Expression Of Platelet Factor Research Articles

The disparity of platelet factor 4 and platelets in individuals of different ages

The aging process profoundly impacts the systemic milieu, with specific blood-borne factors playing critical roles in its regulation. Platelet Factor 4 (PF4), released by platelets, has emerged as a novel blood-borne factor that contributes to the rejuvenation of aging brains in rodents. However, the age-related disparity in PF4 levels in humans remains poorly understood. To explore the relationship between PF4 and the natural aging process in humans, we collected peripheral blood (PB) samples from young (23.40 ± 2.13 years, n = 15) and elderly (75.23 ± 4.19 years, n = 13) individuals, along with cord blood (CB) samples (n = 15). ELISA analysis revealed higher PF4 levels in platelet-rich plasma lysate from young PB compared with that from elderly PB. Consistent with this, qPCR results demonstrated the highest PF4 expression in young PB among the three groups. In addition, FACS analysis showed increased expression of CXCR3 in mononuclear cells of young PB, indicating a greater responsiveness to PF4. Finally, our RNA-sequencing analysis corroborated platelets as a sensitive element during the natural aging process, and indicated platelets play a pivotal role in antioxidant response during aging, as evidenced by significant enrichment of several age-related pathways. These findings reveal that, alongside PF4 levels, platelets undergo substantial alterations during aging. Taken together, our data identified age-related disparities in platelets and PF4-related elements during natural aging and underscored the potential of targeting platelet modulation as an intervention in the aging process.

Proteomic screening identifies PF4/Cxcl4 as a critical driver of myelofibrosis.

Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2V617F mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.

NCOA5 Haploinsufficiency in Myeloid-Lineage Cells Sufficiently Causes Nonalcoholic Steatohepatitis and Hepatocellular Carcinoma

The nuclear receptor coactivator 5 (NCOA5) is a putative type 2 diabetes susceptibility gene. NCOA5 haploinsufficiency results in the spontaneous development of nonalcoholic fatty liver disease (NAFLD), insulin resistance, and hepatocellular carcinoma (HCC) in male mice; however, the cell-specific effect of NCOA5 haploinsufficiency in various types of cells, including macrophages, on the development of NAFLD and HCC remains unknown. Control and myeloid-lineage-specific Ncoa5 deletion (Ncoa5ΔM/+) mice fed a normal diet were examined for the development of NAFLD, nonalcoholic steatohepatitis (NASH), and HCC. Altered genes and signaling pathways in the intrahepatic macrophages of Ncoa5ΔM/+ male mice were analyzed and compared with those of obese human individuals. The role of platelet factor 4 (PF4) in macrophages and the underlying mechanism by which PF4 affects NAFLD/NASH were explored invitro and invivo. PF4 expression in HCC patient specimens and prognosis was examined. Myeloid-lineage-specific Ncoa5 deletion sufficiently causes spontaneous NASH and HCC development in male mice fed a normal diet. PF4 overexpression in Ncoa5ΔM/+ intrahepatic macrophages is identified as a potent mediator to trigger lipid accumulation in hepatocytes by inducing lipogenesis-promoting gene expression. The transcriptome of intrahepatic macrophages from Ncoa5ΔM/+ male mice resembles that of obese human individuals. High PF4 expression correlated with poor prognosis of HCC patients and increased infiltrations of M2 macrophages, regulatory T cells, and myeloid-derived suppressor cells in HCCs. Our findings reveal a novel mechanism for the onset of NAFLD/NASH and HCC initiated by NCOA5-deficient macrophages, suggesting the NCOA5-PF4 axis in macrophages as a potential target for developing preventive and therapeutic interventions against NAFLD/NASH and HCC.

PF4 induces inflammatory response through NF-kB signal pathway in rats with intracerebral haemorrhage.

