Hemostasis is a complex process involving the concerted action of molecular and vascular components. Its basic understanding as well as diagnostic and therapeutic aspects have greatly benefited from ...the use of monoclonal antibodies. Interestingly, camelid‐derived single‐domain antibodies (sdAbs), also known as VHH or nanobodies, have become available during the previous 2 decades as alternative tools in this regard. Compared to classic antibodies, sdAbs are easier to produce and their small size facilitates their engineering and functionalization. It is not surprising, therefore, that sdAbs are increasingly used in hemostasis‐related research. In addition, they have the capacity to recognize unique epitopes unavailable to full monoclonal antibodies. This property can be used to develop novel diagnostic tests identifying conformational variants of hemostatic proteins. Examples include sdAbs that bind active but not globular von Willebrand factor or free factor VIIa but not tissue factor–bound factor VIIa. Finally, sdAbs have a high therapeutic potential, exemplified by caplacizumab, a homodimeric sdAb targeting von Willebrand factor that is approved for the treatment of thrombotic thrombocytopenic purpura. In this review, the various applications of sdAbs in thrombosis and hemostasis‐related research, diagnostics, and therapeutic strategies will be discussed.
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Camelid‐derived single‐domain antibodies (or nanobodies) are endowed with numerous advantages over classical antibodies. They hold the potential to represent next‐generation tools for research, diagnostic and even therapeutic purposes, also in the area of thrombosis and hemostasis. Display omitted
Summary
The laboratory diagnosis of heparin‐induced thrombocytopenia (HIT) is based on an enzyme immunoassay combined with a functional test, and serotonin release assay (SRA) is the gold standard ...for detecting activating HIT antibodies. However, a recent atypical history of HIT prompted us to evaluate whether addition of platelet factor 4 (PF4) during SRA could improve its ability to detect pathogenic HIT antibodies. Using 5B9, a monoclonal antibody to PF4/H with a human Fc fragment, we first defined the optimal PF4 concentration for detecting low amounts of platelet‐activating IgG with SRA. Plasma samples from 50 patients with suspected HIT were then studied, and SRA was positive in 17 cases (Group SRApos), with relatively high levels of PF4‐specific IgG (median optical density = 2·66). SRA was also systematically performed after adding 10 μg/ml of PF4 in the reaction mixture, and significant serotonin release was measured with samples from 9 additional patients (Group PF4‐SRApos). Importantly, levels of PF4‐specific IgG were similar in these samples and those from the 24 persistently SRA negative patients. Moreover, the pre‐test probability of HIT was intermediate/high in all ‘SRApos’ or ‘SRA‐PF4pos’ patients. In conclusion, addition of exogenous PF4 might improve the detection of pathogenic HIT antibodies by SRA.
Les anticorps thérapeutiques en hémostase Gruel, Yves; Kizlik-Masson, Claire; Lenting, Peter
M.S. Médecine sciences,
12/2019, Letnik:
35, Številka:
12
Journal Article
Recenzirano
L’hémostase est un processus complexe qui implique de nombreux acteurs cellulaires et moléculaires. En pathologie, les thromboses d’une part, et les pathologies hémorragiques constitutionnelles ...dominées par l’hémophilie d’autre part, ont bénéficié ces dernières années du développement d’anticorps thérapeutiques qui révolutionnent aujourd’hui la prise en charge des malades.
Heparin-induced thrombocytopenia (HIT) is due to immunoglobulin G (IgG) antibodies, which bind platelet factor 4 (PF4) modified by polyanions, such as heparin (H). IgG/PF4/polyanion complexes ...directly activate platelets via Fc gamma type 2 receptor A (FcγRIIA) receptors. A bacterial protease, IgG-degrading enzyme of Streptococcus pyogenes (IdeS), cleaves the hinge region of heavy-chain IgG, abolishing its ability to bind FcγR, including FcγRIIA. We evaluated whether cleavage of anti-PF4/H IgG by IdeS could suppress the pathogenicity of HIT antibodies. IdeS quickly cleaved purified 5B9, a monoclonal chimeric anti-PF4/H IgG1, which led to the formation of single cleaved 5B9 (sc5B9), without any reduction in binding ability to the PF4/H complex. However, as compared with uncleaved 5B9, the affinity of sc5B9 for platelet FcγRIIA was greatly reduced, and sc5B9 was also unable to induce heparin-dependent platelet activation. In addition, incubating IdeS in whole blood containing 5B9 or HIT plasma samples led to cleavage of anti-PF4/H antibodies, which fully abolished the ability to induce heparin-dependent platelet aggregation and tissue factor messenger RNA synthesis by monocytes. Also, when whole blood was perfused in von Willebrand factor–coated microfluidic channels, platelet aggregation and fibrin formation induced by 5B9 with heparin was strongly reduced after IdeS treatment. Finally, IdeS prevented thrombocytopenia and hypercoagulability induced by 5B9 with heparin in transgenic mice expressing human PF4 and FcγRIIA receptors. In conclusion, cleavage of anti-PF4/H IgG by IdeS abolishes heparin-dependent cellular activation induced by HIT antibodies. IdeS injection could be a potential treatment of patients with severe HIT.
