The NET Effect: Platelet Factor 4 and DNA-Histone Interactions in Sepsis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2197-2197 ◽  
Author(s):  
Kandace Gollomp ◽  
Johnston Ian ◽  
Diarra Fatoumata ◽  
Guohua Zhao ◽  
Sriram Krishnaswamy ◽  
...  
Keyword(s):  
Nuclear Dna ◽  
Conflicts Of Interest ◽  
Calcium Ionophore ◽  
Platelet Factor 4 ◽  
Extracellular Dna ◽  
Microfluidic Channels ◽  
Chromatin Decondensation ◽  
Platelet Factor ◽  
Competitive Binding Assay ◽  
Positively Charged

Abstract In response to infection and inflammation, neutrophils release neutrophil extracellular traps (NETs), web like structures composed of nuclear DNA associated with histones that may have both beneficial and deleterious effects. The formation of NETs alters the course of late-stage sepsis and the associated release of histones has been shown to contribute to many of the observed pathologic complications of sepsis. Histones are octamers comprised of two copies of H2A, H2B, H3 and H4, each of which is highly positively charged. NET formation is dependent on chromatin decondensation mediated by the enzyme peptidylarginine deiminase 4 (PAD4). PAD4 potentiates chromatin decondensation by decreasing the overall positive charge of histones through citrullinating many of their lysines residues, forming Cit-histones, which have a decreased affinity for negatively charged DNA. Platelets also contribute to events in late sepsis by undergoing significant activation and degranulation. We propose that one way platelets may affect outcomes in late sepsis is through the release of large amounts of the highly positively charged chemokine platelet factor 4 (PF4, CXCL4). After its release, we believe that PF4 can displace histones and cit-histones from cell free DNA, altering the composition of NETs. We chose to investigate whether PF4 might liberate cit-histones from NET fibers more effectively than non-citrullinated histones. We initially sought to examine the effect of PF4 on histone attachment to DNA. In a competitive binding assay, we found that PF4 binds to DNA with greater affinity than histones. Of note, cit-histones were approximately 5 times more easily displaced from DNA than non-cit-histones consistent with a model of decreased DNA affinity of cit-histones. Furthermore, using immunofluorescence studies and confocal microscopy, we showed that when NETs are generated in the presence of platelets, endogenous PF4 adheres readily to NET DNA. We have also demonstrated that exogenous PF4 avidly binds to NETs generated from neutrophils isolated with minimal platelet contamination. Based on the results of these experiments, we decided to investigate the interaction between PF4, NETs and histones in a novel microfluidic system that is designed to mimic intravascular flow conditions. We isolated neutrophils from fresh whole blood samples obtained from healthy human donors and stimulated them with TNFα to promote adherence to fibronectin coated microfluidic channels. After the neutrophils had firmly bound to the channels, we exposed them to NET stimuli, including lipopolysaccharide (LPS) and calcium ionophore and visualized NET formation. Extracellular DNA was detected using the cell membrane impermeable dye, SYTOX Green. After NET formation occurred, PF4 was flowed through the channels at 25-100 μg/mL, concentrations similar to those observed in terminal sepsis. Exposure to PF4 at these concentrations lead to the dissolution of NET fibers. Interestingly, although the residual NET fibers continued to stain positive for non-cit-histones, they no longer stained positive for cit-histones. In conclusion, cit-histones are present in NETs and may contribute to the pathobiology of late sepsis. We propose that cit-histones are competitively displaced from NETs by PF4. This may be due to their decreased relative affinity for DNA binding. These studies provide new insights into how histones are released from NET fibers into the circulation during sepsis. This information sheds new light on the interaction of chemokines and NETs and may lead to the identification of new therapeutic strategies in the treatment of sepsis. Disclosures No relevant conflicts of interest to declare.

A Special Role for Neutrophil Extracellular Traps (NETs) and Neutrophils in the Prothombotic Nature of Heparin-Induced Thrombocytopenia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1023-1023
Author(s):  
Kandace Gollomp ◽  
Ian Johnston ◽  
Lubica Rauova ◽  
Douglas B. Cines ◽  
M. Anna Kowalska ◽  
...  
Keyword(s):  
Whole Blood ◽  
Nuclear Dna ◽  
Conflicts Of Interest ◽  
Extracellular Dna ◽  
Molar Ratio ◽  
Microfluidic Channels ◽  
Special Role ◽  
Healthy Human ◽  
Platelet Factor ◽  
Endonuclease Digestion

