Presence Of Platelet Factor 4 Research Articles

Aggregation of Enveloped Viruses By Platelet Factor 4 (PF4) and Neutrophil Extracellular Traps (NETs): Implications for the Anti-Viral Properties of NETs

As part of the innate immune response, neutrophils release neutrophil extracellular traps (NETs), webs of DNA coated with histones that ensnare and kill bacteria. A growing body of literature suggests that NETs can also capture and inactivate enveloped viruses. The ability of NETs to limit the systemic spread of pathogens occurs at the expense of collateral organ damage induced when high concentrations of NETs are digested by DNases to release toxic NET degradation products (NDPs) including cell free DNA and histones. Our group has shown that the positively charged platelet-specific chemokine (PF4, CXCL4), compacts NETs by forming tetramers that electrostatically cross-bind two polyanionic fragments of DNA, similarly to the way it aggregates heparans. PF4 complexed to NETs induces NET resistance to nuclease digestion, preventing the release of toxic NDPs while enhancing NET-bacterial capture by bridging anionic DNA to the negatively charged microbial surface. PF4 has also been shown limit pulmonary injury and enhance survival in mice infected with PR8, a mouse-adapted H1N1 influenza virus. Our group and others have found that both PF4 and NETs are elevated in the setting of SARS-CoV-2, leading us to hypothesize that they may act synergistically to combat the proliferation of enveloped viruses. To investigate the effect of PF4 on the pathogenicity of enveloped viruses, we studied PR8 and murine hepatitis virus (MHV)-1, a betacoronavirus that induces severe pulmonary disease. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) were used to assess the ability of PF4 and human-derived NETs to aggregate MHV-1 and PR8 virions. ChAdOx1, a replication-deficient adenovirus was used as a non-enveloped viral control. The DLS and SEM studies showed that PF4 (5-20 µg/ml) aggregated MHV-1 and PR8, forming complexes with an average diameter of 500nm, the width of 4-5 virions (Figure 1). In contrast, PF4 did not aggregate non-enveloped adenovirus. DLS studies showed that PF4 does not form complexes with coronavirus spike protein. However, the anti-PF4-polyanion antibody KKO, bound to PF4-viral complexes, leading to an additional 50% increase in viral aggregate size, an effect not seen with an isotype control antibody TRA (Figure A). These results suggest that PF4 binds to polyanions on the viral envelop via electrostatic interaction, much in the same way that it binds to heparans and DNA. We preformed subsequent DLS studies in which MHV-1 virions were incubated with both intact and digested NETs, and viral-NET aggregation was only observed in the presence of PF4. To determine if PF4 aggregates virus in vivo, IHC studies were performed on lungs harvested from MHV-1-infected A/J mice inoculated intranasally with 5 x 103 PFU of virus and sacrificed at 48h. The lungs were prepared for immunohistochemistry (IHC), and labeled with antibodies directed against PF4 and the MHV-1 nucleocapsid protein. We calculated the average Pearson's coefficient in 4 randomly selected 20x confocal images of the lungs of MHV-1 infected animals (n=4 mice, 16 images analyzed) using the FIJI JaCoP colocalization plugin. We observed co-localization of PF4 and MHV-1 within the cytoplasm of a subset of cells (r= 0.70225, p<0.0001), suggesting that PF4-MHV-1 aggregation occurs in vivo (Figure B) Lastly, the plaque forming unit assay was used to measure the effect of PF4 on MHV-1 infectivity of L2 cells. We found that incubating MHV-1 with PF4 (0-100µg/ml), leads to a 50% decrease in infectivity(n=5, p=0.023). Our studies demonstrate that PF4 binds to and aggregates MHV-1 and PR8 in vitro likely through electrostatic interactions with the viral envelop rather than adhesion to the viral spike protein. These results also suggest that PF4-mediated aggregation enhances viral capture by NETs to form NETs:PF4:viral complexes that may limit viral spread. IHC studies of lungs from MHV-1 infected mice demonstrate that PF4 colocalizes with the virus, suggesting that aggregation also occurs in vivo. In ongoing studies, we are investigating whether PF4 and NETs act synergistically to reduce coronavirus and influenza infectivity and enhance immune recognition and subsequent clearance of viral aggregates. These findings will help to define the anti-viral properties of NETs and may generate insights relevant to the treatment of coronavirus, influenza, and other enveloped viruses. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia

AbstractSARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Frequency of positive anti-PF4/polyanion antibody tests after COVID-19 vaccination with ChAdOx1 nCoV-19 and BNT162b2.

