Pathogenic Heparin-induced Thrombocytopenia Research Articles

Treatment of thrombocytopenia and thrombosis in HIT in mice using deglycosylated KKO: a novel therapeutic?

AbstractHeparin-induced thrombocytopenia (HIT) is characterized by thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human platelet factor 4 (hPF4) complexed with various polyanions. Although nonheparin anticoagulants are the mainstay of care in HIT, subsequent bleeding may develop, and the risk of developing new thromboembolic events remain. We previously described a mouse immunoglobulin G2bκ (IgG2bκ) antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4–polyanion complexes. KKO, like HIT IgGs, activates platelets through FcγRIIA and induces complement activation. We then questioned whether Fc-modified KKO could be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). Although DGKKO retained binding to PF4–polyanion complexes, it inhibited FcγRIIA-dependent activation of PF4-treated platelets triggered by unmodified KKO, 5B9 (another HIT-like monoclonal antibody), and IgGs isolated from patients with HIT. DGKKO also decreased complement activation and deposition of C3c on platelets. Unlike the anticoagulant fondaparinux, injection of DGKKO into HIT mice lacking mouse PF4, but transgenic for hPF4 and FcγRIIA, prevented and reversed thrombocytopenia when injected before or after unmodified KKO, 5B9, or HIT IgG. DGKKO also reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis induced by IgG from patients with the HIT-related anti-PF4 prothrombotic disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT.

PB2302: HEPARIN-INDUCED THROMBOCYTOPENIA ANTIBODIES CAN MAINTAIN PLATELET-ACTIVATING PROPERTIES AFTER TWO SESSIONS OF THERAPEUTIC PLASMA EXCHANGE

Background: Therapeutic plasma exchange (TPE) has been used as a salvage therapy in a small subset of heparin-induced thrombocytopenia (HIT) patients (severe or refractory HIT, before heparin re-exposure during cardiac surgery, in bleeding patients and in autoimmune HIT). Existing data show that HIT antibodies can lose platelet-activating properties after one or two TPE sessions, while more sessions may be needed to decrease the antibody titer. However, results may be assay dependent. The utility of Heparin-Induced Multiple aggregometry (HIMEA) in this setting has not been evaluated. Aims: To assess the effect of TPE on pathogenic HIT antibodies using HIMEA before an emergency surgery for gynecologic malignancy. Methods: TPE was performed in a 72-year-old patient necessitating emergency surgery for a voluminous uterine sarcoma in the acute phase of HIT (10 days after diagnosis). TPE aimed to reduce post-operative thrombotic risk by decreasing pathogenic HIT antibodies in a patient presenting a high risk of post-operative thrombotic complications. Two consecutive sessions of TPE using Spectra Optia ® Apheresis System were performed immediately before surgery. During each plasma exchange 2 L of plasma were removed and replaced by 5% human albumin. Acid-Citrate-Dextrose (ACD) was used as anticoagulant. Blood samples were taken before and after each TPE session (TPE1 pre, TPE1 after, TPE2 pre, TPE2 after). Removed patient’s plasma collected to the plasmapheresis bag was also tested. IgG anti-PF4/heparin antibodies tested with Enzyme Immunosorbent Assay (EIA) (Hyphen ZYMUTEST™ HIA IgG, Hyphen Biomed) and Heparin-Induced Multiple aggregometry (HIMEA) (Multiplate, Roche) in the absence of heparin (HIMEA saline), in the presence of 1UI/ml unfractionated heparin (UFH) (HIMEA low) and in the presence of 100IU/ml UFH (HIMEA high) were assessed. Results: Patient’s 4T’s score was 7 (2 for platelet count fall, 2 for timing, 2 for catheter associated venous thrombosis and 1 for other causes), strongly positive IgG anti-PF4 antibodies (OD: 2.43, cut-off<0.3 units) and a HIMEA profile favoring HIT (typical sigmoid curve at HIMEA low, >50% decrease of AUC at HIMEA high, absence of platelet aggregation at HIMEA saline). She was treated with fondaparinux at 1.5 mg/ day due to sepsis-associated acute kidney injury requiring hemodialysis. Platelet count (PLT) were 168 G/l before surgery. After two TPE sessions antibody titers remained unchanged (TPE1 pre: 2.63 units, TPE1 after:2.19 units, TPE2 pre:2.51 units, TPE2 after:5.52 units). Moreover, platelet reactivity remained also strong as demonstrated by high area under the curve (AUC) values in HIMEA low (figure 1). Plasma from the collection bag showed strong EIA and platelet activation test reactivity. No thrombosis nor thrombocytopenia were observed in one month follow-up. Image:Summary/Conclusion: Two TPE sessions failed to reduce platelet-activating properties of HIT antibodies as assessed by HIMEA in a cancer patient. TPE could be considered as a therapeutic intervention that can reduce thrombotic risk in patients necessitating an urgent non-cardiac surgery in the acute phase of HIT. However, more data is needed on the clinical benefit and the optimal number of TPE sessions in this clinical setting. Functional assays assessing Fcγ receptor mediated platelet activation may help guide the timing of the surgery.

A Thrombospondin-1 Release Assay (TRA) Coupled to PF4-Treated Cryopreserved Platelets for the Detection of Pathogenic HIT and VITT Antibodies

