Proteins Platelet Factor Research Articles

Association of Autoimmune Diseases and Their Effect on Outcomes of Heparin-Induced Thrombocytopenia: An Analysis of the National Inpatient Sample (2016-2020)

Introduction: Heparin-Induced Thrombocytopenia (HIT) is a prothrombotic reaction to heparin exposure caused by antibodies directed towards heparin-platelet protein platelet factor 4 complexes. This thrombocytopenia is associated with and can lead to fatal venous and arterial thrombosis. This study aims to compare outcomes and assess the association of autoimmune diseases with HIT. Methods: A query of the National Inpatient Sample (2016-2020) was performed for index admissions with a principal diagnosis of HIT and concurrent autoimmune diseases using ICD 10 codes. Patient demographics and outcomes were identified. Univariate analysis with t-test or chi-squared was performed where appropriate. A multivariate regression analysis was used to generate adjusted odds ratios (AOR), adjusting for age, race, sex, and Charlson Comorbidity Index (CCI) score. The P-value significance was set at <0.05. All analysis was performed in STATA MP version 14.2. Results: A total of 72,935 patients with HIT met the inclusion criteria, of which, 2.88% had an underlying autoimmune disease. Those with auto-immune disease had a greater proportion of Caucasians, females, and younger patients. While there were no statistically significant differences in mortality, embolic, or bleeding events, patients with autoimmune disease were 43% less likely to become septic (AOR 0.57, p-value 0.006). Additionally, patients with autoimmune diseases had a lower mean hospital services charge and length of stay (p<0.001). Conclusion: Patients with HIT and autoimmune disease showed better outcomes in terms of sepsis, mean length of stay, and hospital services charge without any significant difference in embolic or bleeding outcomes. Underlying autoimmune disease likely does not confer any additional risk to HIT hospitalizations. This might be speculated to be due to the different underlying pathology driving HIT or accompanying immunosuppressants treatment in autoimmune diseases hindering the aggressiveness of immune response.

Vaccine induced thrombotic thrombocytopenia: development and reactivity of anti-platelet factor 4 antibodies and immune pathogenic mechanisms

In ultrarare cases, patients vaccinated with DNA adenovirus vector vaccine against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), develop a vaccine-induced immune thrombotic thrombocytopenia (VITT), with a high incidence of fatal cases. The causative agent is the development of platelet factor 4 (PF4)-dependent antibodies that resemble heparin-induced thrombocytopenia (HIT) complication, although many differences can be noticed in clinical presentation, antibody reactivity, involved epitopes on the PF4 protein, and pathological mechanisms. From the literature review, and the experience of HIT and testing a few plasmas from patients with VITT, this review analyzes the possible mechanisms, which show the strong immunoglobulin G (IgG) antibody reactivity to PF4 alone, in the absence of heparin, and to a lesser extend to stoichiometric complexes of PF4 and heparin (H-PF4). In addition, much lower heparin concentrations are required for inhibiting antibody binding to PF4. These concentrations are much lower than those required for disrupting the stoichiometric H-PF4 complexes. This confirms that IgG antibodies responsible for HIT bind preferentially to PF4, to epitopes that are readily masked by low concentrations of heparin. These antibodies are at a much higher concentration than the current ones observed for HIT, keeping a strong reactivity even for plasma dilutions as high as 1/500 to 1/5,000, whilst the current dilution for testing heparin-dependent antibodies in HIT is 1/100. Although VITT anti-PF4 antibodies can be detected with the current anti-H-PF4 enzyme-linked immunosorbent assays (ELISAs) designed for HIT, some assays have low sensitivity or are unreactive, like lateral immunofiltration methods or chemiluminescent automated assays. The preferred method should concern the use of capture assays using PF4 coated solid surfaces. This report proposes that the immune response is only targeted to the binding domain of PF4 with the hexons present on the adenovirus vector, through an epitope spreading mechanism, without any exposure of neo-epitopes on PF4 protein.

Biochemical and cellular markers differentiate recovered, in-line filtered plasma, and plasma obtained by apheresis methods.

