scholarly journals Fluid-Phase Immune Complexes Can Assemble and Activate Platelets in Heparin-Induced Thrombocytopenia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3746-3746
Author(s):  
Angela Huynh ◽  
Donald M. Arnold ◽  
James W. Smith ◽  
Taylor D. Elliott ◽  
Hina Bhakta ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Immune Complexes ◽  
Research Funding ◽  
Fluid Phase ◽  
Serotonin Release ◽  
Antibody Binding ◽  
Wild Type ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Heparin Induced Thrombocytopenia

Abstract Introduction: Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy that is caused by antibodies to complexes of platelet factor 4 (PF4) and heparin. Several studies have reported that in order for these immune complexes to be pathogenic, they must assemble on the platelet surface. When bound to the platelet surface, the conformation of PF4 allows for optimal presentation of the epitope for antibody binding and subsequent activation of Fc-receptors on platelets and monocytes. To what degree pathogenic HIT immune complexes can form and activate platelets in fluid-phase as with other immune complex diseases (systemic lupus erythematosus, glomerulonephritis, and rheumatoid arthritis) is not known. We used mutated PF4 proteins that can no longer bind the platelet surface to evaluate anti-PF4/heparin antibody induced platelet activation. We hypothesized that the epitopes required for PF4 binding of HIT antibodies and subsequent platelet activation can be formed in fluid-phase. Methods: Each of the 70 amino acids of PF4 were mutated previously by alanine scanning mutagenesis where non-alanine residues were mutated to alanine or alanine residues to valine. We selected 14 PF4 mutants that affected KKO (a platelet-activating murine monoclonal HIT-like antibody) binding in a heparin-capture assay for this study. Mutant and wild-type PF4 were overexpressed in Escherichia coli and affinity purified. To confirm binding to platelets, biotin-conjugated PF4 mutants were incubated with donor platelets and PF4 platelet binding was measured using streptavidin-FITC by flow cytometry. Platelet activation was measured using a modified 14C-serotonin-release assay, where excess wild-type or mutant PF4 (0, 50 and 100 μg/mL) was added to 14C-serotonin-labelled donor platelets and activation was measured after incubation with KKO. ≥20% 14C-serotonin release was considered positive for platelet activation. Platelet activation was correlated with platelet surface binding to identify mutants that could form surface-bound or fluid-phase antigenic complexes. Results: Of the 14 PF4 mutants tested, 7 bound to platelet surfaces and 11 supported platelet activation by KKO. These PF4 mutants were further characterized into 3 categories: PF4 mutants that bound to the platelet surface and induced platelet activation (n=6); PF4 mutants that did not bind to the platelet surface but induced platelet activation (n=5); and PF4 mutants that bound to the platelet surface but did not induce platelet activation (n=1). These results indicate that certain PF4 mutants were able to bind KKO and induce platelet activation in fluid-phase. These data suggest that specific epitopes in fluid-phase PF4/heparin immune complexes can mediate platelet activation in HIT, without the need for surface assembly on the platelet. Conclusions: Using point mutations of PF4, we have identified that the HIT antigenic complexes can be formed in fluid-phase and induce platelet activation. Further studies are required to investigate the role of a fluid-phase HIT antigen complex in the development of thrombocytopenia, inflammation and thrombosis of HIT. This study was funded by the Canadian Institutes for Health Research. Disclosures Arnold: Bristol Myers Squibb: Research Funding; Amgen: Consultancy, Research Funding; UCB: Consultancy; UCB: Consultancy; Novartis: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding.

