Higher Platelet Factor 4 Research Articles

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.

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 .

Assessment of Serum Platelet Factor 4 as a Predictive Marker for VTE and as a Prognostic Biomarker in Pancreatic, Colorectal, Breast, and Lung Cancer

Introduction: Cancer is the second leading cause of mortality in the United States, with lung, colorectal, breast, and pancreas carcinoma comprising the four leading causes of cancer death. We previously demonstrated that platelet factor 4 (PF4) is an independent prognostic factor in pancreatic adenocarcinoma, with a significant association between elevated serum levels and increased venous thromboembolism (VTE) incidence and death. It is possible that high serum PF4 levels may be a useful biomarker for patient stratification for directing VTE prophylaxis in cancer patients. We sought to determine if PF4 is useful as a biomarker for prognosis and VTE incidence in other common cancers. Methods: We measured PF4 levels in sera from 39 patients with breast cancer, 48 with colorectal cancer, 50 with lung cancer and compared these results with data from 132 patients with pancreatic adenocarcinoma using a commercially available ELISA kit. Results: for all four cancers, age (P < 0.0001) and stage (P < 0.0001) were both significant predictors of survival. When examined in univariate Cox models, serum PF4 was a significant predictor of survival in breast (P = 0.018), colorectal (P = 0.01), and pancreatic (P = 0.003) cancer but not lung cancer (P = 0.65). After correcting for age and stage in multivariate analyses, PF4 remained a significant prognostic indicator in only colorectal (P = 0.008) and pancreatic (P = 0.001) cancers, with resulting hazard ratios for mortality of 1.53 and 1.105 respectively. Serum PF4 levels were significantly elevated in colorectal (P = 0.04) and pancreatic (P = 0.008) cancer subjects with VTE as compared to those without VTE, but this relationship was not present in breast (P = 0.81) or lung (P = 0.29) cancer subjects. When adjusted for age and stage, this association between serum PF4 level and VTE remained significant only in pancreatic cancer (P = 0.015). Conclusions: These initial results suggest PF4 may have utility as a clinical biomarker to predict survival in patients with pancreatic, and colorectal cancer and may be useful for patient stratification for VTE prophylaxis. We hypothesize that colon and pancreatic cancer patients with high serum PF4 levels may benefit from extended VTE prophylaxis. This question should be answered in a clinical trial.

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.

Serum Platelet Factor 4 Is an Independent Predictor of Survival and Venous Thromboembolism in Patients with Pancreatic Adenocarcinoma

Improved diagnostic, predictive, and prognostic biomarkers for pancreatic ductal adenocarcinoma (PDAC) are urgently needed. Platelet factor 4 (PF4) has been proposed as a diagnostic biomarker for PDAC. We assessed the diagnostic and prognostic potential of serum PF4 levels in PDAC patients. Serum PF4 levels were determined by enzyme-linked immunosorbent assay in an initial cohort of 62 PDAC patients, 62 healthy control subjects, and 34 chronic pancreatitis patients. A second validation set consisted of 71 PDAC patients. Linear regression models were used to relate PF4 to class, gender, age, stage, platelet count, and diagnosis. Survival analyses were done using univariate and multivariate Cox models. In the initial cohort, serum PF4 levels distinguished PDAC from chronic pancreatitis patients (P = 0.011), but not from healthy control subjects (P = 0.624). In PDAC patients, high serum PF4 level significantly predicted decreased survival independent of all covariates examined (P < 0.01). The prognostic relationship of serum PF4 levels remained significant in the validation set. Venous thromboembolism (VTE) occurred in 20% of the 133 PDAC patients. The VTE risk was higher in subjects with elevated PF4 levels (P = 0.009). Serum PF4 is shown for the first time to be prognostic for survival in PDAC patients. High PF4 is associated with an increased risk for the development of VTE. Serum PF4 levels may be useful for patient stratification and for directing treatment options in patients with pancreatic cancer including anticoagulation prophylaxis. The relationship between high PF4 levels and poorer outcomes requires further study.

Modulation of Substrate Specificity of the Thrombin-Thrombomodulin Complex for Protein C Versus TAFI Activation by Platelet Factor 4 and Non-Anticoagulant Heparin-Derivatives.

