Platelet Factor 4 Concentrations Research Articles

Long-term stability of frozen platelet-rich plasma under −80 °C storage condition

Platelet rich plasma (PRP) is increasingly used in various fields of medicine, aiming to regeneration and repair damaged tissues, cells and organs. High concentration of bioactive molecules including growth factors, cytokines and chemokines are the rationale of using PRP. The aim of this study is to analyze the effect of frozen on the levels of growth factors. In our study, PRP samples were isolated from 50 healthy volunteers using the Trima Accel blood cell separator. The concentration of growth factors such as platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF-1) and platelet factor 4 (PF-4) were assessed in fresh PRP and frozen PRP stored at −80 °C for one to twelve months. The study found that count of platelet in all fresh and frozen PRP samples was significantly increased compared to whole blood baseline. There was no significant difference in the concentrations of PDGF-BB, bFGF, VEGF, and PF-4 between fresh and frozen samples. The concentrations of EGF and IGF in Frozen-PRP group were significantly higher than those in Fresh-PRP group. And the storage condition of −80 °C is suitable for PRP, which will not lead to a decrease in growth factors concentration for at least 6 months.

Platelet Factor 4 Enhances Antimicrobial Function of the Endothelium and Improves Outcome in a Murine Model of Sepsis

Introduction: In this study, we investigated mechanism(s) by which the platelet-specific chemokine platelet factor 4 (PF4) interacts with bacteria to enhance host-defense function and improve outcome in sepsis. Sepsis, a life-threatening dysregulated response to infection, is the leading cause of mortality worldwide for which there are no targeted treatments. Upon sensing pathogens, platelets release high concentrations of PF4, a positively charged chemokine with high affinity for polyanions including polymers in the bacterial cell wall, the endothelial glycocalyx and cell-free DNA. We previously found that PF4 improves outcomes in the murine lipopolysaccharide endotoxemia and enhances in vitro bacterial capture by neutrophil extracellular traps. We now show that PF4-coating of bacteria reduces coagulopathy, limits bacterial dissemination, and improves survival in a murine model of polymicrobial sepsis, in part through the novel mechanism of enhancing the antimicrobial function of endothelial cells (ECs). Methods: Effects of PF4 on bacterial internalization by ECs was assessed by incubating heat-inactivated Staphylococcus (S) aureus conjugated to pHrodo, a dye that only fluoresces following cellular internalization, with PF4 (0-100mg/mL) and then expose those complexes to human umbilical vein endothelial cells (HUVECs) ± tumor necrosis factor a (TNFa) to simulate inflammation. Internalized S aureus fluorescent signal was visualized 16hr post-exposure. HUVECs were stained for von Willebrand factor (VWF) release to indicate bacteria-induced endothelial activation. Effects of PF4 on EC killing of bacteria was studied by exposing HUVECs to live Escherichia (E) coli pre-treated with PF4 (0-25mg/mL) for 1hr. ECs were washed to remove excess unbound bacteria, and cell lysates were plated on agar plates for 24hr to count colony forming units (CFUs). To investigate the effects of PF4-bacteria interactions on sepsis survival outcomes, contents from the cecum (cecal slurry, CS) of C57BL6 donor mice (100mg/mL) were pre-treated with PF4 (100mg) for 20min and injected intravenously (IV) into wildtype (WT) or PF4 -/-recipient mice to induce immediate bacteremia. Animals were evaluated for sepsis severity for up to 72hr, and survival analysis was performed using the Mantel-Cox test. A separate cohort of WT mice was given IV CS (400mg/mL) pre-incubated with PF4 (8mg). Blood was drawn at 3 to 24hr post-CS infusion and subjected to complete blood count. Thrombin anti-thrombin (TAT) and VWF levels were measured by ELISAs in plasma as indicators of intravascular coagulopathy and endothelial activation. To assess bacterial load, blood and liver and spleen homogenates were plated on agar plates to count for CFUs. Results: PF4 coating of bacteria enhanced bacterial uptake by HUVECs, peaking at 20-50 µg/mL of PF4. TNFa-stimulated HUVECs more readily internalized PF4-coated bacteria at 1-20 µg/mL of PF4. Although PF4 promoted S aureus uptake, it limited bacteria-induced VWF secretion by HUVECs. PF4 also enhanced EC killing of E coli upon bacterial internalization. In the CS model of murine polymicrobial sepsis, PF4 prevented CS-induced mortality in both WT and PF4 -/- mice ( Fig. 1). PF4-pretreatment of CS also reduced leukopenia and thrombocytopenia 3-6hr post-CS challenge in WT mice. Mice given PF4-treated CS exhibited lower TAT and VWF levels as compared to untreated-CS animals. Lastly, PF4 reduced bacterial load in blood, liver, and spleen 24hr post-CS inoculation ( Fig. 2). Conclusion: PF4 enhances bacterial uptake and killing by the endothelium in vitro, promotes bacterial clearance while preventing bacteria-induced coagulopathy and endothelial dysfunction leading to improved survival in the murine CS model of polymicrobial sepsis. These studies suggest that PF4 binding to bacteria may be an important host defense mechanism. Further studies are underway to define the contribution of EC antimicrobial activity to the observed outcomes. The ability of PF4-based therapeutics to enhance clearance of microbes merits further translational studies in sepsis.