Low Level Platelet-Activating Antibodies in Patients Suspected of Having Heparin-Induced Thrombocytopenia but Testing Negative in the ”Gold Standard” Functional Test

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2757-2757
Author(s):  
Ishac Nazi ◽  
Donald M Arnold ◽  
James W Smith ◽  
Theodore E. Warkentin ◽  
Jane C Moore ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Research Funding ◽  
Heparin Therapy ◽  
Serotonin Release ◽  
Patient Specific ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Increased Risk ◽  
Increased Sensitivity ◽  
Clinical Conditions

Abstract Background: Heparin-induced thrombocytopenia (HIT) is a common drug reaction that causes arterial or venous thrombosis as a result of heparin therapy. Platelet-activating antibodies, against complexes of platelet factor 4 (PF4) and heparin, cause intense platelet activation, ultimately leading to an increased risk of thrombosis, limb-loss and even death. Most patients exposed to heparin will produce non-pathogenic anti-PF4/heparin antibodies while only a small number will produce platelet-activating and HIT-causing antibodies (pathogenic HIT antibodies). Among HIT tests, the functional assays, such as the serotonin release assay (SRA), correlate best with the disease because they can specifically identify the pathogenic HIT antibodies whereas the enzyme immunoassays (EIAs) cannot. We have previously shown that anti-PF4/heparin antibody production precedes thrombocytopenia in HIT patients (Warkentin et al., Blood 2009 113: 4963-4969) possibly indicating the need for a threshold plasma level of pathogenic HIT antibody, among other factors, to cause the disease. The objective of this study was to investigate the presence of low levels of pathogenic HIT antibodies in samples from patients suspected of HIT who had detectable anti-PF4/heparin antibodies in the EIA (EIA-positive), but who did not have platelet-activating antibodies in the standard SRA (SRA-negative). Methods: We used an in-house IgG-specific EIA to detect the presence of anti-PF4/heparin antibodies (EIA-positive: OD405nm> 0.45) and the standard SRA to detect the presence of heparin-dependent platelet-activating antibodies (SRA-positive: release >20% with 0.1-0.3 IU/mL of unfractionated heparin). We developed an enhanced SRA (eSRA) by adding increasing concentrations of exogenous PF4 (0-100 μg/mL) to detect sub-threshold levels of platelet activating antibodies undetectable in the standard SRA (eSRA-positive: release >20%). Samples tested were referred for HIT testing by the McMaster Platelet Immunology Laboratory (Hamilton, Canada). Results: Sera from healthy individuals (n=10) and from suspected HIT patients with a negative anti-PF4/heparin EIA (n=15) did not demonstrate platelet activation in the eSRA at any dose of exogenous PF4 added. SRA-positive sera (n = 7), diluted sufficiently that they were non-reactive in the standard SRA, demonstrated PF4 dose-dependent platelet activation in the eSRA. This confirmed the increased sensitivity of the eSRA in detecting low-titre platelet-activating antibodies. Reactivity in the eSRA was inhibited by high heparin (100 U/mL) and by blocking the platelet FcgRIIa receptor with the monoclonal antibody IV.3. We then tested samples (n=24) referred for HIT testing that were positive in the anti-PF4/heparin EIA (optical densities OD405nm 0.7 to 2.4) but negative in the standard SRA. Heparin-dependent platelet activation (20-99% release) was demonstrated in 11 of 24 (46%) in the eSRA. This reactivity directly correlated with the amount of PF4 added to the platelets (optimal concentration of PF4 12.5 - 100 μg/mL) but not with the strength (OD405nm) of the anti-PF4/heparin EIA. In further investigations, we concentrated (4-fold) 7 of the 11 eSRA-positive samples in an attempt to increase the concentration of the antibodies. Of those 7 samples, 5 (71%) became positive in the standard SRA upon testing of the concentrated sample. Conclusions: These data indicate that low-titre platelet-activating antibodies may be found in some patients suspected of having HIT that test negative in the standard SRA irrespective of the strength (OD405nm) of the anti-PF4/heparin EIA. The immune response during heparin therapy can produce both families of pathogenic and non-pathogenic anti-PF4/heparin antibodies but it is the titre of the pathogenic antibody that may be necessary for platelet activation. Perhaps under permissive clinical conditions and with patient-specific factors, the titre of the pathogenic HIT antibodies may increase and lead to HIT. Disclosures Warkentin: Pfizer Canada: Honoraria; Instrumentation Laboratory: Honoraria; GlaxoSmithKline: Consultancy, Research Funding; W.L. Gore: Consultancy, Research Funding.