Abstract 3181 Poster Board III-108 The endothelial cell receptor thrombomodulin (TM) is an essential cofactor for thrombin-mediated activation of protein C and the generation of activated protein C (APC), an enzyme with anticoagulant and multiple cytoprotective activities. TM also stimulates thrombin-mediated activation of Thrombin Activatable Fibrinolysis Inhibitor (TAFI), resulting in anti-fibrinolytic and anti-inflammatory effects. Based on these properties of TM, several potential strategies for therapeutic intervention have been proposed. In hemophilia patients with inhibitory antibodies, stimulation of TM-dependent TAFI activation might diminish bleeding complications by protection of clots against premature lysis. Alternatively, stimulation of endogenous APC generation in patients with severe sepsis might prove to augment the beneficial effects of APC, as pharmacologic applications of APC in these patients increased survival. Platelet Factor 4 (PF4), a platelet alpha-granule protein, acts as a soluble cofactor for protein C activation by the thrombin-TM complex and stimulated endogenous generation of APC in vivo. Here we show that in contrast to stimulation of protein C activation, PF4 dose-dependently inhibited activation of TAFI by thrombin-TM in a purified system, in normal pooled plasma and on TM-expressing cells. Functional consequences of inhibition of TAFI activation by PF4 were determined in hemophilia A plasma by analysis of clot resistance to tPA-induced fibrinolysis. In hemophilia A plasma, activation of TAFI was dependent on TM due to the ineffective amplification of thrombin generation via the tenase complex. PF4 diminished TM-dependent prolongation of clot lysis times but did not affect clot lysis in the absence of TM. Inhibition of TM-dependent prolongation of clot lysis time by PF4 was dependent on both the TM and tPA concentration used, consistent with the proposed threshold mechanism for inhibition of fibrinolysis by TAFIa. To determine the effect of PF4 on TM-dependent TAFI activation in normal plasma, the inactivation of bradykinin (BK) in plasma by TAFIa during tissue factor induced coagulation was analyzed. Addition of TM to plasma accelerated BK cleavage, i.e., generation of des-Arg9-BK, consistent with TM-mediated stimulation of TAFI activation and subsequent BK cleavage by TAFIa. PF4 neutralized the TM-enhanced BK inactivation and reversed BK and des-Arg9-BK levels to those seen in the absence of TM. Thus, PF4 alters the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, such that PF4 prevents generation of TAFIa's anti-fibrinolytic and anti-inflammatory activities. To find an antidote for PF4's inhibitory effects on TAFI activation, heparin-derivatives were screened for high affinity PF4 binding but greatly reduced anticoagulant activity. N-acetylated heparin (NAc-Hep), but not N-desulfated without N-acetylation or N-acetylated and O-desulfated heparins, retained high affinity binding to PF4 and effectively blocked PF4 binding to immobilized TM. Consistent with high affinity binding of NAc-Hep to PF4, sulfylamine group oxygens were relatively minor determinants of the interactions between heparin and PF4 based on the available structural model. NAc-Hep did not show detectable anticoagulant activity in APTT clotting assays but restored BK conversion to des-Arg9-BK by TAFIa in the presence of PF4, similar to that seen in the absence of PF4. In addition, NAc-Hep reversed PF4-mediated inhibition of TAFI activation and TAFIa's antifibrinolytic functions in clot lysis assays on TM-expressing cells using hemophilia A plasma. Thus, NAc-Hep reversed PF4-mediated inhibition of TAFI activation. In summary, PF4 modulates the substrate-specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation. NAc-Hep, a non-anticoagulant heparin derivative with high affinity for PF4, effectively reversed PF4-mediated inhibition of TM-dependent TAFI activation and restored TAFIa's antifibrinolytic and anti-inflammatory functions. Accordingly, NAc-Hep or similar non-anticoagulant heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis. Disclosures No relevant conflicts of interest to declare.

Structural and Functional Studies to Define the Molecular Basis by Which Platelet Factor 4 (PF4) Increases Survival of Mice in Lipopolysaccharide (LPS)-Induced Endotoxicity

