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Monoclonal Antibodies in the Pathogenesis of Heparin-Induced Thrombocytopenia. BACKGROUND: Heparin-induced thrombocytopenia (HIT) is an immune-mediated platelet disorder caused by antibodies that target complexes of platelet factor 4 (PF4) and heparin. HIT has been characterized as a polyclonal immune response; however, studies of other rare anti-PF4 disorders have identified clonally restricted antibodies. METHODS: In this study, we investigated the clonality of pathogenic HIT antibodies. Antibodies against PF4-heparin were affinity-purified with the use of PF4-heparin beads from serum samples obtained from nine patients with clinically and serologically confirmed HIT. Antibody clonality was assessed by means of immunofixation electrophoresis and mass spectrometry. Antibody binding to PF4 was evaluated by an enzyme immunoassay, and functional platelet activation was evaluated with the use of a P-selectin expression assay. HIT antibody epitopes were mapped in two patients with the use of a PF4 mutant library. RESULTS: Serum samples from all nine patients with HIT were positive for platelet-activating antibodies against PF4-heparin by enzyme immunoassay, as well as the P-selectin expression assay, and six samples (67%) had a monoclonal antibody detectable by immunofixation electrophoresis. The affinity-purified antibodies against PF4-heparin from all nine samples activated platelets in the P-selectin expression assay, and mass spectrometry showed monoclonality. After affinity purification, antibody-depleted serum samples lost binding activity in the enzyme immunoassay and functional activity in the P-selectin expression assay, which confirmed the removal of the pathogenic antibodies. The epitopes on PF4 targeted by anti-PF4-heparin antibodies from serum samples were the same as those targeted by the affinity-purified monoclonal antibodies. CONCLUSIONS: The pathogenic antibodies in all nine patients with HIT were found to be monoclonal. This finding provides insight into the pathogenesis of HIT and has implications for improved diagnostics and targeted therapeutics. (Funded by the Canadian Institutes of Health Research and the National Institutes of Health.).

Heparin-induced thrombocytopenia (HIT) is an immune-mediated reaction to heparin, characterized by a reduced platelet count with increased thrombosis risk.1 HIT is caused by IgG antibodies that recognize complexes of platelet factor 4 (PF4, CXCL4) and heparin.1 These immune complexes activate platelets through FcγRIIa receptors,1 leading to the release of procoagulant platelet microparticles and thrombin generation.2,3 The HIT immune response has several unusual features, including rapid occurrence of anti-PF4/heparin IgG antibodies without a preceding IgM response,4 and transient antibodies lasting up to 100 days without evidence of immunologic memory upon heparin re-administration.5

atelet microparticles and thrombin generation.2,3 The HIT immune response has several unusual features, including rapid occurrence of anti-PF4/heparin IgG antibodies without a preceding IgM response,4 and transient antibodies lasting up to 100 days without evidence of immunologic memory upon heparin re-administration.5 Like most immune responses, HIT antibodies have long been considered polyclonal.6–8 This is supported by observations that patients produce a mixture of pathogenic and non-pathogenic anti-PF4/heparin antibodies.9 Additionally, pathogenic HIT antibodies include variable immunoglobulin classes (mostly IgG, with occasional IgA and IgM) and subclasses (mainly IgG1 with some IgG2 and IgG3).4,6,7 However, the antibodies in other rare anti-PF4 disorders have recently been shown to be monoclonal or oligoclonal, including vaccine-induced immune thrombocytopenia and thrombosis (VITT) following adenoviral-vector SARS-CoV-2 vaccination or adenoviral infection.8,10–12 Similar findings have been reported in VITT-like monoclonal gammopathy of thrombotic significance (MGTS), in which the monoclonal IgG protein (M-protein) had anti-PF4 activity.13–15 In light of these observations, we investigated whether HIT, the most common anti-PF4 disorder, may also be caused by clonally restricted anti-PF4/heparin antibodies.