Pathophysiology of Heparin-Induced Thrombocytopenia

2000 ◽  
Vol 124 (11) ◽  
pp. 1657-1666 ◽  
Author(s):  
Fabrizio Fabris ◽  
Sarfraz Ahmad ◽  
Giuseppe Cella ◽  
Walter P. Jeske ◽  
Jeanine M. Walenga ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Thrombin Inhibitors ◽  
Platelet Factor ◽  
Cellular Activation ◽  
Heparin Induced Thrombocytopenia ◽  
New Information ◽  
Study Selection ◽  
Release Assay

Abstract Objective.—This review of heparin-induced thrombocytopenia (HIT), the most frequent and dangerous side effect of heparin exposure, covers the epidemiology, pathophysiology, clinical presentation, diagnosis, and treatment of this disease syndrome. Data Sources and Study Selection.—Current consensus of opinion is given based on literature reports, as well as new information where available. A comprehensive analysis of the reasons for discrepancies in incidence numbers is given. The currently known mechanism is that HIT is mediated by an antibody to the complex of heparin–platelet factor 4, which binds to the Fc receptor on platelets. New evidence suggests a functional heterogeneity in the anti-heparin-platelet factor 4 antibodies generated to heparin, and a “superactive” heparin-platelet factor 4 antibody that does not require the presence of heparin to promote platelet activation or aggregation has been identified. Up-regulation of cell adhesion molecules and inflammatory markers, as well as preactivation of platelets/endothelial cells/leukocytes, are also considered to be related to the pathophysiology of HIT. Issues related to the specificity of currently available and new laboratory assays that support a clinical diagnosis are addressed in relation to the serotonin-release assay. Past experience with various anticoagulant treatments is reviewed with a focus on the recent successes of thrombin inhibitors and platelet GPIIb/IIIa inhibitors to combat the platelet activation and severe thrombotic episodes associated with HIT. Conclusions.—The pathophysiology of HIT is multifactorial. However, the primary factor in the mediation of the cellular activation is due to the generation of an antibody to the heparin-platelet factor 4 complex. This review is written as a reference for HIT research.

scholarly journals Platelet Activation in Heparin-Induced Thrombocytopenia is Followed by Platelet Death via Complex Apoptotic and Non-Apoptotic Pathways

2020 ◽  
Vol 21 (7) ◽  
pp. 2556
Author(s):  
Elmira R. Mordakhanova ◽  
Tatiana A. Nevzorova ◽  
Gulnaz E. Synbulatova ◽  
Lubica Rauova ◽  
John W. Weisel ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Immune Complexes ◽  
Gel Filtration ◽  
Human Platelets ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Mitochondrial Membrane Depolarization ◽  
Low Platelet Count ◽  
Apoptotic Pathways

Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction characterized by thrombocytopenia and a high risk for venous or arterial thrombosis. HIT is caused by antibodies that recognize complexes of platelet factor 4 and heparin. The pathogenic mechanisms of this condition are not fully understood. In this study, we used flow cytometry, fluorimetry, and Western blot analysis to study the direct effects of pathogenic immune complexes containing platelet factor 4 on human platelets isolated by gel-filtration. HIT-like pathogenic immune complexes initially caused pronounced activation of platelets detected by an increased expression of phosphatidylserine and P-selectin. This activation was mediated either directly through the FcγRIIA receptors or indirectly via protease-activated receptor 1 (PAR1) receptors due to thrombin generated on or near the surface of activated platelets. The immune activation was later followed by the biochemical signs of cell death, such as mitochondrial membrane depolarization, up-regulation of Bax, down-regulation of Bcl-XL, and moderate activation of procaspase 3 and increased calpain activity. The results show that platelet activation under the action of HIT-like immune complexes is accompanied by their death through complex apoptotic and calpain-dependent non-apoptotic pathways that may underlie the low platelet count in HIT.