Our previous studies have demonstrated that platelet-released PF4 (chemokine CXCL4) promotes survival in a murine LPS-induced endotoxicity model, although the molecular basis for PF4's protective effects was not fully defined. We hypothesized that enhanced generation of cytoprotective activated protein C (APC) by PF4 might contribute to the molecular mechanism of PF4's beneficial effects in vivo, based on the observation that PF4 stimulates protein C (PC) activation by the thrombin/thrombomodulin complex both in vitro and in vivo. Here we show that PF4 in vitro affects human (h) PC activation in the presence of human thrombomodulin (hTM) in a bell-shaped concentration curve, i.e. stimulation at low, but inhibition at high PF4 concentrations with a peak around 3 μM. This curve is similar to that seen with PF4 for surface-bound heparin-induced thrombocytopenia (HIT) antigenicity, suggesting that similar complexes of PF4 with glycosaminoglycans (GAGs) that occur in HIT (termed ultralarge complexes (ULC)) are relevant to PF4's interaction with the hPC/hTM. Addition of heparin blocks PF4 increase of APC generation in a similar fashion as it does for surface-bound ULC. A PF4 variant PF4K50E that poorly forms PF4 tetramers requires 8-fold higher concentrations to enhance APC generation, supporting that PF4 tetramers are central for APC generation as they are for formation of ULC. Neither PF4 nor PF4K50E accelerated in vitro generation of APC in the presence of hTM that was depleted of its chondroitin sulfate chain, suggesting that PF4 binds to this domain on hTM. In vivo studies involving simultaneous infusions of PF4 and thrombin into PF4 knock out (mPF4−/−) mice showed that PF4 leads to enhanced mouse (m) APC generation not seen with infused PF4K50E, consistent with our in vitro studies. We then asked if surface heparan sulfate on the endothelial lining was necessary for the observed PF4 effect on in vivo mAPC formation. We studied mice with a Tie2-Cre conditional knock out of N-deacetylase-N-sulfotransferase-1 activity (NDST-1−/−) that have only 15% of normal endothelial cell surface heparan sulfate content using a similar thrombin/PF4 infusion model. We found that mAPC generation was accelerated by PF4 to the same extent both in NDST1−/−/mPF4−/− and mPF4−/− mice, suggesting that surface GAGs are not involved in the PF4 effect. We have also tested the in vivo effect of PF4 on mAPC formation in TM mutant (TMpro/pro) mice that have impaired capacity for APC formation to further demonstrate that PF4's positive effect in LPS endotoxic shock survival involves enhanced mAPC generation. Upon injection of high doses of thrombin (40 U/kg), mAPC levels are increased to the same extent in WT and TMpro/pro mice. After injection of low amounts of thrombin (8 U/kg), generation of mAPC was impaired in TMpro/pro as compared to WT mice. Concurrent infusion of PF4 increased mAPC formation in TMpro/pro mice after injection of low doses of thrombin, approximately equal to that seen in WT mice with no PF4 injected. As previously described, TMpro/pro mice had increased mortality after injection of LPS as compared to WT mice; however, with concurrent platelet PF4 overexpression, mortality decreased to that seen in WT mice, suggesting that the biological value of PF4 in LPS endotoxicity is related to its effect on the generation of APC. Thus, these studies support enhanced APC generation as the basis for the positive effect of PF4 on LPS endotoxicity and further define the molecular basis for increased APC generation by PF4 by forming ULC with the chondroitin sulfate domain of TM, but not with heparan sulfate on the vascular surface.

Heparin-induced thrombocytopenia: A stoichiometry-based model to explain the differing immunogenicities of unfractionated heparin, low-molecular-weight heparin, and fondaparinux in different clinical settings

IntroductionHeparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4 (PF4)/heparin complexes. The frequency of HIT is highly variable in different clinical settings, and is more frequent with unfractionated heparin (UFH) than with low-molecular-weight heparin (LMWH), despite the in vitro observation that HIT antibodies activate platelets similarly well with LMWH as with UFH. An important difference between UFH, LMWH, and fondaparinux is their widely differing plasma concentrations. We aimed to provide a model that included anticoagulant concentrations and PF4 availability as risk factors influencing the anti-PF4/heparin immune response. Materials and methodsBy photon correlation spectroscopy we determined the concentrations at which UFH, LMWH, and fondaparinux form complexes optimally with PF4. Plasma concentrations of UFH and LMWH were calculated based on ex vivo pharmacokinetic data, with information on fondaparinux and PF4 concentrations taken from the literature. Results and conclusionsThe main features of our model are: optimal complex formation occurs at prophylactic-dose UFH and high PF4 levels, whereas therapeutic-dose LMWH concentrations are too high for optimal complex formation; in contrast, concentrations of fondaparinux are usually below the optimal stoichiometric range. Thus, immunization should occur more often in situations with major rather than minor platelet activation, and—for a given degree of platelet activation (PF4 availability)—as: prophylactic-dose UFH>therapeutic-dose UFH>prophylactic-dose LMWH, fondaparinux>therapeutic-dose LMWH. Our model provides a framework for explaining empirical observations that LMWH induces less anti-PF4/heparin antibodies than does UFH, and that anti-PF4/heparin antibodies are more often found in patients undergoing major surgery than in medical patients.