Platelet factor 4 induces bone loss by inhibiting the integrin α5-FAK-ERK pathway.

The effect of platelet factor 4 (PF4) on bone marrow mesenchymal stem cells (BMMSCs) and osteoporosis is poorly understood. Therefore, this study aimed to evaluate the effects of PF4-triggered bone destruction in mice and determine the underlying mechanism. First, in vitro cell proliferation and cell cycle of BMMSCs were assessed using a CCK8 assay and flow cytometry, respectively. Osteogenic differentiation was confirmed using staining and quantification of alkaline phosphatase and Alizarin Red S. Next, an osteoporotic mouse model was established by performing bilateral ovariectomy (OVX). Furthermore, the PF4 concentrations were obtained using enzyme-linked immunosorbent assay. The bone microarchitecture of the femur was evaluated using microCT and histological analyses. Finally, the key regulators of osteogenesis and pathways were investigated using quantitative real-time polymerase chain reaction and Western blotting. Human PF4 widely and moderately decreased the cell proliferation and osteogenic differentiation ability of BMMSCs. Furthermore, the levels of PF4 in the serum and bone marrow were generally increased, whereas bone microarchitecture deteriorated due to OVX. Moreover, in vivo mouse PF4 supplementation triggered bone deterioration of the femur. In addition, several key regulators of osteogenesis were downregulated, and the integrin α5-focal adhesion kinase-extracellular signal-regulated kinase (ITGA5-FAK-ERK) pathway was inhibited due to PF4 supplementation. PF4 may be attributed to OVX-induced bone loss triggered by the suppression of bone formation in vivo and alleviate BMMSC osteogenic differentiation by inhibiting the ITGA5-FAK-ERK pathway.

Abstract 12563: The Trajectory of Platelet Factor 4 in Acute Myocardial Infarction and the Impact of PCSK9-Inhibition

Introduction: Increased platelet activity during the peri- and early post-infarction period may contribute to adverse outcomes during that time. Platelet factor 4 (PF4) promotes coagulation and inflammation. The trajectory of PF4 and the impact of proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibition on that trajectory are unknown. Hypothesis: Early administration of the PCSK9 inhibitor evulocumab, added to dual anti-platelet therapy, inhibits a rise in PF4 during the early post-infarction period. Methods: Participants from the Evolocumab in Acute Coronary Syndrome trials (EVACS; NCT03515304, NCT04082442), with a NSTEMI and a troponin-I of > 5 ng/ml or a STEMI were randomized to placebo or to 420 mg SQ of evolocumab within 24 hours of hospitalization. We performed serial ex vivo serum PF4 levels obtained at baseline (prior to study drug administration), and at days 1 and 30 and PF4 values were normalized to total platelet count. Normally distributed data were summarized using means and standard deviations and non-normally distributed data using medians and interquartile ranges. Results: Twenty-three participants were randomized to placebo and 23 to evolocumab. Mean age was 60±13 years, 48% were women, 22% were African American and 96% were on dual anti platelet therapy. Baseline PF4 concentrations (expressed as ng/1k platelets) did not differ between the two groups (placebo: 9.3 [4-12] vs evolocumab 8.0 [4-12], p=0.8). There was no significant change in PF4 in the placebo group from baseline to day 1 but a significant increase to 13.0 [11-14], p<0.01 (baseline vs 30 days) at 30 days (+32%). In contrast, there were no significant changes from baseline in the evolocumab group at either of the timepoints. At 30 days PF4 was higher in the placebo group 13.06 [11-14] than the evolocumab group 10.7 [6-13], p=0.04. Further analysis of results in the evolocumab group did not show any significant changes in PF4 based on the type of P2Y12 inhibitor used and no association with the change in LDL-cholesterol. Conclusion: A rise in PF4 levels was observed during the early post-infarction period in acute myocardial infarction patients. This rise was reversed by a single 420 mg SQ dose of evolocumab administered within 24 hours of hospital admission.

Platelet Inhibition by Low-Dose Acetylsalicylic Acid Reduces Neuroinflammation in an Animal Model of Multiple Sclerosis.

Aside from the established immune-mediated etiology of multiple sclerosis (MS), compelling evidence implicates platelets as important players in disease pathogenesis. Specifically, numerous studies have highlighted that activated platelets promote the central nervous system (CNS)-directed adaptive immune response early in the disease course. Platelets, therefore, present a novel opportunity for modulating the neuroinflammatory process that characterizes MS. We hypothesized that the well-known antiplatelet agent acetylsalicylic acid (ASA) could inhibit neuroinflammation by affecting platelets if applied at low-dose and investigated its effect during experimental autoimmune encephalomyelitis (EAE) as a model to study MS. We found that oral administration of low-dose ASA alleviates symptoms of EAE accompanied by reduced inflammatory infiltrates and less extensive demyelination. Remarkably, the percentage of CNS-infiltrated CD4+ T cells, the major drivers of neuroinflammation, was decreased to 40.98 ± 3.28% in ASA-treated mice compared to 56.11 ± 1.46% in control animals at the disease maximum as revealed by flow cytometry. More interestingly, plasma levels of thromboxane A2 were decreased, while concentrations of platelet factor 4 and glycoprotein VI were not affected by low-dose ASA treatment. Overall, we demonstrate that low-dose ASA could ameliorate the platelet-dependent neuroinflammatory response in vivo, thus indicating a potential treatment approach for MS.