Vaccination using the adenoviral vector COVID-19 vaccine ChAdOx1 nCoV-19 (AstraZeneca) has been associated with rare vaccine-induced immune thrombotic thrombocytopenia (VITT). Affected patients test strongly positive in platelet factor 4 (PF4)/polyanion enzyme immunoassays (EIAs), and serum-induced platelet activation is maximal in the presence of PF4. We determined the frequency of anti-PF4/polyanion antibodies in healthy vaccinees and assessed whether PF4/polyanion EIA+ sera exhibit platelet-activating properties after vaccination with ChAdOx1 nCoV-19 (n = 138) or BNT162b2 (BioNTech/Pfizer; n = 143). In total, 19 of 281 participants tested positive for anti-PF4/polyanion antibodies postvaccination (All: 6.8% [95% confidence interval (CI), 4.4-10.3]; BNT162b2: 5.6% [95% CI, 2.9-10.7]; ChAdOx1 nCoV-19: 8.0% [95% CI, 4.5% to 13.7%]). Optical densities were mostly low (between 0.5 and 1.0 units; reference range, <0.50), and none of the PF4/polyanion EIA+ samples induced platelet activation in the presence of PF4. We conclude that positive PF4/polyanion EIAs can occur after severe acute respiratory syndrome coronavirus 2 vaccination with both messenger RNA- and adenoviral vector-based vaccines, but many of these antibodies likely have minor (if any) clinical relevance. Accordingly, low-titer positive PF4/polyanion EIA results should be interpreted with caution when screening asymptomatic individuals after vaccination against COVID-19. Pathogenic platelet-activating antibodies that cause VITT do not occur commonly following vaccination.

Decontamination of rough implant surfaces colonized by multispecies oral biofilm by application of leukocyte- and platelet-rich fibrin.

Decontamination of biofilm-infected rough implant surfaces is challenging. Platelet rich blood products have been shown to have anti-microbial properties against periodontal pathogens. Our aim was to investigate the effect of a potential biological implant surface disinfectant, leukocyte- and platelet-rich fibrin (L-PRF), on a mature oral multispecies biofilm on a rough titanium surface. Sandblasted, large grit, acid-etched (SLA) titanium disks were inoculated with subgingival dental plaque and cultured anaerobically for 21 days. The L-PRF membranes were collected from 12 donors in three trials (four donors in each trial). The disks were rinsed with 0.9% NaCl and exposed to the cell-rich portion of the L-PRF membranes for 48hours followed by scanning electron microscope (SEM) analysis immediately or after rinsing with 0.9% NaCl prior to fixation. The presence of platelet factor-4 in the rinse samples was analyzed by Western blotting. Remaining bacteria were quantified from SEM images of the implant surfaces and their numbers statistically compared. The L-PRF-treated samples without rinsing displayed numerous cells with multiple pseudopodia in immediate contact with bacteria that appeared perforated and increased in size. The cells were identified as platelets based on morphological criteria and by positive reaction for platelet factor-4 by Western blotting. After post-treatment rinsing, the L-PRF-treated disks displayed a significant reduction in bacterial counts (in average 92% reduction). Application of L-PRF significantly reduced bacterial counts on contaminated SLA titanium surface, most likely through anti-microbial action by platelets.

Cellular immune responses to platelet factor 4 and heparin complexes in patients with heparin‐induced thrombocytopenia

Background Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin characterized by thrombocytopenia and thrombotic complications. HIT is caused by pathogenic antibodies that bind to complexes of platelet factor 4 (PF4) and heparin, leading to platelet activation and inducing a hypercoagulable state. Previous studies have shown immunity to PF4-heparin complexes occurs early in life, even before heparin exposure; however, the immunogenesis of HIT is not well characterized. Objectives To investigate cellular proliferation in response to PF4-heparin complexes in patients with HIT. Patients/Methods Peripheral blood mononuclear cells (PBMCs) from healthy controls (n = 30), postoperative cardiac surgery patients who had undergone cardiopulmonary bypass (CPB) (n = 17) and patients with confirmed HIT (n = 41) were cultured with PF4 and PF4-heparin complexes. Cellular proliferation was assessed by [3 H]thymidine uptake and 5-ethynyl-2'-deoxyuridine detection. Results and Conclusions PBMCs proliferated in the presence of PF4, and this was enhanced by the addition of heparin in all study groups. CPB and HIT patients showed significantly greater proliferative responses than healthy controls. PBMC proliferation was antigen-specific, depended on the presence of platelets, and only CD14+ cells were identified as proliferating cells. Culture supernatants were tested for the levels of regulatory cytokines, and both CPB and HIT patients produced significantly lower levels of interleukin-10 and transforming growth factor-β1 than healthy controls. These findings further demonstrate cellular immune sensitization to PF4-heparin complexes occurs before heparin exposure, and suggests immune dysregulation can contribute to HIT.