Efficacy of Anagrelide Compared with Hydroxyurea in Patients with Essential Thrombocythemia.

The essential thrombocythemia (ET) is characterized by thrombohemorrhagic risk. ET patients receiving HU and anti-aggregants show normal platelets but present platelet coagulant and endothelial aggregation. Anagrelide (ANA) is a platelet-lowering drug that inhibits platelet aggregation. Therefore, we evaluated platelets, platelet factor 4 (PF4), prothrombin fragment 1+2 (F1+2), thrombin antithrombin complex (TAT) and d-dimer (DD) and von Willebrand factor (vWF) as markers of platelet coagulant and endothelial aggregation, respectively, in ET patients treated either with HU or ANA. All measurements were performed before treatment and when complete response, defined as platelet < 400 x109/L for more than 1 month, was achieved. Prolonged thrombocytosis causes thrombohemorrhagic risk. Hence, we investigated the cytoreduction time. The study comprised 36 patients (19 men, 17 women; mean age 65 years) who fulfilled PVSG and WHO. The mean duration of disease was 6 years. 17 patients received HU and 19 patients were on ANA. The average dose of HU was 0.5 g/d. ANA was initially administered at a dose of 0.5 mg/d. The dose was increased by 0.5 mg/d every week until the platelets had decreased to below 400 x 109/L. The average maintenance was 1.7 mg/d. All patients were on aspirin at dose of 100 mg/d and 50 mg/d in HU and ANA group, respectively. None of the patients had acquired or inherited thrombotic risk factors or had inherited coagulopathy. Eleven out of 17 HU patients (5 men, 6 women; mean age 68.6 years) developed thrombosis including six transient ischemic attack (TIA), three myocardial infarct (MI) and two deep venous thrombosis (DVT). The thrombosis occurred at a median time from diagnosis of 2 years. The ANA group did not experience thrombosis. Platelets were determined by automated analyser. PF4, F1+2, TAT and DD and vWF were assayed by ELISA and immunoturbidimetric assay, respectively. Before treatment, all patients had high platelets (1027±297 x 109/L) and elevated PF4, F1+2, TAT and DD (134±48 IU/mL vs 6±2 IU/mL, 3±2 nmol/L vs 0.6±0.2 nmol/L, 25±34 μg/L vs 2.7±1 μg/L, 224±172 μg/L vs 197±43 μg/L, respectively) (p< 0.0001, p<0.0001, p<0.0001 and p<0.0001, respectively) and low vWF (23±9% vs 102±30%) (p<0.0001). After treatment all patients had platelets <400 x 109/L (364 ±77 x 109/L), whereas PF4, F1+2, TAT and DD were higher (147±45 IU/mL, 3±2 nmol/L, 24±41 μg/L and 264±291 μg/L, respectively) and vWF was lower (34±9.9%) in HU group than controls (p<0.0001, p<0.0001, p<0.0001, p=0.001 and p<0.0001, respectively) and normal in ANA group (7±3 IU/mL, 1.1±0.8 nmol/L, 2.4±1.1 μg/L, 121±84 μg/L and 95±37%, respectively). The mean cytoreduction time was 3.2 and 1.8 months in HU and ANA group, respectively. A correlation there was between PF4 and F1+2 and TAT and DD and vWF (p=0.004, p=0.005, p=0.022 and p<0.0001, respectively). vWF did not correlate with F1+2, TAT and DD. Platelets did not correlate with thrombosis, whereas a correlation there was between PF4, F1+2, TAT and DD and thrombosis (p<0.0001, p=0.015, p=0.005 and p=0.034, respectively). A difference was observed in cytoreduction time between HU and ANA (p<0.0001). These results might support the superiority of ANA as antithrombotic drug besides its platelet-lowering effect.

Negative Paracrine Effect of Platelet Factor 4 on Megakaryopoiesis Occurs through Lipoprotein Related Protein Receptor-1 on Megakaryocytes.