Background: Platelet-activating anti-PF4 antibodies that cause thrombocytopenia and thrombosis are implicated in Heparin-induced thrombocytopenia (HIT) and, more recently, in a newly recognized entity, vaccine-induced immune thrombotic thrombocytopenia (VITT). Current frontline PF4-Polyanion ELISAs utilize simple techniques but are highly non-specific. Functional gold-standard assays such as the serotonin-release assay (SRA) and PF4-dependent P-selectin expression assay (PEA) have limited availability due to technical complexity and the requirement for freshly-drawn normal donor platelets which results in significant delays in diagnosis. Thus, there is an unmet need for near-patient, functional assays for HIT/VITT diagnosis.Aim: To develop a highly sensitive and specific functional assay to detect platelet-activating antibodies associated with HIT and VITT using long-term stored platelets. The assay should be technically simple, avoiding current functional HIT testing endpoints such as aggregometry, radioactivity measurement or flow cytometry.Methods: Platelets were subjected to controlled-rate freezing in a trehalose-based buffer. Cryopreserved platelets were then thawed after variable storage periods, incubated with platelet factor 4 (PF4), and sera from HIT or VITT suspected patients. Thrombospondin-1 (TSP1), a protein highly expressed in platelet α-granules, was quantified from activated platelet supernatants in an ELISA-based assay as a measure of platelet activation.Results: In initial studies, p-selectin expression was induced by platelet-activating HIT antibodies using cryopreserved platelets and the TSP1-release assay (TRA) detected HIT antibody-mediated PF4-dependent cryopreserved platelet activation across 20 different platelet lots (data not shown). A representative study using platelets stored for 11 months is shown in Figure 1A. A cohort of thirty-six HIT-suspected patient sera, half with PF4-dependent platelet-activating antibodies (PEA+) and without (PEA-), were used to evaluate the diagnostic accuracy of the TRA (Figure 1B). All 18 PEA+ samples activated platelets strongly with a mean TSP1 concentration of 147 µg/mL measured in the supernatant compared to a mean of 43 µg/mL obtained with non-activating samples. TSP1 release in a subgroup of 7 PEA+/SRA+/ELISA+ and 4 PEA-/SRA-/ELISA+ samples that were matched for ELISA Optical Density was examined (Figure 1C). Results demonstrated that even strong ELISA+ samples that were non-activating (PEA-/SRA-) did not induce platelet activation in the TRA, confirming high specificity of the assay. The TRA was tested for its ability to detect platelet-activating antibodies in four patients who developed VITT after the Janssen vaccine (Ad26.COV2.S). All samples induced high levels of TSP1 release from cryopreserved platelets (Figure 1D).Conclusions: Our results demonstrate that cryopreserved platelets stored for several months are viable and capable of degranulation upon activation. Further, we demonstrate that measurement of TSP-1 released from cryopreserved, PF4-treated platelets is highly accurate for detecting platelet-activating HIT and VITT anti-PF4 antibodies. Specificity and sensitivity were both 100% even with samples that were strongly false-positive in the HIT ELISA. Coupling cryopreserved platelet activation to a simple ELISA endpoint, as demonstrated here, precludes the need for fresh platelets and complex testing techniques. Thus, the TRA has the potential to transform the diagnostic testing paradigm in HIT and VITT by making near-patient in-hospital functional testing available for rapid and accurate diagnosis.Figure Legend:Figure 1. TSP-1 quantification of (A) 3 HIT patient samples (closed circles) relative to 3 normal control samples (open circles) after 11-months of frozen platelet storage. (B) Thirty-six HIT-suspected patient samples: 18 PEA+ samples (closed circles), and 18 PEA- samples (open squares). Data are compared using two-tailed, unpaired Student's t test. **** P<0.0001. (C) Eleven high optical density matched HIT ELISA patient sera: 7 PEA+/SRA+ samples (closed circles) and 4 PEA-/SRA- samples (open squares). (D) TSP-1 quantification of VITT patient sera. NC-normal control. [Display omitted] DisclosuresPruthi: Instrumentation Laboratory: Honoraria; HEMA Biologics: Honoraria; Bayer Healthcare AG: Honoraria; Genentech: Honoraria; CSL Behring: Honoraria; Merck: Honoraria. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

The Identification of the Amino Acids on Platelet Factor 4 That Bind Pathogenic Antibodies in Heparin-Induced Thrombocytopenia

Introduction: Heparin induced thrombocytopenia (HIT) is an adverse drug reaction that occurs when heparin binds to platelet factor 4 (PF4) and forms immunogenic multimolecular complexes. As a result, anti-PF4/heparin IgG antibodies bind to the PF4/heparin complexes, leading to cross-linking of FcγIIa receptors on platelets and FcγRI on monocytes resulting in their activation and an increased risk of thrombosis. Approximately 50% of cardiac surgery patients produce anti-PF4/heparin antibodies, but only a subset (<1%) produces pathogenic HIT antibodies. Current enzyme immunoassays (EIAs) cannot distinguish between pathogenic and non-pathogenic anti-PF4/heparin antibodies and will give a positive test even in the presence of the clinically insignificant non-pathogenic HIT antibodies. Further functional testing, such as the 14C-serotonin release assay (SRA), is required to identify samples containing the pathogenic anti-PF4/heparin antibodies that will lead to HIT. In this study, we hypothesized that different epitopes on PF4 could explain some of the differences between pathogenic and non-pathogenic antibodies.Methods: We used alanine scanning mutagenesis (ASM) to produce 70 mutations of PF4, each with a unique amino acid substitution. Single point mutations were made by mutating non-alanine residues to alanine, and alanine residues to valine. The PF4 mutants were isolated from Escherichia coli and used in an EIA. The binding capacity of the monoclonal pathogenic HIT antibody (KKO) and a non-pathogenic anti-PF4/heparin antibody (RTO) to each of the PF4 mutants, relative to wild-type PF4, was determined. Point mutations of PF4 that resulted in >25% loss of binding were identified. We compared our candidate amino acids to binding sites previously identified in the PF4 and KKO crystal structure. In addition, we screened nine EIA-positive/SRA-positive (pathogenic) and six EIA-positive/SRA-negative (non-pathogenic) patient samples using the PF4 mutants. Student's t-test was used to determine whether the binding capacities of each amino acid was significantly different between the two patient sample groups.Results: We identified 12 amino acid mutations of PF4 that caused ≥25% reduction in KKO binding. Most implicated amino acids cluster in two regions within the PF4 molecule. We identified amino acids on PF4 that were required for KKO binding which were not previously identified based on crystallographic studies. These results are consistent with the previous mutagenesis and crystallographic studies (Cai et al, Nat Commun. 2015;6:8277) of the KKO and PF4 complex but also contradict some previously stated amino acids that were thought to be part of this binding site. ASM of RTO and PF4 revealed only one amino acid that was affected. We used pathogenic and non-pathogenic EIA-positive human sera to further refine the location of the binding sites. We identified 10 amino acids that were required for the binding of human pathogenic HIT sera.Conclusions: Using sequential point mutations, we identified the amino acids that are critical for the binding of pathogenic HIT antibodies to PF4. These amino acids are either directly involved in antibody binding or influence the antigenic conformation of PF4. This library of PF4 mutant proteins can help differentiate pathogenic and non-pathogenic HIT antibodies and this information can improve diagnostic assays.This study was funded by the Canadian Institutes for Health Research. DisclosuresArnold:Amgen: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding; Dova: Consultancy; Novartis: Consultancy, Research Funding; UCB: Consultancy; Rigel: Consultancy.