Intracerebral haemorrhage (ICH) is a lethal cerebrovascular disorder with a high mortality and morbidity. Although it is a major public health problem, there is no effective treatment for ICH. After ICH, the primary and secondary mechanisms are mentioned when discussing brain injury. The transcription factor, nuclear factor-kappa B (NF-kB), is an important regulator of inflammatory responses. The role of platelet factor 4 (PF4) in ICH is unclear. To study the effect of PF4 on inflammatory response of rats in ICH, a rat model of striatum ICH was established by injecting autologous blood from the autogenous femoral artery into the right striatum of rats. Forty-eight hours after ICH, the expression of PF4, NF-kB (P-P65) and inflammatory changes in rats were determined with WB and ELISA. Heme was used to induce PC12 cell damage, simulate the ICH model in vitro, and detect PF4, P-P65 and striatal inflammatory changes. Short hairpin RNA (shRNA-PF4) was used to knock-down the expression of PF4 in PC12 cells to detect changes in inflammatory factors. The results showed that 48 hours after surgery, the behavioural score of cerebral haemorrhage was the lowest. The expression of PF4 and P-P65 in the striatum of the ICH group was significantly higher compared with the sham surgery group. The expression of interleukin (IL)-6 and IL-1b in the ICH group was also greatly improved. After inhibiting NF-kB expression, PF4 expression was decreased. In short, ICH enhances the expression of PF4, which induces an inflammatory response in rats with cerebral haemorrhage through the NF-kB signalling pathway. Reducing the expression of PF4 can attenuate the inflammatory response.

Circular RNA hsa_circ_0069117 suppresses proliferation and migration of osteosarcoma cells lines via miR-875-3p/PF4V1 axis

BackgroundOsteosarcoma (OS) is one of the most common malignant bone tumors in children and adolescents. Circular RNAs (circRNAs) are critical regulators involved in multiple physiological and pathological processes. However, the underlying regulatory mechanisms of circRNA in OS are still not fully understood.MethodsThe circRNA expression profiles were downloaded from the Gene Expression Omnibus (GEO) database and analyzed by GEO2R. Bioinformatics analysis was performed to predict the potential target miRNAs of hsa_circ_0069117 and its downstream mRNAs. The co-expression of hsa_circ_0069117/miR-875-3p/PF4V1 axis was further validated in OS tissue samples via quantitative real-time PCR (qRT-PCR). Luciferase reporter gene plasmids containing the sequence of PF4V1 and hsa_circ_0069117 were constructed to verify the putative sites of miR-875-3p. Gain/loss-of-function assays were performed to verify the effect of hsa_circ_0069117 on miR-875-3p/PF4V1 expression and related pathways via qRT-PCR and Western blot. Cell counting kit-8 (CCK-8) and wound-healing assays were performed to evaluate the effect of hsa_circ_0069117 on cell proliferation and migration of MG63 and U2OS, respectively.ResultsWe identified hsa_circ_0069117 as the most markedly dysregulated circRNA in OS cell lines. Bioinformatics analysis indicated that hsa_circ_0069117 might inhibit the expression of miR-875-3p, thereby promoting the expression of platelet factor 4 variant 1 (PF4V1). The expression of miR-875-3p was negatively correlated to hsa_circ_0069117 and PF4V1 in clinical samples. Luciferase reporter gene assays confirmed the binding sites of miR-875-3p on hsa_circ_0069117 and PF4V1. Gain/loss-of-function and rescue assays further indicated that hsa_circ_0069117 could significantly promote the expression of PF4V1 by sponging miR-875-3p, thereby inhibiting the proliferation and migration of OS cells by suppressing ERK1 and AKT.ConclusionOur study revealed that hsa_circ_0069117 is an anti-OS molecule that could substantially attenuate cell proliferation and migration of OS, which may provide a novel and reliable molecular target for the treatment of OS patients.

Proteomic Discovery of Biomarkers to Predict Prognosis of High-Grade Serous Ovarian Carcinoma.