Platelet Factor 4 (PF4) is a paracrine inhibitor of megakaryopoiesis in vivo, with platelet counts inversely related to platelet PF4 levels in mice. Duration of thrombocytopenia after chemotherapy-induced thrombocytopenia (CIT) was directly related to platelet PF4 levels. Defining the molecular basis by which PF4 has this effect may have clinical implications in patients with CIT. Based on a recent observation that PF4 could activate endothelial cells through the Lipoprotein Related Protein Receptor-1 (LRP-1), we investigated whether a similar receptor might be involved in the negative paracrine effect of PF4 on megakaryopoiesis. We found that the addition of receptor-associated protein (RAP), a general inhibitor of members of the LRP family of receptors by binding to the alpha subunit, could block the inhibitory affect on in vitro megakaryocyte (MEG) colony number per plated marrow cells in a specific and dose-dependent fashion. Thus, at 0.2 μM RAP could prevent ∼50% of the inhibitory affect of the addition of 20 μg/mL of recombinant PF4 and at 0.4 μM RAP, the inhibitory effect of the added PF4 was completely abolished (p = 0.01 of no RAP vs. 0.4 μM RAP). Since a number of LRP receptors are inhibited by RAP, we repeated these studies using a specific anti-LRP-1 monoclonal antibody that blocks LRP-1 activation by binding to its alpha subunit (American Diagnostic). We have previously shown that transgenic mice overexpressing human PF4 by 6-fold have a 50% decrease in MEG-containing colonies in the above colony assay relative to WT littermates due to increase release of PF4 from developing megakaryocytes. We now found that the number of colonies derived from these high PF4 marrows increased to that seen in WT littermates when 25 μg/mL of the anti-LRP-1 antibody was included (p = 0.01). Since there are no prior studies showing that LRP-1 is expressed on human and murine bone marrow and murine fetal liver-derived MEGs, we performed RT-PCR on the appropriate RNAs as well as flow cytometry for the presence of LRP-1. RT-PCR of MEG RNA shows that LRP-1 message is present in MEGS. By flow cytometry, LRP-1 is clearly present on both human and murine MEGs. These studies support that PF4 inhibits megakaryopoiesis through LRP-1 on developing megakaryocytes. Studies were also done on the megakaryocyte-erythroid progenitor cell line G1ME. These cells do not express LRP-1, but when stimulated to differentiate, LRP-1 is detectable by flow cytometry on maturing megakaryocytes, suggesting that PF4 exerts its negative paracrine effect on developing MEGS and not on earlier progenitor cells. Consistent with this finding, we observed that in mice with different levels of platelet PF4, MEG content in the marrow is inversely related to platelet PF4 levels, but the size and ploidy are not correlated to PF4 content. Thus, we believe that we have defined the receptor system involved as well as the target cell of the observed inhibitory effect of PF4 on megakaryopoiesis. Such studies on the mechanism by which PF4 exerts this negative paracrine effect should lead to a more complete understanding of the regulation of megakaryopoiesis and may offer novel strategies to intervene in clinical thrombocytopenia, especially CIT.

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.

Release of High Levels of Platelet Factor 4 (PF4) from Platelets Improves Survival after a Lethal Lipopolysaccharide (LPS) Challenge.

Infusion of activated protein C (APC) improves survival in sepsis. PF4 is a CXC chemokine predominantly expressed during megakaryopoiesis and stored in platelet alpha-granules whose biological function(s) are not well understood. We have shown that recombinant PF4 enhances APC generation by thrombin/thrombomodulin complexes both in vitro and in vivo. Would endogenous PF4 released from platelets activated during an inflammatory state similarly affect APC production? We addressed this issue using mice that were either completely devoid of PF4 (mPF4−/−) or had a 6-fold excess of human PF4 (hPF4+). Using a lethal LPS challenge model (O111:B4, 40 mg/kg IP), we examined whether platelets become activated and release PF4 during endotoxemia. Two hours after LPS injection, the platelet count in mice decreased to ~70% of baseline levels (p=0.006). Serum PF4, as measured by ELISA, also dropped to ~60% of baseline from 47±5 kU/ml to 30±5 kU/ml (p=0.002). At the same time, plasma PF4 level was increased by 20%, consistent with LPS resulting in PF4 release. The smaller increase than expected suggests that much of the released PF4 binds immediately to the surface of vascular cells. Consistent with this, we have observed higher accumulation of PF4 in mouse lungs after LPS injection compared to uninjected mice (990±220 and 660±120 U/mg, respectively, p=0.017). APC generation was assessed 10 min after thrombin infusion (80 U/kg. IV) as a measure of endogenous platelet PF4's effect in an inflammatory/procoagulant state. In mPF4−/− mice APC levels were 72% of that in wild type (WT) mice (p=0.0006) while in hPF4+ mice APC formation increased to 178% (p=0.003). Survival of mice 24 hrs after LPS (25 mg/kg) challenge was then examined. hPF4+ mice had a mortality rate of 9% compared to ~40% in both WT and mPF4−/− (p< 0.001). To examine the role of APC in this improved survival, we performed similar experiments with mice heterozygous for protein C deficiency (PC+/−). More PC+/− mice died 16 hrs after injection of 40 mg/kg LPS than WT mice (61% vs. 29% mortality respectively, p=0.005), while mortality for hPF4+/PC+/− mice was significantly lower (14%, p< 0.001 compared to PC+/− mice), supporting the hypothesis that the protective effect of PF4 is at least in part due to increased APC generation. Next we asked if infusion of platelets with high PF4 is protective in the LPS model. We injected either vehicle buffer or mPF4−/− or hPF4+ platelets (3×108 per 20 g mouse) into WT mice prior to treatment with LPS. Mortality of mice at 24 hrs after LPS injection with mPF4−/−platelet infusion was not significantly different than mice with buffer infusion (86% vs. 96% respectively, p=0.4), but mortality was significantly lower when hPF4+ platelets were infused (58% vs. 96%, p=0.01). Our results suggest that PF4 is released from platelets after an inflammatory stimulus and that this may have a positive physiological role by enhancing APC generation. High endogenous platelet PF4 levels may have a survival advantage after exposure to endotoxins. Infusion of platelets containing high levels of PF4 in sepsis may be a novel therapeutic strategy that warrants further investigation.