In response to infection and inflammation, neutrophils release NETs, histone-decorated nuclear DNA that ensnares bacteria, but also damages host tissues and promotes thrombosis. Platelet Factor 4 (PF4, CXCL4) is a CXC chemokine stored in high concentrations in platelet alpha-granules and released during platelet activation. Tetrameric PF4 has a very high affinity for heparans and other polyanionic molecules, including DNA. At the proper molar ratio, PF4 can form high molecular weight complexes with heparin and other heparans, and these complexes are antigenic targets for pathogenic HIT antibodies. In light of this information, we chose to investigate whether PF4 can interact with NETs and whether these complexes contribute to the prothrombotic nature of HIT. Using an ELISA assay, we confirm that PF4 binds to DNA to form HIT-like complexes just as it does with heparin following a similar bell-shaped curve of HIT antigenicity. Using immunofluorescence studies and confocal microscopy, we found that exogenous PF4 adheres readily to NET DNA. We then investigated PF4-NET interactions under intravascular flow conditions by using neutrophils isolated from healthy human donors to create NET-coated microfluidic channels through which we infused recombinant human PF4. We found that PF4 selectively adhered to extracellular NET fibers but did not bind to the surface of intact neutrophils. We next noted that PF4 infusion led to a change in NET morphology with compaction of the extracellular DNA to approximately 30% of the original area (p<0.001, N=10 per arm). KKO, a monoclonal anti-PF4-heparin antibody, bound readily to NETs following PF4 incubation, indicating that PF4-NET complexes are antigenic. Of note, KKO binding did not induce additional NET compaction. We then observed that while NETs were highly susceptible to endonuclease digestion prior to PF4 incubation, PF4-NET complexes developed resistance to endonuclease digestion. PF4-NET complex incubation with either KKO or HIT IgG isolated from patient samples further enhanced resistance to endonuclease digestion by >2-fold (p<0.001, N=2 studies, 5 NETS per study), while incubation with a polyclonal anti-PF4 antibody did not have this effect. We also show that neutrophils bind readily to endothelial cells when whole blood was flowed through microfluidic channels lined with TNFα-injured endothelium. The number of adherent neutrophils increased markedly when KKO was added to the whole blood, but no significant changes were observed when an isotype control antibody was included. These findings suggest that neutrophils may contribute to thrombosis in HIT through three sequential steps: (1) HIT-antibody activated neutrophils selectively bind to injured endothelium, increasing the numbers of localized neutrophils at sites of thrombus. (2) Subsequently released NETs are bound by PF4 and HIT antibodies, generating immunogenic complexes that are likely prothrombotic, and finally, (3) PF4 and HIT antibody binding induces resistance to endonuclease digestion leading to a prolongation of NET half-life and an increased opportunity to contribute to clot formation. We believe these data support a set of mechanisms by which neutrophils can contribute to the observed prothrombotic nature of HIT, including a novel NET stabilization process. Further in vivo mouse models will now be pursued to confirm these findings. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 530-530
Author(s):  
M. Anna Kowalska ◽  
Guohua Zhao ◽  
George David ◽  
Mortimer Poncz
Keyword(s):  
Severe Sepsis ◽  
Protein C ◽  
Conflicts Of Interest ◽  
Platelet Factor 4 ◽  
Endothelial Protein C Receptor ◽  
Maximal Increase ◽  
Platelet Factor ◽  
Positively Charged ◽  
Formation Of Complexes

Abstract Abstract 530 Platelet 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.

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

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 997-997
Author(s):  
Kandace Gollomp ◽  
Ian Johnston ◽  
Minna Kim ◽  
Li Zhai ◽  
Guohua Zhao ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Cell Damage ◽  
Platelet Factor 4 ◽  
Neutrophil Extracellular Trap ◽  
In Vivo Studies ◽  
Microfluidic Channels ◽  
Platelet Factor ◽  
Osmotic Pumps