Background and ObjectivesAssessment of plasma quality often focuses on the common safety tests for minimizing the risk of transmitting blood‐borne pathogens. Little attention is paid to the possible quality attributes that ensure a consistent biochemical composition of plasma for fractionation. We therefore investigated the suitability of selected biochemical and haematological attributes that could be used as markers of plasma quality obtained by different separation and pre‐treatment procedures.Material and MethodsWe characterized six plasma types, including source plasma, plasma recovered by classic means and in‐line filtered plasma, by determining the analytical attributes protein content, coagulation factors and markers of coagulation, contact and complement activation. Residual cell content and cell‐specific variables were also measured.ResultsWe found relevant differences between the plasma types in complement activation, as indicated by C3a measurements, while thrombin antithrombin complex values and, to a minor extent, activated factor XII concentrations indicated only moderate differences in activation levels of coagulation and contact systems. The most striking differences, however, were detected in residual cell content and concentrations of the platelet‐associated proteins, platelet factor 4 and β‐thromboglobulin. We showed that leucocyte reduction filters disrupt cells. This includes platelets, thereby releasing the platelet‐associated proteins platelet factor 4 and β‐thromboglobulin, and leucocytes as demonstrated by the release of elastase from polymorphonuclear leucocytes. Furthermore, the filtration processing of whole blood can lead to activation of the complement system.ConclusionOur results show that biochemical and cellular surrogate markers are valuable discriminators of plasma types.

Native Mass Spectrometry Sheds Light on Formation of Deadly Heparin-PF4 Complexes

Native Mass Spectrometry Sheds Light on Formation of Deadly Heparin-PF4 Complexes

Mechanism of platelet α-granule biogenesis: study of cargo transport and the VPS33B-VPS16B complex in a model system.

Platelet α-granules play important roles in platelet function. They contain hundreds of proteins that are synthesized by the megakaryocyte or taken up by endocytosis. The trafficking pathways that mediate platelet α-granule biogenesis are incompletely understood, especially with regard to cargo synthesized by the megakaryocyte. Vacuolar-protein sorting 33B (VPS33B) and VPS16B are essential proteins for α-granule biogenesis, but they are largely uncharacterized. Here, we adapted a powerful method to directly map the pathway followed by newly synthesized cargo proteins to reach α-granules. Using this method, we revealed the recycling endosome as a key intermediate compartment in α-granule biogenesis. We then used CRISPR/Cas9 gene editing to knock out VPS33B in pluripotent stem cell-derived immortalized megakaryocyte cells (imMKCLs). Consistent with the observations in platelets from patients with VPS33B mutation, VPS33B-knockout (KO) imMKCLs have drastically reduced levels of α-granule proteins platelet factor 4, von Willebrand factor, and P-selectin. VPS33B and VPS16B form a distinct and small complex in imMKCLs with the same hydrodynamic radius as the recombinant VPS33B-VPS16B heterodimer purified from bacteria. Mechanistically, the VPS33B-VPS16B complex ensures the correct trafficking of α-granule proteins. VPS33B deficiency results in α-granule cargo degradation in lysosomes. VPS16B steady-state levels are significantly lower in VPS33B-KO imMKCLs, suggesting that VPS16B is destabilized in the absence of its partner. Exogenous expression of green fluorescent protein-VPS33B in VPS33B-KO imMKCLs reconstitutes the complex, which localizes to the recycling endosome, further defining this compartment as a key intermediate in α-granule biogenesis. These results advance our understanding of platelet α-granule biogenesis and open new avenues for the study of these organelles.

A CASE OF SPONTANEOUS HEPARIN-INDUCED THROMBOCYTOPENIA AND THROMBOSIS (HITT)

