Addition Of Platelet Factor 4 Research Articles

Neutrophil Extracellular Traps (NETs) Thrombogenicity and Endothelial Cell Toxicity Are Ameliorated By Platelet Factor 4 (PF4): Implications to the Treatment of Sepsis

Sepsis, the dysregulated response to infection, is a leading cause of mortality worldwide for which there are no targeted treatments. Neutrophils play a crucial role in sepsis by releasing NETs, webs of DNA complexed with histones and antimicrobial proteins that capture pathogens. However, when NETs are degraded by circulating nucleases, they release NET-degradation products (NDPs) including cell-free (cf) DNA, histones and myeloperoxidase, which trigger thrombosis, induce complement activation and cause oxidative vascular damage. We propose a novel NET-directed therapy in sepsis, in which NETs are stabilized by the platelet chemokine PF4 (CXCL4), which enhances NET resistance to DNase degradation, promoting NDP sequestration and bacterial capture, improving sepsis outcomes. As NETs are traditionally considered to be prothrombotic, we investigated whether PF4-stabilization alters the thrombogenicity of NETs. We examined the effect of PF4 on both NET- and DNA-mediated thrombus formation and endothelial cell injury using NET and DNA fragments of different lengths. Human neutrophils were stimulated to release NETs and treated with mild DNase I (4U/mL) to liberate high-molecular-weight (hmw) NETs (>50kb), and then digested with restriction enzymes to generate low-molecular-weight (lmw) fragments of ~4kb and ~250bp. We also studied single-stranded (ss) DNA, generated by heating double stranded (ds) DNA fragments to 100°C. We assessed the effects of PF4 on NETs/dsDNA/ssDNA-induced thrombin and fibrin generation in normal and in factor (F) XII- and FXI-depleted plasma. PF4 significantly reduced plasma thrombin and fibrin generation induced by digested NET and DNA fragments and by ssDNA without impairing thrombolysis. PF4 did not exert any effects on DNA-induced fibrin generation in FXII- or FXI-depleted plasma, while supplementation with purified FXII or FXI restored the anticoagulant effects of PF4, suggesting that PF4 binding reduces the ability of cfDNA and NETs to activate the contact pathway of coagulation. We next studied how PF4 alters the ability of DNA to induce a prothrombotic phenotype in human umbilical vein endothelial cells (HUVECs) incubated with dsDNA or ssDNA fragments by measuring von Willebrand factor (VWF) release with immunofluorescence and by quantifying tissue factor (TF) expression with a chromogenic substrate assay. To study the mechanism of endothelial activation, HUVECs were treated with inhibitors of toll-like receptor (TLR) 9, a pattern recognition receptor that binds to dsDNA. Unlike intact NETs or hmwDNA, both digested dsDNA and ssDNA upregulated VWF release and TF expression by endothelial cells via a TLR9-dependent mechanism, which was prevented by the addition of PF4. To study these observations in vivo, hmwDNA was administered intravenously to wildtype (WT) and Cxcl4-/- mice and plasma thrombin-anti-thrombin (TAT) levels were measured 4h post infusion. Cxcl4-/- mice exhibited higher TAT levels as compared to WT mice. We also studied two distinct sepsis-like states: (1) WT mice exposed to lipopolysaccharide (LPS, IP; 35mg/kg) ± human (h)PF4 (IV; 40mg/kg), where plasma cfDNA and TAT levels were measured at 6h, and (2) the cecal slurry model of polymicrobial infection in endothelial-specific TLR9-/- mice, where plasma TAT levels were measured at 4h. In the LPS endotoxemia model (1), TAT and cfDNA levels were elevated at 6h post LPS challenge, which were prevented with the administration of hPF4. In the cecal slurry model (2), endothelial TLR9-/- mice exhibited lower TAT levels at 4h post challenge as compared to WT controls. In summary, while intact NETs help to contain infection, NET degradation liberates lmwDNA and ssDNA that activate the contact pathway and induce a prothrombotic phenotype in endothelial cells via activation of TLR9. PF4-stabilization of NETs decreases the risk of NET-mediated toxicity by (1) inhibiting DNase cleavage of intact NETs and (2) neutralizing released lmwDNA and ssDNA. NET stabilization by PF4 tips the balance, limiting NET- and NDP-induced thrombosis and vascular toxicity, while enhancing NET's ability to fight infection by microbial entrapment. These studies add support to our hypothesis that PF4 stabilization of NETs is protective in sepsis. Strategies that augment the beneficial effects of PF4 merit further investigation.