Abstract When stimulated by infection or inflammation, neutrophils expel NETs, decondensed chromatin coated with histones and antimicrobial proteins that ensnares pathogens but also damages host tissue. Platelet factor 4 (PF4, CXCL4) is a CXC chemokine stored in platelet alpha-granules and released in high concentrations during platelet activation. Tetrameric PF4 has a very high affinity for polyanionic molecules, including DNA, and we have found that PF4 binds and physically compacts NETs, causing them to have increased resistance to endonuclease digestion. Our group has also observed that PF4 expression leads to enhanced survival in a murine model of sepsis. Based on these findings, we chose to investigate whether PF4-mediated NET compaction is protective in endotoxemia. To study PF4-NET interactions, we developed a microfluidic assay in which neutrophils were adhered to fibronectin-coated channels and then stimulated to release NETs with phorbol myristate acetate(PMA). NETs were visualized by staining with the fluorescent nucleic acid stain SYTOX. Changes in NET morphology and fluorescence were quantified in the presence of varying PF4 concentrations. DNase I was then infused through these channels and the extent of digestion was measured. These experiments showed that the presence of PF4 led to NET compaction and decreased NET degradation following DNase infusion. We then performed in vitro studies examining NET-endothelial interactions in which isolated neutrophils were stimulated to release NETs, incubated with buffer alone or buffer containing PF4, and flowed through human endothelial umbilical vein cell (HUVEC) lined microfluidic channels that had been stimulated with tumor necrosis factor (TNF) α. EC viability was assessed 24-hours post NET exposure and revealed that the presence of PF4 protected HUVECs from NET-induced damage. To further investigate PF4-NET interactions in endotoxemia, we conducted in vivo studies using PF4-deficient mice (mPF4-/-) and wildtype (WT) controls injected with lipopolysaccharide (LPS). Plasma NET markers [cell free DNA (cfDNA), citrullinated histones (cit-His), and myeloperoxidase (MPO)] were quantified via ELISA and Western blot at various time points following LPS injection. mPF4-/- mice were also implanted with PF4-containing osmotic pumps and the NET markers were also assessed following LPS exposure. These experiments revealed that compared to WT mice, LPS injected mPF4-/- mice had significantly higher plasma levels of NET components, including cfDNA, cit-His and MPO. When mPF4-/- mice were implanted with PF4-releasing osmotic pumps prior to LPS injection, they had plasma NET component levels comparable to those observed in WT mice. Based on the results of our in vitro and in vivo studies, we propose that PF4 infusion compacts NETs, decreasing their susceptibility to DNAse lysis, and preventing the release of toxic NET degradation products (NDPs) such as cfDNA and cit-His. We posit that PF4-mediated sequestration of NDPs prevents endothelial cell damage in the HUVEC-lined microfluidic model. We believe that the results of our studies in mPF4-/- mice demonstrate that PF4 has a similarly protective effect in vivo, decreasing NET lysis and reducing NDP generation. These findings suggest that in sepsis, the stabilization rather than the lysis of NETs may be therapeutic. Further investigation should be performed to determine if treatment with PF4 or other small positively-charged proteins such as protamine sulfate that can sequester NDPs, may be beneficial the treatment of sepsis. Disclosures No relevant conflicts of interest to declare.

A Proposed Role for Platelet Factor 4 in Histone Pathobiology in Sepsis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 98-98
Author(s):  
M. Anna Kowalska ◽  
Guohua Zhao ◽  
Ian Johnston ◽  
Elsa Treffeisen ◽  
Fatoumata Diarra ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Positive Charge ◽  
Conflicts Of Interest ◽  
Peak Effect ◽  
Platelet Factor 4 ◽  
Vascular Damage ◽  
Platelet Factor ◽  
Competitive Binding Assay ◽  
Activated Platelets ◽  
Bell Shaped Curve

Abstract Sepsis is a high-risk clinical setting often resulting in multi-organ failure and death. Release of chromatin NETS (neutrophil extracellular traps) from neutrophils and the toxic role of highly-positively charged histones in late sepsis have been noted previously. Also, for NET formation to occur, peptidylarginine deiminase 4 activity must be present in the neutrophils, leading to citrullinated (cit) histones formation and loss of a portion of the positive charge. The four histones (H2A, H2B, H3 and H4) alone and as octamers of the four units tightly bind DNA. H3 and H4 histones as well as mixed octameric histones can induce a sepsis-like state in mice. One feature previously noted was that histones could inhibit activated protein C (aPC) production in the presence of thrombomodulin (TM). Since aPC generation is felt to protect against vascular damage, it was felt that this might - in part - account for the deleterious effects of histones in sepsis. We have shown that another highly-positive, small molecule, platelet factor 4 (PF4, CXCL4), which exists as a tetramer and which is stored in high concentrations in platelet alpha granules to be released in large amounts post-platelet activation, binds to the chondroitin sulfate (CS) side-chain of TM (TMCS) and enhanced aPC production along a bell-shaped curve with a peak effect around 25 µg/ml. Non-modified mixed histones had a similar bell-shaped effect on aPC generation and [histones + PF4] are additive on affecting aPC generation via TMCS. We wondered, because of this overlapping biology and the fact that significant levels of free PF4 are available in late sepsis, whether PF4 might affect other histone pathobiological pathways in late sepsis focusing on PF4’s interactions with non-modified and cit-histones. We first asked whether released PF4 might affect the binding of histones to DNA within NETS. We found that PF4 binds to DNA with greater affinity than histones in a competitive binding assay and that this effect was more marked for cit-histone consistent with its decreased positive charge. We then studied PF4 biology in three known targets of histone in sepsis. (1) In aPC generation, we examined cit-histones (either mixed, H3 or H4) relative to non-modified histones in stimulating aPC generation and found that they had a more limited effect on aPC generation with TMCS, but that again, PF4 cooperated in inducing aPC generation along a bell-shaped curve. (2) Histones are known to activate platelets (known to involve the toll-like receptor 4), likely contributing to the observed thrombocytopenia in late sepsis. We affirmed this affect with mixed histones and H4. Cit-mixed histones and cit-H4 also activated platelets in a platelet aggregation system, but much more weakly. PF4 had no effect on platelet activation by non-modified histones, but enhanced platelet activation by both cit-mixed histones and cit-H4. This was especially true for platelet activation studies with cit-H4 which on its own had nearly no affect on platelet activation though in the presence of moderate levels of PF4 (25 µg/ml), cit-H4 activated platelets as well as non-modified histones. (3) Finally, both non-modified and cit-histones activate endothelial cells (EC) by binding to their cell surfaces and likely contribute to the vascular damage of late sepsis. Using a microfluidic system involving controlled photochemical injury of the EC lining we found that PF4 enhanced the observed damage after cit-H4 exposure, but not notably after a comparable H4 exposure so that peak damage (as detected by propidium iodide staining) after cit-H4 approached that seen after H4 alone. In conclusion, NET formation involves citrullination of histones, and these modified histones likely contribute significantly to pathobiology in late sepsis. We now propose that in late sepsis, free histones, especially cit-histones, are mobilized out of NETs by PF4 because the PF4 binds DNA with higher affinity. After the histones and cit-histones are released from DNA, PF4 modifies the biology of these histones, especially the cit-histones enhancing their effects on aPC generation, platelet activation and EC injury. These studies provide additional insights of how histones achieve their pathobiological effects in sepsis. Such new insights may be critical for both understanding and monitoring clinical outcome and may lead to new therapeutic targets in sepsis. Disclosures No relevant conflicts of interest to declare.