A New Assay for Platelet-Activating HIT Antibodies, the PEA, Is Specific, Reproducible and Relatively Independent of the Choice of Platelet Donor

A New Assay for Platelet-Activating HIT Antibodies, the PEA, Is Specific, Reproducible and Relatively Independent of the Choice of Platelet Donor

Platelet Factor 4 Binds to Released Von Willebrand Factor Strands from Injured Endothelium and Forms a Prothrombotic HIT Antigenic Complex

Heparin-induced thrombocytopenia (HIT) is driven by the binding of pathogenic HIT antibodies to platelet factor 4 (PF4) complexed to heparin. We, and others, have shown that PF4 bound to a variety of other polyanionic molecules, like surface glycosaminoglycans, also form HIT antigenic complexes. This is likely due to the formation of high molecular weight aggregates by the PF4. While studying the pathogenesis of HIT in a microfluidic system lined with human umbilical endothelial cells (HUVEC), we noted that PF4 did not bind diffusely to the HUVECs after a hematoporphyrin-induced photochemical injury as one would expect if binding were to the injured surface GAGs, but rather in a linear pattern described previously for von Willebrand Factor (vWF) under flow. We confirmed this by concurrent staining using PF4 and vWF-specific labeled antibodies. We also demonstrated that the HIT-like monoclonal antibody KKO, as well as IgG isolated from patients with likely HIT as determined by clinical assessment and ELISA positivity and positive serotonin release assay, bound to these PF4-vWF complexes. The HIT-like complexes were not uniformly distributed along the vWF, but occurred in a periodic fashion along thicker vWF strands. This suggested that the complexes were composed of multiple molecules of PF4 and vWF forming cross-chained high molecular weight aggregates that induce or enhance binding of HIT antibodies as previously reported for PF4/heparin in solution. We then wanted to identify the potential for therapeutics to intervene with PF4-vWF-KKO complex formation or stability. We tested this by introducing heparin; 2-0,3-0 desulfated heparin (ODSH); ADAMTS13 or n-acetylcysteine into channels post-complex formation. All interventions at therapeutically relevant concentrations were able to significantly remove or disrupt complex formation. When whole human blood + PF4 + KKO were flowed through channels lined with injured endothelium, large platelet aggregates developed within 10 mins along the extended strands of vWF. Significantly fewer platelet aggregates formed in the absence of PF4 or with KKO alone or PF4 plus an isotype control TRA. After 1 hour of flow, aggregates in the PF4 + KKO channels had grown significantly, but had largely disappeared in the other settings. Furthermore, by adding the FcgammaRIIA blocking antibody, IV.3, platelet binding could be reduced to background levels. These studies identify large vWF polymeric strands as a binding site for PF4 and a HIT antigen target. The Fc portions of the bound HIT antibody then contribute to platelet binding to the vWF. We are presently examining whether PF4-vWF-KKO aggregates contribute to thrombotic complications in HIT and defining the vWF domain(s) involved as these targets offer a potential novel intervention target in HIT. [Display omitted] DisclosuresCines:Amgen: Consultancy; Ionic: Consultancy; Juno: Consultancy; Astellas: Consultancy; T2 Biosystems: Research Funding; Rigel: Consultancy; Syntimmune: Research Funding.

New Monoclonal Antibodies Reveal Distinct Specificities for Platelet Factor 4 Bound to Heparin Vs. Platelets: Implications for HIT Pathogenesis

Heparin induced thrombocytopenia (HIT) is a severe adverse reaction to heparin treatment characterized by antibodies to PF4/polyanion complexes. However, HIT antibodies are heterogeneous. Some bind to heparin/polyanion complexes in solid-phase enzyme-linked immunoassay (EIA) but do not activate platelets; other EIA-positive antibodies also activate platelets in assays such as the serotonin release assay (SRA) and in the recently described PF4-dependent p-selectin expression assay (PEA). It is generally thought that only platelet-activating antibodies are pathogenic. On the basis of studies which showed that platelet-activating antibodies, but not those positive only in EIA recognize PF4 bound to platelets (Blood. 2015;125(1):155-61 and others), we hypothesized that pathogenic HIT antibodies may recognize epitopes exposed on PF4 when it binds to a target, possibly chondroitin sulfate, on the platelet surface that are not created when PF4 forms a complex with heparin or other polyanions. Here, we describe studies utilizing newly developed monoclonal antibodies (mAbs) that demonstrate the feasibility of this concept and suggest that HIT antibodies having this specificity could be important in HIT pathogenesis. mAbs were produced in mice immunized with PF4/heparin complexes. We employed a novel dual screening strategy for candidate clones utilizing the PF4/polyanion EIA as well as the PEA. mAbs selected for study were designated 431.3, 431.4 and 447.1 and were compared with KKO, a widely studied HIT-like mAb (generous gift from G Arepally) and an isotype control (IC; Fig 1A). In the EIA, KKO strongly recognized PF4/polyvinylsulfonate (PF4/PVS) but not PF4 alone and was inhibited by high-dose heparin (100U/mL). mAb 447.1 had an EIA profile similar to KKO but with significantly lower optical densities (0.56 vs. 2.43 OD) and 431.3 gave reactions indicating it recognizes PF4 alone but demonstrated much stronger reactivity to PF4/PVS complexes (1.96 OD). However, mAb 431.4, similar to isotype control, was completely non-binding in the EIA (Fig 1A). In contrast, all 4 mAbs strongly recognized PF4-treated platelets (Fig 1B). In addition, all four mAbs produced strong positive reactions in the PEA in which mAbs interact with PF4-primed platelets to induce p-selectin expression (Fig 1C). The EIA requires multiple washing steps, whereas studies performed to assess IgG binding to PF4-treated platelets utilized only a single wash and the PEA is a "no-wash" assay. Thus, failure of 431.4 to react in EIA could be explained by susceptibility of the mAb to being removed by washing, possibly due to low avidity for its target. We therefore studied mAb binding to PF4/heparin complexes in real time using Bio-layer interferometry (Octet system, Pall ForteBio). In this system, mAbs 431.4 and KKO produced a steadily increasing signal, reaching saturation, whereas 431.4 demonstrated no binding to PF4/heparin consistent with results seen in EIA (Fig 1D). These findings indicate that mAb 431.4 binds to PF4 bound to the platelet surface and induces platelet activation as measured by p-selectin expression but fails to react with PF4 in a complex with heparin or PVS. This behavior contrasts strikingly with that of the "HIT-like" mAb KKO that reacts in both assays. Although antibodies from patients with HIT frequently give positive reactions in both platelet-activating assays and in EIA, there are clear examples of antibodies that are positive in one assay and not the other (e.g. EIA-positive, SRA-negative antibodies are common in clinical practice). Thus in HIT patients, multiple antibodies with different specificities are undoubtedly present. Our data support the growing body of evidence that while strength of the reaction in EIA (optical density) is often correlated with platelet-activating antibodies, there are notable exceptions (such as mAb 447.1). These results suggest that studies to isolate and characterize human antibodies according to specificity and reaction strength are needed to define the extent to which antibodies like mAb 431.4 and 447.1 contribute to the pathogenesis of HIT. Disclosures Padmanabhan: Versiti Wisconsin: Patents &amp; Royalties: Related to HIT patents; Retham Technologies: Equity Ownership; Terumo BCT: Consultancy; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen R&amp;D: Consultancy. Jones:Retham Technologies: Equity Ownership; Versiti Wisconsin: Patents &amp; Royalties.