The McMaster Platelet Immunology Reference Laboratory received patient samples for HIT testing from across Canada. Testing was performed using an anti-PF4–polyvinyl sulfonate (PVS) IgG/A/M enzyme immunoassay [EIA; LIFECODES PF4 Enhanced Assay (Immucor GTI Diagnostics, Peachtree Corners, GA, USA)] and the serotonin-release assay (SRA) (Pfizer, New York, NY, USA).16 A positive anti-PF4/heparin EIA was defined as an optical density (OD280nm) ≥0.4. A positive SRA was defined as >20% 14C-serotonin release at 0.1 and 0.3 U/mL of heparin and <20% release at 100 U/mL of heparin. The ability of anti-PF4/heparin antibodies to activate platelets was assessed using the P-selectin expression assay (PEA), as previously described.17 Sera from hospitalized patients recently treated with heparin and healthy volunteers were used as controls. For patients managed at our centre, two hematologists (D.M.A. and J.G.K.) assessed HIT probability using the 4Ts score.18 This study was approved by the Hamilton Integrated Research Ethics Board (HiREB). Anti-PF4/heparin antibodies were affinity purified using PF4/heparin-coated beads, as described in the Supplementary Appendix. Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) were performed to detect M-proteins in patient sera. Clonotype analysis of affinity-purified anti-PF4/heparin antibodies was conducted using mass spectrometry (MS). Epitope mapping was done using a PF4 mutant library. All methods are described in the Supplementary Appendix.

The McMaster Platelet Immunology Reference Laboratory received patient samples for HIT testing from across Canada. Testing was performed using an anti-PF4–polyvinyl sulfonate (PVS) IgG/A/M enzyme immunoassay [EIA; LIFECODES PF4 Enhanced Assay (Immucor GTI Diagnostics, Peachtree Corners, GA, USA)] and the serotonin-release assay (SRA) (Pfizer, New York, NY, USA).16 A positive anti-PF4/heparin EIA was defined as an optical density (OD280nm) ≥0.4. A positive SRA was defined as >20% 14C-serotonin release at 0.1 and 0.3 U/mL of heparin and <20% release at 100 U/mL of heparin. The ability of anti-PF4/heparin antibodies to activate platelets was assessed using the P-selectin expression assay (PEA), as previously described.17 Sera from hospitalized patients recently treated with heparin and healthy volunteers were used as controls. For patients managed at our centre, two hematologists (D.M.A. and J.G.K.) assessed HIT probability using the 4Ts score.18 This study was approved by the Hamilton Integrated Research Ethics Board (HiREB).

Anti-PF4/heparin antibodies were affinity purified using PF4/heparin-coated beads, as described in the Supplementary Appendix. Serum protein electrophoresis (SPE) and immunofixation electrophoresis (IFE) were performed to detect M-proteins in patient sera. Clonotype analysis of affinity-purified anti-PF4/heparin antibodies was conducted using mass spectrometry (MS). Epitope mapping was done using a PF4 mutant library. All methods are described in the Supplementary Appendix.

Of the HIT patients included in this study (n=9), 2 (22%) were female and 7 (78%) were male, with a mean age of 63 years (range: 45–77 years) (Table 1). All HIT sera tested positive in the anti-PF4/heparin EIA, with a mean OD280nm of 1.9 (range, 1.1–2.4) and a positive SRA, with a mean serotonin release of 87.7% (range, 60.0–99.0%) at 0.1 U/mL heparin and 90.7% (range, 74.0–99.0%) at 0.3 U/mL heparin. Platelet activation in the absence of heparin (buffer alone) was observed in 5 of 9 (55.6%) HIT samples, with a mean serotonin release of 71.4% (range 45.0–100.0%). This resulted in a near-equal distribution of heparin-dependent (n=4) and heparin-independent (n=5) platelet activation in vitro among HIT patient samples in this study. Among the 5 (55.6%) HIT patients for whom clinical data were available, the median 4Ts score was 5 (IQR, 4–6) (intermediate or high probability for HIT). Three patients (60%) developed HIT-associated thrombosis, including pulmonary embolism, deep vein thrombosis and right atrial thrombus. None of the HIT patients had a known history of monoclonal gammopathy. None of the samples from hospitalized patients with recent heparin administration (n=4) or healthy volunteers (n=7) had detectable anti–PF4/heparin antibodies.