Anti-Platelet Factor 4/Heparin Antibodies in Patients with Gram Negative Bacteremia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3391-3391
Author(s):  
Georgios Pongas ◽  
Swapan Dasgupta ◽  
Perumal Thiagarajan
Keyword(s):  
Conflicts Of Interest ◽  
Hospital Setting ◽  
Fluorescence Emission ◽  
Cross Reactivity ◽  
Platelet Factor 4 ◽  
Fluorescence Emission Spectrum ◽  
Dependent Manner ◽  
Platelet Factor ◽  
Gram Negative ◽  
Normal Controls

Abstract Abstract 3391 Introduction The anti-platelet factor 4(PF4)/heparin antibodies, arising as a result of previous heparin exposure, are causally related to the procoagulant state due to platelet and monocyte activation. Formation of these antibodies with subsequent thrombocytopenia or thrombosis has also been described in patients, who have not been previously exposed to heparin. The presence of anti-PF4/heparin antibodies in individuals correlates with the severity of periodontal disease, implying that their occurrence may be triggered by periodontal pathogens. In this study, we determined the presence of anti-PF4/heparin antibodies in gram-negative bacteremic patients in a hospital setting and propose a pathophysiologic mechanism of their presence. Method We developed an in house ELISA for quantifying anti-PF4/heparin antibodies using therapeutic heparin and PF4 isolated from platelets. We used serum from a patient with high optical density as a standard and assigned an unit of 100 arbitrarily to construct a standard curve. We tested the sera from gram negative bacteremic patients (n= 34) in the quantitative ELISA along with normal controls (n=10). We also developed an in house ELISA for studying cross reactivity between anti-PF4/heparin antibodies and lipopolysaccharide (LPS)/PF4. We tested the sera from patients (n=5) with heparin induced thrombocytopenia in this cross reactivity ELISA. To test the interaction of LPS with PF4, we labeled PF4 with Alexa488 and measured its binding to LPS by monitoring the changes in fluorescence emission spectrum following excitation at λ480. Results Patients with bacteremia had higher titers of antiPF4/heparin antibodies compared to normal controls (26.4 ± SD 33 units, N=34 versus 6.3 ± SD 2.38 units, N=10, P=0.032). Bacterial LPS interacted with alexa488-labeled PF4 in a concentration-dependent manner, as measured by the quenching of the excitation spectrum. Patients with ant-PF4/heparin antibodies also reacted with LPS/PF4 complex in ELISA. Prior absorption of serum with PF4/heparin complex coated on ELISA plates decreased the reactivity of the serum towards PF4/LPS complex (19–46%) in two out of the five patients tested suggesting some were cross-reaction between PF4/Heparin and PF4/LPS complex. Conclusions PF4 forms a complex with lipopolysaccharide and this complex is immunogenic. Antibodies to PF4/LPS complex can cross-react with PF4/heparin complex raising the possibility that these antibodies may be responsible for the detection of PF4/heparin in individuals never been exposed to heparin previously. These antibodies may also be at least partly responsible for increased thrombosis associated with infection. Disclosures: No relevant conflicts of interest to declare.

