Summary
Fcγ receptors have critical roles in the pathophysiology of heparin-induced thrombocytopenia (HIT), a severe immune-mediated complication of heparin treatment. Activation of platelets, ...monocytes and neutrophils by platelet-activating anti-PF4/heparin IgG antibodies results in thrombocytopenia, hypercoagulability and thrombosis in susceptible patients, effects that depend on FcγRIIA. In addition, FcγRIIIA receptors probably contribute to clearance of platelets sensitised by HIT immune complexes. FcγRI has also been reported to be involved in monocyte activation by HIT IgG antibodies and synthesis of tissue factor. This review focuses on the role of these FcγRs in HIT pathophysiology, including the potential influence of several gene variations associated with variable risk of HIT and related thrombosis. In particular, the 276P and 326Q alleles of CD148, a protein tyrosine phosphatase that regulates FcγRIIA signalling, are associated with a lower risk of HIT, and platelets from healthy donors expressing these alleles are hyporesponsive to anti-PF4/H antibodies. It was also recently demonstrated that the risk of thrombosis is higher in HIT patients expressing the R isoform of the FcγRIIA H131R polymorphism, with HIT antibodies shown to activate RR platelets more efficiently, mainly explained by an inhibitory effect of normal IgG2, which bound to the FcγRIIA 131H isoform more efficiently. Environmental risk factors probably interact with these gene polymorphisms affecting FcγRs, thereby increasing thrombosis risk in HIT.
The rare vaccine-induced immune thrombotic thrombocytopenia that may follow adenovirus-based Covid-19 vaccination resembles heparin-induced thrombocytopenia, but rapid assays for anti-PF4 antibodies ...used to diagnose HIT may be negative in patients with VITT. The PF4–serotonin release assay appears to detect the IgG antibodies to PF4–PVS that mediate this condition.
The steroid receptor RNA activator (SRA) has the unusual property to function as both a non-coding RNA (ncRNA) and a protein SRAP. SRA ncRNA is known to increase the activity of a range of nuclear ...receptors as well as the master regulator of muscle differentiation MyoD. The contribution of SRA to either a ncRNA or a protein is influenced by alternative splicing of the first intron, the retention of which disrupts the SRAP open reading frame. We reported here that the ratio between non-coding and coding SRA isoforms increased during myogenic differentiation of human satellite cells but not myotonic dystrophy patient satellite cells, in which differentiation capacity is affected. Using constructs that exclusively produce SRA ncRNA or SRAP, we demonstrated that whereas SRA ncRNA was indeed an enhancer of myogenic differentiation and myogenic conversion of non-muscle cells through the co-activation of MyoD activity, SRAP prevented this SRA RNA-dependant co-activation. Interestingly, the SRAP inhibitory effect is mediated through the interaction of SRAP with its RNA counterpart via its RRM-like domain interacting with the functional sub-structure of SRA RNA, STR7. This study thus provides a new model for SRA-mediated regulation of MyoD transcriptional activity in the promotion of normal muscle differentiation, which takes into account the nature of SRA molecules present.
Abstract
Background
Heparin-induced thrombocytopenia (HIT) is typically caused by platelet-activating immunoglobulin G (IgG) antibodies (Abs) against platelet factor 4 (PF4) complexed with heparin ...(H). Much less frequent “autoimmune” HIT is distinguished from typical HIT by platelet activation without heparin and the presence of both anti-PF4/H and anti-PF4 IgG. We developed three murine monoclonal anti-PF4 Abs with a human Fc-part, 1E12, 1C12, and 2E1, resembling autoimmune HIT Abs.
Objectives
To characterize 1E12, 1C12, and 2E1 in comparison to the heparin-dependent monoclonal anti-PF4/H Abs 5B9 and KKO, and polyclonal Abs from patients with typical HIT (group-2) and autoimmune HIT (group-3).
Methods
Interactions of Abs with PF4 and PF4/H were studied by enzyme-linked-immunosorbent assay, single-molecule force spectroscopy, isothermal titration calorimetry, and dynamic light scattering. Serotonin release assay and heparin-induced platelet activation assay were used to assess platelet activation. The binding sites of monoclonal Abs on PF4 were predicted in silico (MAbTope method).