Platelets kill bacteria by bridging innate and adaptive immunity via platelet factor 4 and FcγRIIA

Background Activated platelets release the chemokine platelet factor 4 (PF4) stored in their granules. PF4 binds to polyanions (P) on bacteria, undergoes a conformational change and exposes neoepitopes. These neoepitopes induce production of anti-PF4/P antibodies. As PF4 binds to a variety of bacteria, anti-PF4/P IgG can bind and opsonize several bacterial species. Objective Here we investigated whether platelets are able to kill bacteria directly after recognizing anti-PF4/P IgG opsonized bacteria in the presence of PF4 via their FcγRIIA. Methods Using platelet-bacteria suspension co-culture experiments and micropatterns with immobilized viable bacteria, in combination with pharmacological inhibitors and human anti- PF4/P IgG we analyzed the role of platelet-mediated killing of bacteria. Results In the presence of PF4, human anti-PF4/P IgG and platelets, E. coli killing (> 50%) with colony forming units (CFU mL-1 ) 0.71 × 104 ± 0.19 was observed compared with controls incubated only with anti-PF4/P IgG (CFU mL-1 3.4 × 104 ± 0.38). Blocking of platelet FcγRIIA using mAb IV.3 (CFU mL-1 2.5 × 104 ± 0.45), or integrin αIIbβ3 (CFU mL-1 2.26 × 104 ± 0.31), or disruption of cytoskeletal functions (CFU mL-1 2.7 × 104 ± 0.4) markedly reduced E. coli killing by this mechanism. Our observation of E. coli killing by platelets on micropatterned arrays is compatible with the model that platelets kill bacteria by covering them, actively concentrating them into the area under their granulomere and then releasing antimicrobial substances of platelet α-granules site directed towards bacteria. Conclusion These findings collectively indicate that by bridging of innate and adaptive immune mechanisms, platelets and anti-PF4/polyanion antibodies cooperate in an antibacterial host response.

PF4-HIT antibody (KKO) complexes activate broad innate immune and inflammatory responses

IntroductionHeparin-induced thrombocytopenia (HIT) is an immune-mediated complication of heparin anticoagulation therapy resulting in thrombocytopenia frequently accompanied by thrombosis. Current evidence suggests that HIT is associated with antibodies developed in response to multi-molecular complexes formed by platelet factor 4 (PF4) bound to heparin or cell surface glycosaminoglycans. These antibody complexes activate platelets and monocytes typically through FcγRIIA receptors increasing the production of PF4, inflammatory mediators, tissue factor and thrombin. The influence of underlying events in HIT including complex-induced pro-inflammatory cell activation and structural determinants leading to local inflammatory responses are not fully understood. MethodsThe stoichiometry and complex component requirements were determined by incubating fresh peripheral blood mononuclear cells (PBMC) with different concentrations of unfractionated heparin (H), low molecular weight heparin (LMWH), PF4- and anti-PF4-H complex antibodies (KKO). Cytokine mRNA or protein were measured by qRT-PCR or Meso Scale Discovery technology, respectively. Gene expression profile analysis for 594 genes was performed using Nanostring technology and analyzed using Ingenuity Pathway Analysis software. Results and conclusionsThe data show that antibodies magnify immune responses induced in PBMCs by PF4 alone or in complex with heparin or LMWH. We propose that following induction of HIT antibodies by heparin-PF4 complexes, binding of the antibodies to PF4 is sufficient to induce a local pro-inflammatory response which may play a role in the progression of HIT. In vitro assays using PBMCs may be useful in characterizing local inflammatory and innate immune responses induced by HIT antibodies in the presence of PF4 and different sources of heparins. FDA disclaimerThe findings and conclusions in this article are solely the responsibility of the authors and are not being formally disseminated by the Food and Drug Administration. Thus, they should not be construed to represent any Agency determination or policy.

Membrane Remodeling By Pathogenic Antibodies Underlies Monocyte Activation in Heparin-Induced Thrombocytopenia