Initial identification of biomarkers predicting the exact prognosis of high-grade serous ovarian carcinoma (HGSOC) is important in precision cancer medicine. This study aimed to investigate prognostic biomarkers of HGSOC through proteomic analysis. We conducted label-free liquid chromatography-mass spectrometry using chemotherapy-naïve, fresh-frozen primary HGSOC specimens, and compared the results between a favorable prognosis group (progression-free survival (PFS) ≥ 18 months, n = 6) and a poor prognosis group (PFS < 18 months, n = 6). Among 658 differentially expressed proteins, 288 proteins were upregulated in the favorable prognosis group and 370 proteins were upregulated in the poor prognosis group. Using hierarchical clustering, we selected α1-antitrypsin (AAT), nuclear factor-κB (NFKB), phosphomevalonate kinase (PMVK), vascular adhesion protein 1 (VAP1), fatty acid-binding protein 4 (FABP4), platelet factor 4 (PF4), apolipoprotein A1 (APOA1), and α1-acid glycoprotein (AGP) for further validation via immunohistochemical (IHC) staining in an independent set of chemotherapy-naïve primary HGSOC samples (n = 107). Survival analyses revealed that high expression of AAT, NFKB, and PMVK were independent biomarkers for favorable PFS. Conversely, high expression of VAP1, FABP4, and PF4 were identified as independent biomarkers for poor PFS. Furthermore, we constructed models predicting the 18-month PFS by combining clinical variables and IHC results. Through leave-one-out cross-validation, the optimal model was based on initial serum CA-125, germline BRCA1/2 mutations, residual tumors after surgery, International Federation of Gynecology and Obstetrics (FIGO) stage, and expression levels of the six proteins. The present results elucidate the proteomic landscape of HGSOC and six protein biomarkers to predict the prognosis of HGSOC.

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.

Dynamic intercellular redistribution of HIT antigen modulates heparin-induced thrombocytopenia

AbstractHeparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies to platelet factor 4 (PF4)/heparin complexes. PF4 released from platelets binds to surface glycosaminoglycans on hematopoietic and vascular cells that are heterogenous in composition and differ in affinity for PF4. PF4 binds to monocytes with higher affinity than to platelets, and depletion of monocytes exacerbates thrombocytopenia in a murine HIT model. Here we show that the expression of PF4 on platelets and development of thrombocytopenia are modulated by the (re)distribution of PF4 among hematopoietic and endothelial cell surfaces. Binding of PF4 to platelets in whole blood in vitro varies inversely with the white cell count, likely because of the greater affinity of monocytes for PF4. In mice, monocyte depletion increased binding of PF4 to platelets by two- to three-fold. Induction of HIT in mice caused a transient >80-fold increase in binding of HIT antibody to monocytes vs 3.5-fold increase to platelets and rapid transient monocytopenia. Normalization of monocyte counts preceded the return in platelet counts. Exposure of blood to endothelial cells also depletes PF4 from platelet surfaces. These studies demonstrate a dynamic interchange of surface-bound PF4 among hematopoetic and vascular cells that may limit thrombocytopenia at the expense of promoting prothrombotic processes in HIT.

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.

Dual targeting of C-kit and Integrin Linked Kinase abrogates the phosphorylation of S6 ribosomal protein and reduces melanoma cell growth

