Heparin-induced thrombocytopenia and cardiovascular surgery

Clinicians generally counsel patients with a history of heparin-induced thrombocytopenia (HIT) to avoid heparin products lifelong. Although there are now many alternative (nonheparin) anticoagulants available, heparin avoidance remains challenging for cardiac surgery. Heparin is often preferred in the cardiac surgery setting based on the vast experience with the agent, ease of monitoring, and reversibility. To “clear” a patient with a history of HIT for cardiac surgery, hematologists must first confirm the diagnosis of HIT, which can be challenging due to the ubiquity of heparin exposure and frequency of thrombocytopenia in patients in the cardiac intensive care unit. Next, the “phase of HIT” (acute HIT, subacute HIT A/B, or remote HIT) should be established based on platelet count, immunoassay for antibodies to platelet factor 4/heparin complexes, and a functional assay (eg, serotonin release assay). As long as the HIT functional assay remains positive (acute HIT or subacute HIT A), cardiac surgery should be delayed if possible. If surgery cannot be delayed, an alternative anticoagulant (preferably bivalirudin) may be used. Alternatively, heparin may be used with either preoperative/intraoperative plasma exchange or together with a potent antiplatelet agent. The optimal strategy among these options is not known, and the choice depends on institutional experience and availability of alternative anticoagulants. In the later phases of HIT (subacute HIT B or remote HIT), brief intraoperative exposure to heparin followed by an alternative anticoagulant as needed in the postoperative setting is recommended.

An NMR relaxometry approach for quantitative investigation of the transchelation of gadolinium ions from GBCAs to a competing macromolecular chelator

Gadolinium-based contrast agents (GBCAs) have been used in clinical Magnetic Resonance Imaging (MRI) for more than 30 years. However, there is increasing evidence that their dissociation in vivo leads to long-term depositions of gadolinium ions in the human body. In vitro experiments provide critical insights into kinetics and thermodynamic equilibria of underlying processes, which give hints towards the in vivo situation. We developed a time-resolved MRI relaxometry-based approach that exploits distinct relaxivities of Gd3+ in different molecular environments. Its applicability to quantify the transmetallation of GBCAs, the binding of Gd3+ to competing chelators, and the combined transchelation process is demonstrated. Exemplarily, the approach is applied to investigate two representative GBCAs in the presence of Zn2+ and heparin, which is used as a model for a macromolecular and physiologically occurring chelator. Opposing indirect impacts of heparin on increasing the kinetic stability but reducing the thermodynamic stability of GBCAs are observed. The relaxivity of resulting Gd-heparin complexes is shown to be essentially increased compared to that of the parent GBCAs so that they might be one explanation for observed long-term MRI signal enhancement in vivo. In forthcoming studies, the presented method could help to identify the most potent Gd-complexing macromolecular species.

Fc-Modified Kko: A Novel Therapeutic for Heparin-Induced Thrombocytopenia (HIT), Reversing Both the Thrombocytopenia and Thrombosis

Heparin induced thrombocytopenia (HIT) is an immunogenic prothrombotic disorder caused by antibodies that recognize human platelet factor 4 (PF4) complexed to polyanions. We had previously shown using chimeric constructs of hPF4 and mouse (m) PF4 and chimeras with the related chemokine, neutrophil-activating peptide-2 that there is a single antigenic locus on hPF4 in these complexes to which most HIT antibodies bind. KKO is a mouse monoclonal IgG2b k anti-hPF4/polyanion monoclonal antibody that mimics pathogenic antibodies that induce HIT and provokes thrombosis in a murine model of HIT. We previously established that specific hydrolysis of N-linked glycans in the Fc-region of KKO by endoglycosidase from Streptococcus pyogenes EndoS (Genovis) yields >97% deglycosylation on LC-MS/MS generating DGKKO. This modification has no significant effect on binding to PF4-heparin complexes as shown by ELISA and by dynamic light scattering, but abrogates FcgRIIA-mediated binding and platelet activation, and decreases complement activation as evaluated by flow cytometry. To examine if DGKKO reduces prothrombotic effects, we compared DGKKO with KKO in human microfluidic system lined with human umbilical vein cells (HUVECs) that are then photochemically injured and a murine model involving "HIT mice" (mice that express FcgRIIA and human PF4 and lack mouse PF4). Using the microfluidic system described above and infusing blood from healthy donors with added human PF4 (25 µg/ml) and KKO (50 µg/ml) or HIT IgG from three individuals with SRA-positive HIT (1mg/ml) resulted in increased platelet adherence to the injured endothelium (Figure 1). Addition of DGKKO (50 µg/ml) 15 minutes after addition of HIT antibodies eliminated platelet accumulation (Figure 1). In the HIT murine model, we found that intraperitoneal (IP, 200 µg/mice) or intravenous (IV, 20 µg/mice) DGKKO did not induce thrombocytopenia in HIT mice, but reversed the thrombocytopenia induced by either IP KKO (200 µg/mice) or HIT IgG (1 mg/mice) even when the DGKKO is given 6 hrs after HIT induction (Figure 2A). We used an intravital cremaster laser arteriole injury model in HIT mice to study the efficacy of DGKKO as an antithrombotic agent. We found that unlike KKO that enhances growth of established thrombi in these mice, DGKKO significantly reversed the size of the observed thrombi (Figure 2B). These studies suggest that DGKKO obstructs the HIT antigenic site recognized by HIT antibodies and leads to a reversal of thrombocytopenia and thrombus size. Additional studies are underway to examine if DGKKO can be used as a monotherapy or adjunctive therapy in the murine model of HIT thrombosis. Figure 1 Figure 1. Disclosures Cines: Rigel: Consultancy; Dova: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board.