•Cleavage of the anti-PF4/H antibody hinge region by IdeS abolishes FcγRIIA-dependent cellular activation.•IdeS treatment prevents thrombus formation and thrombocytopenia induced by anti-PF4/H antibodies.
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Abstract von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing ...between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A–group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high–molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.
•Nanobody KB-VWF-D3.1 binds to the collagen-binding site in the VWF A3 domain, and it loses its binding upon proteolysis of VWF by ADAMTS13.•KB-VWF-D3.1 identified VWF degradation in patients with ...VWD, which correlated with a loss of larger VWF multimers.
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von Willebrand factor (VWF) is a multimeric protein, the size of which is regulated via ADAMTS13-mediated proteolysis within the A2 domain. We aimed to isolate nanobodies distinguishing between proteolyzed and non-proteolyzed VWF, leading to the identification of a nanobody (designated KB-VWF-D3.1) targeting the A3 domain, the epitope of which overlaps the collagen-binding site. Although KB-VWF-D3.1 binds with similar efficiency to dimeric and multimeric derivatives of VWF, binding to VWF was lost upon proteolysis by ADAMTS13, suggesting that proteolysis in the A2 domain modulates exposure of its epitope in the A3 domain. We therefore used KB-VWF-D3.1 to monitor VWF degradation in plasma samples. Spiking experiments showed that a loss of 10% intact VWF could be detected using this nanobody. By comparing plasma from volunteers to that from congenital von Willebrand disease (VWD) patients, intact-VWF levels were significantly reduced for all VWD types, and most severely in VWD type 2A–group 2, in which mutations promote ADAMTS13-mediated proteolysis. Unexpectedly, we also observed increased proteolysis in some patients with VWD type 1 and VWD type 2M. A significant correlation (r = 0.51, P < .0001) between the relative amount of high–molecular weight multimers and levels of intact VWF was observed. Reduced levels of intact VWF were further found in plasmas from patients with severe aortic stenosis and patients receiving mechanical circulatory support. KB-VWF-D3.1 is thus a nanobody that detects changes in the exposure of its epitope within the collagen-binding site of the A3 domain. In view of its unique characteristics, it has the potential to be used as a diagnostic tool to investigate whether a loss of larger multimers is due to ADAMTS13-mediated proteolysis.
Introduction: The laboratory diagnosis of HIT is difficult and often requires to perform an enzyme immunoassay combined with a functional assay, either serotonin release assay (SRA) or ...heparin-induced platelet aggregation (HIPA), for detecting IgG antibodies that activate platelets in the presence of heparin (anti-PF4/H antibodies). SRA is today considered as the gold standard for HIT diagnosis, but its performances could be improved as suggested by a recent clinical history and subsequent experiments.
Case report: A 74 year-old man was hospitalized for a myeloma and treated with unfractionated heparin (UFH) for right atrium thrombosis. Thrombocytopenia and pulmonary embolism occurred the first day of heparin treatment and HIT was suspected. UFH was replaced by danaparoid sodium, and correction of platelet count was achieved within 3 days. Laboratory tests revealed the presence of significant levels of IgG to PF4/H (OD 1.2) with clearly positive HIPA. However, SRA was also performed to confirm the diagnosis of early onset HIT, but remained negative after testing platelets from two different donors. Unlike HIPA performed with PRP, SRA is done with washed platelets, and we thus hypothesized that PF4 concentration on cell surfaces might be too low for allowing heparin-dependent platelet activation when the patient's sample was tested. We thus performed a new SRA after adding exogenous PF4 (10 μg/ml) in the buffer and positive platelet activation pattern was obtained with a maximal release of 65% with 0.5 IU/ml UFH. This experience therefore prompted us to further evaluate whether addition of exogenous PF4 was beneficial or not on platelet activation induced by PF4/H antibodies when studied in SRA.
Patients and methods: Plasma samples from 45 patients with suspected HIT and significant levels PF4-specific IgG were studied. SRA was systematically performed without and with exogenous PF4 (10 μg/ml). This "optimal" concentration of exogenous PF4 was defined after evaluating the effects of variable concentrations of human PF4 on platelet activation induced by 5B9 a recently developed HIT monoclonal antibody.