Cross-sectional assessment of haemostatic profile and hepatic dysfunction in Fontan patients

BackgroundFontan-associated liver disease is accompanied by a hypercoagulable state. While hepatic dysfunction in Fontan patients is common, its relationship with haemostatic changes and clinical outcomes in this patient population remains...

Platelet factor-4 concentration in adult veno-arterial ECMO patients.

Heparin induced thrombocytopenia (HIT) is reported at a variable rate in extracorporeal membrane oxygenation (ECMO) patients. A critical factor impacting platelet factor-4 (PF4)-heparin antibody formation is plasma PF4 concentration. We hypothesized that PF4 concentration would be increased during veno-arterial (VA) ECMO. Plasma PF4 concentration was measured during the first 5 ECMO days in 20 VA ECMO patients and 10 control plasma samples. PF4-heparin ratios were estimated using an assumed heparin concentration of 0.4 IU/mL. This correlates with an activated partial thromboplastin time of 60 to 80 seconds, which is the anticoagulation target in our center. Twenty VA ECMO patients were enrolled, 10 of which had pulmonary embolism. Median PF4 concentration was 0.03 µg/mL [0.01, 0.13] in control plasma. Median PF4 concentration was 0.21 µg/mL [0.12, 0.34] on ECMO day 1 or 2, 0.16 µg/mL [0.09, 0.25] on ECMO day 3, and 0.12 µg/mL [0.09, 0.22] on ECMO day 5. Estimated median PF4-heparin ratios were 0.04, 0.03, and 0.02 respectively. Two patients (10%) developed HIT that was confirmed by serotonin release assay. PF4 concentration did not differ significantly in these patients compared to non-HIT patients (p = 0.37). No patient had an estimated PF4-heparin ratio between 0.7 and 1.4, which is the reported optimal range for PF4-heparin antibody formation. Our data suggest that PF4 concentration is mildly elevated during VA ECMO compared to control plasma. Estimated PF4-heparin ratios were not optimal for HIT antibody formation. These data support epidemiologic studies where HIT incidence is low during VA ECMO.

Newly Identified HNP-F from Human Neutrophil Peptide-1 Promotes Hemostasis.

Activehemostatic agents can play a crucial role in saving patients' lives during surgery. Active hemostats have several advantages including utilization of natural blood coagulation and biocompatibility. Among them, although human neutrophil peptide-1 (HNP-1) has been previously reported with the hemostatic mechanism, which part of HNP-1 facilitates the hemostatic activity is not known. Here, a partial peptide (HNP-F) promoting hemostasis, originating from HNP-1, has been newly identified by the blood coagulation ability test. HNP-F shows the best hemostatic effect between the anterior half and posterior half of peptides. Moreover, microscopic images show platelet aggregation and an increase in the concentration of platelet factor 4, and the scanning electron microscope image of platelets support platelet activation by HNP-F. Thromboelastography indicates decreased clotting time and increased physical properties of blood clotting. Mouse liver experiments demonstrate improved hemostatic effect by treatment of peptide solution. Cell viability and hemolysis assays confirm the HNP-F's biosafety. It is hypothesized that the surface charge and structure of HNP-F could be favorable to interact with fibrinogen or thrombospondin-1. Collectively, because HNP-F as an active peptide hemostat has many advantages, it could be expected to become a potent hemostatic biomaterial, additive or pharmaceutical candidate for various hemostatic applications.

Optimizing Measurement of Vascular Endothelial Growth Factor in Small Blood Samples of Premature Infants

To establish a method that allows for the reliable assessment of vascular endothelial growth factor (VEGF-A) concentrations in very small blood samples of preterm infants. Systemic VEGF measurements are important in view of the most appropriate Anti-VEGF drug to be used for the treatment of acute retinopathy of prematurity (ROP). Cord blood samples from preterm (n = 6) infants, blood samples from preterm infants with treatment requiring ROP (n = 12), and blood samples from healthy adults (n = 10) were collected. Serum, citrate plasma, and serum from recalcified citrate blood were obtained. Levels of VEGF-A and platelet factor-4 (PF-4) were quantified by ELISA or AlphaLISA immunoassay. VEGF-A levels could be detected by both assays, with the AlphaLISA generating slightly lower levels in healthy adults, but not in cord blood of preterm infants. In plasma samples, VEGF levels ranged from non detectable to 181 pg/ml. PF-4 concentrations were between 0.16–3.88 µg/ml. Values of VEGF-A and PF-4 in serum and recalcified serum were significantly higher compared to plasma through the release of these cytokines after platelet activation. In plasma samples of infants with ROP, VEGF-A could always be detected and its values ranged from 19.50 to 245.91 pg/ml and PF-4 concentrations were between 0.1 and 3.3 µg/ml. Using the AlphaLISA kit, we were able to detect VEGF in small sample volumes (5 µl plasma or serum/well) in premature infants with treatment requiring ROP and to monitor platelet activation by PF-4 detection. Minimal blood probe volumes reduce phlebotomy losses avoiding the risk of iatrogenic anemia, thus allowing close monitoring of the cytokine levels in these very fragile infants.

