expressed counter-receptor, P-selectin glycoprotein ligand 1 (PSGL-1), on leukocytes. Monocyte-platelet aggregates are a more sensitive marker of platelet activation than platelet surface P-selectin. Detection of leukocyte-platelet aggregates is relatively simple by whole-blood flow cytometry. Light scatter and at least one leukocyte-specific antibody are used to gate the desired population, and the presence of associated platelets is detected by immunostaining for abundant platelet-specific markers. © 2016 by John Wiley & Sons, Inc.

Modification of Pulsed Electric Field Conditions Results in Distinct Activation Profiles of Platelet-Rich Plasma.

BackgroundActivated autologous platelet-rich plasma (PRP) used in therapeutic wound healing applications is poorly characterized and standardized. Using pulsed electric fields (PEF) to activate platelets may reduce variability and eliminate complications associated with the use of bovine thrombin. We previously reported that exposing PRP to sub-microsecond duration, high electric field (SMHEF) pulses generates a greater number of platelet-derived microparticles, increased expression of prothrombotic platelet surfaces, and differential release of growth factors compared to thrombin. Moreover, the platelet releasate produced by SMHEF pulses induced greater cell proliferation than plasma.AimsTo determine whether sub-microsecond duration, low electric field (SMLEF) bipolar pulses results in differential activation of PRP compared to SMHEF, with respect to profiles of activation markers, growth factor release, and cell proliferation capacity.MethodsPRP activation by SMLEF bipolar pulses was compared to SMHEF pulses and bovine thrombin. PRP was prepared using the Harvest SmartPreP2 System from acid citrate dextrose anticoagulated healthy donor blood. PEF activation by either SMHEF or SMLEF pulses was performed using a standard electroporation cuvette preloaded with CaCl2 and a prototype instrument designed to take into account the electrical properties of PRP. Flow cytometry was used to assess platelet surface P-selectin expression, and annexin V binding. Platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), endothelial growth factor (EGF) and platelet factor 4 (PF4), and were measured by ELISA. The ability of supernatants to stimulate proliferation of human epithelial cells in culture was also evaluated. Controls included vehicle-treated, unactivated PRP and PRP with 10 mM CaCl2 activated with 1 U/mL bovine thrombin.ResultsPRP activated with SMLEF bipolar pulses or thrombin had similar light scatter profiles, consistent with the presence of platelet-derived microparticles, platelets, and platelet aggregates whereas SMHEF pulses primarily resulted in platelet-derived microparticles. Microparticles and platelets in PRP activated with SMLEF bipolar pulses had significantly lower annexin V-positivity than those following SMHEF activation. In contrast, the % P-selectin positivity and surface P-selectin expression (MFI) for platelets and microparticles in SMLEF bipolar pulse activated PRP was significantly higher than that in SMHEF-activated PRP, but not significantly different from that produced by thrombin activation. Higher levels of EGF were observed following either SMLEF bipolar pulses or SMHEF pulses of PRP than after bovine thrombin activation while VEGF, PDGF, and PF4 levels were similar with all three activating conditions. Cell proliferation was significantly increased by releasates of both SMLEF bipolar pulse and SMHEF pulse activated PRP compared to plasma alone.ConclusionsPEF activation of PRP at bipolar low vs. monopolar high field strength results in differential platelet-derived microparticle production and activation of platelet surface procoagulant markers while inducing similar release of growth factors and similar capacity to induce cell proliferation. Stimulation of PRP with SMLEF bipolar pulses is gentler than SMHEF pulses, resulting in less platelet microparticle generation but with overall activation levels similar to that obtained with thrombin. These results suggest that PEF provides the means to alter, in a controlled fashion, PRP properties thereby enabling evaluation of their effects on wound healing and clinical outcomes.

The Antiplatelet Effect of Clopidogrel Decreases With Patient Age.

Recent data suggest that clopidogrel-mediated platelet inhibition is age dependent. However, so far the effect of age on adenosine diphosphate (ADP)-inducible platelet reactivity has only been investigated by test systems measuring surrogate markers of platelet aggregation. We therefore sought to study the impact of age on platelet inhibition by clopidogrel by whole-blood flow cytometry. Platelet surface P-selectin expression, activated glycoprotein (GP) IIb/IIIa, and monocyte-platelet aggregate (MPA) formation were determined by flow cytometry in 302 patients with dual antiplatelet therapy after successful angioplasty and stenting. Patient age was independently associated with ADP-inducible P-selectin expression, GPIIb/IIIa, and MPA formation (all P < .05). Moreover, platelet surface expressions of P-selectin and activated GPIIb/IIIa were significantly higher in patients ≥75 years compared with younger patients (both P ≤ .004). Likewise, MPA formation was significantly more pronounced in patients ≥75 years ( P = .02). Finally, high P-selectin and high GPIIb/IIIa were significantly more frequent in patients ≥75 years compared with younger patients (both P < .001). Further, high MPA ADP occurred more frequently in patients ≥75 years compared to younger patients ( P < .05). In conclusion, the antiplatelet effect of clopidogrel decreases with patient age.