The cytokine platelet factor 4 successfully replaces bovine serum albumin for the in vitro culture of porcine embryos

The cytokine platelet factor 4 (PF4) enhances differentiation and cell viability of different stem cells lines in vitro. This study investigated whether PF4 addition to customary pig embryo semi-defined culture media can improve their developmental outcome (Experiment 1) and ultimately replace the need for bovine serum albumin (BSA, Experiment 2). Experiment 1 added PF4 (100–1000 ng/mL, 0 = control) to NCSU-23 with 0.4 mg/mL BSA culturing 3430 presumptive zygotes. Experiment 2 added PF4 (100–1000 ng/mL, 0 = Control-PVA) to a BSA-free medium (NCSU-23 with 0.3 mg/mL PVA) culturing 3820 presumptive zygotes. Zygote culture in NCSU-23 with 0.4 mg/mL BSA was used as overall control. All groups of Experiment 1 displayed similar rates of day 2-cleavage (range: 65.0 ± 10.9 to 70.0 ± 5.8%); of day 7-blastocyst rates (range: 46.6 ± 10.0 to 56.4 ± 8.2%) and of total day 7-blastocyst efficiency (range: 32.3 ± 8.3 to 37.2 ± 7.3%). Addition of PF4 did not affect total cell numbers of day 7 blastocysts (range: 44.1 ± 23.2 to 50.5 ± 26.4). In Experiment 2, PF4 accelerated embryo development, increasing (P < 0.01) blastocyst yield compared to 0-PF4, and blastocyst formation by day 5 adding PF4 100–500 ng/mL (range: 29.9 ± 7.8 to 31.8 ± 5.5%; P < 0.05) compared with BSA-control (17.2 ± 8.2%) and PF4 1000 ng/mL (15.5 ± 7.9%); showing similar blastocyst rates (range: 42.0 ± 11.5 to 49.3 ± 10.0%), total efficiency (28.0 ± 8.2 to 32.3 ± 7.1%) total cell numbers (range: 42.6 ± 19.3 to 45.7 ± 23.9) as BSA-controls. In conclusion, although PF4 did not show additive improvement under usual semi-defined, BSA-supplemented embryo media, it successfully replaced BSA sustaining porcine blastocyst production in chemically defined conditions.

