Structural Characterization of a Pathogenic Antibody Underlying Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but dangerous side effect of adenoviral-vectored COVID-19 vaccines. VITT had been linked to production of autoantibodies recognizing platelet factor 4 (PF4). Here, we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact mass measurements indicate that a significant fraction of these antibodies represent a limited number of clones. MS analysis of large antibody fragments (the light chain and the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire and reveals the presence of a mature complex biantennary N-glycan within the Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS was used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to the IgG2 subclass and verifies that the light chain belongs to the λ-type. Incorporation of enzymatic de-N-glycosylation into the peptide mapping routine allows the N-glycan in the Fab region of the antibody to be localized to the framework 3 region of the VH domain. This novel N-glycosylation site is the result of a single mutation within the germline sequence. Peptide mapping also provides information on lower-abundance (polyclonal) components of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1-IgG4) and both types of the light chain (λ and κ). This case study demonstrates the power of combining the intact, middle-down, and bottom-up MS approaches for meaningful characterization of ultralow quantities of pathogenic antibodies extracted directly from patients' blood.

Reverse Engineering of a Pathogenic Antibody Reveals the Molecular Mechanism of Vaccine-Induced Immune Thrombotic Thrombocytopenia.

The massive COVID-19 vaccine roll-out campaign illuminated a range of rare side effects, the most dangerous of which─vaccine-induced immune thrombotic thrombocytopenia (VITT)─is caused by adenoviral (Ad)-vectored vaccines. VITT occurrence had been linked to the production of pathogenic antibodies that recognize an endogenous chemokine, platelet factor 4 (PF4). Mass spectrometry (MS)-based evaluation of the ensemble of anti-PF4 antibodies obtained from a VITT patient's blood indicates that the major component is a monoclonal antibody. Structural characterization of this antibody reveals several unusual characteristics, such as the presence of an N-glycan in the Fab segment and high density of acidic amino acid residues in the complementarity-determining regions. A recombinant version of this antibody (RVT1) was generated by transient expression in mammalian cells based on the newly determined sequence. It captures the key properties of VITT antibodies such as their ability to activate platelets in a PF4 concentration-dependent fashion. Homology modeling of the Fab segment reveals a well-defined polyanionic paratope, and the docking studies indicate that the polycationic segment of PF4 readily accommodates two Fab segments, cross-linking the antibodies to yield polymerized immune complexes. Their existence was verified with native MS by detecting assemblies as large as (RVT1)3(PF4)2, pointing out at FcγRIIa-mediated platelet activation as the molecular mechanism underlying VITT clinical manifestations. In addition to the high PF4 affinity, RVT1 readily binds other polycationic targets, indicating a polyreactive nature of this antibody. This surprising promiscuity not only sheds light on VITT etiology but also opens up a range of opportunities to manage this pathology.

Performance characteristics of platelet autoantibody testing for the diagnosis of immune thrombocytopenia using strict clinical criteria.

Misclassification of immune thrombocytopenia (ITP) is common, which might undermine the value of platelet autoantibody testing. We determined the sensitivity and specificity of platelet autoantibody testing using the direct antigen capture assay for anti-glycoprotein (GP) IIb/IIIa or anti-GPIbIX in patients with 'definite ITP', defined as those with a documented treatment response. Sensitivity of platelet autoantiboody testing increased from 48·3% [95% confidence interval (CI) 43·5-53·2] for all ITP patients to 64·7% (95% CI 54·6-73·9) for definite ITP patients. Specificity was unchanged [75·3% (95% CI 67·5-82·1)]. High optical density values (>0·8) improved the specificity of platelet autoantibody testing but lowered sensitivity. In patients with a high pretest probability, platelet autoantibodies can aid in the diagnosis of ITP and may be most prevalent in certain patient subsets.

A platelet viability assay (PVA) for the diagnosis of heparin-induced thrombocytopenia.