Understanding How Fc-Modification Transforms a Pathogenic Heparin-Induced Thrombocytopenia (HIT)-like Monoclonal Antibody into a Novel Treatment for Sepsis

Arepally: Biokit: Patents & Royalties; Apotex Pharmaceuticals: Consultancy; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Pathogenic Antibodies in Heparin-Induced Thrombocytopenia Specifically Target an Immunodominant Region on Platelet Factor 4

Introduction: Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that occurs when heparin binds to platelet factor 4 (PF4) forming immunogenic complexes. Anti-PF4/heparin IgG antibodies bind PF4/heparin complexes, leading to cross-linking of FcγRIIa receptors on platelets and FcγRI on monocytes, resulting in platelet activation, thrombocytopenia, and thrombosis. The current diagnostic challenge is that the majority of patients suspected of HIT yield false-positive results in immunoassays, since up to 50% of patients will make anti-PF4/heparin antibodies but will not develop HIT. The antibody response in HIT patients is polyclonal, making it difficult to identify a common pathogenic epitope. The disparities between anti-PF4/heparin antibodies that activate platelets (pathogenic HIT antibodies) and those that do not (non-pathogenic anti-PF4/heparin antibodies) present a significant challenge in diagnosing HIT. The objective of this study was to map and characterize the critical immunodominant region on PF4 for the binding of pathogenic antibodies in confirmed HIT patients. Methods: We used sera with anti-PF4/heparin antibodies from patients with confirmed HIT (n=10). Post-cardiopulmonary bypass patients (CPB; n=10) and healthy individuals (n=10) were used as controls. Confirmed HIT patients met clinical criteria (4Ts ≥ 4) and tested positive in both the anti-PF4 IgG/A/M immunoassay (OD &amp;gt; 0.4; range 2.33 - 3.90) and in the serotonin release assay (SRA release &amp;gt; 20%; range 88-100%). CPB patients all received heparin but did not develop HIT, tested positive in the anti-PF4 IgG/A/M immunoassay (OD &amp;gt; 0.4; range 0.42 - 2.73), and tested negative in the SRA (SRA release &amp;lt; 20%; range 0-18%). We previously used alanine scanning mutagenesis and identified 30 amino acids that were on the surface of PF4 and were likely a part of the region essential for the binding of pathogenic HIT antibodies. From those results, we used the panel of 30 PF4 mutants and tested their ability to bind to HIT, CPB, and healthy control sera. Loss of binding to PF4 mutants was applied to in-silico structural analysis to determine binding regions specific for pathogenic and non-pathogenic antibodies. We also determined binding affinities of pathogenic and non-pathogenic anti-PF4/heparin antibodies using biolayer interferometry (BLI). Results: When 30 PF4 mutants were used to test the effect of the amino acid changes on the binding of HIT and CPB patient sera, an average of 8 different PF4 mutants resulted in more than 35% loss of binding to confirmed HIT sera when compared to wild-type PF4. None of the 30 PF4 mutants resulted in more than 35% loss of binding to CPB sera. Structural analysis demonstrated that the amino acids of PF4 that significantly affected the binding of HIT sera, but not CPB sera, were clustered to a specific region on PF4, similar to the region of KKO, but with varying epitopes. Using BLI, anti-PF4/heparin antibodies of confirmed HIT patients had a stronger binding response to PF4 and PF4/heparin than that of CPB patients and healthy controls. Overall, we were able to show a significant difference between confirmed HIT sera and the false-positive antibodies of CPB patients that did not develop HIT (P &amp;lt; 0.01). Conclusion: This work shows that among the polyclonal response in HIT, pathogenic HIT antibodies must bind to the critical immunodominant region on PF4 with high affinity. This ensures the proper spatial configuration of the antibodies for Fc-receptor cross-linking, platelet activation, and subsequently HIT. This study has implications for the development of novel epitope-targeted diagnostic and therapeutic approaches for HIT. Disclosures Arnold: Novartis: Honoraria, Research Funding; Rigel: Consultancy, Research Funding; Principia: Consultancy; Bristol-Myers Squibb: Research Funding.