Pathogenic Mechanisms of Vaccine-Induced Immune Thrombotic Thrombocytopenia in People Receiving Anti-COVID-19 Adenoviral-Based Vaccines: A Proposal

VITT is a rare, life-threatening syndrome characterized by thrombotic symptoms in combination with thrombocytopenia, which may occur in individuals receiving the first administration of adenoviral non replicating vectors (AVV) anti Covid19 vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) is characterized by high levels of serum IgG that bind PF4/polyanion complexes, thus triggering platelet activation. Therefore, identification of the fine pathophysiological mechanism by which vaccine components trigger platelet activation is mandatory. Herein, we propose a multistep mechanism involving both the AVV and the neo-synthetized Spike protein. The former can: i) spread rapidly into blood stream, ii), promote the early production of high levels of IL-6, iii) interact with erythrocytes, platelets, mast cells and endothelia, iv) favor the presence of extracellular DNA at the site of injection, v) activate platelets and mast cells to release PF4 and heparin. Moreover, AVV infection of mast cells may trigger aberrant inflammatory and immune responses in people affected by the mast cell activation syndrome (MCAS). The pre-existence of natural antibodies binding PF4/heparin complexes may amplify platelet activation and thrombotic events. Finally, neosynthesized Covid 19 Spike protein interacting with its ACE2 receptor on endothelia, platelets and leucocyte may trigger further thrombotic events unleashing the WITT syndrome.

Recombinant binder of sperm protein1 (rec-BSP1) as a potent fertility factor mediates its effect on embryo production

Objective: To understand the effect of recombinant BSP1 (rec-BSP1) on in vitro capacitation of sperm and fertilization study Method(s): Articles were screened for reports including rec-BSP1, Capacitation, in vitro fertilization Intervention: None Main Outcome Measure(s): Reproductive outcomes, effect on gametes and embryos Result(s): Here we report an optimization of condition for rec-BSP1 production which was used for in vitro capacitation and enhancement of buffalo embryo production. The sequence of the protein was used for multiple sequence alignment which has 99% similarity with PDC 109 protein. The expression of rec-BSP1 was carried out successfully with 1 mM IPTG at 160 C for 22 hrs and purified it in soluble form. The structure of rec-BSP1 was generated using 3D modelling and analysed its mode of binding with heparin and PC by molecular docking and the structural stability of rec-BSP1-PC and rec-BSP1-heparin complexes by using molecular dynamic (MD) simulation. The effect of rec-BSP1 was observed on in vitro capacitation of spermatozoa and buffalo blastocyst production. It was found that the rec-BSP1 enhanced the sperm motility at a concentration of 50 microg/ml for 1 h of incubation without having any detrimental effect on the sperm morphology and a significant (P<0001) increase in blastocyst production at a concentration of 50 microg/ml rec-BSP1. Hence this finding represents a new insight and advance the prospective approach to developing a potential fertility factor in reproduction. Conclusion(s): The purified rec-BSP1 may affect on male fertility and mediated its effect on in vitro embryo production

Anti-SARS-CoV-2 Spike Protein and Anti-Platelet Factor 4 Antibody Responses Induced by COVID-19 Disease and ChAdOx1 nCov-19 vaccination