Diagnosing heparin-induced thrombocytopenia (HIT) requires functional assays measuring platelet activation as they are highly specific and sensitive. A useful functional test for diagnosing HIT is the serotonin release assay (SRA), but this assay is technically demanding and requires a radioactive marker. We describe an alternate functional HIT assay, the platelet viability assay (PVA), that overcomes the need for a radioactive marker by using a viability dye endpoint to measure platelet activation. We compared the performance characteristics of the PVA to the SRA. Serum samples from 76 patients with suspected HIT were tested in both the PVA and the SRA. The PVA uses calcein-AM as a marker of platelet viability, with decreases in fluorescence and cell size as surrogate markers for platelet activation. A significant linear correlation (Spearman correlation, r = -0.78, P < 0.0001) was observed between the PVA and SRA. Calcein-AM fluorescence decreased in a negative linear relationship with platelet activation as measured by 14C-serotonin release. The PVA detected all positive SRA samples, with an overall sensitivity of 100% and a specificity of 97% in comparison to the SRA. The measurement of platelet viability using the PVA provided similar results to the SRA when testing suspected HIT patient samples.

Autoantibodies to thrombopoietin and the thrombopoietin receptor in patients with immune thrombocytopenia.

Autoantibodies to thrombopoietin (TPO, also termed THPO) or the TPO receptor (cMpl, also termed MPL) could play a pathological role in immune thrombocytopenia (ITP). In this study, we tested for autoantibodies against TPO, cMpl, or the TPO/cMpl complex in ITP and other thrombocytopenic disorders. Using an inhibition step with excess TPO in fluid-phase to improve binding specificity, the prevalence of anti-TPO autoantibodies was: active ITP: 9/32 (28%); remission ITP: 0/14 (0%); non-immune thrombocytopenias: 1/10 (10%); and healthy controls: 1/11 (9%). Similarly, using an inhibition step with excess cMpl, the prevalence of specific anti-cMpl autoantibodies was: active ITP: 7/32 (22%); remission ITP: 1/14 (7%); non-immune thrombocytopenias: 3/10 (30%); and healthy controls: 0/11 (0%). Two active ITP patients had autoantibodies against the TPO/cMpl complex, but not against TPO or cMpl alone. Anti-TPO or anti-cMpl autoantibodies were found in 44% of ITP patients, and in 40% of patients with other thrombocytopenic disorders. These autoantibodies did not correlate with ITP disease severity or number of ITP treatments received; however, in this cohort, 3 patients failed to respond to TPO receptor agonist medications, and of those, 2 had anti-TPO autoantibodies. This suggests that anti-TPO and anti-cMpl autoantibodies are associated with thrombocytopenia, and may be clinically relevant in a subset of ITP patients.

Megakaryocyte apoptosis in immune thrombocytopenia.

The mechanisms of platelet underproduction in immune thrombocytopenia (ITP) remain unknown. While the number of megakaryocytes is normal or increased in ITP bone marrow, further studies of megakaryocyte integrity are needed. Megakaryocytes are responsible for the production of platelets in the bone marrow, and they are possible targets of immune-mediated injury in ITP. Since the biological process of megakaryocyte apoptosis impacts platelet production, we investigated megakaryocyte DNA fragmentation as a marker of apoptosis from ITP bone marrow biopsies. Archived bone marrow biopsy specimens from ITP patients, bone marrow specimens from controls with normal platelet counts, and bone marrow specimens from thrombocytopenic controls with myelodysplastic syndrome (MDS) were evaluated. Sections were stained with anti-CD61 for megakaryocyte enumeration, and terminal deoxynucleotidyl transferase dUTP nick-end labeling was used as an apoptotic indicator. In ITP patients, megakaryocyte apoptosis was reduced compared to nonthrombocytopenic controls. Megakaryocyte apoptosis was similarly reduced in thrombocytopenic patients with MDS. These results suggest a link between megakaryocyte apoptosis and platelet production.

The effect of rituximab on anti-platelet autoantibody levels in patients with immune thrombocytopenia.