scholarly journals Detection of Platelet-Activating Antibodies Associated with Heparin-Induced Thrombocytopenia

2020 ◽  
Vol 9 (4) ◽  
pp. 1226 ◽  
Author(s):  
Brigitte Tardy ◽  
Thomas Lecompte ◽  
François Mullier ◽  
Caroline Vayne ◽  
Claire Pouplard
Keyword(s):  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Serotonin Release ◽  
Membrane Expression ◽  
Platelet Factor ◽  
Strict Adherence ◽  
Heparin Induced Thrombocytopenia ◽  
Functional Assays ◽  
Healthy Donors ◽  
Sensitivity Specificity

Heparin-induced thrombocytopenia (HIT) is a prothrombotic immune drug reaction caused by platelet-activating antibodies that in most instances recognize platelet factor 4 (PF4)/polyanion complexes. Platelet activation assays (i.e., functional assays) are more specific than immunoassays, since they are able to discern clinically relevant heparin-induced antibodies. All functional assays used for HIT diagnosis share the same principle, as they assess the ability of serum/plasma from suspected HIT patients to activate fresh platelets from healthy donors in the presence of several concentrations of heparin. Depending on the assay, donors’ platelets are stimulated either in whole blood (WB), platelet-rich plasma (PRP), or in a buffer medium (washed platelets, WP). In addition, the activation endpoint studied varies from one assay to another: platelet aggregation, membrane expression of markers of platelet activation, release of platelet granules. Tests with WP are more sensitive and serotonin release assay (SRA) is considered to be the current gold standard, but functional assays suffer from certain limitations regarding their sensitivity, specificity, complexity, and/or accessibility. However, the strict adherence to adequate preanalytical conditions, the use of selected platelet donors and the inclusion of positive and negative controls in each run are key points that ensure their performances.

scholarly journals Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3232-3239 ◽  
Author(s):  
JG Kelton ◽  
JW Smith ◽  
TE Warkentin ◽  
CP Hayward ◽  
GA Denomme ◽  
...  
Keyword(s):  
Minimum Degree ◽  
Equilibrium Dialysis ◽  
Fluid Phase ◽  
Platelet Factor 4 ◽  
Minimum Size ◽  
Sulfated Polysaccharides ◽  
Platelet Factor ◽  
Platelet Surface ◽  
Heparin Induced Thrombocytopenia

Abstract Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.

The Impact of Non-H:PF4 Antibodies on Results of the Serotonin Release Assay for Heparin-Induced Thrombocytopenia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3952-3952
Author(s):  
M. Margaret Prechel ◽  
Maura A. Woznica ◽  
Meredith K. McDonald ◽  
Walter P. Jeske ◽  
Jeanine M. Walenga
Keyword(s):  
Platelet Activation ◽  
Immune Complexes ◽  
Hla Class I ◽  
Serotonin Release ◽  
Class I ◽  
Heparin Induced Thrombocytopenia ◽  
Platelet Antibodies ◽  
Group I ◽  
Release Assay ◽  
Group Ii