Murine In Vivo Studies Support Platelet Factor 4 as a Negative Autocrine of Megakaryopoiesis with Clinical and Therapeutic Implications.

Platelet Factor 4 (PF4) is a member of the CXC family of chemokines that appears to act as an autocrine regulator of megakaryopoiesis in vitro. To demonstrate that PF4 plays a physiological role in vivo, we examined megakaryopoiesis in animals that over- or under-expressed PF4. We generated PF4−/− knockout animals as well as transgenic animals that overexpress human (h) PF4 to a level that is ~6-fold greater than control human platelet samples (hPF4+). Relative to wildtype (WT) littermate controls (n=26), platelet counts were elevated by ~15% in knockout animals (n=18, p=0.001) and reduced by ~33% in hPF4+ animals (n=13, p=0.0004). Platelet survival studies insured that the observed changes were not due to a consumptive process. The hypothesis that PF4 results in a decrease in megakaryocyte and/or platelet production was supported by in vitro evidence that growth of megakaryocyte-containing colonies was inversely related to the PF4 phenotype. The hPF4+ mice formed 52%±15% fewer megakaryocyte colonies compared to WT (n=8 each arm, p<0.00002). This inhibition was blocked by the inclusion of a rabbit polyclonal anti-hPF4 antibody. Based on these observations, we hypothesized that lysis of megakaryocytes induced by cytolytic chemotherapy with resultant release of PF4 may contribute to post-cytolytic-therapy thrombocytopenia. Exposing mice to intraperitoneal 5-fluorouracil (5-FU) decreased WT platelet counts below normal for 10.8±1.3 days (n=23). PF4−/− mice had a significantly more rapid recovery of 8.2±1.4 days (n=14, p<0.0001), while hPF4+ mice had significantly delayed recovery of 15.3±1.7 days (n=15, p<0.000006). Importantly, the duration of thrombocytopenia in WT animals could by shortened to that seen in PF4−/− mice by administration of 50 mg/kg of F(ab)2 fragments of a polyclonal rabbit anti-murine PF4 antibody, but not by rabbit non-specific polyclonal IgG. Specific anti-PF4 therapy improved recovery of platelet count in WT mice to 8.8±1.5 days vs. 10.7±1.0 days in non-treated WT mice (n=10 and 8, respectively, p<0.008). Forty percent of the specific-treated animals recovered by day 7 post injection of 5-FU, while none of the control animals recovered before day 10. Similar interventions in hPF4+ animals resulted in a marked improvement in platelet count recovery compared to controls with the anti-hPF4 antibody-treated group recovering by 9.0±1 vs. 13.6±1.2 for the control mice (n=8 and 5, respectively, p<0.002). Our data suggest that efforts to block the effect of PF4 during cytotoxic therapy may limit the duration of thrombocytopenia. These observations have clinical implications, particularly for patients who express high endogenous levels of PF4. To that end, we studied 250 well adults and found that 10% of these individuals have 2–4.5 times the average platelet PF4 content. We propose that these individuals with high platelet PF4 levels may be particularly sensitive to developing thrombocytopenia after cytolytic therapy and may specifically benefit from therapy that blocks the effects of released PF4.