SESSION TITLE: Critical Care 2 SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/09/2018 01:15 PM - 02:15 PM INTRODUCTION: Spontaneous heparin-induced thrombocytopenia and thrombosis (HITT) is established with positive serum platelet factor 4 (PF4) antibody, thrombosis, and without prior heparin exposure. Only a handful of spontaneous HITT cases have been reported. Due to history of no exposure to heparin products, diagnosis is often challenging and treatment delayed leading to increased patient morbidity and mortality. Here we present a case of spontaneous HIT and thrombosis after a left total knee arthroplasty (TKA). CASE PRESENTATION: A 71 year old male presented on postoperative day (POD) 7 from left TKA with acute onset abdominal pain, bright red blood per rectum, and coffee ground emesis. Prior to his TKA, he was noted to have a platelet count of 185 x 103/uL. Post-operatively he was discharged on Clindamycin 600mg and Aspirin 325mg twice daily for deep venous thrombosis (DVT) prophylaxis. On admission, he was found to have a platelet count of 47 x 103/uL and further work-up revealed splenic vein and bilateral lower extremity deep venous thrombosis. Due to acute bleeding patient was not started on anticoagulation and an inferior vena cava filter was placed. On hospital day 3, patient became acutely short of breath and computed tomography angiogram of the chest revealed bilateral pulmonary artery emboli. Serum PF4 [define PF4 here] antibody resulted strongly positive at 3.004 optical density (OD) (Range: 0.0 – 0.4 OD) indicating HITT associated thrombosis. Once the serum PF4 antibody titer was resulted positive, patient was started on Argatroban drip with eventual increase in platelet count past 150 x 103/uL. DISCUSSION: HIT is a complication of exposure to heparin that could be life threatening. It is caused by autoantibodies that recognize and bind to highly antigenic multi-molecular complexes formed of heparin binding to endogenous protein PF4 and causing platelet activation.1 Multiple theories have been proposed but the exact mechanism behind spontaneous HIT remains unclear. In vitro studies have shown that heparin antibodies are not specific to heparin and can also recognize PF4 bound to other polyanions and have shown to be elicited by reaction with lipopolysaccharides from gram negative bacteria.2 This may explain why cases of spontaneous HIT are seen in patient following total knee or hip arthroplasty where significant limb ischemia and tissue injury occur. CONCLUSIONS: Although exceedingly rare, spontaneous HITT is important to be recognized early to reduce patient morbidity and mortality. Patients without prior exposure to heparin who have undergone orthopedic procedures and present with worsening platelet count or new arterial/venous thrombosis should be considered to have spontaneous HITT and treated accordingly. Reference #1: Rauova L, Zhai L, Kowalska MA, et al. Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications. Blood 2006; 107:2346. Reference #2: G. Pongas, S. K. Dasgupta, and P. Thiagarajan, “Antiplatelet factor 4/heparin antibodies in patients with gram negative bacteremia,” Thrombosis Research, vol. 132, no. 2, pp. 217–220, 2013. Reference #3: Warkentin T.E., Cook R.J., Marder V.J., Greinacher A. Anti-PF4/heparin antibody formation postorthopedic surgery thromboprophylaxis: the role of nondrug risk factors and evidence for a stoichiometry-based model of immunization. J Thromb Haemost. 2010; 8:504. DISCLOSURES: No relevant relationships by Bryan Anderson, source=Web Response No relevant relationships by Chinthaka Bulathsinghala, source=Web Response No relevant relationships by Palla De Silva, source=Web Response No relevant relationships by Ahmed Hassan, source=Web Response No relevant relationships by aftab mahmood, source=Web Response No relevant relationships by Chirag Patel, source=Web Response Speaker/Speaker's Bureau relationship with Sunovian Please note: $5001 - $20000 Added 11/20/2017 by Salim Surani, source=Web Response, value=Honoraria Advisory Committee Member relationship with Astra Zeneca Please note: $5001 - $20000 Added 11/20/2017 by Salim Surani, source=Web Response, value=Consulting fee No relevant relationships by Deanna Yamamura, source=Web Response

Plasma protein profiling in patients undergoing coronary artery bypass grafting surgery and clinical significance.

This study was designed to identify the protein profiling in patients with triple vessel coronary artery disease (CAD) undergoing CABG, in order to detect CAD-related differential proteins in these patients. CABG patients with triple vessel disease with/without left main stenosis (n =160) were compared to normal coronary angiographic subjects (n =160). Plasma samples of 20 males and 20 females in each group were analyzed with iTRAQ technique. ELISA test was used to test the chosen proteins from iTRAQ results in plasma samples from a new cohort of the CABG group (n=120, male/femal=61/59) and control (n =120, male/female=60/60). iTRAQ detected 544 proteins with 35 up-regulated and 41 down-regulated (change fold > 1.2 or < 0.83, p < 0.05). Three proteins including platelet factor 4 (PF4), coagulation factor XIII B chain (F13B), and secreted frizzled-related protein 1 (sFRP1) were selected for validation by using ELISA that demonstrated significant up-regulation of PF4 and sFRP1 (p < 0.05). There was a positive correlation between these proteins and CAD (p < 0.05) and myocardial infarction history (p < 0.05). Thus, we for the first time have found 76 proteins differentially expressed in plasma of CABG patients. The thrombotic disease/inflammation progress-related protein PF4 and sFRP1, a member of the Wnt/fz signal-transduction pathway and related to myocardial repair, are significantly up-regulated in triple-vessel disease with/without left main stenosis. PF4 may be developed as a biomarker for the diagnosis of the severity of CAD requiring CABG procedure.

Biophysical tools to assess the interaction of PF4 with polyanions.