Abstract Background: The Serotonin Release Assay (SRA) is frequently used to test for the presence of heparin-platelet factor 4 (H:PF4) antibodies as part of a diagnosis of Heparin-Induced Thrombocytopenia (HIT). In this test, immune complexes formed when antibodies bind to soluble or membrane-bound H:PF4 complexes, activate platelets via FcγIIa receptors. A HIT-positive SRA demonstrates donor platelet activation when test sera are incubated with low concentrations (0.1–1.0 U/ml) of heparin, but not when the incubation includes excess, neutralizing levels (10–100 U/ml) of heparin. Platelet activation at both low and high heparin is designated an indeterminate result. This heparin-independent platelet activation can be caused by immune complexes unrelated to HIT, by other anti-platelet antibodies or by platelet agonists. The present studies were conducted to study the occurrence of anti-platelet antibodies in specimens with marginal or inconclusive SRA activity. Methods: The study included two populations of specimens that had been previously tested by both SRA and H:PF4 antibody ELISA (GTI, Brookfield, WI). Group I included 44 specimens that tested SRA positive in spite of the absence of measurable H:PF4 antibodies. Most were relatively weak in the SRA: 51% ± 3.4 % (S.E.) serotonin release with 0.1 U/ml heparin. Group II consisted of 18 specimens that gave an indeterminate SRA response: heparin-independent platelet activation. Of these, 5 were positive for H:PF4 antibodies and 13 were negative. All specimens were analysed by PakPlus ELISA screening (GTI, Brookfield, WI) to determine if antibodies to HLA Class I and/or to common platelet specific glycoproteins (GPs) were present. Results: In Group I, 19 of the 44 (43%) specimens tested positive for one or more anti-platelet antibody. 18 of the 19 (95%) had either anti-GP IIb/IIIa (n=10)(53%) and/or anti- HLA Class I (n=11)(58%) antibodies. One specimen had antibodies to GP Ib/IX and to GP IV. The remaining 25 (57%) specimens tested negative. In Group II, 13 of the 18 (72%) SRA-indeterminate specimens had detectable anti-platelet antibodies. All but one of the 13 (92%) included antibody to HLA Class I. The anti-glycoprotein antibodies were less frequent in this group: anti-GP IIb/IIIa (n=2)(15%), GP Ia/IIa (n=3)(23%), GP Ib/IX (n=2)(15%) or GP IV (n=3)(23%). Conclusion: Non-H:PF4 anti-platelet antibodies, especially antibodies to GP IIb/IIIa or to HLA Class I, are not uncommon in sera referred for SRA testing. Specimens containing anti-platelet antibodies can give a positive or an indeterminate response in the two-point SRA. Specimens without H:PF4 antibodies that test positive in the SRA should be scrutinized for anti-platelet antibodies as an alternative to the diagnosis of HIT. Also, an indeterminate SRA should not be considered a negative test result. Anti-platelet antibodies alone can cause a non-heparin dependent platelet activation: however, their presence may also mask a positive, heparin-dependent, SRA response to H:PF4 antibodies. Finally, it is not uncommon for specimens to test positive by the 2-point SRA in the absence of antibodies to H:PF4 or to other platelet antigens. The mechanism for this response, and its significance to diagnosis of HIT, requires further investigation.

Spontaneous Heparin-Induced Thrombocytopenia Syndrome: Two New Cases and a Proposal For Defining This Disorder

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2328-2328 ◽  
Author(s):  
Theodore E. Warkentin ◽  
Paul Andrew Basciano ◽  
Richard A. Bernstein
Keyword(s):  
Platelet Count ◽  
Platelet Activation ◽  
Research Funding ◽  
Left Vertebral Artery ◽  
Platelet Factor ◽  
Shoulder Hemiarthroplasty ◽  
Heparin Induced Thrombocytopenia ◽  
Activation Assay ◽  
Count Recovery ◽  
Heparin Exposure