Results: SRA was consistently negative when 5B9 was tested at concentrations lower than 5 μg/ml (1 or 2.5 μg/ml). In contrast, SRA was positive with these low 5B9 concentrations when 10 μg/ml of PF4 were present in the platelet buffer. When samples from suspected HIT were studied, conventional SRA (without PF4) was positive in 14 of 45 cases (Group SRA+), which all contained relatively high levels of PF4-specific IgG (median OD = 2.6). After addition of exogenous PF4, SRA was positive in 8 additional cases (Group SRAPF4+), with maximal release between 32% and 64% and a complete inhibition in the presence of 10 IU/ml UFH. Interestingly, levels of PF4-specific IgG were similar in this group and the 23 samples with whom SRA remained negative (median OD 1.6 vs. 1.3 respectively, p = 0.8). On the other hand, the pre-test probability of HIT (4Ts score) was always intermediate or high in 'SRA+' and 'SRAPF4+' groups, although it was low in more than 30% of patients with negative SRA.
Discussion: Addition of PF4 in the platelet buffer might improve the detection of pathogenic HIT antibodies using SRA. Our results are in agreement with a recent study (I Nazi et al, Journal of Thrombosis and Haemostasis, 2015; 13: 1900-7), although they were obtained with a lower concentration of exogenous PF4, but which may be observed in vivo.
No relevant conflicts of interest to declare.
Heparin-induced thrombocytopenia (HIT) is a severe drug-adverse event due to platelet-activating antibodies (Abs) directed against platelet factor 4 (PF4)/heparin (H) complexes. In most patients, HIT ...Abs are IgG that directly activate platelets and monocytes in the presence of heparin via FcγRIIA receptors. The interaction between the Fc fragment of anti-PF4/H IgG and FcγRIIA is thus a key step for cellular activation in HIT. Several bacterial proteases such as IdeS (IgG-degrading enzyme of Streptococcus pyogenes) are cleaving IgG in the lower hinge region of heavy chain leading to the formation of single cleaved IgG (scIgG) and then of Fab'2. Importantly, cleavage of IgG by IdeS can abolish their ability to bind FcγR and suppress the cellular effects resulting from this interaction.
The aim of this study was therefore to evaluate whether anti-PF4/H IgG cleavage by IdeS could inhibit cell activation induced by HIT antibodies, and their pathogenicity. To achieve this objective, we studied the effects of IdeS on platelet responses to 5B9, a monoclonal chimeric anti-PF4/H IgG1 recently developed in our laboratory, and which fully mimics the effects of human HIT antibodies (Kizlik-Masson et al, J Thromb Haemost, 2017).
IdeS was demonstrated to quickly (6 minutes) cleave purified 5B9 IgG, leading to the formation of sc5B9, without any reduction in its binding ability to PF4/H complex. However, flow cytometry experiments showed that heparin-dependent binding of sc5B9 to platelets and FcγRIIA was dramatically reduced compared to those of uncleaved 5B9. In addition, functional assays (serotonin release assays and platelet aggregation tests) also confirmed that sc5B9 was unable to induce platelet activation and aggregation in the presence of heparin. Incubation of IdeS (0.02 U/µg of IgG; 6 minutes) in whole blood containing 5B9 IgG or HIT plasma samples also lead in every sample tested to the cleavage of anti-PF4/H Abs, which fully abolished their capacity to induce heparin-dependent platelet aggregation, as demonstrated by impedance aggregometry (Multiplate analyzer). As expected, no effect of IdeS was observed on platelet aggregation induced by collagen (1 µg/mL), or ADP (10 µM). Moreover, tissue factor (TF) gene expression induced in monocytes by 5B9 in the presence of heparin was also completely abolished after addition of IdeS (0.02 U/µg of IgG; 6 minutes) in whole blood, whereas no inhibitory effect of this protease on TF expression induced by LPS was evidenced. We also showed that platelet aggregation and fibrin formation induced by 5B9 with heparin was completely inhibited after IdeS treatment when whole blood was perfused in vWF-coated microfluidic channels with shear rates similar to those of venous flow (500s-1). Finally, IdeS was also showed to prevent efficiently thrombocytopenia and hypercoagulability (with no increase in thrombin/anti-thrombin plasma levels) induced by 5B9 in transgenic mice expressing human PF4 and FcγRIIA receptors, when previously treated by this protease (0.5 µg/g) before IV injection of heparin.