Rituximab is an effective therapy resulting in a platelet count improvement in 60% of patients with immune thrombocytopenia (ITP). Rituximab depletes B cells; thus, a reduction in platelet autoantibody levels would be anticipated in patients who achieve a clinical response to this treatment. The objectives of this study were to determine whether rituximab was associated with a reduction in platelet autoantibody levels, and to correlate the loss of autoantibodies with the achievement of a treatment response. We performed a case-control study nested within a previous randomized controlled trial of standard therapy plus adjuvant rituximab or placebo. We measured platelet-bound anti-glycoprotein (GP) IIbIIIa and anti-GPIbIX using the antigen capture test. Of 55 evaluable patients, 25 (45%) had a detectable platelet autoantibody at baseline. Rituximab was associated with a significant reduction in anti-GPIIbIIIa levels (P = 0·02) but not anti-GPIbIX levels (P = 0·51) compared with placebo. Neither the presence of an autoantibody at baseline nor the loss of the autoantibody after treatment was associated with a response to rituximab. The subset of patients with persistent autoantibodies after treatment failed to achieve a platelet count response, suggesting that persistence of platelet autoantibodies can be a marker of disease severity.

Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin.

The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis-like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin-elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca(2+) activity as well as Ca(2+) -dependent proteolytic processing of µ-calpain. Pyocyanin further up-regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin-induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl-ester labelling, pyocyanin-treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis-inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection.

Producing megakaryocytes from a human peripheral blood source.

BACKGROUND: Cultured megakaryocytes could prove useful in the study of human diseases, but it is difficult to produce sufficient numbers for study. We describe and evaluate the use of an expansion process to develop mature megakaryocytes from peripheral blood-derived human hematopoietic stem and progenitor cells (HSPCs). STUDY DESIGN AND METHODS: HSPCs (CD34+) were isolated from peripheral blood by positive selection and expanded using an optimal CD34+ expansion supplement. We evaluated megakaryocyte growth, maturation, and morphology in response to thrombopoietin (TPO) stimulation using flow cytometry and electron microscopy. TPO demonstrated a dose-dependent stimulatory effect on both megakaryocyte number and maturation. RESULTS: From 90 to 120 mL of unmanipulated peripheral blood, we isolated a mean of 1.5 × 10(5) HSPCs (1.5 × 10(3) cells/mL of whole blood). HSPCs expanded nine-fold after a 4-day culture using an expansion supplement. Expanded cells were cultured for an additional 8 days with TPO (20 ng/mL), which resulted in a 2.9-fold increase in megakaryocytic cells where 83% of live cells expressed CD41a+, a marker of megakaryocyte commitment, and 50% expressed CD42b+, a marker for megakaryocyte maturation. The expanded HSPCs responded to TPO stimulation to yield more than 1.0 × 10(6) megakaryocytes. This cell number was sufficient for morphologic studies that demonstrated these expanded HSPCs produced mature polyploid megakaryocytes capable of forming proplatelet extensions. CONCLUSIONS: Peripheral blood HSPCs can be expanded and differentiated into functional, mature megakaryocytes, a finding that supports the use of this process to study inherent platelet (PLT) production disorders as well as study factors that impair normal PLT production.

Antibody binding to megakaryocytes in vivo in patients with immune thrombocytopenia.