Abstract 505: Circulating Monocyte-Platelet Aggregates are Different Across Phenotypes of Vascular Disease

Background: Platelets are a major culprit in the pathogenesis of cardiovascular disease (CVD). Although vascular disease in different arterial beds share common risk factors, the role of platelet activity may differ by vascular bed. Clinical trials suggest that more potent antiplatelet therapy is needed in lower extremity peripheral artery disease (PAD) than coronary artery disease (CAD). We investigated circulating monocyte platelet aggregates (MPA), a reproducible ex vivo measurement of platelet activity, in patients with CVD (PAD, CAD, carotid artery stenosis [CAS], and abdominal aortic aneurysm [AAA]) and disease controls. Methods: Subjects with CVD (n=351) and disease controls (n=73) had MPA measured using strict quality control, all of whom were on aspirin monotherapy. MPA were identified by CD14/CD61 positivity. Data is represented as median (IQR, interquartile range). Wilcoxon rank sum, Wilcoxon signed rank, and multivariable linear regression were used to analyze data. Results: Platelets aggregated with monocytes had a higher platelet surface expression of PAC-1, P-selectin, and CD40 versus platelets not aggregated with monocytes (P&lt;0.05 for each comparison). Compared with controls, MPA was significantly higher in CVD (14.5 [10.3, 27.8] vs. 9.4 [8.2, 11.5], P&lt;0.001) which remained significant after multivariable adjustment for demographics and risk factors (β=9.1 (SER=3.9), P=0.02). For each vascular disease phenotype, MPA was higher than controls ( Figure ). In a multivariable linear regression model including demographics, risk factors and each vascular bed, PAD was the only vascular disease associated with a higher value of MPA (β=10.2 (SER=2.4), P&lt;0.001). Conclusions: Platelets aggregated with monocytes have increased platelet activity and are significantly elevated in subjects with PAD. Future studies understanding the platelet profile in PAD and its clinical relevance are needed.

Abstract 113: Platelet and Monocyte Activity in Carotid Artery Stenosis Treated with Carotid Endarterectomy

Background: Carotid artery stenosis (CAS) is a marker of atherosclerosis, a disease mediated by abnormalities in platelet and monocyte function, and a significant cause of stroke. Moreover, the effect of carotid artery revascularization via carotid endarterectomy (CEA) on platelet and monocyte markers is unknown. Objective: This study aims to investigate platelet activity, monocyte subsets and monocyte platelet aggregates (MPA) in CAS and changes with CEA. Methods: This prospective cohort study evaluated 48 patients who underwent non emergent CEA. Peripheral venous blood samples were obtained before, immediately postoperative and at 24 hours postoperative. Twenty healthy subjects served as controls. Platelet surface expression of P-selectin and PAC-1, monocyte subsets, and MPA were assessed using flow cytometry. Three distinct monocyte subsets were measured: anti-inflammatory (i.e. classical CD14 ++ CD16 - ) and pro-inflammatory (i.e. intermediate CD14 ++ CD16 + and nonclassical CD14 + CD16 ++ ) monocytes. Differences between two matched samples and between the study and control groups were statistically analyzed. Results: Compared to healthy subjects, CAS subjects had significantly greater markers of platelet activity (P-selectin [p=0.003] and PAC-1 [p=0.01]), pro-inflammatory monocytes (intermediate [p&lt;0.0001] and nonclassical [p=0.009]) and MPA (p=0.0002). Following CEA, anti-inflammatory monocytes increased and pro-inflammatory monocytes decreased (Figure 1A). Platelet expression of P-selectin and MPA did not change, while PAC-1 transiently increased but then returned to baseline by 24 hours postoperative (Figure 1B &amp;C). Conclusions: Subjects with CAS have elevated markers of thrombosis, inflammation, and their interface. However, only the pro-inflammatory monocytes are significantly reduced following CEA. Future studies investigating the clinical consequences of this reduction are warranted.

Lovastatin induces platelet apoptosis.