thrombosis, including pulmonary embolism, deep vein thrombosis and right atrial thrombus. None of the HIT patients had a known history of monoclonal gammopathy. None of the samples from hospitalized patients with recent heparin administration (n=4) or healthy volunteers (n=7) had detectable anti–PF4/heparin antibodies. SPE demonstrated a normal gamma globulin distribution in all HIT samples, with no detectable M-protein. IFE identified a non-quantifiable (<1 g/dL) IgG M-protein in 6 of 9 (66.7%) HIT samples (Supplementary Figure 1A–B) and an additional non-quantifiable IgM M-protein in 1 of the 6 (16.7%) samples, indicating a biclonal gammopathy. All control sera from hospitalized patients (n=4) and healthy volunteers (n=7) showed normal gamma globulin distributions in both SPE and IFE. Patient sera were fractionated using PF4-coated heparin-Sepharose beads to isolate PF4/heparin-binding antibodies, yielding affinity-purified antibody fractions and corresponding antibody-depleted sera. EIA of the depleted sera showed absent or greatly reduced anti-PF4/heparin antibody levels compared to the original sera (mean OD280: 0.3, range 0.1–0.7; compared with 1.8, range 0.7–2.7), confirming antibody depletion (Figure 1A). The affinity-purified fraction retained PF4/heparin-binding activity with a mean OD280 of 1.0 (range, 0.1–2.0), representing a 20-fold increase compared to eluates from control beads.

ared to the original sera (mean OD280: 0.3, range 0.1–0.7; compared with 1.8, range 0.7–2.7), confirming antibody depletion (Figure 1A). The affinity-purified fraction retained PF4/heparin-binding activity with a mean OD280 of 1.0 (range, 0.1–2.0), representing a 20-fold increase compared to eluates from control beads. The platelet functional activity of the antibodies was assessed using the PEA. HIT sera induced PF4-dependent platelet activation with a mean activation of 106.6% ± 29.2% in the presence of exogenous PF4 (37.5 μg/mL), which was inhibited by high-dose heparin (125 U/mL) and by IV.3, an FcγRIIa-blocking antibody (Figure 1B). In contrast, antibody-depleted sera demonstrated absent or greatly reduced platelet activation with a mean activation of 28.6% ± 17.1%. The affinity-purified antibody fraction retained platelet activation with a mean activation of 121.4% ± 26.7%, confirming the isolated antibody fraction contained the platelet-activating component (Figure 1B).

leted sera demonstrated absent or greatly reduced platelet activation with a mean activation of 28.6% ± 17.1%. The affinity-purified antibody fraction retained platelet activation with a mean activation of 121.4% ± 26.7%, confirming the isolated antibody fraction contained the platelet-activating component (Figure 1B). Of the HIT patient samples with a single M-protein detected by IFE (n=5), the M-protein was no longer detectable by IFE in the antibody depleted sera (Supplementary Figure 2), indicating the affinity-purified anti–PF4/heparin antibodies corresponded to the M-proteins. An IgG M-protein remained detectable in the affinity purified fraction in 4 of 6 (66.7%) patients that had an M-protein in their serum. For the patient with a biclonal gammopathy, only the IgG M-protein was purified, confirming it as the pathogenic anti–PF4/heparin antibody, while the IgM M-protein remained in the depleted serum (Supplementary Figure 3).