Platelet Factor 4 Attenuates Experimental Acute Liver Injury in Mice.

Platelet factor 4 (PF4) is a pleiotropic inflammatory chemokine, which has been implicated in various inflammatory disorders including liver fibrosis. However, its role in acute liver diseases has not yet been elucidated. Here we describe an unexpected, anti-inflammatory role of PF4. Serum concentrations of PF4 were measured in patients and mice with acute liver diseases. Acute liver injury in mice was induced either by carbon tetrachloride or by D-galactosamine hydrochloride and lipopolysaccharide. Serum levels of PF4 were decreased in patients and mice with acute liver diseases. PF4-/- mice displayed increased liver damage in both models compared to control which was associated with increased apoptosis of hepatocytes and an enhanced pro-inflammatory response of liver macrophages. In this experimental setting, PF4-/- mice were unable to generate activated Protein C (APC), a protein with anti-inflammatory activities on monocytes/macrophages. In vitro, PF4 limited the activation of liver resident macrophages. Hence, the systemic application of PF4 led to a strong amelioration of experimental liver injury. Along with reduced liver injury, PF4 improved the severity of the pro-inflammatory response of liver macrophages and induced increased levels of APC. PF4 has a yet unidentified direct anti-inflammatory effect in two models of acute liver injury. Thus, attenuation of acute liver injury by systemic administration of PF4 might offer a novel therapeutic approach for acute liver diseases.

Serum platelet factor 4 is a promising predictor in newly diagnosed patients with multiple myeloma treated with thalidomide and VAD regimens

ABSTRACTObjective: Frequent loss of expression of platelet factor 4 (PF4) in multiple myeloma (MM) was revealed in several previous researches. The predictive analysis of serum PF4 level in newly diagnosed MM has not been well elucidated. This study is to assess if serum PF4 could be a prognostic factor in predicting treatment response and survival of MM treated with thalidomide and VAD regimens.Methods: Sera of 122 MM were gained pre- and post-treatment of chemotherapy and oral thalidomide. Serological PF4 measurements were performed by ELISA. Kaplan–Meier method was employed for survival analysis. Log rank test was used significance analysis. Multivariate analysis of overall survival used Cox-regression.Results: Our data showed that the median serum PF4 concentration was negatively associated with MM response and a significant correlation between serum PF4 level and unfavorable clinical features (β2-microglobulin, ISS stage, del17p and creatinine). MM with lower serum PF4 concentration at diagnosis were prone to gain complete remission and very good partial remission after two courses of chemotherapy. Besides del17p, β2-microglobulin, treatment response, the low serum PF4 concentration was an independent variable associated with a poor overall survival by univariate analysis and multivariate analysis.Conclusions: We speculate serum PF4 is a promising response and prognostic factor in newly diagnosed MM treated with thalidomide and VAD regimens.

Effect of short-term water restriction on oxidative and inflammatory status of sheep (Ovis aries) reared in Southern Italy

Water restriction (WR) may impair productive and reproductive performances in livestock. Our major aim was to assess physiological changes associated with a short-term WR in sheep reared in Southern Italy, in order to develop novel methods for evaluating/monitoring the biological implications of such environmental stress. Control animals received water ad libitum (24 h/day) throughout the experimental period. Treated animals were exposed to progressive water restriction (WR) (days 0–4: 12–6 h/day; days 4–8: 3 h/day), followed by a re-adaptation phase (days 9–10). Plasma concentrations of nitro-tyrosine (N-Tyr) and carbonyl (PC) derivatives were measured as markers of protein oxidation. Lipid hydroperoxides (LPOs) were titrated as index of lipid oxidation. Haptoglobin (Hpt) and cortisol were titrated as markers of systemic inflammation and endocrine stress, respectively. Polypeptide profiling of serum samples was performed by MALDI-TOF-MS analysis. Plasma N-Tyr concentration was higher in WR group at day 8 and 10, and significantly increased from day 4–8. Both PC and LPOs levels were higher in WR animals at each time point, and increased until day 8. The concentration of the inflammatory marker Hpt was higher in WR at day 4 and 8, and progressively increased until day 8. MALDI-TOF-MS ascertained decreased levels of apolipoprotein A-II (ApoA-II) and increased levels of platelet factor 4 (PF4) in WR animals at day 8. Taking into consideration the limitations due to the involvement of these redox status and inflammatory biomarkers in other stressing events, these results suggest that N-Tyr concentration might contribute to assess whether a condition of severe WR is occurring, while PC and LPOs may be used for identifying WR animals, and for evaluating the extent of deprivation. The concentration of Hpt, PF4 and ApoA-II might be used in combination for identifying dehydrated animals, and for monitoring the restoring of homeostasis.