Heparin induced thrombocytopenia (HIT) is an iatrogenic antibody-mediated disorder with a paradoxically high propensity for thrombosis. We have shown previously that human HIT IgGs and the HIT-like monoclonal antibody (MAb) KKO bind to platelet factor 4 (PF4) complexed with glycosaminoglycans (GAGs) on the surface of platelets and monocytes, initiating cell activation in vitro, thrombocytopenia in a transgenic mouse model, and thrombus formation in a laser microvascular injury model in vivo even in the absence of exogenous heparin. Monocytes bind PF4 and HIT Ab more readily than platelets because they express higher affinity GAGs, heparan sulfate and dermatan sulfate, in addition to chondroitin sulfate found on both cell types. To study changes in the structure of the monocytes that accompany HIT, we used scanning electron microscopy, confocal microscopy and flow cytometry to characterize the morphology and function of isolated human monocytes and mouse transgenic Fcg receptor IIA positive (FcγRIIA+) or wt (FcγRIIA-) monocytes in the absence or presence of platelets. We show by scanning electron microscopy that upon binding of pathogenic HIT Abs to PF4/GAG complexes on FcgRIIA expressing monocytes, they initiate profound remodeling of the cell membrane. Addition of 100 μg/ml recombinant human PF4 in the absence of HIT Abs initiates the activation process with the appearance of 177 ± 53 nm "knobs" on the surface of 70% of monocytes. Subsequent addition of the HIT-like monoclonal antibody KKO at 50 μg/ml dramatically alters the cellular surface with the appearance of large 701 ± 208 nm membrane "blebs" that were not seen on FcγRIIA-mouse monocytes. These large, membrane-associated structures likely engage FcγRIIA, clustering them in proximity to cell-bound immune complexes, which promotes cell activation that leads to thrombosis. These blebs increase in size over time and are then shed from the cells as monocyte-derived microparticles, which self-aggregate. As a result of shedding of these blebs, the monocytes lose much of their typical ruffled surface (only 67% of monocytes maintain ruffles in the presence of PF4 plus KKO, compared to 97% of control monocytes) and appear smoother, sometimes with pores indicating degranulation. In the presence of platelets, monocytes exposed to PF4 and KKO formed heterocellular aggregates in addition to these subcellular changes. In contrast to KKO, addition of the non-pathogenic MAb RTO not only did not induce blebbing, but largely inhibited PF4-induced changes in the monocyte surface. This suggests that RTO might prevent monocyte activation by interfering with PF4 tetramerization. Structural analysis of the shed microparticles by microscopy revealed that they had an average diameter of 356 ± 307 nm, with many larger particles and aggregates. Flow cytometry confirmed that the shed particles contain cell membrane lipids and receptors. Confocal microscopy showed uniform binding of labeled PF4 to the monocyte cell membrane followed by rapid clustering into large complexes after the addition of KKO, but not RTO. These studies affirm the centrality of cell surface PF4/GAG complexes in the pathogenesis of HIT and provide quantitative morphometric characteristics of the changes in the monocyte membrane structure. We propose that PF4 released from activated platelets binds to the surface of GAG-expressing monocytes in vivo, forming clusters of PF4/GAG complexes that likely promote antibody binding and cause monocyte activation through FcγRIIA along with large-scale remodeling of the cell membrane and shedding of procoagulant microparticles. DisclosuresNo relevant conflicts of interest to declare.

Imaging Morphologic Changes On Platelet and Monocyte Surfaces In Heparin-Induced Thrombocytopenia (HIT)

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). Unlike most other immune thrombocytopenias, HIT is markedly prothrombotic. We have proposed that this prothrombotic tendency is due to binding of pathogenic antibodies to PF4 complexes attached to the surface GAGs expressed by all intravascular cells, including platelets with their relatively low affinity surface GAGs, chondroitin sulfates, and monocytes with their higher affinity membrane GAGs, heparan and dermatan sulfates. Using isolated monocytes from healthy volunteers, we show by scanning electron microscopy that the addition of 10-50 µg/ml of recombinant human PF4 causes the appearance of ∼200 nm “knobs” on the cell surface. Subsequent addition of a HIT-like monoclonal antibody KKO at 50 µg/ml to the PF4-coated cells markedly alters their surface with the appearance of larger, up to 1-2 µm, membrane “blebs”. These blebs increase in size over time (15-60 minutes) and are shed from the cells. After shedding of these blebs, the monocytes lose their typical ruffled surface and appear spherical. These surface changes in the presence of KKO and PF4 are not seen in the presence of PF4 and 50 µg/ml of the anti-PF4 monoclonal antibody RTO, which does not induce the prothrombotic state of HIT. Platelets in suspension exposed to PF4 and KKO show by scanning electron microscopy similar knobs on their surface, but only minimally form blebs or microvesiculate. Platelets spread on fibrinogen in culture medium stimulated with PF4 and KKO and observed by hopping probe ion conductance microscopy progressively developed surface protrusions over a period of an hour, becoming more spherical. This morphological change was also observed in platelets exposed to IgG purified from 5 patients with HIT, but not when the KKO Fab fragment was tested. Neither PF4 alone nor with RTO antibody induced this morphological transition. Exposure to KKO plus PF4 for an hour induced minimal microvesiculation of platelets as measured by flow cytometry. Platelets adherent to fibrinogen underwent a similar morphological transition and did not microvesiculate after adding 50 mM of a thrombin-receptor activating peptide, whereas ADP-stimulated platelets rapidly microvesiculated during the same period of time. We believe that the “knobs” observed on monocytes and platelets represent aggregates of PF4-GAG complexes that are the targets of HIT antibodies. Bleb formation on monocytes and morphological transition of platelets result from clustering of knobs caused by bivalent HIT antibodies which cross-link Fc receptors. These blebs are released from monocytes, potentially becoming the microparticles found in the plasma of patients with HIT. In contrast, platelets treated with KKO plus PF4 showed minimal microvesiculation. This finding differs from reported high platelet microparticle counts in the plasma of HIT patients, suggesting that additional factors may be required to induce platelets to microvesiculate. Our images represent the first visualization of surface events when platelets and monocytes assume an active prothrombotic state. Whether these are unique to HIT or have wider applicability to the changes that occur in other prothrombotic, proinflammatory states needs to be addressed. Disclosures:No relevant conflicts of interest to declare.