in the affinity purified fraction in 4 of 6 (66.7%) patients that had an M-protein in their serum. For the patient with a biclonal gammopathy, only the IgG M-protein was purified, confirming it as the pathogenic anti–PF4/heparin antibody, while the IgM M-protein remained in the depleted serum (Supplementary Figure 3). Monoclonal antibodies were identified in purified antibody fractions from all patients by MS, which showed a single light chain species and/or discrete F(ab)2 fragment peaks. The mean molecular weight of the antibodies was 149.1 kDa (range, 147.0–152.0 kDa), indicating each HIT patient had a distinct monoclonal anti–PF4/heparin antibody (Supplementary Table 1). Specifically, a monoclonal light chain peak was detected in 8 of 9 (88.9%) HIT patients. Among them, kappa light chains were identified in 5 of 8 (62.5%) HIT patient samples and lambda light chains in 2 of 8 (25.0%) patient samples. The mean molecular weight of the light chains was 23.1 kDa (range, 22.7–23.4 kDa) (Supplementary Table 1). In one HIT sample, the light chain mass fell within the overlapping range of kappa and lambda light chain distributions and could not be definitively classified; however, IFE showed lambda restriction (data not shown). All nine HIT samples exhibited discrete F(ab)2 fragment peaks, confirming monoclonality in the one sample lacking a detectable light chain and supporting monoclonality in the eight with identifiable light chains. In 4 of 9 (44.4%) HIT samples, glycoform-resolved heavy chain peaks were also detected, providing further confirmation of monoclonality (Supplementary Table 1). Representative mass distributions showing monoclonal light chain, F(ab)2 fragment and heavy chain peaks are shown in Figure 1C–E.

th identifiable light chains. In 4 of 9 (44.4%) HIT samples, glycoform-resolved heavy chain peaks were also detected, providing further confirmation of monoclonality (Supplementary Table 1). Representative mass distributions showing monoclonal light chain, F(ab)2 fragment and heavy chain peaks are shown in Figure 1C–E. Paired epitope mapping was performed on serum and affinity-purified anti–PF4/heparin antibodies from two HIT patients. For both patients, epitopes of sera and affinity-purified antibodies were congruent, with 10 of 11 (90.9%) overlapping amino acid residues in one patient and 7 of 10 (70.0%) overlapping amino acid residues in the other patient. This indicated the same antibody was present in both the serum and the purified fractions (Figure 2). These epitope profiles were different than KKO, a murine HIT-like monoclonal antibody, which showed only 2 of 11 (18.2%) overlapping amino acids for one patient, and 3 of 10 (30.0%) for the other. They also differed from VITT antibodies, with 1 of 11 (9.1%) and 4 of 10 (40.0%) overlapping amino acids. These results indicate that pathogenic HIT antibodies target a distinct region on PF4 (Figure 2).

SPE demonstrated a normal gamma globulin distribution in all HIT samples, with no detectable M-protein. IFE identified a non-quantifiable (<1 g/dL) IgG M-protein in 6 of 9 (66.7%) HIT samples (Supplementary Figure 1A–B) and an additional non-quantifiable IgM M-protein in 1 of the 6 (16.7%) samples, indicating a biclonal gammopathy. All control sera from hospitalized patients (n=4) and healthy volunteers (n=7) showed normal gamma globulin distributions in both SPE and IFE.

Patient sera were fractionated using PF4-coated heparin-Sepharose beads to isolate PF4/heparin-binding antibodies, yielding affinity-purified antibody fractions and corresponding antibody-depleted sera. EIA of the depleted sera showed absent or greatly reduced anti-PF4/heparin antibody levels compared to the original sera (mean OD280: 0.3, range 0.1–0.7; compared with 1.8, range 0.7–2.7), confirming antibody depletion (Figure 1A). The affinity-purified fraction retained PF4/heparin-binding activity with a mean OD280 of 1.0 (range, 0.1–2.0), representing a 20-fold increase compared to eluates from control beads. The platelet functional activity of the antibodies was assessed using the PEA. HIT sera induced PF4-dependent platelet activation with a mean activation of 106.6% ± 29.2% in the presence of exogenous PF4 (37.5 μg/mL), which was inhibited by high-dose heparin (125 U/mL) and by IV.3, an FcγRIIa-blocking antibody (Figure 1B). In contrast, antibody-depleted sera demonstrated absent or greatly reduced platelet activation with a mean activation of 28.6% ± 17.1%. The affinity-purified antibody fraction retained platelet activation with a mean activation of 121.4% ± 26.7%, confirming the isolated antibody fraction contained the platelet-activating component (Figure 1B).