Prevalence of Anti-Heparin Platelet Factor 4 Antibodies in Patients with Disseminated Intravascular Coagulation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2094-2094
Author(s):  
Jawed Fareed ◽  
He Zhu ◽  
Josephine Cunanan ◽  
Walter Jeske ◽  
Debra Hoppensteadt ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Disseminated Intravascular Coagulation ◽  
Conflicts Of Interest ◽  
Platelet Factor 4 ◽  
Additional Analysis ◽  
Plasma Samples ◽  
Platelet Factor ◽  
Intravascular Coagulation ◽  
Activation Products ◽  
Low Levels

Abstract Abstract 2094 Poster Board II-71 Disseminated intravascular coagulation (DIC) represents a complex syndrome with multiple pathophysiologic components. Most patients with DIC exhibit thrombocytopenic responses due to endogenous consumption of platelets. A systematic study on the prevalence on anti-heparin platelet factor 4 (AHPF4) antibodies and HIT syndrome in DIC patients has not been presented. To determine the prevalence of AHPF4 antibodies in patients with suspected DIC syndrome a total of 25 plasma samples were retrospectively analyzed utilizing the two commercially available methods (GTI, Brookfield, WI and Hyphen Biomedical, Paris, France). Out of 25 patients, 24 samples were positive for the AHPF4 antibody in the GTI method (OD>0.400), whereas only 16 were positive in the Hyphen Biomedical assay (OD>0.500). Interestingly, only 9 samples were positive in both of these assays. None of the positive samples in either the GTI or the Hyphen assay exhibited a positive 14C serotonin response. Additional analysis of these samples revealed that only 8 of these patients were previously exposed to heparin. Only 4 of the baseline samples were found to contain low levels of heparin as measured by anti-Xa method (< 0.2 U/ml). Additional analysis of these samples revealed the presence of platelet activation products such as platelet factor 4 (PF4), selectin and p-selectin. These studies suggest that circulating AHPF4 antibodies are non-functional and do not produce any thrombocytopenic responses. The elevated circulating PF4 levels and other cytokines may be contributory to the generation of these antibodies in the DIC patients. Disclosures: No relevant conflicts of interest to declare.

Influence of Protein Tyrosine Phosphatase Polymorphisms on the Development of Antibodies to Platelet Factor 4 and the Risk of Heparin-Induced Thrombocytopenia.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3683-3683
Author(s):  
Jerôme Rollin ◽  
Claire Pouplard ◽  
Dorothee Leroux ◽  
Marc-Antoine May ◽  
Yves Gruel
Keyword(s):  
Immune Response ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Platelet Factor 4 ◽  
Control Group ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Protein Tyrosine ◽  
Significant Difference

Abstract Abstract 3683 Introduction. Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of platelet-activating IgG antibodies that activate platelets via FcgRIIa receptors. The reasons explaining why only a subset of patients treated with heparin develop IgG to PF4/H complexes, and why most patients who synthesize these antibodies do not develop HIT, have not been fully defined. The immune response in HIT involves both B and T cells, and protein tyrosine kinases (PTKs) and phosphatases (PTPs) are crucial for regulating antigen receptor-induced lymphocyte activation. Moreover, some PTPs such as CD148 and low-molecular-weight PTP (LMW-PTP) could also have a critical role in platelet activation. Dysregulation of the equilibrium between PTK and PTP function could therefore have pathologic consequences and influence the pathogenesis of HIT. Aim of the study. To investigate an association between polymorphisms affecting genes encoding 4 different PTPs i.e. CD45 (PTPRC), CD148 (PTPRJ), LYP (PTPN22) and LMW-PTP (ACP1) and the development of heparin-dependent antibodies to PF4 and HIT. Patients and methods. A cohort of 89 patients with definite HIT (positive PF4-specific ELISA and positive serotonin release assay) and two control groups were studied. The first control group (Abneg) consisted of 179 patients who had undergone cardiopulmonary bypass (CBP) with high doses of heparin and who did not develop Abs to PF4 post-operatively. The second control group (Abpos) consisted of 160 patients who had also undergone cardiac surgery with CPB and heparin, who had all developed significant levels of PF4-specific antibodies but without HIT. Genotypes of PTPRC 77C/G (rs17612648), PTPN22 1858C/T (rs2476601), PTPRJ 2965 C/G (rs4752904) and PTPRJ 1176 A/C (rs1566734) were studied by a PCR-HRM method using the LightCycler 480 (Roche). In addition, the ACP1 A, B, C alleles were defined by combining the analysis of T/C transition at codon 43 of exon 3 (rs11553742) and T/C transition at codon 41 of exon 4 (rs11553746). Results. The frequency of PTPRC 77G and PTPN22 1858T alleles was not different in HIT patients and controls, whether they had developed antibodies to PF4 or not. The third PTP gene analyzed was ACP1, in which three alleles (A, B and C) were previously associated with the synthesis of distinct active LMW-PTP isoforms exhibiting different catalytic properties. The percentage of subjects in our study carrying the AC, BB and BC genotypes was significantly higher in the HIT and the Abpos groups than in patients without antibodies to PF4 after CPB (Abneg). In addition, the ACP1 A allele was less frequent in patients with antibodies to PF4, whether they had developed HIT (25%) or not (27.5% in Abpos controls), than in Abneg subjects (37%). The AC, BB and BC genotypes (associated in Caucasians with the highest LMW-PTP enzyme activity) therefore appeared to increase the risk of antibody formation in heparin-treated patients (OR 1.8; 95% CI 1.2–2.6, p=0.004 after comparing Abpos + HIT vs. Abneg). We also evaluated 2 SNPs affecting PTPRJ encoding CD148. No significant difference was found concerning the 2965 C/G polymorphism, but the frequency of PTPRJ 1176 AC and CC genotypes was significantly lower in the HIT (17%) than in the Abneg and Abpos groups (35%, p=0.003 and 29.5%, p=0.041, respectively). The C allele therefore appeared to provide a significant protection from the risk of HIT (OR 0.52; 95%CI 0.29–0.94, p=0.041) in patients with antibodies to PF4. Discussion-Conclusion. Recent studies have demonstrated that CD148 is a positive regulator of platelet activation by maintaining a pool of active SFKs in platelets. This non-synonym PTPRJ 1176 A/C SNP is associated with a Q276P substitution inducing a torsional stress of a fibronectin domain that is critical for the activity of CD148 and may influence the pathogenic effects of HIT Abs. This study supports the hypothesis that PTPs such as LMW-PTP and CD148 influence the immune response to heparin and the risk of HIT in patients with antibodies to PF4. Disclosures: No relevant conflicts of interest to declare.