Endogenous Platelet PF4 Promotes In Vivo Activated Protein C (APC) Generation and Survival after Lethal Lipopolysaccharide Challenge in Mice: A Potential Physiologic Role for PF4.

Pharmacologic infusion of activated protein C (APC) improves survival in human sepsis and genetically induced deficiencies in APC generation increase mortality in a murine lipopolysaccharide (LPS) model of inflammation/sepsis. We have shown that the platelet α-granule-specific chemokine, platelet factor 4 (PF4), accelerates thrombin·thrombomodulin complex APC generation in vitro and in vivo when recombinant PF4 is pharmacologically co-infused in a primate thrombin infusion model system. Hypothesizing a potential physiologic role for PF4 in stimulating APC formation in vivo, we tested whether variations in the level of endogenous platelet PF4 content affect APC generation and also influence survival in the murine LPS challenge model. To stimulate APC formation and platelet PF4 release in mice, we injected murine thrombin (80U/kg IV). Ten min after thrombin injection into wildtype (WT) C57BL/6J mice, plasma APC levels, assessed by enzyme capture amidolytic assay, increased ~10-fold over basal levels. Injection of concurrent murine PF4 (7.5 mg/kg IV) further increased APC levels 2-2.6-fold. We then studied APC generation in mice that were either completely deficient in PF4 (PF4−/−) or transgenic mice that had 6-fold normal levels of human PF4 (hPF4+), both of which had been crossbred onto the same C57BL/6J background >10 times. Plasma APC generation by thrombin was 74 ± 6% in the PF4−/− mice (n = 7) compared to WT littermates (n = 15, p<0.1) and increased to 178 ± 49% in hPF4+ mice (n= 22, p< 0.003 compared to WT (n= 36)). Heterozygous protein C deficient (PC+/−) mice had 65 ± 21% of WT APC generation (n= 5, p= 0.05 compared to WT (n=7)), but APC generation was restored to 96 ± 6% level in hPF4+/PC+/− mice (n= 6, p= 0.88 compared to WT (n=12)). Only PF4−/− and PC+/− mice died during this thrombin injection trial with mortality rates of 30% and 58%, respectively. A lethal LPS challenge model (25 mg/kg IP) was used to test the susceptibility of mouse strains with varying PF4 levels. Significant numbers of WT and PF4−/− mice died before the end of the first day [(18 of 44 (40% mortality) and 6 of 21 (28% mortality), respectively], while none of the 19 hPF4+ animals died (p< 0.001). However, after the first day, mortality did not differ significantly between the groups and final mortality for the three groups was ~70%. In summary, these studies of mice with varying platelet PF4 suggest that endogenous PF4 plays a physiologic role to stimulate APC production. Moreover, mice with high platelet PF4 content appear to be protected from both thrombin- and LPS-provoked deaths. This suggests that PF4 plays a physiologic role to enhance APC generation, that patients with high PF4 levels may be intrinsically protected from sepsis and supports the possibility that PF4 infusions in patients with sepsis may be a viable therapeutic strategy.

Treatment of Essential Thrombocythemia with Anagrelide: A Novel Antiangiogenic Agent?.