PF4-Related Kinocidins Mediate Plasmodicidal Activity of Platelets

Abstract 1140 Malaria is a thrombocytopenic syndrome. Platelet activation occurs after binding to the knobs of Plasmodium-infected erythrocytes (Srivastava et al 2008). Severely thrombocytopenic thrombopoetin receptor knock-out mice infected with murine malaria have significantly higher mortality as compared to wild-type mice (McMorran et al 2008). Platelets have significant antibacterial properties when added to the bacterial cultures (Yeaman et al 2002). These are mediated by platelet microbicidal proteins, or kinocidins, alpha granule proteins related to the chemokine Platelet Factor 4 (PF4). PF4 possesses antibacterial properties itself. Additionally, plasma PF4 levels are significantly elevated in malaria. We hypothesized that the antimalarial properties of platelets are mediated by PF4-related thrombocidins, and studied growth-inhibition of platelet and PF4 on Plasmodium falciparum, in vitro. Plasmodium falciparum (ITG-2F2 strain) was cultivated in O negative human erythrocytes in a media comprised of RPMI 1640 plus glutamine, hypoxanthine and human serum, at low O2 (1%) and 37°C (Trager and Jensen 1972). Parasite growth was monitored by estimating percent parasitemia of stained smears by light microscopy. The parasite growth cycle was synchronized using gelatin floatation prior to the experiments. To ensure similar baseline parasitemia same stock culture was split between experimental and control cultures. Exp. 1 Washed human platelets were added to the culture of mature stage P. falciparum at a ratio of 1:1 of platelets to erythrocytes, and growth compared to control cultures at 12 and 24 hours. Platelet activation was studied by measuring supernatant PF4 levels (human PF4-specific ELISA [Hyphen Biomed]), and compared to platelet controls, where washed platelets were added to culture media containing only uninfected erythrocytes. Exp. 2 Recombinant human PF4 was added to P. falciparum cultures at a concentration of 2500 IU/ml, and growth compared to control cultures at 12 and 24 hours. Chloroquine-treated cultures represented positive controls in all experiments. The results of the experiments are reported as proportional change in baseline parasitemia, averaged for repeat experiments. These data are based on three sets of experiments. The addition of platelets to parasite cultures synchronized to mature stages resulted in significant growth inhibition at 24 hours as compared to the controls (Decrease to 0.44 of baseline versus 1.5 fold rise in controls; Figure 1). The PF4 levels (IU/ml) were significantly higher in experimental cultures than the platelet controls (mean 440, SD 80 vs. mean 265, SD 35; p=0.032), indicating platelet activation in the presence of Plasmodia. Finally, addition of PF4 to parasite cultures synchronized to mature stages resulted in significant growth inhibition at 24 hours as compared to the controls (Decrease to 0.5 of baseline versus 2.1 fold rise in control; Figure 1). An additional experiment was performed to study the effect of platelets and PF4 on immature stages of parasites. No differences in growth of parasites were observed between experimental and control cultures at 24 hours (1.4, 1.3 and 1.3 fold growth in control, platelet-treated, and PF4-treated cultures).Figure 1.Average change in parasitemia with timeFigure 1. Average change in parasitemia with time The results of this study indicate that the platelets are inhibitory to the growth of Plasmodium falciparum, in vitro. This is accompanied by platelet activation as evidenced by significantly higher PF4 levels in the presence of infected erythrocytes. Furthermore, we demonstrate that PF4 is growth-inhibitory to P. falciparum, likely mediating the plasmodicidal action of the platelets. This effect of platelets is dependent on the growth stage of parasites and appears to affect primarily mature stages. Currently available data do not exclude an intra-erythrocytic effect of PF4, but our data support the more likely hypothesis that the cytotoxic effect of PF4 occurs following the release of merozoites from erythrocytes. Although, supra-physiologic platelet and PF4 concentrations were used in this study, platelets are known to rosette Plasmodium-infected erythrocytes in vivo, particularly in microcirculation, indicating that the ‘local’ concentrations of PF4 are likely higher. Therefore, our data provide additional experimental evidence in support of the role of platelets in malaria innate immunity. Disclosures: No relevant conflicts of interest to declare.

Visualizing the Molecular and Cellular Basis of Heparin Induced Thrombocytopenia.

Abstract 228▪▪This icon denotes an abstract that is clinically relevant.Heparin-induced thrombocytopenia (HIT) is an iatrogenic complication of heparin therapy caused by antibodies that recognize the platelet chemokine, Platelet Factor 4 (PF4), complexed to heparin or to cellular glycosaminoglycans (GAG). The origin of the sentinel feature of HIT - high propensity for thrombosis in the setting of mild thrombocytopenia - remains undefined. We have shown previously that monocytes are particularly susceptible to activation by HIT antibody because unlike platelets, whose surface glycosaminoglycans (GAGs) consist almost entirely of chondroitin sulfates (CS), monocytes express heparan and dermatan sulfates that bind to PF4 with greater affinity. Therefore, antigenic complexes form at lower concentrations of PF4 on monocytes than on platelets and are more resistant to removal by exogenous heparin. We now provide 2 lines of visual evidence in support of this model of the prothrombotic state in HIT: 1) We demonstrate by scanning electron microscopy that spheroidal particles indicative of PF4/GAG complexes are present on the surface of human monocytes upon addition of PF4. 2) Thrombus formation has been visualized in a previously described murine HIT model involving the infusion of the HIT-like monoclonal antibody KKO into mice double-transgenic for human PF4 (hPF4+) and FcγRIIA receptor (FcγRIIA+) on their platelets. After KKO infusion, hPF4+/FcγRIIA+ mice developed vigorous and concurrent accumulation of HIT antigenic complexes and fibrin at sites of laser-induced endothelial injury, not present in similarly treated single transgenic mice. Accumulation of PF4 and fibrin at sites of injury was not increased by a polyclonal anti-hPF4 antibody that does not induce thrombocytopenia in hPF4+/FcγRIIA+ mice. Monocytes and/or monocyte microparticles accumulated at sites of laser-induced injury in KKO-infused hPF4+/FcγRIIA+ mice, but not in KKO-infused single transgenic mice or hPF4+/FcγRIIA+ mice infused with an isotype control, beginning 2.5 min after formation of HIT antigenic complexes and fibrin deposition, accumulating preferentially at the upstream end of developing thrombi. These results provide new insights into the potential role of monocytes in the prothrombotic sequelae of HIT. Antigenic complexes form on monocytes exposed to PF4 and accumulate in HIT antibody-induced thrombi. Monocytes appear to reinforce thrombus formation initiated by injured endothelial cells and activated platelets. Disclosures:Poncz:Diagnostica Stago: Patents & Royalties.