Platelet Factor 4 (PF4)-Mediated Neutrophil Extracellular Trap Compaction Limits Endothelial Injury and Promotes Survival Following Lipopolysaccharide Challenge

When stimulated by infection or inflammation, neutrophils expel NETs, decondensed chromatin coated with histones and antimicrobial proteins that ensnares pathogens but also damages host tissue. Platelet factor 4 (PF4, CXCL4) is a CXC chemokine stored in platelet alpha-granules and released in high concentrations during platelet activation. Tetrameric PF4 has a very high affinity for polyanionic molecules, including DNA, and we have found that PF4 binds and physically compacts NETs, causing them to have increased resistance to endonuclease digestion. Our group has also observed that PF4 expression leads to enhanced survival in a murine model of sepsis. Based on these findings, we chose to investigate whether PF4-mediated NET compaction is protective in endotoxemia. To study PF4-NET interactions, we developed a microfluidic assay in which neutrophils were adhered to fibronectin-coated channels and then stimulated to release NETs with phorbol myristate acetate(PMA). NETs were visualized by staining with the fluorescent nucleic acid stain SYTOX. Changes in NET morphology and fluorescence were quantified in the presence of varying PF4 concentrations. DNase I was then infused through these channels and the extent of digestion was measured. These experiments showed that the presence of PF4 led to NET compaction and decreased NET degradation following DNase infusion. We then performed in vitro studies examining NET-endothelial interactions in which isolated neutrophils were stimulated to release NETs, incubated with buffer alone or buffer containing PF4, and flowed through human endothelial umbilical vein cell (HUVEC) lined microfluidic channels that had been stimulated with tumor necrosis factor (TNF) α. EC viability was assessed 24-hours post NET exposure and revealed that the presence of PF4 protected HUVECs from NET-induced damage. To further investigate PF4-NET interactions in endotoxemia, we conducted in vivo studies using PF4-deficient mice (mPF4-/-) and wildtype (WT) controls injected with lipopolysaccharide (LPS). Plasma NET markers [cell free DNA (cfDNA), citrullinated histones (cit-His), and myeloperoxidase (MPO)] were quantified via ELISA and Western blot at various time points following LPS injection. mPF4-/- mice were also implanted with PF4-containing osmotic pumps and the NET markers were also assessed following LPS exposure. These experiments revealed that compared to WT mice, LPS injected mPF4-/- mice had significantly higher plasma levels of NET components, including cfDNA, cit-His and MPO. When mPF4-/- mice were implanted with PF4-releasing osmotic pumps prior to LPS injection, they had plasma NET component levels comparable to those observed in WT mice. Based on the results of our in vitro and in vivo studies, we propose that PF4 infusion compacts NETs, decreasing their susceptibility to DNAse lysis, and preventing the release of toxic NET degradation products (NDPs) such as cfDNA and cit-His. We posit that PF4-mediated sequestration of NDPs prevents endothelial cell damage in the HUVEC-lined microfluidic model. We believe that the results of our studies in mPF4-/- mice demonstrate that PF4 has a similarly protective effect in vivo, decreasing NET lysis and reducing NDP generation. These findings suggest that in sepsis, the stabilization rather than the lysis of NETs may be therapeutic. Further investigation should be performed to determine if treatment with PF4 or other small positively-charged proteins such as protamine sulfate that can sequester NDPs, may be beneficial the treatment of sepsis. DisclosuresNo relevant conflicts of interest to declare.

Novel ELISA-Based Assay for Detection of Complement Activation By PF4/Heparin Complexes

The immune response to platelet factor 4 (PF4)/heparin complexes is a frequent iatrogenic complication of heparin therapy associated with development of heparin-induced thrombocytopenia (HIT). Our recent studies indicate that PF4/heparin complexes potently activate complement (C’) in healthy donors and patients receiving heparin therapy. In these studies, we also show that C’ mediates selective antigen-binding to circulating B cells via the complement receptor 2, CD21 (Khandelwal, Blood 2016). In the course of performing these studies, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based technique for measuring C3 subunit generation by protein/heparin complexes in plasma. For this assay, monoclonal antibodies to PF4/heparin (KKO; Arepally, Blood 2000) or protamine (PRT)/heparin complexes (ADA, Lee unpublished data) are incubated overnight on a microtiter plate, followed by washing and blocking with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 2 hours. To examine C’ activation, plasma is incubated with buffer or antigen (PF4, 25µg/mL ± heparin or PRT 31 µg/mL ± 4U/ml heparin) for 60 minutes at 37°C followed by addition of 10mM EDTA to inhibit further C’activation. Plasma containing antigen and activated C’ fragments is next added to the antibody coated plate for 1 hour at 40C followed by three washes. C’ activation is detected using a biotinylated antibody to C3c (Quidel Corporation, San Diego, CA) followed by streptavidin-HRP (BD Bio Sciences San Jose, CA). Using this assay, we show that plasma incubated with PF4/heparin complexes, but not PF4 alone or heparin alone trigger C’ activation as measured by C3 binding (1:50 plasma dilution shown; Figure 1A). Similarly, using ADA, a recently developed monoclonal antibody to PRT/heparin complexes, we show that PRT/heparin complexes, but not PRT alone or heparin alone, robustly activate C’ (1:10 plasma dilution shown; Figure 1B). To show that C3 generation is dependent on ultra-large complex (ULC) formation, we performed experiments using a fixed dose of UFH (0. 5 U/mL) and varying doses of PF4 (5-200 µg/mL) or fixed dose of PF4 (25 µg/mL) and varying doses of UFH (0.0005-5.0 U/mL), LMWH (0.01-100 µg/mL) and fondaparinux (0.05-100µg/mL). Consistent with published observations (Khandelwal, Blood 2016), we note that changes in PF4 concentration (Figure 2A) or UFH/LMWH/fondaparinux concentration (Figure 2B) results in a bell-shaped curve of C’activation that mirrors ULC formation. Because the immune response to PF4/heparin is highly variable among heparin-exposed patients, we examined inter-individual variation in C’ activation by PF4/heparin complexes. For these studies, we analyzed C’ activation using a fixed dose of PF4/heparin (25 µg/mL PF4 and 0.25U/mL heparin) in freshly collected plasma from 10 healthy donors . As shown in Figure 3, we noted that C’ activation is highly variable among donors, with some donors showing significant C’ activation (donors A,B,C, E ,G,I), while others show minimal C3 generation (donors D,F,H,J) in response to same dose of PF4/heparin complexes. Together, these studies show that the ELISA-based C3 generation assay is a simple, robust assay for detecting C’ activation by PF4/heparin or PRT/heparin complexes and can be useful in studying mechanisms of C’ activation and biologic effects of commercial heparins. [Display omitted] [Display omitted] [Display omitted] DisclosuresArepally:Biokit: Patents & Royalties.