Emphasis on the Role of PF4 in the Incidence, Pathophysiology and Treatment of Heparin Induced Thrombocytopenia

Heparin Induced Thrombocytopenia (HIT) is caused by antibodies that recognize platelet factor 4 (PF4) associated with polyanionic glycosaminoglycan drugs or displayed on vascular cell membranes. These antibodies are elicited by multimolecular complexes that can occur when heparin is administered in clinical settings associated with abundant PF4. Heparin binding alters native PF4 and elicits immune recognition and response. While the presence of heparin is integral to immunogenesis, the HIT antibody binding site is within PF4. Thus HIT antibodies develop and function to cause thrombocytopenia and/or thrombosis only in the presence of PF4. Future emphasis on understanding the biology, turnover and regulation of PF4 may lead to insights into the prevention and treatment of HIT.

Histones, Like Platelet Factor 4 (PF4), Affect Generation of Activated Protein C: Implications for the Pathogenesis of Severe Sepsis

Abstract 530Platelet factor 4 (PF4) increases aPC generation by the thrombin (IIa)/thrombomodulin (TM) complex and may impact outcome in sepsis. PF4’s effect on aPC generation follows a biphasic curve when tested in solution, on human TM expressing HEK293, and on primary endothelial cells (ECs) with a peak concentration at around 25 μg/ml. Formation of complexes at a specific molar ratio between positively-charged tetramers of PF4 and negatively-charged chondroitin sulfate (CS) on the TM glycosaminoglycan (GAG) is crucial for the increase in aPC generation. Other positively-charged molecules like protamine sulfate (PRT) affect aPC generation in a similar manner, and heparin, which is known to bind PF4 and PRT more avidly than CS, lowers effective PF4 or PRT concentrations. Here we examined whether histones, that are also small positively-charged molecules, affect aPC generation. Histones released from cells in sepsis are cytotoxic toward ECs and lethal when injected into mice, and aPC reverses this lethality. May histones affect aPC generation by the same mechanism as other positively-charged molecules, and how does the presence of PF4 or heparin influence this effect? We have addressed these questions both in solution and with TM-expressing cells, in the absence or presence of endothelial protein C receptor. We found that individual, or mixed histones affect aPC formation following a similar biphasic curve seen with PF4 with a peak effect at around 10 μg/ml but to lesser extent (2-fold maximal increase compared to 6-fold for PF4). Histones and PF4 are additive at low concentrations; however, more importantly, histones only decreased aPC generation when tested in the presence of optimal or higher PF4 concentration (>25 μg/ml). Just as with PF4, added heparin decreased effective histone concentration and shifted the curve for aPC generation to the right, both in the absence or presence of PF4. We hypothesize that normally PF4 released from platelets augments aPC generation and low concentration of histones have similar effect. But when histones are released in sepsis in high concentrations, their interaction with CS on TM blocks formation of complexes between PF4 and TM’s CS that are optimal for maximal increase of aPC generation. Further we tested the effect of histones on aPC generation in vivo. Injection of histones in mice increased IIa-induced (2U/kg) aPC generation in plasma. This increase was concentration dependent (at 1 to 20 mg/kg increasing aPC generation up to 10-fold), but injection of higher amount of histones (40 mg/kg) became lethal. Mice that were overexpressing human PF4 had an increased lethality when histones at 40 mg/kg were co-injected with thrombin (2U/kg) over the littermate mice deficient in murine PF4 (60% vs. 0% mortality, respectively, n=5 for each group) suggesting that in vivo histones may also act additively with PF4 on aPC generation. We propose that in severe septic patients, especially those with high levels of released PF4, concurrently available histones suppress aPC generation. By binding to the excess of PF4 and/or histones, heparin may be beneficial in severe sepsis by allowing improved aPC generation. Disclosures:No relevant conflicts of interest to declare.

Antibodies associated with heparin-induced thrombocytopenia (HIT) inhibit activated protein C generation: new insights into the prothrombotic nature of HIT

AbstractHeparin-induced thrombocytopenia (HIT) is caused by antibodies that recognize complexes between platelet factor 4 (PF4) and heparin or glycosaminoglycan side chains. These antibodies can lead to a limb- and life-threatening prothrombotic state. We now show that HIT antibodies are able to inhibit generation of activated protein C (aPC) by thrombin/thrombomodulin (IIa/TM) in the presence of PF4. Tetrameric PF4 potentiates aPC generation by formation of complexes with chondroitin sulfate (CS) on TM. Formation of these complexes occurs at a specific molar ratio of PF4 to glycosaminoglycan. This observation and the finding that the effect of heparin on aPC generation depends on the concentration of PF4 suggest similarity between PF4/CS complexes and those that bind HIT antibodies. HIT antibodies reduced the ability of PF4 to augment aPC formation. Cationic protamine sulfate, which forms similar complexes with heparin, also enhanced aPC generation, but its activity was not blocked by HIT antibodies. Our studies provide evidence that complexes formed between PF4 and TM's CS may play a physiologic role in potentiating aPC generation. Recognition of these complexes by HIT antibodies reverses the PF4-dependent enhancement in aPC generation and may contribute to the prothrombotic nature of HIT.