Cross Reactivity of Heparin-Induced PF4 Antibodies with Heparin- Bound IL-8.

Background: Heparin-Induced Thrombocytopenia (HIT) is caused by polyclonal antibodies, formed to conformational neoepitopes exposed in platelet factor 4 (PF4) bound to heparin (H). HIT antibodies are commonly measured by one of several commercially available ELISAs. The literature reports cases of clinical HIT and/or positive HIT antibody-mediated platelet activation tests in patients without antibodies detectable in PF4 based ELISAs, and suggests that antibodies to other chemokines, such as interleukin-8 (IL-8), may be involved. The current study was undertaken to characterize the frequency of such antibodies in a population of consecutive serum specimens submitted for HIT diagnosis by 14C-serotonin release assay (SRA), and also in a group of retrospectively selected SRA-positive specimens.Methods: Specimens (n=90) analyzed by SRA between March and July 2008 were subsequently tested by PF4 Enhanced ELISA (GTI, Waukesha, WI), and the Zymutest HIA ELISA (Hyphen BioMed, Neuville sur Oise, France). An additional ELISA was carried out on the specimens using the Zymutest heparin-bonded plate coated with purified, recombinant human interleukin-8 (IL-8) (72 aa form, Prospec, Rehovot, Israel). The second group consisted of archived specimens (n=32) that tested positive by SRA but negative by GTI HAT ELISA; these specimens were subsequently tested by the Zymutest HIA and the H:IL-8 ELISA.Results: Recombinant IL-8 (1μg/well) added to heparin-bound ELISA plates was recognized by anti-IL-8 (BD Biosciences), but not anti-PF4 (Leinco Technologies, Inc.) antibodies and was not displaced from the plate by addition of PF4 from normal serum at the dilution (1:100) used in the ELISA. Of the 90 consecutive specimens, 21 (23.3%) tested positive in both the GTI and the Zymutest HIA tests; 15 of the 90 (16.6%) tested positive by one but not both of the PF4-based ELISAs (Table 1). In this group of 90 specimens, 17 (18.9%) had OD > 0.500 on the heparin-bound IL-8 plate. Only 3 of the specimens bound to the H:IL-8 exclusively, while 14 were also positive in the GTI and/or Zymutest HIA ELISA. Thus, of the 36 specimens that tested positive in one or both of the PF-4-based ELISAs, 14 (38.9%) had antibodies that also targeted heparin-bound IL-8. Ten of the 90 specimens tested positive in the SRA. Six of SRA-positives had PF4-specific antibodies that did not cross react with H:IL-8, 3 had H:IL-8 reactive antibodies in addition to PF4 reactive antibodies, and 1 SRA positive specimen tested negative in all ELISAs.Table 1. Comparison of GTI HAT vs Zymutest HIA results: n = 90 consecutive patientsNumbers in parentheses indicate how many specimens in each subgroup contained antibodies that bound to H:IL-8.Zymutest HIA+−Total+21 (11)9 (1)30GTI HAT−6 (2)54 (3)60Total276390Among the set of 32 specimens that tested positive by SRA and negative by GTI ELISA, 8 tested positive in the Zymutest ELISA, including 4 that had OD > 0.500 on the H:IL-8 plate. The 24 specimens that tested negative by both HIT ELISAs ranged in percent serotonin release (tested with 0.1 U/ml H) between 21–84% with a median of 45.6%, and did not contain antibodies that bound the H:IL-8 plate.Conclusions: The data agree with other studies that show differences between results of commercially available HIT ELISA kits. The study also shows that over one third of specimens that test positive in the HIT ELISAs contain antibodies that are not specific for PF4, but also recognize heparin-bound IL-8. These results do not prove that the antibodies that bind PF4 targets are the same ones that bind IL-8. However, the significant degree of homology (40%) between the peptides suggests that the same antibodies may cross react with either target. The relevance of these antibodies is as yet unclear. Only 3% of specimens bound H:IL-8 plates but not PF4 plates, and these were not associated with SRA activity. Also HIT ELISA negative specimens with SRA activity, which showed only moderate percent serotonin release, did not contain H:IL-8-binding antibodies. The study did not find evidence of a role for H:IL-8 antibodies in HIT-related platelet activation tests.