OBJECTIVES: Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder caused by increased platelet destruction and impaired platelet production. Antibody binding to megakaryocytes may occur in ITP, but in vivo evidence of this phenomenon is lacking. METHODS: We determined the proportion of megakaryocytes bound with immunoglobulin G (IgG) in bone marrow samples from primary patients with ITP (n = 17), normal controls (n = 13) and thrombocytopenic patients with myelodysplastic syndrome (MDS; n = 10). Serial histological sections from archived bone marrow biopsies were stained for CD61 and IgG. IgG binding and the number of bone marrow megakaryocytes were determined morphologically by a hematopathologist with four assessors after a calibration exercise to ensure consistency. RESULTS: The proportion of ITP patients with high IgG binding (>50% of bone marrow megakaryocytes) was increased compared with normal controls [12/17 (71%) vs. 3/13 (23%), P = 0.03]. However, the proportion of ITP patients with high IgG binding was no different than thrombocytopenic patients with MDS [12/17 (71%) vs. 7/10 (70%), P = 1.00]. IgG binding was associated with increased megakaryocyte numbers. Like platelet-associated IgG, megakaryocyte-associated IgG is related to thrombocytopenia but may not be specific for ITP. CONCLUSION: Mechanistic studies in ITP should focus on antibody specificity and include thrombocytopenic control patients.

Pitfalls in the diagnosis of heparin-Induced thrombocytopenia: A 6-year experience from a reference laboratory.

Heparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies against complexes of platelet factor 4 (PF4) and heparin. The diagnosis of HIT is contingent on accurate and timely laboratory testing. Recently, alternative anticoagulants for the treatment of HIT have been introduced along with algorithms for better HIT diagnosis. However, the increased reliance on immunoassays for the diagnosis of HIT may have harmful consequences due to the high rate of false positive results. To compare trends and implications of current HIT testing approaches, we analyzed results over a six-year period from the McMaster University Platelet Immunology Reference Laboratory. From 2008 to 2013, 8,546 samples were investigated for HIT using both an in-house IgG-specific anti-PF4/heparin enzyme immunoassay (EIA) and the serotonin-release assay (SRA). Of 8,546 samples tested, 13.4% were true-positives (positive in both assays); 65.6% were true-negatives (negative in both assays); 20.9% were presumed false positive for HIT (EIA-positive/SRA-negative); and 0.2% were EIA-negative/SRA-positive. The frequency of EIA-positive/SRA-negative results increased over time (from 12.9% in 2008 to 22.9% in 2013). We found that the number of SRA-negative samples was reduced from referring centers that used an immunoassay as an initial screen; however, 41% of those samples tested negative in the immunoassay and in the SRA at the reference laboratory. The suspicion of HIT continues at a high rate and the agreement between the EIA and SRA test results remains problematic.

Structural characterization of a pathogenic antibody underlying vaccine-induced immune thrombotic thrombocytopenia (VITT).

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but extremely dangerous side effect that has been reported for several adenoviral (Ad)-vectored COVID-19 vaccines. VITT pathology had been linked to production of antibodies that recognize platelet factor 4 (PF4), an endogenous chemokine. In this work we characterize anti-PF4 antibodies obtained from a VITT patient's blood. Intact-mass MS measurements indicate that a significant fraction of this ensemble is comprised of antibodies representing a limited number of clones. MS analysis of large antibody fragments (the light chain, as well as the Fc/2 and Fd fragments of the heavy chain) confirms the monoclonal nature of this component of the anti-PF4 antibodies repertoire, and reveals the presence of a fully mature complex biantennary N-glycan within its Fd segment. Peptide mapping using two complementary proteases and LC-MS/MS analysis were used to determine the amino acid sequence of the entire light chain and over 98% of the heavy chain (excluding a short N-terminal segment). The sequence analysis allows the monoclonal antibody to be assigned to IgG2 subclass and verify that the light chain belongs to the λ-type. Incorporation of enzymatic de- N -glycosylation into the peptide mapping routine allows the N -glycan in the Fab region of the antibody to be localized to the framework 3 region of the V H domain. This novel N -glycosylation site (absent in the germline sequence) is a result of a single mutation giving rise to an NDT motif in the antibody sequence. Peptide mapping also provides a wealth of information on lower-abundance proteolytic fragments derived from the polyclonal component of the anti-PF4 antibody ensemble, revealing the presence of all four subclasses (IgG1 through IgG4) and both types of the light chain (λ and κ). The structural information reported in this work will be indispensable for understanding the molecular mechanism of VITT pathogenesis.