Background: Activating mutations in c-kit have been observed in distinct subtypes of melanoma and are associated with disease progression and the development of resistance to treatment. Mutant c-kit uses mainly the phosphatidylinositol 3 kinase (PI3k)/Akt pathway and to a lesser extent the mitogen activated protein kinase (MAPK) pathway in order to promote characteristics of the cancer phenotype, such as excessive proliferation, angiogenesis and invasiveness. However, clinical studies on the inhibition of c-kit in melanoma patients have yielded disappointing results. The reactivation of these pathways have been reported in treatment resistant melanoma cells. Previously we showed that downregulation of integrin linked kinase (ILK), an intracellular adapter molecule that promotes tumor progression and angiogenesis when dysregulated, suppressed Akt phosphorylation and inhibited tumor growth. Therefore we investigated the effects of dual inhbition of c-kit and ILK on tumor cell growth and angiogenesis. Methods: The effects of dual blocking of c-kit and ILK-down regulation using siRNA on tumor cell growth were evaluated in an in vitro co-culture system that contained human vein endothelial cells (HUVECs) and melanoma cells. To investigate the effects of blocking c-kit and ILK on angiogenesis, the migration and invasion of HUVECs in response to conditioned medium from melanoma cells was studied using the xCELLigence system, while endothelial cell tube formation was studied using a collagen-based assay. Protein array studies were employed to investigate the expression patterns of angiogenic proteins in mono- and co-cultured cells. Further marker expression was determined using immunocytochemistry. Results: Significant inhibition of melanoma cell growth was observed in treated co-cultures, and the inhibition correlated with reduced expression of S6 ribosomal protein and S6 kinase. ILK-knockdown inhibited endothelial cell migration and invasion, as well as endothelial cell morphogenesis induced by melanoma conditioned medium. Results also revealed an increase in the expression of Platelet Factor 4 (PF4), and a suppression of proangiogenic factors, including leptin, vascular endothelial growth factor and basic fibroblast growth factor. Conclusions: Findings from this study suggest that the cross-talk between melanoma and endothelial cells which supports tumor cell growth, can be disrupted by dual targeting of c-kit and ILK. Furthermore, dual targeting of c-kit and ILK which also resulted in the altered expression profile of angiogenesis markers to favor antiangiogenesis, may imply that such dual targeting could contribute to enhancing complimentary approaches for melanoma therapy. Legal entity responsible for the study: Angiogenesis Laboratory, University of Pretoria Funding: National Research Foundation, University of Pretoria Disclosure: All authors have declared no conflicts of interest.

Fractalkine/CX3CR1 Signaling Promotes Angiogenic Potentials in CX3CR1 Expressing Monocytes

Introduction: Myelo-monocytic cells expressing CD11b are involved in angiogenesis, but their specific roles and underlying mechanisms are unclear. CX3CR1 is the only known receptor for fractalkine (Fkn). CD11b+CX3CR1+ cells isolated from ischemic muscles expressed F4/80, which is a common marker for mouse macrophages (Tissue (T)-CD11b+CX3CR1+ macrophages). T-CD11b+CX3CR1+ macrophages exert pro-angiogenic effect by platelet factor-4 (PF-4) production. PF-4 did not promote angiogenesis by itself but magnified the angiogenic activity of VEGF. Although T-CD11b+CX3CR1+ macrophages show definite pro-angiogenic effects, their clinical implementation is limited because they require muscle excision for cell harvesting. Therefore, we focused on angiogenic potential of more accessible bone marrow (BM)-CD11b+CX3CR1+ monocytes, because the CD11b+CX3CR1+monocytes migrate from BM into ischemic muscles via Fkn-mediated chemotaxis and differentiate into macrophages upon tissue damage.Materials and methods: CD11b+CX3CR1+cells were isolated by MoFlo™ XDP Cell Sorter (Beckman Coulter, Brea, CA). Isolated CD11b+CX3CR1+ cells were fixed and stained with anti-PF-4 Ab with PE-conjugated IgG for immunofluorescence staining. For aortic tissue sprouting assay, thoracic aortas were embedded in Collagen Type-1. Aortic segments were incubated with CD11b+CX3CR1+ monocytes in the presence or absence of Fkn, and their conditioned medium (CM) at 37°C in a humidified 5% CO2 atmosphere with triweekly media replacement. For Fkn treatment, CD11b+CX3CR1+monocytes were incubated with Fkn (50ng/mL) for 30 minutes. Morphometric analysis of sprouting was performed using Image J software (National Institute Health, Bethesda, MD, USA).Results: T-CD11b+CX3CR1+ macrophages greatly increased sprouting from mouse aortic tissue segments, while T-CD11b+CX3CR1- macrophages showed only modest effects (p<0.05). CM harvested from T-CD11b+CX3CR1+ macrophages showed equivalent effects as T-CD11b+CX3CR1+ macrophages in inducing sprouting. Their high angiogenic potential largely attributed to increased expression of PF-4, according to angiogenic protein array. Depletion of PF-4 in CM of T-CD11b+CX3CR1+ macrophages strongly reduced vascular sprouting compared to control (p <0.001). In contrast to T-CD11b+CX3CR1+ macrophages, PF-4 expression was not detected in BM-CD11b+CX3CR1+ monocytes. However, activation of Fkn/CX3CR1 interaction by treating the cells with Fkn (50ng/mL for 30 minutes) induces high PF-4 expression. Heightened expression of PF-4 mRNA in Fkn-treated BM-CD11b+CX3CR1+ monocytes was confirmed by RT-PCR. Aortic tissue sprouting assay confirmed Fkn-treated BM-CD11b+CX3CR1+ monocytes achieve a similar angiogenic potential to that of T-CD11b+CX3CR1+ macrophages. CD11b+CX3CR1+ monocytes were also found in human blood. CM of Fkn-treated human CD11b+CX3CR1+ monocytes (50ng/mL) greatly increased vascular sprouting, while CM of human CD11b+CX3CR1+monocytes without Fkn treatment only showed modest effects on inducing sprouting (p<0.05).Conclusion; Fkn triggers angiogenic potential of CD11b+CX3CR1+ monocytes by Fkn/CX3CR1 signaling. Thus, Fkn-treated CD11b+CX3CR1+monocytes may be of potential therapeutic use to accelerate recovery of blood perfusion in ischemic diseases. DisclosuresBroxmeyer:CordUse: Other: SAB Member .