Platelet Factor 4 (PF4) Is Selectively Recycled During Megakaryopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 388-388
Author(s):  
Michele Lambert ◽  
Liqing Xiao ◽  
Ronghua Meng ◽  
Michael S. Marks ◽  
Mortimer Poncz
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Culture Media ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Platelet Factor 4 ◽  
Complex Life Cycle ◽  
Platelet Factor ◽  
Receptor Associated Protein ◽  
Density Lipoprotein Receptor

Abstract Abstract 388 Platelet factor 4 (PF4), a member of the C-X-C family of chemokines, is abundantly expressed in megakaryocytes (Megs) and was thought to be stored in alpha-granules until released by activated platelets. We now show that PF4 has a more complex life-cycle, being released during megakaryopoiesis and then undergoing reuptake into Megs. This recycling may underlie PF4's physiologic role as a negative paracrine regulator of megakaryopoiesis and contribute to the regulation of platelet count at steady state or after recovery from bone marrow injury. When cultured in serum-free media, both murine and human bone marrow CD34+-derived Megs release detectable PF4 into the culture media starting at Day 3 of culture. We examined whether released PF4 is taken up by Megs in culture and whether this process is low-density lipoprotein receptor related protein-1 (LRP1)-dependent, since our previous studies had identified LRP1 as a potential receptor for PF4 on the megakaryocyte surface. Consistent with the known role of PF4 as a negative paracrine regulator, exposure of PF4null murine (m) Megs in culture to hPF4 decreases final cell numbers (8 × 104 hPF4 exposed cells/mL vs. 14 × 104 control cells/mL, p<0.01) and increases LRP1 expression on the surface of megakaryocytes/megakaryocyte precursors by >2-fold after 48 hrs in culture (p<0.01). Treatment of cells with anti-PF4 or anti-LRP1 antibodies blocked the PF4 effect on megakaryopoiesis but did not interfere with the increased expression of LRP1 on Megs after PF4 exposure. Upon exposing PF4null bone marrow-derived Megs to hPF4 (25 μg/ml) for 24 hrs, hPF4 bound to the surface of the cells; however, washing these cells with 1000 units/mL of heparin removed this surface-bound hPF4 and allowed us to measure uptake of hPF4 into these PF4null Megs. PF4 uptake by Megs was LRP1-dependent, as treatment with anti-LRP1 antibody or Receptor Associated Protein (RAP, an inhibitor of LRP family proteins) markedly decreased uptake of PF4 (95±32 IU/106 cells for PF4 treatment alone vs. 32±13 IU/106 cells for anti-LRP1 treatment, p<0.05 and 49±26 IU/106 cells for RAP treatment, p<0.01). Knockdown of LRP1 by an anti-LRP1 shRNA, which decreases LRP1 but does not completely eliminate it, also decreased PF4 uptake by Megs by 79% (41±33 IU/106 cells for control treated cells vs. 9±8 IU/106 cells for anti-LRP1 treated cells, p=0.01) and partially blocked the PF4-mediated increase in surface LRP1 expression. Using immunofluorescence microscopy, we show that internalized hPF4 in PF4null Megs co-localizes with P-selectin, an alpha-granule marker. When the Megs are treated with thrombin, the internalized PF4 is released into the medium and depleted from the cell lysates. Platelet basic protein (PBP), a closely related chemokine that is also stored in alpha granules, was not taken up by Megs in similar analyses. In summary, our study demonstrates that PF4 is not only synthesized by Megs but is also released and available for LRP1-dependent reuptake into alpha-granules. Notably, this cycling was not observed for another alpha granule chemokine, PBP. Whether the uptake is related to PF4's negative paracrine effect on megakaryopoiesis, whether other alpha granule proteins undergo similar recycling, and whether newly synthesized PF4 is first stored in alpha granules or is constitutively secreted will be addressed in future studies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Heparin Re-Exposure in Patients with Heparin-Induced Thrombocytopenia (HIT)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4265-4265 ◽  
Author(s):  
Prajwal Dhakal ◽  
Vijaya R. Bhatt
Keyword(s):  
Conflicts Of Interest ◽  
Small Sample Size ◽  
Small Sample ◽  
Platelet Factor 4 ◽  
Systemic Review ◽  
Enzyme Linked Immunosorbent Assay ◽  
Platelet Factor ◽  
Platelet Recovery ◽  
Risk Of Recurrence ◽  
History Of