Platelet Factor 4 Inhibits Thrombomodulin-dependent Activation of Thrombin-activatable Fibrinolysis Inhibitor (TAFI) by Thrombin

Thrombomodulin (TM) is a cofactor for thrombin-mediated activation of protein C and thrombin-activatable fibrinolysis inhibitor (TAFI) and thereby helps coordinate coagulation, anticoagulation, fibrinolysis, and inflammation. Platelet factor 4 (PF4), a platelet α-granule protein and a soluble cofactor for TM-dependent protein C activation, stimulates protein C activation in vitro and in vivo. In contrast to stimulation of protein C activation, PF4 is shown here to inhibit activation of TAFI by thrombin-TM. Consequences of inhibition of TAFI activation by PF4 included loss of TM-dependent prolongation of clot lysis times in hemophilia A plasma and loss of TM-stimulated conversion of bradykinin (BK) to des-Arg(9)-BK by TAFIa in normal plasma. Thus, PF4 modulates the substrate specificity of the thrombin-TM complex by selectively enhancing protein C activation while inhibiting TAFI activation, thereby preventing the generation of the antifibrinolytic and anti-inflammatory activities of TAFIa. To block the inhibitory effects of PF4 on TAFI activation, heparin derivatives were tested for their ability to retain high affinity binding to PF4 despite having greatly diminished anticoagulant activity. N-acetylated heparin (NAc-Hep) lacked detectable anticoagulant activity in activated partial thromboplastin time clotting assays but retained high affinity binding to PF4 and effectively reversed PF4 binding to immobilized TM. NAc-Hep permitted BK conversion to des-Arg(9)-BK by TAFIa in the presence of PF4. In a clot lysis assay on TM-expressing cells using hemophilia A plasma, NAc-Hep prevented PF4-mediated inhibition of TAFI activation and the antifibrinolytic functions of TAFIa. Accordingly, NAc-Hep or similar heparin derivatives might provide therapeutic benefits by diminishing bleeding complications in hemophilia A via restoration of TAFIa-mediated protection of clots against premature lysis.

Heparin-Induced Thrombocytopenia Antibodies Inhibit PF4-Dependent Enhancement of Activated Protein C Formation by Binding to Antigenic Complexes Formed with the Chondroitin Sulfate Side-Chain of Thrombomodulin

AbstractAbstract 721Previous studies have shown that platelet factor 4 (PF4) increases activated protein C (aPC) generation both in vitro and in vivo. PF4 increase of aPC generation by thrombin (IIa) and thrombomodulin (TM) complex followed a bell-shaped curve when tested in solution, on human TM expressing HEK293 and on endothelial cells. PF4 failed to enhance aPC in the presence of chondroitin sulfate (CS)-free TM. These results were consistent with PF4 binding to the CS on the TM glycosaminoglycan (GAG) domain and forming complexes that are similar to PF4/GAG antigenic complexes seen in heparin-induced thrombocytopenia (HIT). We tested the hypothesis that PF4 forms a HIT-like antigenic complex with the TM-CS using the HIT-like monoclonal antibody KKO. KKO abolished the potentiating effects of PF4 on aPC formation measured with TM in solution or with a TM-expressing cell line. To further address the nature of complexes formed between PF4 and TM, we used a mutant of PF4, PF4T38Q, which forms complexes with GAGs that are not recognized by KKO and a subgroup of HIT antibodies. Similar to PF4, PF4T38Q potentiated TM-dependent aPC generation in a bell-shaped manner, but this potentiation was not blocked by KKO. Moreover, KKO did not have any effect when PF4 was replaced with protamine sulfate (PS), which can also form macromolecular complexes with heparin/GAGs and can also enhance aPC generation. We also tested HIT antibodies isolated from patients that developed HIT with thrombocytopenia and thromboembolism developing >4 days after the last exposure to heparin. Patient IgGs specific for PF4/GAG complex were purified using PF4 bound to heparin columns. Specific binding of antibodies to PF4/heparin complexes was checked by ELISA. Complex-specific antibodies were then tested in an aPC generation assay in the presence IIa and TM and near peak concentration of PF4 and compared to a control human IgG. Three of four patient's antibodies significantly inhibited the increase in aPC generation in the presence of PF4. These studies provide evidence that HIT-like PF4/GAG complexes develop naturally in vivo. In this case, the ability of HIT or HIT-like antibodies to specifically inhibit the PF4-dependent increase in aPC formation may contribute to the prothrombotic state in HIT. Disclosures:No relevant conflicts of interest to declare.