Abstract Introduction Heparin-induced thrombocytopenia (HIT) is a transient, autoimmune-like, prothrombotic disorder caused by heparin-dependent, platelet-activating IgG reactive against platelet factor 4/heparin (PF4/H). There is an emerging literature (Am J Med 2008;121:632-6. J Thromb Haemost 2008;6:1598-1600; Thromb Haemost 2013;109:669-75) pointing to rare instances of “spontaneous” HIT in patients without preceding heparin. We report 2 new cases and propose a definition for this controversial disorder. CASE #1. A 62-y.o. man presented with left middle cerebral artery stroke and thrombocytopenia (platelet count, 65×109/L). There was no previous history of thrombocytopenia, surgery, hospitalization, or heparin exposure. Clot extraction performed with heparin was complicated by further platelet count decline to 27 (nadir) and progressive thrombosis of the carotid artery. Aspirin was started, and the platelets recovered to >150 by day 13. CASE #2. A 54-y.o. female developed right leg swelling, left-upper extremity weakness/paresthesias, and thrombocytopenia (61×109/L) 15 days post-shoulder hemiarthroplasty; no intra-/postoperative heparin had been given. Brain MRI demonstrated acute infarct in the left posterior inferior cerebellar artery territory; angiography showed non-visualization of the left vertebral artery. Ultrasound revealed right lower-limb deep-vein thrombosis. Heparin treatment resulted in further platelet count fall to 37 (nadir). Treatment with argatroban, followed by fondaparinux, was associated with platelet count recovery to >150 by day 39. Methods Testing for HIT antibodies was performed by commercial EIA-IgG/A/M (Immucor GTI Diagnostics), in-house EIA-IgG (McMaster), and serotonin-release assay (SRA). Results Both patients’ sera (obtained before any heparin administration) tested strongly positive for HIT antibodies (Table), including strong platelet activation at 0.1 and 0.3 IU/mL heparin, as well as at 0 U/mL heparin, with no platelet activation at 100 IU/mL heparin: these serological features are characteristic of “delayed-onset HIT” (Ann Intern Med 2001;135:502-6). Antibody reactivity declined markedly by 2 to 4 weeks (including loss of platelet-activating properties at 0 IU/mL heparin), in keeping with the usual transience of HIT antibodies (N Engl J Med 2001;344:1286-92), and paralleling both patients’ platelet count recovery. Discussion These cases further support spontaneous HIT as an unusual explanation for acute arterial stroke and thrombocytopenia. One patient had preceding orthopedic surgery, an event previously reported with spontaneous HIT (Thromb Haemost 2013;109:669-75). The strong serum-dependent platelet activation at 0 IU/mL heparin helps to explain how thrombocytopenia and thrombosis can occur in a patient not receiving heparin. RECOMMENDATION. Based on the serological findings of these and previous cases, we propose that a definitive diagnosis of spontaneous HIT syndrome should be based upon all of the following criteria: thrombocytopenia, thrombosis, lack of proximate heparin exposure, strong-positive PF4-dependent immunoassay(s), and a strong-positive platelet activation assay featuring both heparin-dependent (e.g., high heparin neutralization) and heparin-independent platelet activation (at 0 IU/mL heparin). Disclosures: Warkentin: Pfizer Canada: Honoraria; Paringenix: Consultancy; Immucor GTI Diagnostics: Research Funding; WL Gore: Consultancy; GSK: Research Funding.

scholarly journals A Humanized Anti FcγRIIa Scfv Inhibits Platelet Activation, Aggregation and Thrombus Formation in Heparin-Induced Thrombocytopenia (HIT)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 10-10
Author(s):  
Jose Perdomo ◽  
Jaa Yien New ◽  
Zohra Ahmadi ◽  
Xing-Mai Jiang ◽  
Beng H Chong
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Immune Complexes ◽  
Whole Blood ◽  
Conflicts Of Interest ◽  
Dependent Manner ◽  
Single Chain ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Micro Fluidics