Platelet factor 4 is produced by subsets of myeloid cells in premetastatic lung and inhibits tumor metastasis.

Bone marrow-derived myeloid cells can form a premetastatic niche and provide a tumor–promoting microenvironment. However, subsets of myeloid cells have also been reported to have anti-tumor properties. It is not clear whether there is a transition between anti- and pro- tumor function of these myeloid cells, and if so, what are the underlying molecular mechanisms. Here we report platelet factor 4 (PF4), or CXCL4, but not the other family members CXCL9, 10, and 11, was produced at higher levels in the normal lung and early stage premetastatic lungs but decreased in later stage lungs. PF4 was mostly produced by Ly6G+CD11b+ myeloid cell subset. Although the number of Ly6G+CD11b+ cells was increased in the premetastatic lungs, the expression level of PF4 in these cells was decreased during the metastatic progression. Deletion of PF4 (PF4 knockout or KO mice) led an increased metastasis suggesting an inhibitory function of PF4. There were two underlying mechanisms: decreased blood vessel integrity in the premetastatic lungs and increased production of hematopoietic stem/progenitor cells (HSCs) and myeloid derived suppressor cells (MDSCs) in tumor-bearing PF4 KO mice. In cancer patients, PF4 expression levels were negatively correlated with tumor stage and positively correlated with patient survival. Our studies suggest that PF4 is a critical anti-tumor factor in the premetastatic site. Our finding of PF4 function in the tumor host provides new insight to the mechanistic understanding of tumor metastasis.

Ginkgolide B inhibits platelet release by blocking Syk and p38 MAPK phosphorylation in thrombin-stimulated platelets

IntroductionAtherosclerosis is a chronic vascular inflammatory disease. Platelets play a critic role in the initiation of vascular inflammation in atherosclerosis. In the present study, we investigated the effects of ginkgolide B on the inhibition of platelet release and the potential mechanisms. MethodsExperiments were performed in freshly human platelets. Platelet aggregation and ATP release were measured with a Lumi-aggregometer. Thrombin (0.5 U/ml) was used to induce platelet activation. Protein expression and phosphorylation was examined by Western blotting. ResultsThe results showed that ginkgolide B significantly suppressed ATP release by 50.8% in thrombin-activated platelets. Ginkgolide B completely abolished the expression of platelet factor 4 (PF4) and CD40 Ligand (CD40L). Moreover, ginkgolide B fully attenuated the phosphorylation of Syk and p38MAPK. Similarly, R788 (a syk inhibitor) and SB203580 (a p38 MAPK inhibitor) inhibited the expression PF4 and CD40L, respectively. Furthermore, the combination of low concentrations of ginkgolide B and R788 or SB203580 has synergistic inhibition on the expression of PF4 and CD40L. Ginkgolide B partially reduced calcium efflux by 52.7% in thrombin-stimulated platelets. ConclusionGinkgolide B potently inhibited the expression of PF4 and CD40L in thrombin-activated platelets. Ginkgolide B partially decreased ATP release and Ca2+ efflux. The mechanism might be associated with the inhibition of Syk and p38 MAPK phosphorylation. These results demonstrated that ginkgolide B might be a promising drug on inhibiting platelet function and reducing inflammation in atherosclerosis.