In conclusion, the cleavage of anti-PF4/H IgG by IdeS prevents heparin-dependent cellular activation induced by HIT antibodies, thereby reducing their pathogenicity. Therefore, injection of IdeS could be considered as a potential treatment in patients with severe HIT, particularly in those who necessitate emergent cardiac surgery with cardiopulmonary bypass and thus anticoagulation with unfractionated heparin, which remains the safest and easiest anticoagulant to be used in this specific surgical procedure.
No relevant conflicts of interest to declare.
Heparin-induced thrombocytopenia (HIT) is a frequent drug-adverse event caused in the majority of patients by platelet-activating antibodies (Abs) directed against complexes of heparin (H) bound to ...platelet factor 4 (PF4). In most cases, HIT Abs are IgG which are potentially pathogenic as they are able to activate platelets directly in the presence of heparin via FcgRIIA receptors. The diagnosis of HIT is based on both clinical and biological criteria. However, despite recent improvements in HIT laboratory assays, a standard is always lacking for both immunological and functional assays. First, platelets from healthy donors exhibit wide variability in their response to HIT antibodies. Second, many patients who synthesize significant levels of antibodies to PF4/H while being treated with heparin do not develop HIT and the mechanisms that regulate the pathogenicity of HIT antibodies have not been fully defined. However, several studies suggested that epitope specificity of IgG antibodies is critical for the pathophysiology of HIT, especially by influencing the stability of PF4 tetramers.
We therefore aimed to develop a monoclonal anti-PF4/H antibody with a human Fc fragment using transgenic mice homozygous for Cg gene of a G-class human immunoglobulin and that directly produce chimeric IgG antibodies.
After immunization, several clones were found to synthesize anti-PF4 IgG antibodies but only one (5B9) produced an IgG1 that specifically bind PF4/H complexes without reactivity against PF4 alone. 5B9 was able to induce dose-dependent platelet activation and aggregation in the presence of low concentrations of UFH (0.1 and 0.5 IU/mL), and this effect was not observed without UFH or with high concentration of UFH (10 IU/ml), and was fully inhibited by IV.3 antibody. In addition, 5B9 with UFH (0.5 IU/mL) induced strong TF mRNA synthesis in isolated monocytes. Injection of 5B9 with UFH to transgenic mice expressing human PF4 and FcgRIIA receptors was followed by significant thrombocytopenia, similar to that observed with KKO, a murine IgG2b anti-PF4/H antibody. Plasma levels of thrombin/anti-thrombin (TAT) also significantly increased after injection of heparin in all mice having received 5B9 or KKO, but this effect was more pronounced on day 1 in 5B9-treated mice (mean value= 47 vs. 5 ng/mL at day 0) than in mice injected with KKO (33 vs. 11 ng/mL, respectively).
Competitive immunoassays were also developed and 15 of 25 plasma samples (60%) collected in HIT patients were shown to inhibit (by at least 20%) the binding of 5B9 to PF4/H complexes compared to 3/25 (12%) samples containing non pathogenic anti-PF4/H antibodies (obtained in patients without HIT). Similar experiments were performed with KKO and its binding to PF4/H was also inhibited by 32% of HIT plasma samples (8/25), and none of the non-HIT plasmas. However, the levels of 5B9 and KKO binding inhibition to PF4/H were highly correlated (R2=0.85), thereby suggesting that the epitopes recognized by the antibodies are similar. Noticeably, KKO was also shown to inhibit in a concentration-dependent manner the binding of 5B9 to PF4/H complexes, further supporting this hypothesis.
5B9 was then sequenced and a docking model was elaborated based on VH and VL sequences of 5B9 obtained and a recently described crystal structure of KKO/PF4 tetramer complex. According to this model, 5B9 Fab likely interacts with the B and D monomers of PF4, thus possibly contributing to the stability of tetramers. In addition, the binding of 5B9 to PF4 involves 28 aa, 16 in the B monomer including Asp-7, Gln-9, Pro-34 and Pro-37 and 12 in the D monomer including Gln-9. Importantly, 13 of these 28 aa have also been identified as critical in the formation of PF4/KKO complex (Cai et al, Nat Commun. 2015;6:8277). Three regions (Asp-7 to Thr-15 and Ala-32 to Lys-41 in the B monomer and Gln-9 to Asp-18 in the D monomer) therefore appear particularly important in the binding of both 5B9 and KKO on PF4 tetramers.
In conclusion, 5B9 is the first anti-PF4/H monoclonal antibody that has a human Fc fragment, and fully mimics the effect of HIT human antibodies. 5B9 could therefore be used as a standard in HIT laboratory assays. Moreover, our results support that three regions both recognized by 5B9 and KKO within PF4 tetramers are critical for the binding and pathogenicity of HIT antibodies.
No relevant conflicts of interest to declare.
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