The antigen in heparin-induced thrombocytopenia (HIT) is expressed on platelet factor 4 (PF4) when PF4 complexes with polyanions. In recent years, biophysical tools (e. g. circular dichroism spectroscopy, atomic force microscopy, isothermal titration calorimetry, x-ray crystallography, electron microscopy) have gained an important role to complement immunological and functional assays for better understanding the interaction of heparin with PF4. This allowed identification of those features that make PF4 immunogenic (e. g. a certain conformational change induced by the polyanion, a threshold energy of the complexes, the existence of multimeric complexes, a certain number of bonds formed by PF4 with the polyanion) and to characterize the morphology and thermal stability of complexes formed by the protein with polyanions. These findings and methods can now be applied to test new drugs for their potential to induce the HIT-like adverse drug effect by preclinical in vitro testing. The methods and techniques applied to characterize the antigen in HIT may also be helpful to better understand the mechanisms underlying other antibody-mediated disorders in thrombosis and hemostasis (e. g. acquired hemophilia, thrombotic thrombocytopenic purpura). Furthermore, understanding the mechanisms making the endogenous protein PF4 immunogenic may help to understand the mechanisms underlying other autoimmune disorders.

Quantitative description of thermodynamic and kinetic properties of the platelet factor 4/heparin bonds.

Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetrameric protein platelet factor 4 (PF4) forming PF4/heparin complexes which may cause a severe immune-mediated adverse drug reaction, so-called heparin-induced thrombocytopenia (HIT). Although new heparin drugs have been synthesized to reduce such a risk, detailed bond dynamics of the PF4/heparin complexes have not been clearly understood. In this study, single molecule force spectroscopy (SMFS) is utilized to characterize the interaction of PF4 with heparins of defined length (5-, 6-, 8-, 12-, and 16-mers). Analysis of the force-distance curves shows that PF4/heparin binding strength rises with increasing heparin length. In addition, two binding pathways in the PF4/short heparins (≤8-mers) and three binding pathways in the PF4/long heparins (≥8-mers) are identified. We provide a model for the PF4/heparin complexes in which short heparins bind to one PF4 tetramer, while long heparins bind to two PF4 tetramers. We propose that the interaction between long heparins and PF4s is not only due to charge differences as generally assumed, but also due to hydrophobic interaction between two PF4s which are brought close to each other by long heparin. This complicated interaction induces PF4/heparin complexes more stable than other ligand-receptor interactions. Our results also reveal that the boundary between antigenic and non-antigenic heparins is between 8- and 12-mers. These observations are particularly important to understand processes in which PF4-heparin interactions are involved and to develop new heparin-derived drugs.

Platelet Factor 4 Enables Platelets to Suppress Early Parasite Growth in a Mouse Model of Blood Stage Malarial Infection

Platelets are best known for their roles in hemostasis, but there is accumulating evidence for their importance in immunity and host defence against infection. We have shown that platelets are required for host protection during malarial infection and are a major determinant for survival. Platelets control the growth of intraerythrocytic Plasmodium parasites by directly binding to infected erythrocytes and killing the parasite inside 1. We 2 and others 3 have also described the parasite killing mechanism exerted by platelets. Upon binding to parasite-infected red cells, platelets release a protein with dual chemokine and antimicrobial functions called platelet factor 4 (PF4). PF4 binds to the red cell Duffy antigen chemokine receptor, and is then absorbed into the parasite where it lyses the parasite food vacuole and kills the parasite.This work aimed to further investigate the role of PF4 in vivo using mice a PF4 knockout mouse strain (Pf4tm1Mako/tm1Mako; 4) and blood stage infection with a murine malaria model parasite, P. chabaudi. We confirmed that the homozygous mutant mice did not produce any PF4 protein. Following challenge with P. chabaudi (1 x 104 parasites administered intravenously), rates of survival amongst cohorts of age and sex matched wild type and homozygous mice (both on a C57BL/6 background) were not significantly different (approximately 30% for females and 10% for males). However, a deficiency of PF4 resulted in significant differences in parasite growth during the pre-peak parasitemia phase of the infection. Between days 5 and 8 following infection, proportions of parasite-infected red cells in the circulation of PF4-null mice were double those in wild-type mice. This coincided with a 50-60% reduction in the frequency of growth arrested or dead parasites (detected using terminal deoxynucleotidyl transferase dUTP nick end labelling, also known as TUNEL labelling).Together these data indicate that platelet-derived PF4 has an important in vivo malaria-protective role. The protein is necessary for the platelet to control parasite growth during the early stage of erythrocytic malarial infection. However, the highly virulent nature of the P. chabaudi strain used in these experiments precluded detection of any predicted survival disadvantage in the PF4-null mice. 1.McMorran BJ, Marshall VM, de Graaf C, et al. Platelets kill intraerythrocytic malarial parasites and mediate survival to infection. Science. 2009;323(5915):797-800.2.McMorran BJ, Wieczorski L, Drysdale KE, et al. Platelet factor 4 and Duffy antigen required for platelet killing of Plasmodium falciparum. Science. 2012;338(6112):1348-1351.3.Love MS, Millholland MG, Mishra S, et al. Platelet Factor 4 Activity against P. falciparum and Its Translation to Nonpeptidic Mimics as Antimalarials. Cell host & microbe. 2012;12(6):815-823.4.Eslin DE, Zhang C, Samuels KJ, et al. Transgenic mice studies demonstrate a role for platelet factor 4 in thrombosis: dissociation between anticoagulant and antithrombotic effect of heparin. Blood. 2004;104(10):3173-3180. DisclosuresNo relevant conflicts of interest to declare.