Abstract Introduction. Heparin is widely used as an anticoagulant to prevent thrombosis and to treat venous thromboembolism and myocardial infarction. A complication of heparin use is the development of heparin-induced thrombocytopenia (HIT), which is a limb- and life-threatening disorder due to associated thrombotic events. HIT arises through the formation of immune complexes between heparin, platelet factor 4 and HIT autoantibodies. These immune complexes engage with FcγRIIa receptors on platelets, leading to platelet activation and aggregation and subsequent initiation of the coagulation pathway. Current HIT treatment consists of cessation of heparin administration and substitution with parenteral anticoagulants such as argatroban and danaparoid. While these anticoagulants are generally beneficial in reducing thrombocytopenia, they are only partially effective since the risk of thrombosis continues due to the underlying FcγRIIa-mediated platelet activation. Thus, alternative anticoagulants do not reduce morbidity and mortality rates, highlighting the need for more effective HIT interventions. Methods. IV.3 is a monoclonal antibody that recognizes and blocks the FcγRIIa receptor and is used in assays to confirm the presence of HIT antibodies. We derived the VH and VL sequences of IV.3 and constructed a single-chain variable fragment (scFv) antibody in the form of VH-linker-VL. Using a complementarity determining region grafting and point mutation approach the scFv was humanized with the aim of reducing potential immunogenicity for future clinical applications. The molecule was expressed in E. coli and purified by FPLC. We reconstituted the HIT condition in a micro-fluidics device on a Vena8 Fluoro+ biochip coated with vWf using whole blood flowing at 20 dyne/cm2 at 37oC. Whole blood was stained with DiOC6 and the formation of platelet aggregates was monitored by fluorescence microscopy. Video images were acquired at 1 frame every 2 sec for 460 sec. Results. The purified scFv interacts with FcγRIIa on platelets. Platelet aggregation and serotonin release assays show that the scFv effectively prevents aggregation and activation induced by HIT immune complexes. We demonstrate that in the HIT condition reconstituted in a micro-fluidics system the scFv precludes thrombus deposition in a dose-dependent manner as determined by thrombus coverage area and mean thrombus diameter (Figure 1). Conclusions. These data provide evidence that a humanized scFv binds and neutralizes FcγRIIa on platelets. This interaction prevents HIT immune complex-induced platelet aggregation and activation in vitro and stops thrombus deposition ex vivo. This molecule, therefore, inhibits a critical initiating event in HIT and may serve as a potential treatment for this condition. Disclosures No relevant conflicts of interest to declare.

scholarly journals Clinical Features of Heparin-Induced Thrombocytopenia Diagnosed By Lumi-Aggregometry

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4185-4185
Author(s):  
Elona Turley ◽  
Artur J. Szkotak ◽  
Irwindeep Sandhu ◽  
Cynthia M Wu
Keyword(s):  
Platelet Activation ◽  
Light Flash ◽  
Patient Characteristics ◽  
Serotonin Release ◽  
Radioactive Isotopes ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Platelet Counts ◽  
Dense Granules ◽  
Heparin Exposure

Abstract Background: Heparin-induced thrombocytopenia (HIT) is associated with anti-platelet factor-4 (PF4)/heparin antibodies that activate platelets, resulting in thrombocytopenia and a pro-thrombotic state. At our institution antibody-mediated platelet activation is demonstrated by lumi-aggregometry, which is a method previously validated against the gold standard serotonin-release assay (SRA). Lumi-aggregometry does not involve radioactive isotopes, which is its major advantage over the SRA. The clinical course of HIT diagnosed via SRA and ELISA has been previously described, and clinical prediction tools such as the 4-T score were validated using these diagnostic tests. However, the clinical picture of HIT diagnosed by lumi-aggregometry has not been previously described. Aims: The objective of this study is to describe the clinical and laboratory presentation of patients diagnosed with HIT by lumi-aggregometry. Methods: Patients with clinically suspected HIT and quantitative anti-PF4 IgG-specific ELISA OD ≥0.400 (Gen-Probe, San Diego) received confirmatory HIT testing by lumi-aggregometry. Briefly, HIT antibody-induced activation of washed healthy donor platelets was tested at therapeutic (0.1U/mL and 0.5U/mL) and high (100U/mL) porcine heparin concentration. The degree of platelet activation was quantitated luminographically based on the light flash reaction of ATP (released from platelet dense-granules) with luciferin luciferase reagent. A ratio of therapeutic to high heparin luminescence amplitude of >5.0 and platelet aggregation at therapeutic, but not high, concentrations was considered a positive result. The results of assays performed by our regional HIT testing referral laboratory from June 2009 to July 2012 were reviewed to identify patients with positive HIT testing by lumi-aggregometry. Patient records were retrospectively reviewed to obtain predefined data on baseline patient characteristics, heparin exposure, platelet counts, and thrombotic events occurring in the 5 days preceding or the 30 days following the date of positive HIT testing. Results: We identified 43 patients diagnosed with HIT by lumi-aggregometry (median age 68.0, 49% male) while under the care of local academic (46%) or urban community hospitals (37.2% medical; 53.5% surgical; 9.3% intensive care). Median baseline platelet count was 187 (14-349). Median date of platelet drop post-heparin exposure was 6 days (range 3-14) in patients without prior heparin exposure or platelet transfusions (Figure 1). Platelet drop >50% and platelet nadir ≥20x109/L were present in the majority of patients (Table 1). Thrombocytopenia occurred prior to (70.5%) or the same day (23.5%) as thrombosis in 16/17 patients with serial platelet counts who developed HIT-associated thromboembolism. Conclusion: Patients diagnosed with HIT by lumi-aggregometry present with similar findings to those described in SRA-confirmed HIT. These findings lend support to the use of lumi-aggregometry as an accurate diagnostic assay for the clinico-pathologic syndrome of HIT. Figure 1 Figure 1. Table 1. Percentage platelet drop from baseline and platelet nadir Percent platelet drop Platelet nadir >50% 30-50% <30% ≥20 x 109/L 28 3 2 10-19 x 109/L 5 1 0 <10 x 109/L 1 1 0 Disclosures Szkotak: Alexion Pharmaceuticals: Research Funding. Sandhu:Celgene: Honoraria; Jansen: Honoraria; Novartis: Honoraria.