Optimization of hypolipidemic therapy in patients with ischemic stroke and diabetes mellitus

Evaluation of effectiveness of Mexidol in optimization of hypolipidemic therapy in ischemic stroke and diabetes mellitus patients. Authors analyzed the indicators of lipid status: total cholesterol, low-density lipoproteins, high density lipoproteins, triglycerides and concentration of platelet factor-4, β-tromboglobulin, von Willebrand factor in 68 patients with acute ischemic stroke and diabetes mellitus. Authors investigate the dynamics of these parameters (1(st), 21(st), 3-d and 6(th) month after onset stroke) depending on timing and dose of Mexidol. Long time therapy of Mexidol may optimize of hypolipidemic therapy in ischemic stroke and diabetes mellitus patients.

Heparin-induced thrombocytopenia in patients undergoing cardiac surgery

To investigate the incidence for heparin-induced thrombocytopenia (HIT) in patients undergoing cardiac surgery and to evaluate the risk factors for the generation of HIT-antibody. A total of 315 patients undergoing cardiac surgery in the Department of Cardiothoracic Surgery, Xiangya Hospital between December, 2013 and July, 2014 were enrolled for this study. Among them, 120, 154 and 41 were for surgery of congenital heart defect, valve and coronary artery bypass graft, respectively. There were 170 male patients and 69 patients were under 18 years old. Platelet counts, HIT-antibody and concentration of platelet factor 4 (PF4) were tested before and after the surgery. Diagnosis of HIT was based on "4Ts" (Pretest Clinical Scoring System). HIT was diagnosed in 11 patients (3.5%, 11/315). And thromboembolic events occurred in 2 of 11 patients with HIT. The positive ratio for HIT-antibody was 36.5% (115/315). The coronary artery disease patients had a higher incidence of HIT than that of either the valve disease or the congenital heart defect (17.1%, 7/41 versus 1.9%, 3/154 or 0.8%, 1/120; P<0.05). The congenital heart defect patients had a higher positive ratio for HIT-antibody than that of both the valve disease and the coronary artery disease. The valve disease patients had a higher positive ratio for HIT-antibody than that of the coronary artery disease (51.7%, 62/120 versus 30.5%, 47/154 versus 14.6%, 6/41; P<0.05). Major postoperative complications occurred more frequently in HIT patients (36.4%, 4/11 versus 10.5%, 32/304; P<0.05). Age was a risk factor for HIT (P=0.030, OR=1.083, 95% CI 1.008-1.163). Cardiopulmonary bypass (CPB) (P=0.037, OR=3.113, 95% CI 1.071-9.050) and age (P<0.001, OR=0.970, 95% CI 0.959-0.982) were risk factors for HIT-antibody. The incidence of HIT is low during cardiac surgery, but HIT is a highly risk factor for the major postoperative complications. More attentions should be paid to these severe complications and the risk factors for HIT.

Low Level Platelet-Activating Antibodies in Patients Suspected of Having Heparin-Induced Thrombocytopenia but Testing Negative in the ”Gold Standard” Functional Test