Laboratory diagnosis of heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a clinical-pathological disorder; thus, laboratory testing for the pathogenic platelet-activating antiplatelet factor 4 (PF4)/heparin antibodies is central for diagnosis. The "iceberg" model summarizes the inter-relationship between platelet activation assays and PF4-dependent immunoassays, with platelet-activating antibodies comprising a subset of anti-PF4/heparin antibodies. The platelet serotonin-release assay (SRA), performed by reference laboratories, has high sensitivity and specificity for HIT (~95% each), and is especially suited for detecting highly pathogenic HIT sera containing both heparin-dependent and heparin-independent platelet-activating antibodies; this latter subgroup of antibodies explains "autoimmune HIT" disorders (delayed-onset, persisting, spontaneous, heparin "flush," fondaparinux-associated). Recently, SRA-negative HIT has become recognized, in which serum from some HIT patients contains subthreshold levels of platelet-activating antibodies (by SRA) that become detectable using a PF4-enhanced platelet activation assay. Unusual immunologic features of HIT include early antibody detectability (at onset of platelet count fall) and antibody transience (seroreversion). Widely available PF4-dependent enzyme immunoassays (EIAs) have high sensitivity but poor specificity for HIT, although specificity is enhanced with IgG-specific EIAs and strong positive results; unfortunately, EIA results are usually not available in real time. Automated rapid immunoassays, such as the chemiluminescence immunoassay (CLIA) and latex immunoturbidimetric assay (LIA), facilitate real-time laboratory diagnosis. Recently available likelihood ratio (LR) data for positive (LR+) and negative (LR-) test results allow clinicians to adjust their pretest probabilities for HIT, using Bayesian analysis, into real-time posttest probabilities that are dramatically increased (test positive) or decreased (test negative). Moreover, (semi-)quantitative CLIA- and LIA-positive results (weak, moderate, strong positive) can further refine the posttest probability of HIT.

An international external quality assessment for laboratory diagnosis of heparin‐induced thrombocytopenia

Essentials A pilot study for External Quality Assessment for testing of HIT is described. The qualitative accordance for the PF4/heparin IgG test was 97.6%. The qualitative accordance for functional HIT tests was considerably lower. External Quality Assessment for functional HIT tests is required. SUMMARY: Objective Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening complication of heparin exposure. Diagnosis is most reliable using a combination of an enzyme immunoassay (EIA) that detects antibodies against platelet factor 4 (PF4)/heparin complexes ("antigen" assay) and a "functional" assay that detects platelet-activating properties of the pathogenic HIT antibodies. No External Quality Assessment (EQA) is available for a combination of the tests. Here we report on the results of the first international EQA. Methods The pilot EQA was organized by the Department of Transfusion Medicine, Universitätsmedizin Greifswald, Germany. Six serum samples of patients, which were referred to Greifswald for HIT diagnosis, and one negative control sample were distributed to seven participants in Germany, Canada, and Singapore. Participants were asked to report the optical density (OD) values of their local EIA test for IgG-specific antibodies against the PF4/heparin complexes and the results for a functional assay (HIPA or SRA). Consensus was defined as a minimum 70% agreement, i.e., agreement among at least five of the seven participating laboratories. Results and conclusion Six out of seven participants reported results for EIA, with a high quantitative accordance (97.6%). For the functional assay, consensus was reached for all samples except the negative control, for which some participants reported nonspecific reactivity. All HIT-negative samples were correctly diagnosed by all participants; for HIT-positive samples, consensus of 70% was reached. Although the limited availability of sample material is an obstacle to overcome, an EQA combining both EIA and functional testing is feasible.

Characterization of platelet factor 4 amino acids that bind pathogenic antibodies in heparin‐induced thrombocytopenia

Essentials Many patients produce antibodies but few lead to heparin-induced thrombocytopenia (HIT). Pathogenic epitopes are difficult to identify as HIT antibodies are polyclonal and polyspecific. KKO binding to platelet factor 4 (PF4) depends on 13 amino acids, three of which are newly observed. Five amino acids in PF4 can help distinguish pathogenic from non-pathogenic antibodies. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is an adverse drug reaction that results in thrombocytopenia and, in some patients, thrombotic complications. HIT is mediated by antibodies that bind to complexes of platelet factor 4 (PF4) and heparin. The antigenic epitopes of these anti-PF4/heparin antibodies have not yet been precisely defined, because of the polyspecific immune response that characterizes HIT. Objectives To identify PF4 amino acids essential for binding pathogenic HIT antibodies. Methods Alanine scanning mutagenesis was utilized to produce 70 single point mutations of PF4. Each PF4 mutant was used in an enzyme immunoassay (EIA) to test their capacity to bind a platelet-activating murine monoclonal anti-PF4/heparin antibody (KKO) and HIT patient sera (n=9). Results and Conclusions We identified 13 amino acids that were essential for binding KKO because they directly affected either the binding site or the antigenic conformation of PF4. We also identified 10 amino acids that were required for the binding of HIT patient sera and five of these amino acids were required for binding both KKO and the HIT patient sera. The 10 amino acids required for binding HIT sera were further tested to differentiate pathogenic HIT antibodies (platelet activating, n=45) and non-pathogenic antibodies (EIA-positive but not platelet activating, n=28). We identified five mutations of PF4 that were recognized to be essential for binding pathogenic HIT antibodies. Using alanine scanning mutagenesis, we characterized possible binding sites of pathogenic HIT antibodies on PF4.

Frequency of Anti-PF4/Heparin Antibodies in Patients Treated with Extra-Corporeal Membrane Oxygenation and Detection By Sensitized Serotonin Release Assay of Pathogenic Heparin-Induced Thrombocytopenia Antibodies