Essential thrombocythemia is a myeloproliferative disorder complicated by thrombotic phenomena as a result of the platelet-clotting hyperactivation linked to increased angiogenesis. It is reported that vascular endothelial growth factor (VEGF) generates thrombin and promotes platelet activation and that activated megakaryocytes and platelets release VEGF. Anagrelide (ANA) is a newer cytoreductive agent which decreases megakaryocyte mass and inhibits the platelet activation. Therefore, we studied platelet factor 4 (PF4), as indicator of platelet activation, VEGF and prothrombin fragment 1+2 (F1+2) and d-dimer (DD), as indicators of thrombin generation, in 22 patients (12 males and 10 females, mean age 50 years) affected by ET diagnosed according to PVSG criteria. Their mean duration of disease was 4.8 years. All patients were on either HU (6 patients) or IFN-α (4 patients) and ANA (12 patients). HU was used at doses ranging between 1 and 1.5g/day. The dosage of IFN-α was 3 MIU, 3 days/week. ANA was initially administered in dose of 0.5 mg/day, with increases of 0.5 mg/day every 7 days until the platelets decreased below 500×109/L and with a average maintenance dosage of 2.2 mg/day. All patients recieved antiplatelets either aspirin (ASA) (15 patients) or indobufen (IND) (6 patients) and dipirydamole (DYP) (1 patient). Platelets, PF4, VEGF, F1+2 and DD were measured before cytoreduction and to complete response defined as platelets≤ 500×109/L. Platelets were determined by automated analyser. PF4, VEGF and F1+2 were assayed by ELISA and DD by immunoturbimetric latex agglutination. Before treatment all patients had marked platelets (1060±356×109/L) and high PF4 and VEGFPLT (157±83 IU/ml vs 1.6±0.9 IU/ml and 1.2±1 pg/ml vs 0.3±0.2 pg/ml, respectively) (p<0.0001 and p= 0.002, respectively). F1+2 and DD were elevated 3.6±3.7nml/L and 205±83 μg/L, respectively) than controls (0.6±0.3nmol/L and 75±21 μg/L, respectively) (p<0.0001 and p<0.0001, respectively). After a median time of 4.5 months from cytoreduction all patients had platelets ≤ 500 × 109/L (430±70 × 109/L). PF4 and VEGFPLT remained high (121±72 IU/ml and 3.5±4.6 pg/ml, respectively) such as F1+2 and DD (3.5±3.8 nmol/L and 197±55 μg/L, respectively) in the HU- and IFN-α-treated patients. PF4 and VEGFPLT normalised (7.4±3 IU/ml and 0.9±0.5 pg/ml, respectively) in the ANA-treated patients such as F1+2 and DD (1.1±0.4 nmol/L and 98±62 μg/L, respectively). These measurements were repeated in another sample collected after 1 o 2 months and showed concordance for PF4, VEGFPLT, F1+2 and DD concentrations. A positive correlation we found between PF4 and F1+2 and DD (p=0.023 and p=0.019, respectively) and between VEGF and F1+2 (p=0.020, respectively). Interestingly, these data might suggest that ANA has antiangiogenic property and hence antiplatelet and anticoagulant effects.

Human Platelet Factor 4 and Its C-Terminal Peptides: Heparin Binding and Clearance from the Circulation

Human platelet factor 4 (PF4), a high affinity heparin binding protein, is released from stimulated platelets and stored at vascular sites, predominantly in liver, from where it can be brought back into circulation by heparin. We attempted to define structural requirements for PF4 binding to heparin and for the pattern of its clearance from the circulation. Intact PF4 bound strongly to heparin agarose and was eluted at 1.4 M NaCl, while reduced PF4 and PF4 C-terminal peptides PF4 (47-70) and PF4 (58-70) bound weakly and were eluted at 0.2-0.5 M NaCl. 125I-radiolabeled intact PF4, reduced PF4 and C-terminal PF4 peptides injected into rabbits were cleared from the circulation in a biphasic pattern with components having half-life time of 1-2 min and 20-140 min. Heparin eliminated the fast component of PF4 clearance, but it did not affect clearance of reduced PF4 or C-terminal PF4 peptides. In contrast to reduced PF4 and PF4 (47-70), intact PF4 that accumulated in the liver and spleen, was displaced by heparin into circulating blood. In conclusion, specific binding sites and native conformation of the molecule are critical for high affinity PF4 binding to insolubilized heparin and for a pattern of PF4 clearance from the circulation in the presence of heparin.

Human platelet factor 4 and protamine sulphate interaction with glycosaminoglycans in the rabbit.

We studied the action of protamine sulphate on human platelet factor 4 (PF4) kinetics in rabbits in the presence of various glycosaminoglycans (CAGs). The animals pretreated with heparin showed high initial PF4 levels with a subsequent slow monoexponential clearance. The PF4 kinetics, in the presence of heparan and dermatan sulphate, reflected the different affinities that PF4 has for these GAGs. Protamine, at dosages that totally neutralized 1000 USP units of heparin pretreatment, caused an immediate disappearance in the circulating PF4. However, a second heparin injection 10 min after protamine, induced a PF4 peak release. It is possible that protamine displaced the PF4 from the binding sites of heparin, releasing it for storage in the body 'pool', from where it can be harvested again. The action of protamine on PF4 kinetics in the rabbits pretreated with 30 mg heparan sulphate was similar to that obtained with heparin pretreatment; however, a higher dose of protamine was required to obtain the optimal effect. After a 20 mg dose of protamine, unexpectedly, a bolus of heparin did not produce any peak release of PF4 in the rabbits pretreated with 30 mg of dermatan sulphate. Although part of the protamine will displace PF4 from the binding sites of the dermatan sulphate molecule, the remaining part of protamine could probably be bound to this GAG without losing its activity so that, upon subsequent heparin injection, it is immediately neutralized, rendering it unavailable for further PF4 harvesting.