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.

Monocytes Are a Particularly Favorable Target for Surface Platelet Factor 4 (PF4) Antigenic Complex Formation in Heparin-Induced Thrombocytopenia: New Insights into the Thrombotic Risk in HIT

Monocytes are a Favored Target for Surface Platelet Factor 4 (PF4) Antigenic Complex Formation in Heparin-Induced Thrombocytopenia: New Insights into the Thrombotic Risk in HIT Lubica Rauova, Gowthami Arepally, Douglas Cines and Mortimer Poncz HIT is a drug-induced autoimmune thrombocytopenia caused by antibodies to heparin/PF4 complexes that predispose to thrombotic complications. The studies described below examine how monocytes (Mo) may contribute to the thrombotic risk. We demonstrated previously that glycosaminoglycans (GAG) on the surface of platelets bind PF4, forming complexes that are recognized by HIT antibody, leading to platelet activation via the platelet FcγRIIA receptor in vitro and thrombocytopenia/thrombosis in vivo. However, heparin not only induces antibodies to develop against the PF4/GAG surface antigenic complexes, but also rapidly removes the same PF4/GAG complexes from the platelet surface, which may limit the likelihood of developing HIT and help limit its duration. This led us to study the involvement of Mo, which are a rich potential source of tissue factor and are known to be activated in HIT. Moreover, unlike platelets, which are coated with GAG composed almost entirely of chrondroitin sulfate (CS), Mo also express heparan sulfate, which has the capacity to bind PF4 with greater avidity and be resistant to the effect of plasma heparin. We found that Mo bind PF4 with greater avidity than platelets and higher concentrations of UFH are needed to remove PF4/GAG complexes and reduce the binding of a HIT monoclonal antibody KKO. In contrast to platelets, dissociation of PF4/GAG complexes from monocytes requires heparinases in addition to chondroitinases. In addition, macrophages GAG undergo hypersulfation during inflammation. Because clinical studies have shown inflammation predisposes to HIT, we examined the binding of KKO to unstimulated and bacterial lipopolysaccharide (LPS, E. coli serotype 011) stimulated cultured macrophages. Macrophages were derived from primary human Mo or murine bone marrow, cultured in the presence of M-CSF and stimulated with 0–500 ng/mL of LPS for 72 hrs. LPS increased KKO binding in the presence of PF4 2.7±0.7-fold compared to unstimulated cells (p<0.002) and the stimulated cells required ~2-fold higher concentrations of heparin to remove surface PF4/GAG complexes. Addition of [35S]sulfate during the last 24 hrs of incubation lead to a 4.1±0.1-fold increase in the incorporation of 35S into surface GAG after LPS stimulation (p<0.0001). These results provide important insights into the potential role of Mo in the prothrombotic sequelae of HIT. Compared to platelets, Mo are relatively resistant to “antigen down-regulation” by heparin and are more likely to bind anti-PF4/GAG HIT antibodies and become activated. The relative resistance of Mo to the dissociation PF4/GAG complexes from the cell surface also suggest a role in the development of Delayed-Onset HIT after heparin withdrawal.

Dual Effect of Platelet Factor Four on the Activities of Factor Xa

Platelet Factor 4 (PF4) is a cationic molecule that binds to heparin with high affinity and neutralises the activity of the latter. Our recent studies indicate that heparin can promote an interaction of fXa with PF4 since neutralization of heparin activity by PF4 was dependent on the concentration of protease. To examine the contribution of PF4 in protease function, fXa activity was determined in chromogenic assays. Upon preincubation with fXa and heparin, PF4 (at a concentration of 100 nM) decreased the kcat of S2765 peptide hydrolysis 4-fold and that of prothrombin activation about 2-fold. These results suggested an effect of PF4 on the primary specificity of the protease. In fact, PF4 exerted a mild effect (30 % decrease) on the Na+ dependence of fXa, consistent with linkage between Na+ and S1. PF4 preincubation with fXa also prevented the binding of the S1 probe p-aminobenzamidine (pAB) while simultaneous addition of PF4 and pAB diminished the contribution of PF4. In the presence of excess fVa (relative to fXa), kinetic parameters measuring fXa amidolytic activity in the presence of PF4 were restored to control values in the absence of PF4. Interestingly, high concentrations of PF4 (> 1 μM) totally restored fXa activity toward peptidyl substrate and strongly enhanced prothrombin activation, indicating a dual effect of PF4 on fXa activities. The inhibitory contribution of PF4 during prothrombin activation was due to a three-fold decreased affinity of fXa for fVa while enhancement of prothrombin activation was accompanied by a three-fold increase in fVa-dependent cofactor activity. Thus, the effects of PF4 possibly involved a region of the heparin/fVabinding exosite that is linked to the S1 and Na+ sites. These findings suggest that PF4 is a probe of fVa-dependent changes occurring in the active site of fXa and provide an explanation for the in vivo paradoxical effects of PF4 reported in the literature.