Abstract Introduction Patients with a history of HIT, who require cardiopulmonary bypass (CPB), have limited anticoagulation options. Unfractionated heparin (UFH) is preferred by surgeons during CPB because of their extensive experience, short half-life of the drug, and the availability of protamine sulfate to reverse its effect. However, heparin re-challenge may be associated with a risk of recurrence of HIT. A number of instances of successful heparin re-exposure during CPB have been reported in HIT patients. However, small sample size of these reports and a lack of systemic review have prevented better understanding of the potential complications. The objective of this study was to determine the safety of heparin re-exposure in HIT patients and various strategies utilized to reduce the recurrence of HIT. Methods Using several search terms, all cases of heparin re-exposure in HIT patients published and indexed in English language in Pubmed by June 2014 were reviewed. The bibliography of each relevant article was searched for additional related reports. The diagnosis of HIT was based on the clinical probability or 4T scoring system and laboratory tests. The exposure to either UFH or low molecular weight heparin (LMWH) in patients with a history of HIT was considered a re-exposure. In two cases, heparin was used multiple times for repeated cardiovascular surgeries after an initial diagnosis of HIT. Each re-exposure was determined as a different instance of re-exposure during analysis. Results A total of 136 patients with a history of HIT had 141 instances of heparin re-exposure. Median age was 56 years (6 weeks -87 yrs) and 67% were males. Regarding the original HIT diagnosis, UFH (98%) and nadroparin (2%) were the causative agents. Thrombotic complications occurred in 23%. The pretest probability score was high in 79% and moderate in 21%. Platelet aggregation studies (66%), enzyme linked immunosorbent assay (ELISA)/enzyme immunoassay (EIA) (20%), serotonin release assay (SRA) (2%), and both SRA and EIA (12%) were performed for diagnosis. Cardiac (76%) and vascular surgeries (11%) were the most common indications for heparin re-exposure. Although 67% were re-exposed to heparin after 3 months of HIT diagnosis, 11%, 8% and 15% were re-exposed within 1 week, between 1 week to 1 month, and 1 month to 3 months of HIT diagnosis respectively. Anti-platelet factor 4/heparin antibodies were positive in 63% before re-exposure. UFH (93%) or LMWH (7%) were the utilized agents during re-exposure. Sixteen patients (11%) underwent plasmapheresis to lower the level of anti-platelet factor 4/heparin antibodies before the re-exposure. Non-heparin anticoagulants such as bivalirudin, fondaparinux, danaparoid, r-hirudin, argatroban, lepirudin, and warfarin were used singly or in combination after the exposure in 63% of patients. With heparin re-exposure, 4.2% had complications, which included recurrence of HIT (2.1%), and bleeding (2.1%). Among the patients with HIT recurrence (n=3), one patient was re-exposed to UFH within a week of HIT diagnosis and shortly after platelet recovery with LMWH (Intensive Care Med. 1991;17(3):185-6.). The other two patients were initially diagnosed with HIT more than 5 years back and tested negative for anti-platelet factor 4/heparin antibody prior to heparin re-exposure. Conclusion A review of the published reports indicates that intra-operative heparin re-exposure in patients with HIT has a small risk of developing thrombocytopenia or recurrence of HIT. The use of pre-exposure plasmapheresis in patients with positive anti-platelet factor 4/heparin antibody and post-exposure non-heparin anticoagulants may have reduced the risk of recurrence of HIT. Given several limitations of such retrospective review, prospective studies are needed to validate these results. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Detection of ANTI-Platelet Factor 4 /Heparin Antibodies during the Post Chirurgical Period of LUNG Transplantation Is Associated with a Higher Frequency of ACUTE Cellular Rejection