Treatment of orthotopic malignant peripheral nerve sheath tumors with oncolytic herpes simplex virus

Malignant peripheral nerve sheath tumors (MPNSTs) are an aggressive and often lethal sarcoma that frequently develops in patients with neurofibromatosis type 1 (NF1). We developed new preclinical MPNST models and tested the efficacy of oncolytic herpes simplex viruses (oHSVs), a promising cancer therapeutic that selectively replicates in and kills cancer cells. Mouse NF1(-) MPNST cell lines and human NF1(-) MPNST stemlike cells (MSLCs) were implanted into the sciatic nerves of immunocompetent and athymic mice, respectively. Tumor growth was followed by external measurement and sciatic nerve deficit using a hind-limb scoring system. Oncolytic HSV G47Δ as well as "armed" G47Δ expressing platelet factor 4 (PF4) or interleukin (IL)-12 were injected intratumorally into established sciatic nerve tumors. Mouse MPNST cell lines formed tumors with varying growth kinetics. A single intratumoral injection of G47Δ in sciatic nerve tumors derived from human S462 MSLCs in athymic mice or mouse M2 (37-3-18-4) cells in immunocompetent mice significantly inhibited tumor growth and prolonged survival. Local IL-12 expression significantly improved the efficacy of G47Δ in syngeneic mice, while PF4 expression prolonged survival. Injection of G47Δ directly into the sciatic nerve of athymic mice resulted in only mild symptoms that did not differ from phosphate buffered saline control. Two new orthotopic MPNST models are described, including in syngeneic mice, expanding the options for preclinical testing. Oncolytic HSV G47Δ exhibited robust efficacy in both immunodeficient and immunocompetent MPNST models while maintaining safety. Interleukin-12 expression improved efficacy. These studies support the clinical translation of G47Δ for patients with MPNST.

Expression of angiogenesis regulatory proteins and epithelial-mesenchymal transition factors in platelets of the breast cancer patients.

Platelets play a role in tumor angiogenesis and growth and are the main transporters of several angiogenesis regulators. Here, we aimed to determine the levels of angiogenesis regulators and epithelial-mesenchymal transition factors sequestered by circulating platelets in breast cancer patients and age-matched healthy controls. Platelet pellets (PP) and platelet-poor plasma (PPP) were collected by routine protocols. Vascular endothelial growth factor (VEGF), platelet-derived growth factor BB (PDGF-BB), thrombospondin-1 (TSP-1), platelet factor 4 (PF4), and transforming growth factor-β1 (TGF-β1) were measured by enzyme-linked immunosorbent assay. Angiogenesis-associated expression of VEGF (2.1 pg/106 platelets versus 0.9 pg/106 platelets, P < 0.001), PF4 (21.2 ng/106 platelets versus 10.2 ng/106 platelets, P < 0.001), PDGF-BB (42.9 pg/106 platelets versus 19.1 pg/106 platelets, P < 0.001), and TGF-β1 (15.3 ng/106 platelets versus 4.3 ng/106 platelets, P < 0.001) differed in the PP samples of cancer and control subjects. In addition, protein concentrations were associated with clinical characteristics (P < 0.05). Circulating platelets in breast cancer sequester higher levels of PF4, VEGF, PDGF-BB, and TGF-β1, suggesting a possible target for early diagnosis. VEGF, PDGF, and TGF-β1 concentrations in platelets may be associated with prognosis.

RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4gene expression in combination with ETS family proteins

Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt-related transcription factor 1 (RUNX1) of FPD patients. To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype. We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFβ), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells. We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFβ, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFβ, and FLI-1. RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.