Background: Heparin-induced thrombocytopenia (HIT) is a common drug reaction that causes arterial or venous thrombosis as a result of heparin therapy. Platelet-activating antibodies, against complexes of platelet factor 4 (PF4) and heparin, cause intense platelet activation, ultimately leading to an increased risk of thrombosis, limb-loss and even death. Most patients exposed to heparin will produce non-pathogenic anti-PF4/heparin antibodies while only a small number will produce platelet-activating and HIT-causing antibodies (pathogenic HIT antibodies). Among HIT tests, the functional assays, such as the serotonin release assay (SRA), correlate best with the disease because they can specifically identify the pathogenic HIT antibodies whereas the enzyme immunoassays (EIAs) cannot. We have previously shown that anti-PF4/heparin antibody production precedes thrombocytopenia in HIT patients (Warkentin et al., Blood 2009 113: 4963-4969) possibly indicating the need for a threshold plasma level of pathogenic HIT antibody, among other factors, to cause the disease. The objective of this study was to investigate the presence of low levels of pathogenic HIT antibodies in samples from patients suspected of HIT who had detectable anti-PF4/heparin antibodies in the EIA (EIA-positive), but who did not have platelet-activating antibodies in the standard SRA (SRA-negative).Methods: We used an in-house IgG-specific EIA to detect the presence of anti-PF4/heparin antibodies (EIA-positive: OD405nm> 0.45) and the standard SRA to detect the presence of heparin-dependent platelet-activating antibodies (SRA-positive: release >20% with 0.1-0.3 IU/mL of unfractionated heparin). We developed an enhanced SRA (eSRA) by adding increasing concentrations of exogenous PF4 (0-100 μg/mL) to detect sub-threshold levels of platelet activating antibodies undetectable in the standard SRA (eSRA-positive: release >20%). Samples tested were referred for HIT testing by the McMaster Platelet Immunology Laboratory (Hamilton, Canada).Results: Sera from healthy individuals (n=10) and from suspected HIT patients with a negative anti-PF4/heparin EIA (n=15) did not demonstrate platelet activation in the eSRA at any dose of exogenous PF4 added. SRA-positive sera (n = 7), diluted sufficiently that they were non-reactive in the standard SRA, demonstrated PF4 dose-dependent platelet activation in the eSRA. This confirmed the increased sensitivity of the eSRA in detecting low-titre platelet-activating antibodies. Reactivity in the eSRA was inhibited by high heparin (100 U/mL) and by blocking the platelet FcgRIIa receptor with the monoclonal antibody IV.3. We then tested samples (n=24) referred for HIT testing that were positive in the anti-PF4/heparin EIA (optical densities OD405nm 0.7 to 2.4) but negative in the standard SRA. Heparin-dependent platelet activation (20-99% release) was demonstrated in 11 of 24 (46%) in the eSRA. This reactivity directly correlated with the amount of PF4 added to the platelets (optimal concentration of PF4 12.5 - 100 μg/mL) but not with the strength (OD405nm) of the anti-PF4/heparin EIA. In further investigations, we concentrated (4-fold) 7 of the 11 eSRA-positive samples in an attempt to increase the concentration of the antibodies. Of those 7 samples, 5 (71%) became positive in the standard SRA upon testing of the concentrated sample.Conclusions: These data indicate that low-titre platelet-activating antibodies may be found in some patients suspected of having HIT that test negative in the standard SRA irrespective of the strength (OD405nm) of the anti-PF4/heparin EIA. The immune response during heparin therapy can produce both families of pathogenic and non-pathogenic anti-PF4/heparin antibodies but it is the titre of the pathogenic antibody that may be necessary for platelet activation. Perhaps under permissive clinical conditions and with patient-specific factors, the titre of the pathogenic HIT antibodies may increase and lead to HIT. DisclosuresWarkentin:Pfizer Canada: Honoraria; Instrumentation Laboratory: Honoraria; GlaxoSmithKline: Consultancy, Research Funding; W.L. Gore: Consultancy, Research Funding.

Serum Platelet Factor 4 Is a Promising Therapeutic Response and Prognostic Marker in Patients with Newly Diagnosed Multiple Myeloma