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2445-2445
Author(s):  
Francois Parquin ◽  
Antoine Roux ◽  
Patrick Van Dreden ◽  
Elisabeth Longchampt ◽  
Dominique Francois ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Thrombotic Event ◽  
Immunosuppressive Regimen ◽  
Acute Cellular Rejection ◽  
Platelet Factor 4 ◽  
First Year ◽  
Platelet Factor ◽  
Median Level ◽  
Surgical Sample ◽  
Cellular Rejection

Abstract Background: Heparin-induced thrombocytopenia (HIT) is characterized by the presence of antibodies directed against a complex of Platelet Factor 4 and heparin (PF4/H). These antibodies usually appear within 3 to 6 days after the beginning of heparin therapy, belong predominantly to IgG isotype, and activate platelets, leading to a thrombotic tendency. Aims: To analyze prospectively the incidence of PF4/H Ab in patients with bipulmonary transplantation (BPT) and to identify a possible consequence of the presence these antibodies on thrombotic events and graft dysfunction. Patients & Methods: Citrated blood samples of 85 consecutive patients (median age 37 years, range : 15-65) with BTP [51 (60 %) cases of cystic fibrosis (CF), 34 (40 %) cases of chronic obstructive pulmonary disease (COPD)] were analyzed on a first sample before the transplantation (D0) and on a second sample collected within 10 and 17 days after the surgery [median level: 13 days, range : 7 - 17). A third sample was analysed at least 3 months later in case of positivity of PF4/H Ab. For 12 patients, extracorporeal membrane oxygenation (ECMO) was needed up to 10 post-operative days. Patients were treated at least by a triple immunosuppressive regimen (tacrolimus, mycophenolic acid and corticosteroids) and usually by a low molecular weight heparin. PF4/H Ab were detected using ELISA (Asserachrom HPIA, Diagnostica Stago, France). Bronchoscopic transbronchial biopsies (BTB) were performed 10 days after the surgery, then every month during the first year of follow-up (FU). Acute cellular rejection (ACR) is defined by perivascular or peribronchiolar lymphocytic infiltrates in the absence of infection and are expressed as grade A0 (none), grade A1 (minimal), grade A2 (mild), grade A3 (moderate) and qrade A4 (severe). As recommended, in order to quantify the ACR intensity during first year after BPT, we summed the numbers characterizing the intensity of the rejection. Results: Eight patients (9.4 %) had PF4/H Ab at D0, which were either IgA or IgM,but not of IgG subtype. In 5 cases, the antibodies remaind present on the post-surgical sample. One patient had a plasma exchange the day before the post surgical sample and was excluded. Presence of PF4/H Ab was observed in 27 cases (32.1%) on the second sample, but disappeared in all cases on the third sample. The duration of the surgery was similar in both group, as well as the use of post-surgical ECMO. Frequency of PF4/H Ab was similar in patients with CF or COPD. PF4/H Ab were of IgG isotype in 52.2% of cases. Seventeen-patients (21.5 %) experienced a thrombotic event within the 3 months following the BPT: 8 of them (29.6 %) had PF4/H Ab, this was not significantly different from the frequency of PF4/H Ab in patients without thrombosis (17.3 %, p = 0.33). Because of frequent pulmonary infections, median leucocyte count was increased at D0 in patients with PF4/H Ab (12.5 x109/L) as well as in patients without these antibodies (10.9 x109/L), and remained elevated on the second sample (respectively 12.7 and 13.4 x109/L), but there was no significant differences between patients with or without PF4/H Ab. Platelet counts were not different at D0 between both groups (median 304 x109/L for patients with PF4/H Ab versus 300 x109/L for patients without PF4/H Ab) but during the FU, patients with PF4/H Ab had a median level of platelets (515 x109/L) significantly higher (p = 0,034) than patients without PF4/H Ab (429 x109/L). Lastly, tacrolimus levels were not significantly different in both group of patients (6.8 ng/mL in patients with PF4/H Ab versus 6.7 ng/mL in patients without PF4/H Ab) making unlikely an insufficient immunosuppression in patients with PF4/H Ab. A one-year FU was obtained for 73 patients. Eighteen patients (69.2%) with PF4/H Ab presented at least one episode of ACR versus 40.4% in patients without PF4/H Ab (p = 0.035). In addition, the mean score of ACR (indicating the severity or the repetition of the ACR) was significantly (p = 0.02) higher in patients with PF4/H Ab than in patients without PF4/H Ab (1.21 versus 0.59). Conclusion: As for cardiac or hepatic transplantations, despite a strong immunosuppressive regimen, a high frequency of transient PF4/H Ab is observed in patients undergoing BLT. Their appareance is not related to thrombocytopenia and/or thrombotic events. However, they could be an early marker of a cellular reaction againts the graft. Disclosures No relevant conflicts of interest to declare.

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