Introduction: Extra-Corporeal Membrane Oxygenation (ECMO) provides circulatory support in case of severe cardiac failure and is associated, as cardiopulmonary bypass, with strong activation of platelets, which likely leads to the release in patient blood of large amounts of platelet factor 4 (PF4). Therefore, systematic use of unfractionated heparin (UFH) for maintaining patency of circuits and to prevent thrombosis, exposes patients to heparin-induced thrombocytopenia (HIT). However, the risk of HIT in this particular clinical setting remains poorly documented.Objectives: In this monocentric study, the frequency of development of antibodies to PF4 modified by heparin (anti-PF4/H Abs) was prospectively evaluated in patients who had underwent ECMO, and the impact of these Abs on platelet count (PC) and clinical course was assessed.Patients and Methods: From February 2014 to January 2018, we enrolled in the University Hospital of Tours all consecutive adult patients with ECMO and treated with UFH for at least 5 days. Plasma samples were collected daily, and PF4-specific Abs were systematically detected using HAT45® (GTI), an ELISA assaying IgG, IgA and IgM isotypes, and another kit specific for IgG to modified PF4 (HAT45G®; GTI). Serotonin release assay (SRA) was also performed to detect platelet-activating Abs (and therefore potentially pathogenic) in plasma samples containing significant levels of anti-PF4/H IgG. Noticeably, SRA was also done after adding 10 μg/ml of PF4 in the reaction mixture (PF4-SRA), since we recently showed this modification might improve the detection of pathogenic HIT antibodies (Vayne et al, Brit J Haematol, 179(5):811-819, 2017). Demographics, PC, and clinical events were collected in all patients until the end of ECMO.Results: Fifty-seven patients with a median age of 57 years (range: 24-76) and who were mainly men (sex ratio W/M: 0.3) were enrolled. ECMO was performed due to medical issues (dilated cardiomyopathy, myocardial infarction, acute myocarditis) in 70 % of cases and in surgical settings in the others. Significant titres of anti-PF4/H IgG/A/M Abs were detected before ECMO in 6 of 57 patients (10.5%), with anti-PF4/H IgG present in only 3 cases (5%). After ECMO was initiated, 28 patients (49%) developed IgG/A/M anti-PF4/H Abs, with anti-PF4/H IgG present in 17 of them (30%). Half of these patients were immunized within 5 days after ECMO initiation and 95% of them within 10 days, with a median delay of 8 days before Abs detection (range: 4-26 days). Only 3 patients (5%) exhibited pathogenic anti-PF4/H IgG with positive SRA and the level of platelet release was always stronger when the test was performed with exogenous PF4 (SRA-PF4). Moreover, in one patient, pathogenic HIT Abs were detected earlier by PF4-SRA, i.e. 2 days before conventional SRA was positive. An abnormal PC pattern was evidenced in 2 of the 3 patients with positive SRA, with a dramatic PC fall after day 5 (of 49% and 31.5 % between days 9-10 and 5-6 respectively). The diagnosis of HIT was confirmed in these 2 cases, which also had suggestive clinical manifestations (arterial thrombosis and cutaneous necrosis). In the third patient, HIT was not suspected, because of low PC before ECMO with persistent thrombocytopenia post-operatively and early death on day 9. In the patients with negative SRA, the development of anti-PF4/H IgG did not appear to impact the biological and clinical evolution, with similar rates of thrombosis and death than in non-immunized patients. Therefore, patients with non-pathogenic anti-PF4/H IgG displayed the same PC pattern than non-immunized patients, with decreasing PC from ECMO initiation until day 5, followed by a slow PC recovery.Conclusion: The development of anti-PF4/H Abs is frequent during ECMO but these antibodies are rarely pathogenic, and not associated with a less favourable prognosis. PC evolution analysis during ECMO appears reliable for suspecting HIT, and a dramatic PC fall or a non-recovery of PC after day 5 should prompt to look for platelet-activating anti-PF4/H Abs. Therefore, IgG specific immunoassays should always be combined with platelet activation tests to improve the specificity of HIT diagnosis in this particular clinical condition. Interestingly, the addition of exogenous PF4 when performing SRA could allow an earlier detection of HIT pathogenic antibodies in patients undergoing ECMO. DisclosuresNo relevant conflicts of interest to declare.

Is There an Alternative to Serotonin Release Assay for the Diagnosis of Heparin Induced Thrombocytopenia (HIT)? Evaluation of 5 Other Functional Methods Using 5B9 a Monoclonal IgG That Mimics HIT Human Antibodies

Introduction: Serotonin Release Assay (SRA) is today considered as the “gold standard” to detect pathogenic Heparin-Induced Thrombocytopenia (HIT) antibodies. However, this method is time-consuming, expensive and necessitates the use of 14C-radio-labelled serotonin, this implicating a specific agreement and secured premises, with a non-negligible environmental impact. These limitations explain that the use of SRA is restricted to a few laboratories worldwide. Finding a more accessible method with similar performances is therefore a challenge, and other different functional assays, such as Heparin-Induced Multiple Electrode Aggregometry (HIMEA), Light Transmission Aggregometry (LTA) using platelet rich plasma (PRP) or washed platelet (WP), ATP release, and Flow Cytometry (FC), are available. However, the sensitivity of these assays has never been comparatively evaluated with a standardized reagent.Objectives:The objective of our study was therefore to evaluate the sensitivity of these 5 functional methods for the detection of HIT antibodies in comparison with SRA, using 5B9, a monoclonal chimeric anti-PF4/H IgG recently developed in our laboratory, which fully mimics the effects of human HIT antibodies (Kizlik-Masson et al, J Thromb Haemost, 2017).Material and Methods: Platelet activation induced by 5B9 with heparin was assessed by the 6 following methods with blood samples from 10 consecutive unselected healthy donors:•HIMEA performed with whole blood (Multiplate Analyzer® Roche),•LTA performed with PRP (Chronolog®, Chrono-Log corporation),•FC based on the assessment of P-selectin expression and performed with PRP (HIT Confirm®, Emosis on AccuriC6 plus®, Becton Dickinson),•ATP release performed with WP (Chronolog®, Chrono-Log corporation),•LTA performed with WP (Chronolog®, Chrono-Log corporation),•SRA performed with WP (LSC scintillation counter, Perkin Elmer).For each method, different concentrations of 5B9 (10-20-50 µg/mL) were tested without heparin, and with “therapeutic” or high concentrations of unfractionated heparin (ranging from 0.1 to 1 and from 10 to 200 IU/mL respectively, according to the functional assay performed). The 3 concentrations of 5B9 were previously defined as “low” (10 µg/mL inducing in most cases a serotonin release <50% and no platelet aggregation in PRP), “high” (50 µg/mL always inducing a serotonin release >50% and platelet aggregation in PRP) or “intermediate” (20 µg/mL yielding variable results).Results: With the highest concentration of 5B9 (50 µg/mL), a strong platelet activation was detected with all methods and donors tested. HIMEA exhibited similar sensitivity (Ss 100%) than SRA to detect the activation induced by 20 μg/mL 5B9. FC was also able to detect the effect induced by 20 μg/mL 5B9 with 9/10 donors tested (90%). Alternatively, the measurement of ATP release, and LTA performed with WP or PRP failed to detect the effect of 20 μg/mL 5B9 in 30, 30 and 40 % of donors tested, respectively. SRA was the only method able to detect platelet activation induced by 10 μg/mL 5B9 with all donors tested, and the other methods were less sensitive (table). LTA performed with PRP was always negative (Ss= 0%). Platelet washings increased LTA sensitivity for detecting 10 or 20 μg/mL 5B9 (40% and 70% with WP vs. 0 and 60% with PRP, respectively), and the measurement of ATP release exhibited similar sensitivity. When platelet activation was evaluated in whole blood by HIMEA or in PRP using FC, the sensitivity to detect HIT antibodies was also improved (60% and 50%, respectively).Conclusion: These results confirm that SRA is likely the more sensitive functional assay to detect low concentrations of HIT antibodies. Indeed, apart from SRA, none of the other methods was able to detect the lowest concentration of 5B9 with 100% of donors. Interestingly, FC or HIMEA, which are rapid assays, also exhibit a high sensitivity, close to 100%, for detecting “intermediate” concentrations of HIT antibodies (i.e. corresponding to 20 μg/mL 5B9). We will further study the performances of these functional tests, including their specificity, by assessing patient's samples with confirmed HIT or having developed non-pathogenic antibodies (study in progress). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Platelet Factor-4 (PF4)-Dependent p-Selectin Expression Assay (PEA) Is Highly Accurate for the Detection of Pathogenic HIT Antibodies: Results of a 440-Sample Prospective Blinded Study