Monoclonal antibodies were identified in purified antibody fractions from all patients by MS, which showed a single light chain species and/or discrete F(ab)2 fragment peaks. The mean molecular weight of the antibodies was 149.1 kDa (range, 147.0–152.0 kDa), indicating each HIT patient had a distinct monoclonal anti–PF4/heparin antibody (Supplementary Table 1). Specifically, a monoclonal light chain peak was detected in 8 of 9 (88.9%) HIT patients. Among them, kappa light chains were identified in 5 of 8 (62.5%) HIT patient samples and lambda light chains in 2 of 8 (25.0%) patient samples. The mean molecular weight of the light chains was 23.1 kDa (range, 22.7–23.4 kDa) (Supplementary Table 1). In one HIT sample, the light chain mass fell within the overlapping range of kappa and lambda light chain distributions and could not be definitively classified; however, IFE showed lambda restriction (data not shown). All nine HIT samples exhibited discrete F(ab)2 fragment peaks, confirming monoclonality in the one sample lacking a detectable light chain and supporting monoclonality in the eight with identifiable light chains. In 4 of 9 (44.4%) HIT samples, glycoform-resolved heavy chain peaks were also detected, providing further confirmation of monoclonality (Supplementary Table 1). Representative mass distributions showing monoclonal light chain, F(ab)2 fragment and heavy chain peaks are shown in Figure 1C–E.

Paired epitope mapping was performed on serum and affinity-purified anti–PF4/heparin antibodies from two HIT patients. For both patients, epitopes of sera and affinity-purified antibodies were congruent, with 10 of 11 (90.9%) overlapping amino acid residues in one patient and 7 of 10 (70.0%) overlapping amino acid residues in the other patient. This indicated the same antibody was present in both the serum and the purified fractions (Figure 2). These epitope profiles were different than KKO, a murine HIT-like monoclonal antibody, which showed only 2 of 11 (18.2%) overlapping amino acids for one patient, and 3 of 10 (30.0%) for the other. They also differed from VITT antibodies, with 1 of 11 (9.1%) and 4 of 10 (40.0%) overlapping amino acids. These results indicate that pathogenic HIT antibodies target a distinct region on PF4 (Figure 2).

Our study suggests that the pathogenic antibodies in HIT are monoclonal. In 6 patients, serum M-proteins were detected by IFE, and in all nine HIT patients, affinity-purified anti-PF4/heparin IgG antibodies activated platelets in the PEA and were confirmed to be monoclonal by MS. Depleted serum did not retain pathogenic anti-PF4/heparin antibodies. Epitope mapping showed serum samples and purified anti-PF4/heparin antibody fractions bound to the same amino acids on PF4, indicating that they represent the same antibodies. These findings showed the isolated, platelet activating anti-PF4/heparin antibodies from all HIT patients studied are monoclonal. Most antibody responses to pathogens or self-antigens are polyclonal, engaging multiple B-cell clones that produce diverse antibodies.19 In autoimmune diseases, loss of tolerance results in polyclonal activation and broad autoantibody repertoires.20 Monoclonal antibodies that appear as a result of a primary malignant plasma cell clone can cause disease, as seen with multiple myeloma or Waldenström macroglobulinemia; however, our findings indicate HIT, a non-malignant immunologic disorder, is caused by a monoclonal IgG antibody.21