Mechanism of platelet factor 4 (PF4) deficiency with RUNX1 haplodeficiency: RUNX1 is a transcriptional regulator of PF4

Platelet factor 4 (PF4) is an abundant protein stored in platelet α-granules. Several patients have been described with platelet PF4 deficiency, including the gray platelet syndrome, characterized by a deficiency of α-granule proteins. Defective granule formation and protein targeting are considered to be the predominant mechanisms. We have reported on a patient with thrombocytopenia and impaired platelet aggregation, secretion, and protein phosphorylation, associated with a mutation in the transcription factor RUNX1. Platelet expression profiling showed decreased transcript expression of PF4 and its non-allelic variant PF4V1. To understand the mechanism leading to PF4 deficiency associated with RUNX1 haplodeficiency, we addressed the hypothesis that PF4 is a transcriptional target of RUNX1. Chromatin immunoprecipitation and gel-shift assays with phorbol 12-myristate 13-acetate-treated human erythroleukemia (HEL) cells revealed RUNX1 binding to RUNX1 consensus sites at -1774/-1769 and -157/-152 on the PF4 promoter. In luciferase reporter studies in HEL cells, mutation of each site markedly reduced activity. PF4 promoter activity and PF4 protein level were decreased by small interfering RNA RUNX1 knockdown and increased by RUNX1 overexpression. Our results provide the first evidence that PF4 is regulated by RUNX1 and that impaired transcriptional regulation leads to the PF4 deficiency associated with RUNX1 haplodeficiency. Because our patient had decreased platelet albumin and IgG (not synthesized by megakaryocytes) levels, we postulate additional defects in RUNX1-regulated genes involved in vesicular trafficking. These studies advance our understanding of the mechanisms in α-granule deficiency.

Platelet Factor 4 Potently Inhibits Tumor Cell Growth and Angiogenesis In Multiple Myeloma

AbstractAbstract 4173Multiple myeloma (MM) is a B-cell derived plasma cell malignancy characterized by accumulation of clonal plasma cells in bone marrow (BM). Platelet factor 4 (PF4), a potent antiangiogenic chemokine, not only inhibits endothelial cell proliferation and migration in vitro but also inhibits solid tumor growth in vivo. Our group previously demonstrated loss of PF4 expression in patient multiple myeloma (MM) samples and MM cell lines. Here, we characterized the effects of PF4 on both MM cells and endothelial cells in the BM milieu. We found that PF4 inhibits cell growth in MM cell lines (U266 and NCI-H929) with an IC50 4μM at 96 hours by the WST-1 assay. Cell apoptosis by Annexin V-7 AAD staining showed that percentages of apoptotic cells increased from 15.6% to 16.5%, 23.6% and 39.2% for U266 cells and from 19.8% to 20.1%. 26.8% and 71.0% for NCI-H929 cells when incubated with 2, 4, and 8μM PF4, respectively. PF4 also has direct effects on endothelial cells isolated from patient's BM aspirates (MMECs). Our results showed that PF4 suppresses MMECs proliferation (IC50 8μM) and capillary-like tube formation on matrigel in a dose-dependent manner. It is known that BM endothelial cells promote MM cell growth, survival, and drug resistance in BM microenvironment. Therefore, we further examined whether the proliferation of MM cell is influenced by the presence of endothelial cells. U266 cells were cultured for 96 hours with or without MMECs, in the presence or absence of PF4. We found that adhesion of MM cell to MMECs up regulates cell proliferation (about 1.5 fold), which is markedly inhibited by PF4 (>4uM). Given the ability of PF4 to suppress MM cell growth and angiogenesis in vitro, we evaluated its tumor suppressive function in vivo. In SCID-rab mouse model, 1× 106 U266 MM cells were directly injected into the rabbit bone which was subcutaneously implanted into the NOD-SCID mice. Two weeks after injection, SCID mice were treated with various dose of PF4 (20 or 200 ng per injection, three times per week) or vehicle control by tail vein injection. ELISA assay with hIg (Lambda) showed that tumor growth in PF4-treated mice is markedly reduced by 2.5 fold compared with the control group, which is further confirmed by immunohistochemistry analysis of CD138 staining on rabbit bone section. Consistent with the in vitro results, MM cells' proliferation and angiogenesis are also significantly inhibited by PF4 in vivo, as evidenced by ki67 and CD31 staining on rabbit bone sections from treated versus control mice. Moreover, PF4 improves the survival rate of mice. The survival rate of PBS treated mice was 80% after 3 weeks and less than 30% after 12 weeks, while PF4-treated groups had 100% survival rate after 12 weeks. Taken together, our findings confirm that PF4 is a critical regulator of MM pathogenesis, which targets both MM cells and MMECs in the BM milieu in vitro and in vivo and prolongs survival in the SCID-rab mice model of human MM. These studies provide an important framework for critical clinical studies of PF4 to improve patient treatment outcome in MM. Disclosures:No relevant conflicts of interest to declare.