Statins are widely used in the prevention of atherosclerosis and treatment of coronary artery disease because of pleiotropic effects on thrombosis. Thrombocytopenia and hemorrhage occurred in some statin-treated patients, but the reason remains unclear. In the current study, we show that lovastatin dose-dependently induces depolarization of mitochondrial inner transmembrane potential, leading to up-regulation of Bak, down-regulation of Bcl-XL, and activation of caspase-3/8/9. Lovastatin treatment did not increase the surface expression of P-selectin or PAC-1 binding but led to strongly reduced collagen- and thrombin-induced platelet aggregation. The integrin αIIbβ3 antagonist, RGDS, inhibited lovastatin-induced apoptosis in both human platelets and Chinese hamster ovary (CHO) cells stably expressing integrin αIIbβ3. The number of circulating platelets in mice was significantly reduced after intraperitoneal injections with lovastatin. Taken together, these data indicate that lovastatin induced caspase-dependent platelet apoptosis. Lovastatin does not incur platelet activation, whereas impairs platelet function and reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia and hemorrhage in patients treated with statins.

Dok-1 negatively regulates platelet integrin αIIbβ3 outside-in signalling and inhibits thrombosis in mice.

Adaptor proteins play a critical role in the assembly of signalling complexes after engagement of platelet receptors by agonists such as collagen, ADP and thrombin. Recently, using proteomics, the Dok (downstream of tyrosine kinase) adapter proteins were identified in human and mouse platelets. In vitro studies suggest that Dok-1 binds to platelet integrin β3, but the underlying effects of Dok-1 on αIIbβ3 signalling, platelet activation and thrombosis remain to be elucidated. In the present study, using Dok-1-deficient (Dok-1-/-) mice, we determined the phenotypic role of Dok-1 in αIIbβ3 signalling. We found that platelets from Dok-1-/- mice displayed normal aggregation, activation of αIIbβ3 (assessed by binding of JON/A), P-selectin surface expression (assessed by anti-CD62P), and soluble fibrinogen binding. These findings indicate that Dok-1 does not affect "inside-out" platelet signalling. Compared with platelets from wild-type (WT) mice, platelets from Dok-1-/- mice exhibited increased clot retraction (p < 0.05 vs WT), increased PLCγ2 phosphorylation, and enhanced spreading on fibrinogen after thrombin stimulation (p < 0.01 vs WT), demonstrating that Dok-1 negatively regulates αIIbβ3 "outside-in" signalling. Finally, we found that Dok-1-/- mice exhibited significantly shortened bleeding times and accelerated carotid artery thrombosis in response to photochemical injury (p < 0.05 vs WT mice). We conclude that Dok-1 modulates thrombosis and haemostasis by negatively regulating αIIbβ3 outside-in signalling.

Specific cell-derived microvesicles: Linking endothelial function to carotid artery intima-media thickness in low cardiovascular risk menopausal women

BackgroundDecreases in endothelial function measured by reactive hyperemic index (RHI) correlated with increases in carotid intima-media thickness (CIMT) in recently menopausal women with a low risk cardiovascular profile. Factors linking this association are unknown. ObjectiveAssess, longitudinally, markers of platelet activation and cell-derived, blood-borne microvesicles (MV) in relationship to RHI and CIMT in asymptomatic, low risk menopausal women. MethodsRHI by digital pulse tonometry (n = 93), CIMT by ultrasound (n = 113), measures of platelet activation and specific cell-derived, blood-borne MV were evaluated in women throughout the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic. ResultsCIMT, but not RHI, increased significantly over 4 years. The average change in CIMT correlated significantly with the average follow-up values of MV positive for common leukocyte antigen [CD45; ρ = 0.285 (P = 0.002)] and VCAM-1 [ρ = 0.270 (P = 0.0040)]. Using principal components analysis (PC) on the aggregate set of average follow-up measures, the first derived PC representing numbers of MV positive for markers of vascular endothelium, inflammatory cells (leukocyte and monocytes), pro-coagulant (tissue factor), and cell adhesion molecules (ICAM-1 and VCAM-1) associated with changes in RHI and CIMT. Changes in RHI associated with another PC defined by measures of platelet activation (dense granular ATP secretion, surface expression of P-selectin and fibrinogen receptors). ConclusionsMV derived from activated endothelial and inflammatory cells, and those expressing cell adhesion and pro-coagulant molecules may reflect early vascular dysfunction in low risk menopausal women. Assays of MV as non-conventional measures to assess cardiovascular risk in asymptomatic women remain to be developed.