Abstract 451: Platelet Activation and Thrombosis by Antibodies against VEGF and CD40 Ligand: Extending the mechanism of HIT

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Ali Amirkhosravi ◽  
Todd Meyer ◽  
Florian Langer ◽  
Theresa Robson ◽  
Liza Robles ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Cd40 Ligand ◽  
Serotonin Release ◽  
Heparin Binding ◽  
Wild Type ◽  
Platelet Factor ◽  
Cardiovascular Comorbidity ◽  
Platelet Aggregometry

Introduction: The vascular endothelial growth factor (VEGF) monoclonal antibody, bevaci-zumab (Avastin), has been associated with arterial thromboembolic events in some cancer patients. Another therapeutic antibody, hu5c8, which targets CD40L, also produced unexpected thrombosis in lupus clinical trials. Because platelets play a crucial role in arterial thrombosis, we hypothesized that antibodies against VEGF and CD40L may activate platelets via a mechanism similar to that responsible for thrombosis in Heparin-Induced Thrombocytopenia (HIT). Methods: Immune complexes (ICs) were prepared by combining these monoclonal IgG antibodies with their antigens: M90 (or hu5c8) with CD40L (“M90+CD40L”), or bevacizumab with VEGF and heparin (“BVH”). VEGF binds heparin (as does platelet factor 4, the HIT antigen). We measured platelet activation by serotonin release assay, platelet aggregometry, and flow cytometry. We also evaluated IC-induced thrombosis in hFc mice, transgenic for the human IgG receptor, CD32. Results: Similar to HIT antibodies, these ICs potently induced platelet activation dependent on CD32, IC concentration (>10nM) and optimal stoichiometry. Intravenous injection of M90+CD40L or BVH into hFc, but not wild-type mice rapidly produced signs of thrombotic shock, thrombocytopenia and pulmonary thrombosis. However, wild-type control mice (lacking platelet CD32) were unaffected by IC injection. Similarly as with HIT antibodies, bevacizumab IC activity was reduced in the absence or excess of heparin both in vitro and in vivo, whilst M90+CD40L-induced platelet activation was abolished in vitro by blockade of the platelet CD40L receptor, CD40, demonstrating a requirement for Fab-dependent anchoring. Furthermore, VEGF 121 (which lacks the heparin-binding domain of VEGF 165 ) and bevacizumab with or without heparin failed to activate platelets or cause thrombosis in hFc mice. Conclusions: Together, these findings demonstrate that Fab-dependent anchoring of anti-CD40L and anti-VEGF ICs is required for potent platelet activation and thrombosis, as is the case in HIT, suggesting common mechanistic elements. Clinical implications may apply in patients with cardiovascular comorbidity receiving immunotherapy.

Sign in / Sign up

Export Citation Format

Share Document