Platelet Factor 4 Mediates Activated Protein C Resistance by Impairment of Protein S Cofactor Enhancement

Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.

Platelet Factor 4 and Interleukin-8 CXC Chemokine Heterodimer Formation Modulates Function at the Quaternary Structural Level

The apparent complexity of biology increases as more biomolecular interactions that mediate function become known. We have used NMR spectroscopy and molecular modeling to provide direct evidence that tetrameric platelet factor-4 (PF4) and dimeric interleukin-8 (IL8), two members of the CXC chemokine family, readily interact by exchanging subunits and forming heterodimers via extension of their antiparallel beta-sheet domains. We further demonstrate using functional assays that PF4/IL8 heterodimerization has a direct and significant consequence on the biological activity of both chemokines. Formation of heterodimers enhances the anti-proliferative effect of PF4 on endothelial cells in culture, as well as the IL8-induced migration of CXCR2 vector-transfected Baf3 cells. These results suggest that CXC chemokine biology, and perhaps cytokine biology in general, may be functionally modulated at the molecular level by formation of heterodimers. This concept, in turn, has implications for designing chemokine/cytokine variants with modified biological properties.

A Spliced Variant of CXCR3 (CXCR3B) Is Differentially Expressed on CD4+CD25+ Regulatory vs. CD4+CD25− Non-Regulatory T Cells Exposed to Platelet Factor 4 (PF4).

Heparin-induced thrombocytopenia (HIT), without or with thrombosis, is a serious complication of heparin therapy. Work done in several laboratories including ours has shown that patients with HIT almost invariably have antibodies specific for a platelet derived CXC chemokine, platelet factor 4 (PF4; CXCL4), modified by an external agent, heparin. In the past decade, much has been learned about the pathogenesis of HIT. However, knowledge of the molecular nature of the immune-response is far from complete. We have recently reported that human PF4 modulates, in an opposite manner, CD4+CD25+ regulatory vs. CD4+CD25− non-regulatory T cells and have proposed that a transient impairment of the T regulatory (Tr) cell suppressor activity might be postulated in otherwise healthy individuals, experiencing acute and severe platelet destruction, as it is the case in HIT, systemic lupus erythematosus (SLE), and autoimmune thrombocytopenic purpura (AITP), in which the trigger for the primary immune response is poorly understood. For many years identification of a receptor for the chemokine PF4 eluded discovery. Recently, Lasagni et al. have identified a spliced variant of CXCR3 (CXCR3B) to which PF4 binds with high affinity. While it is not known which CXCR3 isoform is expressed on CD4+CD25+ Tr cells, Lasagni and al. (J Exp Med. 2003; 197:1537–49) reported the intriguing observations that engagement of CXCR3A by their ligands (CXCL9, CXCL10, and CXCL11), is associated with a proliferative response, whereas an opposite effect, e.g. inhibition of cell proliferation, is seen for CXCR3B engagement by the same ligands. In addition, as already noted, PF4, that binds with high affinity to the CXCR3B variant only, has been reported to mediate inhibition of different cell types including hematopoietic progenitors, endothelial cells, and activated T cells. We assessed the relative abundance of CXCR3B (the receptor for PF4) and CXCR3A (not a receptor for PF4) in CD4+CD25+ Tr cells and CD4+CD25− T cells in the presence or the absence of PF4 at different time points. We used a TaqMan quantitative real time PCR (qRTPCR) to determine the mRNA transcription profiles of CXCR3B and A in CD4+CD25+ and CD4+CD25− T cells in the absence or the presence of PF4. mRNA levels were quantitated by comparing experimental levels to standard curves generated using serial dilutions of the same amount of the cloned plasmid DNA for human CXCR3B and A. In CD4+CD25+ Tr cells we found a significant increase of the cDNA copy number encoding CXCR3B (4.67x104 vs. 1.93x104 copies, respectively) in the presence of PF4, while the levels of expression were substantially unchanged for CXCR3A (4.74x104 vs. 4.91x104 copies, respectively). On the contrary, in CD4+CD25− T cells, under the same culture conditions in the presence or the absence of PF4, the expression levels were unchanged for CXCR3B (7.98x104 vs. 7.66x104, respectively) and only slightly increased for CXCR3A (8.77x104 vs. 1.02x105, respectively). Because commercially available antibodies cannot distinguish between the non-spliced (CXCR3A) and the B form of CXCR3, we developed rabbit IgG specific for selected peptides corresponding to the 51 N-terminus AA residues unique to CXCR3B. Using these antibodies we confirmed by flow cytometry that the surface expression of CXCR3B is enhanced on CD4+CD25+ Tr cells primed by PF4. Our data suggest that CXCR3B expression varies differentially according to both cell type and activation status.