Background: Angiogenesis is closely related with progression and prognosis of multiple myeloma (MM). Imbalance between pro- and anti-angiogenic factors leads to aberrant angiogenesis in MM. Platelet factor 4(PF4) is a potent antiangiogenic factor. It has been shown that PF4 and its p17-70 peptide can inhibit myeloma proliferation and angiogenesis both in vitro and in vivo. In addition, PF4 also directly induces cell apoptosis by inhibition of STAT3 via up-regulation of SOCS3 expression in MM. We identified the peptide (m/z7763.24) with decreased relative intensity in newly diagnosed MM as PF4. Downregulation of PF4 in MM was revealed in several previous researches. However, the predictive analysis of serum PF4 level in newly diagnosed MM has not been well elucidated. Thus in the current study, we proposed to assess if serum PF4 could be a therapeutic response and prognostic marker in patients with newly diagnosed MM.Materials and methods: 62 newly diagnosed MM patients (35 male: 27 female) with a median age of 56.5 years (range 44-73) were recruited. The median follow-up duration was 21 months (range 6–42 months). After two courses of chemotherapy and oral thalidomide, 30 patients failed to achieve complete remission and very good partial remission (non-CR&VGPR) and 30 cases gained CR&VGPR. The relative intensity of PF4 was compared among different MM patients and healthy controls. Results were validated by western blot. Sera of 62 MM patients were gained pre- and post-treatment. Sera from 60 healthy donors were used as controls. Serum PF4 was quantified by ELISA. Independent sample t-test and linear regression were employed to do statistic analyses. Kaplan-Meier method was employed for survival analysis. Log rank test was used for significance analysis. Multivariate analysis of overall survival (OS) used Cox-regression.Results: The relative intensity of PF4 were significantly decreased in newly diagnosed and non-CR&VGPR MM patients, comparing with CR&VGPR patients and healthy controls(p<0.05). Weak PF4 immunoreactive bands were seen in newly diagnosed and non-CR&VGPR MM cases. ELISA demonstrated that mean serum PF4 concentrations in the newly diagnosed group (0.6676±0.2755μg/L) and non-CR&VGPR group (0.6294±0.2732μg/L) significantly differed from the healthy control group (2.4329±0.9709μg/L) and CR&VGPR group (2.4179±0.9605μg/L). Linear regression analysis showed no correlation between PF4 content and platelet count. The PF4 serum concentration was lower in patients with elevated β2-microglobulin (p<0.001), advanced ISS (p=0.003), p53 mutation (p=0.001) and higher level of creatinine (p<0.001). Newly diagnosed MM patients with higher PF4 serum concentration were prone to achieve CR&VGPR (p<0.001).We did not identify any significant distinction in PF4 serum concentration when grouping the patients by age (p=0.988), lactate dehydrogenase (p=0.755), albumin (p=0.305) or hemoglobin (p=0.962). The median survival time of newly diagnosed MM patients in lower serum concentration group (<median serum concentration) was 20.00 months (95%CI: 16.84, 23.16), while in higher serum concentration group (≥median serum concentration) 28.00 months (95%CI: 26.37, 29.63). Kaplan–Meier analyses of OS showed that patients with higher serum concentration of PF4 had a significantly superior outcome and lower serum concentration of PF4 was associated with an unfavorable OS (14.1±8.3% versus 23±8.2%, P=0.001). In univariate survival analysis, the presence of β2-microglobulin, p53 mutation, treatment response and creatinine were also significant predictors of survival, whereas survival was independent of gender, age, lactate dehydrogenase, ISS, albumin, hemoglobin. In multivariate analysis with a Cox regression model, the independent variables associated with a poor OS were p53 mutation (p=0.0296), β2-microglobulin (p=0.0393), treatment response(p=0.031). In addition to these known prognostic factors, MM patients with the low serum PF4 concentration had a significantly inferior outcome (p=0.006).Conclusion: We speculatethatserum PF4 is a promising therapeutic response and prognostic marker in patients with newly diagnosed MM. DisclosuresNo relevant conflicts of interest to declare.

Bacteriostatic effect of equine pure platelet-rich plasma and other blood products against methicillin-sensitive Staphylococcus aureus. An in vitro study.

1) To evaluate the bacteriostatic in vitro effect of pure platelet-rich plasma (P-PRP), pure platelet-rich gel (P-PRG), leukocyte-poor gel (LPG), platelet-poor plasma (PPP), and heat inactivated plasma (IP) against methicillin-sensitive Staphylococcus aureus (MSSA) over a period of 24 hours. 2) To determine the degradation of platelet factor-4 (PF-4), transforming growth factor beta 1 (TGF-β1), and platelet-derived growth factor isoform BB (PDGF-BB) in these equine blood components. 3) To establish correlations between platelet and leukocyte counts, PF-4 concentrations, and MSSA growth. Fourteen horses were used. Blood components were obtained by a manual protocol. Every blood component was mixed with MSSA and Müller-Hinton Broth and cultured at 37°C for 24 hours. Samples for the determination of bacterial growth (colony-forming units) and PF-4, TGF-β1 and PDGF-BB concentrations were taken at one, four, eight, 12 and 24 hours. The bacterial growth was significantly (p = 0.01) inhibited for P-PRP, P-PRG, LPG and PPP in comparison with IP and, the positive control group during the first 12 hours. The P-PRG had higher and sustained TGF-β1 and PDGF-BB concentrations over time in comparison with the other blood components. The plasma complement could be one of the most responsible components of the in vitro bacteriostatic effect of P-PRP, P-PRG, LPG and PPP against MSSA. Additionally, P-PRG was the better biomaterial because it had an acceptable bacteriostatic effect and the highest concentration of growth factors.

Increased plasma concentration of vascular endothelial growth factor in patients with atopic dermatitis and its relation to disease severity and platelet activation

BackgroundOverproduction of vascular endothelial growth factor (VEGF) in atopic dermatitis (AD) lesions has previously been observed. It is also known that platelet is an important source of VEGF and platelet factor 4 (PF-4), a potential marker of AD severity.AimTo evaluate concentrations of VEGF and its soluble receptors (sVEGF-R1 and sVEGF-R2) in the plasma of AD patients and to examine its possible correlation with disease severity and plasma concentrations of PF-4, a platelet activation marker.MethodsPlasma concentrations of VEGF and its receptors and levels of PF-4 were measured by an immunoenzymatic assay in 51 AD patients and in 35 healthy non-atopic controls. The severity of the disease was evaluated using the eczema area and severity index.ResultsAD patients showed significantly increased VEGF and PF-4 plasma concentrations as compared with the controls. Plasma concentrations of sVEGF-R1 and sVEGF-R2 did not differ between the groups. There were no remarkable correlations between plasma VEGF concentration and disease severity or between VEGF and PF-4 concentration.ConclusionsThis study shows that plasma concentration of VEGF may be increased in patients suffering from AD. It seems that plasma VEGF concentration is not a useful marker of disease severity and, apart from platelets, other cells might also release the cytokine.