Results
1C12, 1E12, and 2E1 displayed higher affinity for PF4/H complexes than 5B9 and KKO, comparable to human group-3 Abs. Only 1C12, 1E12, 2E1, and group-3 Abs formed large complexes with native PF4, and activated platelets without heparin. The predicted binding sites of 1C12, 1E12, and 2E1 on PF4 differed from those of KKO and 5B9, but were close to each other. 2E1 exhibited unique bivalent binding, involving its antigen recognition site to PF4 and charge-dependent interactions with heparin.
Conclusion
1C12, 1E12, and 2E1 are tools for studying the pathophysiology of autoimmune HIT. 2E1 provides evidence for a new binding mechanism of HIT Abs.
Thrombosis results in heparin-induced thrombocytopenia (HIT) from cellular activation involving Fc receptors. In this study, the FcγRIIA 131RR genotype was found to increase the risk of thrombosis in ...HIT patients (odds ratio: 5.9; 95% confidence interval: 1.7-20). When platelet aggregation tests (PATs) were performed with platelet-rich plasma (PRP), a shorter lag time was measured in 131RR donors compared to individuals with the HR and HH genotypes in response to HIT plasma or 5B9, a recently developed humanized monoclonal antibody to PF4/heparin. Importantly, this difference was no longer detectable when PATs were performed with washed platelets or immunoglobulin (Ig)G-depleted PRP. Moreover, polyclonal IgG or monoclonal IgG1 added to IgG-depleted PRP increased the lag time in response to 5B9. HH platelets were also sensitive to IgG2, which in contrast, failed to inhibit the response of 131RR platelets to 5B9. Finally, higher tissue factor messenger RNA levels were measured in the whole blood of 131RR donors after activation by HIT antibodies, with increased phospholipid procoagulant activity. These results demonstrate that HIT patients homozygous for the FcγRIIA 131R allele have a higher risk of thrombosis, probably due to increased cell activation by antibodies to PF4/heparin, with a lower inhibitory effect of endogenous IgG, especially from the IgG2 subclass.
•Normal IgG and IgG2 differentially inhibit HIT antibody-dependent platelet activation according to the FcγRIIA H131R polymorphism.•This variable effect of IgG and IgG2 probably explains the higher risk of thrombosis in patients homozygous for the FcγRIIA 131R allele.
Abstract
Introduction/Objectives
Extracorporeal membrane oxygenation (ECMO) provides circulatory support in patients with severe heart failure, but the frequent use of unfractionated heparin exposes ...patients to high risk of heparin-induced thrombocytopenia (HIT). We prospectively evaluated the development and clinical impact of platelet factor 4 (PF4)-specific antibodies (Abs) during ECMO and whether specific biological characteristics could predict HIT.
Materials and Methods
From 2014 to 2018, we studied 57 adults who underwent an ECMO for at least 5 days. The plasma samples collected daily were tested for PF4-specific Abs using immunoassays to detect immunoglobulin (Ig) G, A, and M isotypes or only IgG. Serotonin release assay was performed without and with PF4 to detect pathogenic Abs.
Results
Twenty-nine patients (50%) were positive for PF4-specific Abs (IgG, A, M), with IgG in 17/57 (30%) and 16 of them (94%) were immunized within 10 days. PF4-specific IgG Abs did not affect the clinical or biological course of most patients. HIT was suspected in only two patients with ECMO circuit dysfunction and unexpected platelet count decrease after day 5. High levels of PF4-specific IgG were detected in both patients, and HIT was confirmed by a serotonin release assay, which was also more sensitive when exogenous PF4 was present.
Conclusion
PF4-specific Abs are common during ECMO but are mostly non-pathogenic and not associated with a less favorable prognosis. However, an abnormal platelet count evolution, in particular if associated with ECMO circuit dysfunction, should prompt the search for pathogenic PF4-specific IgG.
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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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.
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