Binding of anti–platelet factor 4/heparin antibodies depends on the thermodynamics of conformational changes in platelet factor 4

AbstractThe chemokine platelet factor 4 (PF4) undergoes conformational changes when complexing with polyanions. This can induce the antibody-mediated adverse drug effect of heparin-induced thrombocytopenia (HIT). Understanding why the endogenous protein PF4 becomes immunogenic when complexing with heparin is important for the development of other negatively charged drugs and may also hint toward more general mechanisms underlying the induction of autoantibodies to other proteins. By circular dichroism spectroscopy, atomic force microscopy, and isothermal titration calorimetry we characterized the interaction of PF4 with unfractionated heparin (UFH), its 16-, 8-, and 6-mer subfractions, low-molecular-weight heparin (LMWH), and the pentasaccharide fondaparinux. To bind anti-PF4/heparin antibodies, PF4/heparin complexes require (1) an increase in PF4 antiparallel β-sheets exceeding ∼30% (achieved by UFH, LMWH, 16-, 8-, 6-mer), (2) formation of multimolecular complexes (UFH, 16-, 8-mer), and (3) energy (needed for a conformational change), which is released by binding of ≥11-mer heparins to PF4, but not by smaller heparins. These findings may help to synthesize safer heparins. Beyond PF4 and HIT, the methods applied in the current study may be relevant to unravel mechanisms making other endogenous proteins more vulnerable to undergo conformational changes with little energy requirement (eg, point mutations and post-translational modifications) and thereby predisposing them to become immunogenic.

Editorial introductions

Current Opinion in Hematology was launched in 1994. It is part of a successful series of review journals whose unique format is designed to provide a systematic and critical assessment of the literature as presented in the many primary journals. The field of hematology is divided into nine sections that are reviewed once a year. Each section is assigned a Section Editor, a leading authority in the area, who identifies the most important topics at that time. Here we are pleased to introduce the Journal's Section Editors for this issue. SECTION EDITORS Narla MohandasNarla MohandasDr Narla Mohandas is Vice President for Research and Director of the Lindsley F. Kimball Research Institute of New York Blood Center, USA. He received his doctoral degree from Washington University in St. Louis, USA, in Chemical Engineering. After completing post-doctoral training in hematology research with Dr Marcel Bessis, Institute of Cellular Pathology in Paris, France, he joined the faculty of the Department of Laboratory Medicine at University of California, USA, where he spent 13 years. In 1989, he moved to Lawrence Berkeley National Laboratory, University of California to head the Hematopoiesis group. During his 12 year tenure at the Berkeley Laboratory he also served as Interim Director of the Human Genome Project for three years. In 2001, he moved to the New York Blood Center, USA. Dr Mohandas’ scientific interests have long focused on red cell physiology and pathology. In particular, his efforts have contributed to improved understanding of the molecular and structural basis for red cell membrane disorders, developing detailed mechanistic insights into pathophysiology of thalassemias and sickle cell anemia, characterizing structural and functional changes induced in red cells by the malarial parasite, plasmodium falciparum. More recently his research efforts are focused on molecular understanding of normal and disordered erythropoiesis including Diamond-Blackfan Anemia and Myelodysplasia. Dr Mohandas served as a member of the National Institute of Health Hematology study section for 13 years and has been a member of numerous committees including the Executive committee of the American Society of Hematology. He served as Associate Editor of Blood from 2003–2012. He is currently Editor-in-Chief of Blood Cells, Molecules and Diseases and is on the Editorial Board of Journal of Biological Chemistry and Biochemistry. Thomas F. DeuelThomas F. DeuelThomas F. Deuel, M.D., is Professor of Molecular and Experimental Medicine and Cell Biology at The Scripps Research Institute, USA. He has formally held positions at the University of Chicago, Chicago, USA, Washington University School of Medicine, USA and Harvard Medical School, Boston, USA. He is a graduate of Princeton University, USA and Columbia University, College of Physicians and Surgeons, USA. He trained in internal medicine at the University of Chicago, USA, and the Massachusetts General Hospital, USA and in hematology/oncology at Barnes Hospital, USA. Dr Deuel trained in research under the mentorship of Charles Huggins at the Ben May Laboratory for Cancer Research, USA, Eugene Kennedy at Harvard Medical School, USA, and Earl Stadtman at the National Heart Lung and Blood Institute, USA. His early research focused on the purification and biochemical characterization of the platelet secretory proteins platelet factor 4 (PF4) and the platelet-derived growth factor (PDGF). He established the relationship of platelet secretory proteins as mediators of the inflammatory response and discovered the relationship of PDGF and the v-sis oncogene, opening the field of how oncogenes function in cancer cells. He discovered the downstream functions of PDGF and that PDGF activates its receptor before secretion to stimulate autocrine growth. More recently, he discovered pleiotrophin, described its unique signaling mechanism and its roles in angiogenesis, growth and differentiation, and the progression of human neoplasms. Dr Deuel is a member of the Institute of Medicine and a recipient of the Dameshek Prize and the American Society of Hematology and the Scientific Councils Distinguished Achievement Award. Apart from the Editorial Board of Current Opinion in Hematology, he has been a member of the Editorial Committee of Journal of Clinical Investigation, the Editorial Board of Bio Factors and Circulation, the Editorial Committee of Journal of Clinical Investigation, an Editor of Oncogene Research, and an Associate Editor of Blood.