Platelet Factor 4 Enhances the Binding of Oxidized Low-density Lipoprotein to Vascular Wall Cells

Accumulation of low-density lipoprotein (LDL)-derived cholesterol by macrophages in vessel walls is a pathogenomic feature of atherosclerotic lesions. Platelets contribute to lipid uptake by macrophages through mechanisms that are only partially understood. We have previously shown that platelet factor 4 (PF4) inhibits the binding and degradation of LDL through its receptor, a process that could promote the formation of oxidized LDL (ox-LDL). We have now characterized the effect of PF4 on the binding of ox-LDL to vascular cells and macrophages and on the accumulation of cholesterol esters. PF4 bound to ox-LDL directly and also increased ox-LDL binding to vascular cells and macrophages. PF4 did not stimulate ox-LDL binding to cells that do not synthesize glycosaminoglycans or after enzymatic cleavage of cell surface heparan and chondroitin sulfates. The effect of PF4 on binding ox-LDL was dependent on specific lysine residues in its C terminus. Addition of PF4 also caused an approximately 10-fold increase in the amount of ox-LDL esterified by macrophages. Furthermore, PF4 and ox-LDL co-localize in atherosclerotic lesion, especially in macrophage-derived foam cells. These observations offer a potential mechanism by which platelet activation at sites of vascular injury may promote the accumulation of deleterious lipoproteins and offer a new focus for pharmacological intervention in the development of atherosclerosis.

Platelet factor 4 binds to low-density lipoprotein receptors and disrupts the endocytic itinerary, resulting in retention of low-density lipoprotein on the cell surface

The influence of platelets on the cellular metabolism of atherogenic lipoproteins has not been characterized in detail. Therefore, we investigated the effect of platelet factor 4 (PF4), a cationic protein released in high concentration by activated platelets, on the uptake and degradation of low-density lipoprotein (LDL) via the LDL receptor (LDL-R). LDL-R-dependent binding, internalization, and degradation of LDL by cultured cells were inhibited 50%, 80%, and 80%, respectively, on addition of PF4. PF4 bound specifically to the ligand-binding domain of recombinant soluble LDL-R (half-maximal binding 0.5 microg/mL PF4) and partially (approximately 50%) inhibited the binding of LDL. Inhibition of internalization and degradation by PF4 required the presence of cell-associated proteoglycans, primarily those rich in chondroitin sulfate. PF4 variants with impaired heparin binding lacked the capacity to inhibit LDL. PF4, soluble LDL-R, and LDL formed ternary complexes with cell-surface proteoglycans. PF4 induced the retention of LDL/LDL-R complexes on the surface of human fibroblasts in multimolecular clusters unassociated with coated pits, as assessed by immuno-electron microscopy. These studies demonstrate that PF4 inhibits the catabolism of LDL in vitro in part by competing for binding to LDL-R, by promoting interactions with cell-associated chondroitin sulfate proteoglycans, and by disrupting the normal endocytic trafficking of LDL/LDL-R complexes. Retention of LDL on cell surfaces may facilitate proatherogenic modifications and support an expanded role for platelets in the pathogenesis of atherosclerosis.