Antibodies against platelet factor 4 and the risk of cerebral venous sinus thrombosis in patients with vaccine-induced immune thrombotic thrombocytopenia.

BACKGROUND: Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare complication of adenoviral vector-based vaccines against SARS-CoV-2. This syndrome is caused by antibodies against platelet factor 4 (PF4; CXCL4) that lead to platelet activation and is characterized by thrombocytopenia and thrombosis in unusual locations, including cerebral venous sinus thrombosis (CVST). VITT can be classified based on anti-PF4 antibodies properties in vitro: those that require PF4 to activate platelets (PF4-dependent) and those that can activate platelets without additional PF4 (PF4-independent) in the serotonin release assay. OBJECTIVES: We aim to characterize the relationship of VITT platelet-activating profiles with CVST. METHODS: We conducted a retrospective cohort study involving patients with confirmed VITT who were tested between March and June 2021. Data were collected with an anonymized form and cases were identified as VITT with high clinical suspicion according to platelet activation assays. Anti-PF4 antibody binding regions on PF4 were further characterized with alanine scanning mutagenesis. RESULTS: Of the patients with confirmed VITT (n = 39), 17 (43.6%) had PF4-dependent antibodies and 22 (56.4%) had PF4-independent antibodies. CVST occurred almost exclusively in PF4-independent patients (11 of 22 vs 1 of 17; P < .05). Additionally, PF4-independent antibodies bound to 2 distinct epitopes on PF4, the heparin-binding region and a site typical for heparin-induced thrombocytopenia antibodies, whereas PF4-dependent antibodies bound to only the heparin-binding region. CONCLUSION: These findings suggest that VITT antibodies that cause PF4-independent platelet activation represent a unique subset of patients more likely to be associated with CVST, possibly due to the 2 different types of anti-PF4 antibodies.

Vaccine-induced immune thrombotic thrombocytopenia: Updates in pathobiology and diagnosis.

Coronavirus disease 2019 (COVID-19) is a viral respiratory infection caused by the severe acute respiratory syndrome virus (SARS-CoV-2). Vaccines that protect against SARS-CoV-2 infection have been widely employed to reduce the incidence of symptomatic and severe disease. However, adenovirus-based SARS-CoV-2 vaccines can cause a rare, thrombotic disorder termed vaccine-induced immune thrombotic thrombocytopenia (VITT). VITT often develops in the first 5 to 30 days following vaccination and is characterized by thrombocytopenia and thrombosis in unusual locations (e.g., cerebral venous sinus thrombosis). The diagnosis is confirmed by testing for anti-PF4 antibodies, as these antibodies are capable of platelet activation without any cofactor. It can be clinically challenging to differentiate VITT from a similar disorder called heparin-induced thrombocytopenia (HIT), since heparin is commonly used in hospitalized patients. VITT and HIT have similar pathobiology and clinical manifestations but important differences in testing including the need for PF4-enhanced functional assays and the poor reliability of rapid immunoassays for the detection of anti-platelet factor 4 (PF4) antibodies. In this review we summarize the epidemiology of VITT; highlight similarities and differences between HIT and VITT; and provide an update on the clinical diagnosis of VITT.

The clinical and laboratory diagnosis of vaccine-induced immune thrombotic thrombocytopenia.