Characterization of bonds formed between platelet factor 4 and negatively charged drugs using single molecule force spectroscopy

Immunogenicity (i.e., the ability to initiate immune reactions) is one of the major challenges for the development of new drugs, as it may turn the developed drug therapeutically ineffective or cause severe immune-related effects. Using single molecule force spectroscopy, we study rupture forces between the positively charged, endogenous protein platelet factor 4 (PF4; also known as CXC chemokine ligand 4, CXCL4) and the antithrombotic drug heparin and other negatively charged glycosaminoglycans (GAGs), which are known to form immunogenic PF4/GAG-complexes (e.g., heparin and dextran sulfate) as well as non-immunogenic complexes (e.g., chondroitin sulfate A). Our measurements suggest that the average number of sulfate groups per monosaccharide unit (i.e., the degree of sulfation DS) does not affect the unbinding characteristics of single PF4/GAG-bonds (reaction coordinate x0 = 2.2 ± 0.2 Å, energy barrier ΔG ≈ -1 kBT). However, the average number of GAG bonds formed to a single PF4 molecule increases with increasing DS as indicated by a rising frequency of unbinding events, suggesting a multivalent binding scheme between PF4 and GAGs. Our studies show that at least three GAG bonds have to be formed to each PF4 molecule to induce epitope formation on the PF4/GAG-complex to which PF4/GAG-complex specific antibodies bind. Hence, GAG-based drugs that form less than three bonds per PF4 molecule are unlikely to constitute PF4/drug-complexes that are of immunologic relevance.

Potential biomarkers for adult acute myeloid leukemia minimal residual disease assessment searched by serum peptidome profiling

BackgroundPost treatment minimal residual disease (MRD) determination contributes to impending relapse prediction, chemotherapy response and clinical outcomes assessment, guiding clinicians to develop reasonable and effective individual chemotherapy options after induction/consolidation. This study was to identify serum candidate peptides for monitoring adult acute myeloid leukemia (AML) MRD.Results47 statistically different expressed peptide peaks were obtained in the molecular weight range of 700-10000 Da. Quick classifier (QC) model had optimal distinction efficiency, in the training set with a sensitivity of 90% and a specificity of 93.33%. Peptides were identified as ubiquitin-like modifier activating enzyme 1(UBA1), isoform 1 of fibrinogen alpha chain precursor and platelet factor 4(PF4). The peptide up-regulated in newly diagnosed AML patients were decreased to the normal level after CR. When refractory & relapsed, relative intensity was elevated again. Results were contrary to down-regulated peptide peaks. Western blot demonstrated that levels of the UBA1 protein did not differ between the leukemia and normal cells. Levels of isoform 1 of fibrinogen alpha chain precursor protein and PF4 protein were both decreased in leukemia cells comparing with normal cells. The serum levels of the PF4 in the newly diagnosed AML patients and healthy controls were significantly different. Further correlation analysis did not indicate the correlated relation between platelet counts and PF4 content, the correlation coefficient was 0.097. Kaplan–Meier analyses of overall survival showed that relative intensity of peptides was correlated with patient’s clinical outcome.ConclusionsWe speculate the peptides can be used as potential markers for monitoring minimal residual disease and clinical outcome assessment.