Background: Some recipients of ChAdOx1 nCoV-19 COVID-19 Vaccine AstraZeneca develop antibody-mediated vaccine-induced thrombotic thrombocytopenia (VITT), associated with cerebral venous and other unusual thrombosis resembling autoimmune heparin-induced thrombocytopenia. A prothrombotic predisposition is also observed in Covid-19. We explored whether antibodies against the SARS-CoV-2 spike protein induced by Covid-19 cross-react with platelet factor 4 (PF4/CXLC4), the protein targeted in both VITT and autoimmune heparin-induced thrombocytopenia.Methods: Immunogenic epitopes of PF4 and SARS-CoV-2 spike protein were compared via prediction tools and 3D modelling software (IMED, SIM, MacMYPOL). Sera from 222 PCR-confirmed Covid-19 patients from five European centers were tested by PF4/heparin ELISA, heparin-dependent and PF4-dependent platelet activation assays. Immunogenic reactivity of purified anti-PF4 and anti-PF4/heparin antibodies from patients with VITT were tested against recombinant SARS-CoV-2 spike protein. Results: Three motifs within the spike protein sequence share a potential immunogenic epitope with PF4. Nineteen of 222 (8.6%) Covid-19 patient sera tested positive in the IgG-specific PF4/heparin ELISA, none of which showed platelet activation in the heparin-dependent activation assay, including 10 (4.5%) of the 222 Covid-19 patients who developed thromboembolic complications. Purified anti-PF4 and anti-PF4/heparin antibodies from two VITT patients did not show cross-reactivity to recombinant SARS-CoV-2 spike protein. Conclusions: The antibody responses to PF4 in SARS-CoV-2 infection and after vaccination with COVID-19 Vaccine AstraZeneca differ. Antibodies against SARS-CoV-2 spike protein do not cross-react with PF4 or PF4/heparin complexes through molecular mimicry. These findings make it very unlikely that the intended vaccine-induced immune response against SARS-CoV-2 spike protein would itself induce VITT.

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 & 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&D: Consultancy. Jones:Retham Technologies: Equity Ownership; Versiti Wisconsin: Patents & Royalties.

Fibronectin modulates formation of PF4/heparin complexes and is a potential factor for reducing risk of developing HIT

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating anti–platelet factor 4 (PF4)/heparin antibodies. Platelet activation assays that use “washed” platelets are more sensitive for detecting HIT antibodies than platelet-rich plasma (PRP)–based assays. Moreover, heparin-exposed patients vary considerably with respect to the risk of PF4/heparin immunization and, among antibody-positive patients, the risk of subsequent “breakthrough” of clinical HIT with manifestation of thrombocytopenia. We used washed platelets and PRP, standard laboratory HIT tests, and physicochemical methods to identify a plasma factor interfering with PF4/heparin complexes and anti-PF4/heparin antibody–platelet interaction, thus explaining differences in functional assays. To investigate a modulating risk for PF4/heparin immunization and breakthrough of HIT, we also tested 89 plasmas from 2 serosurveillance trials. Fibronectin levels were measured in 4 patient groups exhibiting different degrees of heparin-dependent immunization and expression of HIT. The heat-labile plasma protein, fibronectin, inhibited PF4 binding to platelets in a dose-dependent fashion, particularly in washed (vs PRP) systems. Fibronectin also inhibited PF4/heparin binding to platelets, anti-PF4/heparin antibody binding to PF4/heparin complexes, and anti-PF4/heparin antibody–induced platelet activation as a result of PF4/heparin complex disruption. In addition, plasma fibronectin levels increased progressively among the following 4 patient groups: enzyme-linked immunosorbent assay (ELISA)+/serotonin-release assay (SRA)+/HIT+ &lt; ELISA+/SRA+/HIT− ∼ ELISA+/SRA−/HIT− &lt; ELISA−/SRA−/HIT−. Altogether, these findings suggest that fibronectin interferes with PF4/heparin complex formation and anti-PF4/heparin antibody–induced platelet activation. Reduced fibronectin levels in washed platelet assays help to explain the greater sensitivity of washed platelet (vs PRP) assays for HIT. More importantly, lower plasma fibronectin levels could represent a risk factor for PF4/heparin immunization and clinical breakthrough of HIT.

Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway

The mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder “phenotype” (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels (r = 0.898; P &lt; .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.