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare but serious adverse syndrome occurring 5 to 30 days after adenoviral vector COVID-19 vaccination. Therefore, a practical evaluation of clinical assessments and laboratory testing for VITT is needed to prevent significant adverse outcomes as the global use of adenoviral vector vaccines continues. We received the clinical information and blood samples of 156 patients in Canada with a suspected diagnosis of VITT between April and July 2021. The performance characteristics of various diagnostic laboratory tests were evaluated against the platelet factor 4 (PF4)-14C-serotonin release assay (SRA) including a commercial anti-PF4/heparin immunoglobulin G (IgG)/IgA/IgM enzyme immunoassay (EIA, PF4 Enhanced; Immucor), in-house IgG-specific anti-PF4 and anti-PF4/heparin-EIAs, the standard SRA, and the PF4/heparin-SRA. Of those, 43 (27.6%) had serologically confirmed VITT-positive based on a positive PF4-SRA result and 113 (72.4%) were VITT-negative. The commercial anti-PF4/heparin EIA, the in-house anti-PF4-EIA, and anti-PF4/heparin-EIA were positive for all 43 VITT-confirmed samples (100% sensitivity) with a few false-positive results (mean specificity, 95.6%). These immunoassays had specificities of 95.6% (95% confidence interval [CI], 90.0-98.6), 96.5% (95% CI, 91.2-99.0), and 97.4% (95% CI, 92.4-99.5), respectively. Functional tests, including the standard SRA and PF4/heparin-SRA, had high specificities (100%), but poor sensitivities for VITT (16.7% [95% CI, 7.0-31.4]; and 46.2% [95% CI, 26.6-66.6], respectively). These findings suggest EIA assays that can directly detect antibodies to PF4 or PF4/heparin have excellent performance characteristics and may be useful as a diagnostic test if the F4-SRA is unavailable.

Characteristics of Anti-SARS-CoV-2 Antibodies in Recovered COVID-19 Subjects.

Coronavirus Disease 2019 (COVID-19) is a global pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While detection of SARS-CoV-2 by polymerase chain reaction with reverse transcription (RT-PCR) is currently used to diagnose acute COVID-19 infection, serological assays are needed to study the humoral immune response to SARS-CoV-2. Anti-SARS-CoV-2 immunoglobulin (Ig)G/A/M antibodies against spike (S) protein and its receptor-binding domain (RBD) were characterized in recovered subjects who were RT-PCR-positive (n = 153) and RT-PCR-negative (n = 55) using an enzyme-linked immunosorbent assay (ELISA). These antibodies were also further assessed for their ability to neutralize live SARS-CoV-2 virus. Anti-SARS-CoV-2 antibodies were detected in 90.9% of resolved subjects up to 180 days post-symptom onset. Anti-S protein and anti-RBD IgG titers correlated (r = 0.5157 and r = 0.6010, respectively) with viral neutralization. Of the RT-PCR-positive subjects, 22 (14.3%) did not have anti-SARS-CoV-2 antibodies; and of those, 17 had RT-PCR cycle threshold (Ct) values > 27. These high Ct values raise the possibility that these indeterminate results are from individuals who were not infected or had mild infection that failed to elicit an antibody response. This study highlights the importance of serological surveys to determine population-level immunity based on infection numbers as determined by RT-PCR.

The role of fluid-phase immune complexes in the pathogenesis of heparin-induced thrombocytopenia.

Immune complexes assemble on the platelet surface and cause Fc-mediated platelet activation in heparin-induced thrombocytopenia (HIT); however, it is not known if fluid-phase immune complexes contribute to HIT. The objective of this study was to understand the role of fluid-phase immune complexes in platelet activation and HIT. Binding of wild-type and 15 platelet factor 4 (PF4) mutants to platelets was measured using flow cytometry. Platelet activation was measured using the PF4-dependent 14C-serotonin release assay (PF4-SRA) with KKO and a HIT-patient plasma in the presence of wild-type or PF4 mutants. To activate platelets, we found that a minimal level of wild-type PF4 is required to bind the platelet surface in the presence of KKO (2.67 relative MFI) or HIT-patient plasma (1.71 relative MFI). Only a subset of PF4 mutants was able to support platelet activation, despite having lower surface binding than the minimum binding required of wild-type PF4 (9 mutants with KKO and 2 mutants with HIT-patient plasma). Using individual PF4 mutants, we identified that HIT immune complexes can be formed in fluid-phase and induce platelet activation. Further studies are required to investigate the role of fluid-phase HIT immune complexes in the development of thrombocytopenia and thrombosis associated with clinical HIT.