Quantitative pattern analysis of the N‐terminally processed isoforms of platelet factor‐4 in serum

Platelet factor 4 (PF4) is a small cytokine belonging to the CXC chemokine family which has been shown to play a role in inflammation and in the regulation of angiogenesis. In general, chemokines are susceptible to proteolytic cleavage in amino and carboxy terminal regions, which usually results in dramatic changes to the chemokine bioactivity. The purpose of this study was to identify various platelet factor-4 (PF4) isoforms caused by proteolytic processing and to quantify their levels in normal serum. First, we identified the N-terminally truncated PF4 isoforms from a standard purified PF4 protein sample by using mass spectrometry (MS) and tandem mass spectrometry (MS/MS) analysis. Then, we used high-performance liquid chromatography (HPLC) to semi-purify PF4 from serum samples, and the levels of the four most abundant PF4 isoforms were quantitatively determined using selected reaction monitoring (SRM) assays on a nano-LC/triple-quadrupole mass spectrometer. We have identified seven N-terminally processed PF4 isoforms and compared the levels of major PF4 isoforms from nine serum samples. Pro-p1 (EAEEDGDLQCLCVK-; average MW, 7765.2) is the major PF4 isoform in serum whereas the PF4 isoforms, designated Prot-p4 (FASAEAEEDGDLQCLCVK-;average MW, 8141.5), Prot-p3 (SAEAEEDGDLQCLCVK-; average MW, 7923.3), and Prot-p2 (AEEDGDLQCLCVK- ; average MW, 7836.3), are at about 16%, 3%, and 2% levels of the major one, respectively. This study is the first report on the levels of N-terminally processed PF4 isoforms in serum. Also, this study shows the usefulness of SRM in determining concentrations of protein isoform variants, which can be often overlooked in immunoassay analysis.

Safety of IVC Filters with Heparin-Induced Thrombocytopenia: A Retrospecive Study

Abstract 2225 IntroductionHeparin-induced thrombocytopenia (HIT) results from antibody formation to a complex of the platelet-derived protein platelet factor 4(PF4) and heparin. Thrombocytopenia is not usually severe, and not associated with bleeding. In fact, HIT is associated with markedly increased risk of thrombosis from activated platelets and thrombin generation. Inferior Vena Cava filters (IVC filter) have been used to prevent pulmonary embolization with deep vein thrombosis. In a patient with thrombosis, the best established indications for IVC filter placement are (1) contraindication to pharmacologic anticoagulation, such as thrombocytopenia, and (2) failure of anticoagulant therapy. While HIT might be viewed as meeting these criteria, actually the syndrome mandates, rather than contraindicates, aggressive alternative anticoagulation. Further, anecdotes support that the foreign body can precipitate devastating thromboses in this extreme hypercoagulable milieu. We present data on IVC filter use in HIT from 2003 to 2010 observed in a tertiary academic hospital in the Texas Medical Center, Houston, Texas. MethodsWe retrospectively reviewed patient charts from those discharged with ICD-9 diagnosis code for HIT (289.84) from our institution from 2008 to 2010. We also reviewed patient charts from those discharged with either ICD-9 diagnosis code for thrombocytopenia unspecified (287.5) or secondary thrombocytopenia (287.4) and ICD-9 procedure code for interruption of the vena cava (38.7). Of note, ICD-9 diagnosis code for HIT was newly introduced in late 2007. We collected data regarding appropriateness of diagnosis, heparin PF4 antibody titer, the use of alternative anticoagulation, the use of IVC filter, incidence of thrombosis and incidence of new thrombosis after IVC filter placement. ResultsOf one hundred and twenty four patients discharged with the ICD-9 diagnosis code for HIT. chart review revealed that fifty-five patients did not have true HIT. These cases were seen by a hematologist at the time of diagnosis and determined not to have HIT and/or they had negative heparin PF4 ELISA antibody test. Sixty-nine patients were confirmed to have HIT by hematologist's evaluation and/or high heparin PF4 antibody titer with use of alternative anticoagulation. Among these 69 patients with HIT, ten patients had IVC filter placement either some time prior to or at the time of diagnosis of HIT. Nine of these ten patients developed new thrombosis related to IVC filters. Among 59 patients who had HIT and no IVC filter, 14 patients had thrombosis at the time of HIT diagnosis and four more developed new thrombosis after the diagnosis (three of these while receiving direct thrombin inhibitor). We identified 193 patients eligible for chart review from 2003 to 2007 using ICD-9 diagnosis and procedure code. The data review is underway and will be presented at the ASH 2011 annual meeting. ConclusionHeparin-Induced Thrombocytopenia (HIT) is associated with high risk of thrombosis from activated platelets and thrombin generation. Placement of IVC filter in the setting of HIT or even prior insertion of IVC filter is associated with higher risk of thrombosis, and could lead to devastating anticoagulation-resistant limb-threatening thrombosis. Disclosures:Rice:GSK: Speakers Bureau; Canyon Pharma: Membership on an entity's Board of Directors or advisory committees.