broad autoantibody repertoires.20 Monoclonal antibodies that appear as a result of a primary malignant plasma cell clone can cause disease, as seen with multiple myeloma or Waldenström macroglobulinemia; however, our findings indicate HIT, a non-malignant immunologic disorder, is caused by a monoclonal IgG antibody.21 Our findings are consistent with reports from VITT where oligoclonal or monoclonal anti-PF4 antibodies have been identified.10 In VITT, studies have shown complementarity-determining region (CDR) sequence convergence of anti-PF4 antibodies across unrelated individuals.11 A related disorder, VITT-like MGTS, has been described in patients with primary monoclonal gammopathy of undetermined significance (MGUS), where the M-protein exhibits anti-PF4 activity and induces thrombocytopenia and thrombosis.13–15

on (CDR) sequence convergence of anti-PF4 antibodies across unrelated individuals.11 A related disorder, VITT-like MGTS, has been described in patients with primary monoclonal gammopathy of undetermined significance (MGUS), where the M-protein exhibits anti-PF4 activity and induces thrombocytopenia and thrombosis.13–15 HIT has traditionally been described as polyclonal due to variability in PF4 epitopes, antibody classes and IgG subclasses.6–8 Anti-PF4/heparin antibody studies using cross-inhibition and PF4 chimeras and mutants supported this view, showing antibodies from different patients recognize distinct binding sites.8,22,23 The detection of multiple IgG subclasses, predominantly IgG1 with smaller amounts of IgG2 and IgG3, further reinforced the polyclonal model.6,7 The findings described in this report demonstrate although HIT patients have polyclonal, non-pathogenic anti–PF4/heparin antibodies, the platelet activation and the disease itself is caused by a monoclonal antibody. We postulate this monoclonal antibody might have remained undetected due to it being admixed with broader polyclonal populations.

this report demonstrate although HIT patients have polyclonal, non-pathogenic anti–PF4/heparin antibodies, the platelet activation and the disease itself is caused by a monoclonal antibody. We postulate this monoclonal antibody might have remained undetected due to it being admixed with broader polyclonal populations. Pathogenicity in HIT cannot be determined by isotype, subclass, affinity, titer, or epitope alone; only antibodies that activate platelets cause clinical symptoms, emphasizing the importance of functional platelet activation assays.4,5,8 The variability in sensitivity of different test methods to detect the M-protein likely reflects differences in antibody titer and traditional techniques like SPE and IFE may miss lower levels of monoclonal antibodies. In contrast, MS and epitope mapping offer enhanced sensitivity and resolution. Anti-PF4/heparin epitopes showed >70% overlap in PF4-binding residues between purified antibodies and serum, indicating they are the same antibodies. Monoclonal affinity-purified anti-PF4/heparin antibodies identified by MS corresponded to the M-proteins detected in the patient serum using IFE, which were undetectable following depletion. Each HIT monoclonal antibody had a distinct molecular weight, suggesting differences among patients; although conserved motifs may exist, as seen in VITT antibodies with shared CDRs.11

s identified by MS corresponded to the M-proteins detected in the patient serum using IFE, which were undetectable following depletion. Each HIT monoclonal antibody had a distinct molecular weight, suggesting differences among patients; although conserved motifs may exist, as seen in VITT antibodies with shared CDRs.11 Approximately one-third of HIT patients demonstrate platelet reactivity in the SRA even without the addition of heparin.24 This pattern, referred to as heparin-independent platelet activation in vitro or autoimmune HIT, contrasts with the heparin-dependent activation of classic HIT.24 M-proteins were detectable by IFE in all 5 autoimmune HIT patients, compared with 1 of 4 classic HIT patients, suggesting higher M-protein titres may explain stronger platelet activation and more severe disease. Diluting autoimmune HIT sera yielded a heparin-dependent profile (Supplementary Figure 4), supporting a model in which disease severity is dictated by monoclonal anti-PF4/heparin antibody titres. Current test methods for HIT rely on anti-PF4/heparin EIAs, which are sensitive but lack specificity since they cannot differentiate between pathogenic and non-pathogenic antibodies,9 often yielding false-positive results. Functional platelet activation assays are accurate but technically demanding and not widely accessible.9 Our findings provide new insights into the pathogenesis of HIT with implications for improved diagnostic tests and therapeutics targeting the pathogenic monoclonal antibody.