Platelet Factor 4 Is Highly Upregulated in Dendritic Cells after Severe Trauma

Dendritic cells (DCs) represent an important linkage between the innate and adaptive immune system and express proinflammatory transcriptomic products early after trauma. The use of a genomic approach recently revealed that platelet factor 4 (PF4) is significantly upregulated in DCs in patients after multiple trauma. However, knowledge about subsequent PF4 alteration and its potential clinical relevance in the context of multiple trauma is still limited. We used quantitative reverse transcription-polymerase chain reaction to analyze PF4 expression in both myeloid DCs (MDCs) and plasmocytoid DCs (PDCs) isolated from 10 patients after multiple trauma. Intracellular PF4 as well as HLA-DR expression were detected by flow cytometry. Furthermore, DCs and peripheral blood mononuclear cells were incubated on a monolayer of human umbilical endothelial cells and their adhesion properties were analyzed. The ratio of the DC subtypes (MDC and PDC) was assessed by flow cytometry. PF4 expression significantly increased on d 1 and d 2 as measured by reverse transcription-polymerase chain reaction. Intracellular PF4 content in MDCs and PDCs was significantly elevated in trauma patients compared with healthy controls. In addition, the surface antigen HLA-DR on MDCs was significantly elevated on d 1 and d 4 after trauma in patients compared with controls. However, cell adhesion of DCs did not show any significant differences between patients and controls. PF4 concentration in MDCs and PDCs significantly correlated with the injury severity score. These results confirm an early and subsequent posttraumatic activation of PF4 in DCs. PF4 also participates in the posttraumatic activation of DCs in relation to injury severity, a role that might be preferably based on the modification of receptor expression, whereas adhesion properties are largely unaffected.

Heparin‐induced thrombocytopenia: An autoimmune disorder regulated through dynamic autoantigen assembly/disassembly

Heparin-induced thrombocytopenia (HIT) is the most common drug-induced, antibody-mediated cause of thrombocytopenia and thrombosis. HIT is caused by IgG antibodies that bind to epitopes on platelet factor 4 (PF4) released from activated platelets that develop when it forms complexes with heparin. Anti-PF4/antibodies develop in over 50% of patients undergoing surgery involving cardiopulmonary bypass (CPB), an incidence 20-fold higher than HIT. Why might this occur? Binding of HIT IgG occurs only over a narrow molar ratio of reactants, being optimal at 1 mol PF4 tetramer to 1 mol unfractionated heparin (UFH). At these ratios, PF4 and UFH form ultralarge (>670 kD) complexes that bind multiple IgG molecules/complex, are highly antigenic, and promote platelet activation. Low molecular weight heparin (LMWH), which is less antigenic, forms ultralarge complexes less efficiently and largely at supratherapeutic concentrations. In transgenic mice that vary in expression of human PF4 on their platelets, antigenic complexes form between PF4 and endogenous chondroitin sulfate. Binding of HIT IgG to platelets and induction of thrombocytopenia in vivo is proportional to PF4 expression. Heparin prolongs the duration and exacerbates the severity of the thrombocytopenia. High doses of heparin, as used in CPB, or protamine, which competes with PF4 for heparin, disrupts antigen formation and prevents thrombocytopenia induced by HIT antibody. These studies may help explain the disparity between the incidence of antibody formation and clinical disease and may help identify patients at risk for HIT (high platelet PF4). They also demonstrate that this autoimmune disease can be modulated at the level of autoantigen formation and point to rational means to intervene proximal to thrombin generation.