Introduction: Heparin-induced Thrombocytopenia (HIT) is a potentially life and limb threatening pro-thrombotic syndrome triggered by antibodies to platelet factor 4 (PF4)/polyanion complexes. Commonly used screening tests such as the PF4/Heparin ELISA (PF4 ELISA), while easy to perform in the near-patient setting, are highly non-specific. In contrast, more accurate tests such as the serotonin release assay (SRA) are technically complex, require radioactivity and are performed in only a handful of reference laboratories. These diagnostic challenges lead to both unnecessary anticoagulant use as well as avoidable cases of severe bleeding. Recent reports suggest that a relatively simple three-hour flow cytometry-based test that uses PF4-treated normal platelets, the PEA (PF4-dependent p-selectin Expression Assay), is at least as accurate as the SRA for identifying patients who have HIT (Thromb Haemost. 2015 Nov 25;114(6):1322-3; Chest. 2016 Sep;150(3):506-15; Chest. 2017 Oct;152(4):e77-e80). Here we describe results of a large prospective study designed to compare the accuracy of the SRA and PEA for HIT diagnosis in a clinical setting.Methods: The PEA and SRA were performed on serum samples obtained from adult patients (≥18 yrs.) from 2 large tertiary care academic hospitals from May 2016 to Sep 2017. All inpatients for whom PF4 ELISA testing was ordered were included in the study. The PEA was performed with platelets pretreated with PF4 at 3 concentrations: 30mcg/mL (PEA30), 10mcg/mL (PEA10) and 3mcg/mL (PEA3). Sera were batch-tested in both assays (SRA and PEA) using the same target platelets. The central laboratory was blinded to clinical history and to the results of the PF4 ELISA performed by participating hospitals at the time of diagnosis. Both centers performed IgG-specific PF4 ELISAs and utilized manufacturer-recommended cut-offs for positivity. Predefined “more-stringent” and “less stringent” criteria based on clinical findings (4Ts score) and results of PF4 ELISA testing were used to define the likelihood of a patient having HIT (Table 1A). Receiver Operating Curve (ROC) analysis was performed and Area Under the Curve (AUC) of each assay was calculated to assess their diagnostic accuracy. Since high test specificity was desired, positive cut-offs that maximized the sum of sensitivity and specificity in a specificity range of ≥90% and ≥95% were derived. To evaluate non-inferiority of the PEA to the SRA, we assessed whether the lower limit of the one-sided 95% confidence interval of the ΔAUC (PEA-SRA) was higher than a non-inferiority margin of 5% and 10%.Results: A total of 440 patient samples were tested with both assays. Seventeen and 28 patients were classified as having true HIT based on the use of the more- and less-stringent criteria, respectively. When classified by more-stringent criteria, the AUC was numerically greater for each of the three PEA tests than for the SRA (Table 1B). All tests had excellent accuracy (AUC>0.90, “excellent” by the Traditional academic point system). Using less-stringent criteria, the PEA30 had the highest AUC estimate followed by the SRA (Table 1C). Positive cut-offs generated at test specificities of ≥90% and ≥95% using data classified by more stringent criteria are shown in Table 1D. PEA3 was non-inferior to the SRA at a 5% non-inferiority margin, and all PEA tests were non-inferior at the 10% margin using the more stringent classification for true HIT (Fig 1A). In the less-stringently classified data set, the PEA30 was non-inferior to the SRA at a 10% margin (Fig 1B).Conclusions: The PEA, a 3-hour, non-radioactive functional HIT assay demonstrated high diagnostic accuracy and was non-inferior to the SRA using commonly accepted non-inferiority margins for diagnostic testing (5-10%). The technical advantages of the PEA over the SRA may encourage its widespread adoption as a rapid-turnaround, accurate and near-patient diagnostic for patients with suspected HIT. [Display omitted] DisclosuresJones:BloodCenter of Wisconsin: Patents & Royalties. Curtis:Ionis Pharmaceuticals: Consultancy. Bougie:BloodCenter of Wisconsin: Patents & Royalties. Aster:BloodCenter of Wisconsin: Patents & Royalties. Garcia:Shingoi: Consultancy; Retham Technologies LLC: Consultancy; Incyte: Research Funding; Daiichi Sankyo: Research Funding; Bristol Meyers Squibb: Consultancy; Portola: Research Funding; Janssen: Consultancy, Research Funding; Pfizer: Consultancy; Boehringer Ingelheim: Consultancy. Padmanabhan:BloodCenter of Wisconsin: Patents & Royalties: Patent/royalties for HIT diagnostic testing; Veralox Therapeutics: Other: Advisory Board; Retham Technologies LLC: Equity Ownership, Other: Retham seeks to develop HITDx(TM), a new assay for HIT diagnosis; TerumoBCT: Consultancy; GE Healthcare: Other: Material support for Clinical Quality Improvement Initiative; Janssen Pharmaceuticals: Consultancy.