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

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

Platelet factor 4 binds to bacteria, inducing antibodies cross-reacting with the major antigen in heparin-induced thrombocytopenia

AbstractA clinically important adverse drug reaction, heparin-induced thrombocytopenia (HIT), is induced by antibodies specific for complexes of the chemokine platelet factor 4 (PF4) and the polyanion heparin. Even heparin-naive patients can generate anti-PF4/heparin IgG as early as day 4 of heparin treatment, suggesting preimmunization by antigens mimicking PF4/heparin complexes. These antibodies probably result from bacterial infections, as (1) PF4 bound charge-dependently to various bacteria, (2) human heparin-induced anti-PF4/heparin antibodies cross-reacted with PF4-coated Staphylococcus aureus and Escherichia coli, and (3) mice developed anti-PF4/heparin antibodies during polymicrobial sepsis without heparin application. Thus, after binding to bacteria, the endogenous protein PF4 induces antibodies with specificity for PF4/polyanion complexes. These can target a large variety of PF4-coated bacteria and enhance bacterial phagocytosis in vitro. The same antigenic epitopes are expressed when pharmacologic heparin binds to platelets augmenting formation of PF4 complexes. Boosting of preformed B cells by PF4/heparin complexes could explain the early occurrence of IgG antibodies in HIT. We also found a continuous, rather than dichotomous, distribution of anti-PF4/heparin IgM and IgG serum concentrations in a cross-sectional population study (n = 4029), indicating frequent preimmunization to modified PF4. PF4 may have a role in bacterial defense, and HIT is probably a misdirected antibacterial host defense mechanism.

Investigation of a Sequence-Modified Antimicrobial Peptide

Antimicrobial peptides serve as one of the first lines of defense in the immune systems of higher organisms. These peptides specifically target and neutralize infecting bacteria in the host organism while exhibiting little or no toxic effect on host cells. The peptide C18G is a highly cationic, amphiphilic peptide derived from the C-terminal sequence of the human protein platelet factor 4 (involved in blood coagulation and wound repair) exhibited antibacterial activity against both gram positive and gram negative bacteria. Using a modified C18G sequence that did not significantly affect antimicrobial efficacy (Tyr3 changed to Trp and all Lys changed to Arg (C18G Y3W K R)). The binding affinity was measured with fluorescence spectroscopy using the W in the peptide sequence as a probe of peptide environment. Small unilamellar lipid vesicles were used to investigate the binding affinity of the peptide to bilayers composed of variable amounts of DOPC, POPG, and POPE. DOPC and POPE have a zwitterionic head group, whereas POPG has an anionic charged head group. These studies showed binding affinity had a dramatic dependence on lipid composition. The effect of pH on peptide binding and behavior was also examined and, as expected, also impacted binding affinity. Quenching of the Trp fluorescence by acrylamide was performed to confirm that the Trp was located in the membrane. Likewise circular dichroism (CD) spectroscopy was used to determine the structure of the peptide upon interaction with the lipid vesicles. Additionally, in an assay monitoring membrane permeabilization of E. coli the C18G Y3W K R peptide was shown to permealize bacterial membranes in a concentration dependent manner.

Markers of Blood Cell Activation and Complement Activation in Factor VIII and von Willebrand Factor Concentrates

Background: Preparations of commercially available clotting factor VIII are complex protein mixtures. Most of them contain either von Willebrand factor or human serum albumin as stabilizers. The aim of the study was to quantify further proteins in twelve concentrates either of recombinant origin or derived from human plasma. Methods: Proteins were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Some proteins were quantified by ELISA. Results: Recombinant clotting factor preparations showed fewer protein spots in the 2D-PAGE, than plasma-derived preparations. Proteins identified in some of the plasma-derived concentrates included up to 90 ng/IU of the anaphylatoxin C3a, up to 40 ng/IU of the platelet Α-granule protein thrombospondin-1, up to 0.85 ng/IU of the platelet agranule protein platelet factor 4, 3.5 ng/IU myeloperoxidase secreted by leukocytes and up to 0.05 ng/IU of the leukocyte-secreted protein a-defensin. The protein content differed between concentrates from different manufacturers. Conclusion: The origin of the plasma used to prepare the factor concentrates might influence the protein impurities in these products. It is unknown whether the impurities observed have long-term consequences for chronic inflammatory conditions.