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

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. DisclosuresArnold: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.

A Novel PF4-Dependent P-Selectin Expression Assay (PEA) Facilitates Early Detection of Pathogenic HIT Antibodies: Implications for Diagnosis and Treatment of HIT

The PF4-dependent P-Selectin Expression Assay (PEA) is a novel assay for diagnosis of heparin-induced thrombocytopenia (HIT) which, in preliminary testing, demonstrates higher diagnostic accuracy than the "gold standard" serotonin release assay (SRA) (Padmanabhan et al. Chest, 2016 epub). Here, we describe two cases of severe thrombotic HIT in which the PEA detected pathogenic HIT antibodies (abs) before the SRA tested positive, and in one case, earlier than the highly sensitive PF4 ELISA. These findings have implications for diagnosis and monitoring of the response to therapy in HIT.Patient 1, a 67 yr old woman was admitted for treatment of a Bochdalek hernia and was started on unfractionated heparin (UFH). Her course was complicated by a declining platelet count and on the eighth day of admission PF4 ELISA and SRA tests were strongly positive (Fig 1A). Vascular imaging demonstrated venous thrombosis in both upper and lower extremities. Pre- and post-diagnosis patient samples were tested in the PF4 ELISA, SRA and PEA (Fig 1A). The PEA was positive at 152 hrs (~6 days) post-heparin exposure (69% expression), but the PF4 ELISA was only weakly positive (OD 0.67) and the SRA was negative (0%) at a time when the platelet count was 52% of baseline (Fig 1A). The SRA was still negative (18%) 16 hours later (168 hrs). Thirty hours after the first positive PEA test result, the SRA became positive (100% release). The patient's condition was then further complicated by sepsis-associated multisystem organ failure (MSOF) and therapeutic plasma exchange (TPE) was instituted. TPE treatment led to a rapid reduction in the SRA but only a gradual decline in PEA and ELISA test results (Fig 1B), in line with gradual improvement in platelet counts. Dissociation between SRA and PF4 ELISA test results following TPE has been described previously (Blood. 2015;125:195-8). In our patient, a PEA-SRA dissociation was also observed. Early in this patient's course, PEA-positive/SRA-negative abs were clearly "pathogenic," causing a significant decrease in platelet counts. Thus, it is plausible that PEA-positive/SRA-negative abs detected during the course of TPE were also "pathogenic". PEA results during the course of TPE appeared to correlate better with clinical status in light of the gradual improvement in platelet counts. Unfortunately, the patient expired after the 4th TPE secondary to complications of MSOF.Patient 2, a 57 yr old man was admitted for a creation of a thigh flap in preparation for knee replacement. Postoperatively, the flap became pale with poor pulses and UFH was started. On day 5 platelets dropped 25% from baseline and PF4 ELISA and SRA were both negative (Fig 1C). A strong suspicion of HIT prompted repeat testing on day 6, and yielded positive SRA (79%) and PF4 ELISA (2.8 OD) test results. Notably, the PEA was positive (74%) on Day 5 when both PF4 ELISA and SRA were negative. All tests remained strongly positive through day 12. Thrombocytopenia persisted (Fig 1C) and extensive venous thrombosis of the right upper extremity developed on day 15. The patient was transitioned to Coumadin after platelet recovery and discharged on Day 30.Previous studies indicate that the PEA is at least as accurate as the SRA for detection of pathogenic abs (Padmanabhan et al Thromb Haemost. 2015;114:1322-3, Padmanabhan et al. Chest, 2016 epub). Experience with these two cases suggests that the PEA may in some cases detect pathogenic antibodies before the SRA becomes positive and may be superior to the SRA for monitoring response to treatment. Because of its technical simplicity, the PEA has the potential to be performed on demand, rather than through a referral laboratory, facilitating early diagnosis and treatment. [Display omitted] DisclosuresPadmanabhan:Fenwal (Fresenius Kabi): Research Funding; Schlesinger & Associates: Consultancy, Honoraria; LEK Consulting: Consultancy, Honoraria; Mallinckrodt Pharmaceuticals: Consultancy, Honoraria; Terumo BCT: Consultancy, Honoraria; Bloodcenter of Wisconsin: Patents & Royalties: A patent application has been filed on a Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591. Jones:Bloodcenter of Wisconsin: Patents & Royalties: A patent application has been filed on a Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591. Bougie:Bloodcenter of Wisconsin: Patents & Royalties: A patent application has been filed on a Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591. Aster:Bloodcenter of Wisconsin: Patents & Royalties: A patent application has been filed on a Method of detecting platelet activating antibodies that cause heparin-induced thrombocytopenia/thrombosis; PCT/US14/62591.

Atomic features of an autoantigen in heparin-induced thrombocytopenia (HIT).

Autoantigen development is poorly understood at the atomic level. Heparin-induced thrombocytopenia (HIT) is an autoimmune thrombotic disorder caused by antibodies to an antigen composed of platelet factor 4 (PF4) and heparin or cellular glycosaminoglycans (GAGs). In solution, PF4 exists as an equilibrium among monomers, dimers and tetramers. Structural studies of these interacting components helped delineate a multi-step process involved in the pathogenesis of HIT. First, heparin binds to the 'closed' end of the PF4 tetramer and stabilizes its conformation; exposing the 'open' end. Second, PF4 arrays along heparin/GAG chains, which approximate tetramers, form large antigenic complexes that enhance antibody avidity. Third, pathogenic HIT antibodies bind to the 'open' end of stabilized PF4 tetramers to form an IgG/PF4/heparin ternary immune complex and also to propagate the formation of 'ultralarge immune complexes' (ULCs) that contain multiple IgG antibodies. Fourth, ULCs signal through FcγRIIA receptors, activating platelets and monocytes directly and generating thrombin, which transactivates hematopoietic and endothelial cells. A non-pathogenic anti-PF4 antibody prevents tetramer formation, binding of pathogenic antibody, platelet activation and thrombosis, providing a new approach to manage HIT. An improved understanding of the pathogenesis of HIT may lead to novel diagnostics and therapeutics for this autoimmune disease.

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

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

The transfusion‐related acute lung injury controversy: lessons from heparin‐induced thrombocytopenia

The transfusion‐related acute lung injury controversy: lessons from heparin‐induced thrombocytopenia