Monoclonal and Oligoclonal Anti-PF4 Antibodies Mediate VITT

Background: ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen Johnson & Johnson) vaccines against COVID-19 have been associated with thrombotic thrombocytopenic reactions referred to as vaccine-induced immune thrombotic thrombocytopenia (VITT) characterized by the presence of platelet-activating, anti-PF4 antibodies. While VITT shares key clinical features with a similar but separate entity, Heparin-induced thrombocytopenia (HIT), there appear to be important differences: 1) VITT patients have extremely high thrombosis rates and are very strongly positive in PF4-polyanion ELISAs, and 2) Many patients with VITT frequently present with refractoriness to therapy or have disease recurrence that suggests distinct antibody characteristics due to a strong autoimmune anti-PF4 response. Aims: The goal of this study was to characterize anti-PF4 antibodies in VITT. Methods: Five VITT patients were studied, one after ChAdOx1 nCoV-19 vaccination and four after Ad26.COV2. Reactivity of VITT anti-PF4 antibodies to uncomplexed PF4, PF4-Polyvinyl sulfonate (PVS), and PF4-heparin targets was evaluated, and the platelet-activating ability of these antibodies was examined in the PF4-dependent P-selectin Expression assay (PEA). Anti-PF4 antibodies were isolated from patient blood samples using PF4-treated heparin sepharose beads, and isolated antibodies were subject to mass spectrometric evaluation (Liquid Chromatography Electrospray Ionization Quadrupole time-of-flight mass spectrometry [LC-ESI-QTOF MS]). Results: Antibodies from all VITT patients recognized both uncomplexed and complexed PF4 (Fig. 1A). Interestingly, recognition of PF4 by VITT antibodies was lower if PF4 targets were complexed with polyanions, PVS, or heparin (Fig. 1A). These results contrasted with those obtained in a "classical" HIT patient which showed reactivity to PF4/polyanion complexes, but not to uncomplexed PF4 (Fig 1A). All samples activated platelets in the PEA (data not shown). Mass spectrometric evaluation of anti-PF4 antibodies isolated from VITT patients demonstrated monoclonal anti-PF4 antibodies in three patients, and bi- and tri-clonal antibodies in one patient each (a representative monoclonal antibody anti-PF4 antibody is shown in Fig 1B). Consistent with current dogma, polyclonal anti-PF4/polyanion antibodies were seen in "classical" HIT (Fig 1C). Evaluation of anti-PF4 antibodies in spontaneous HIT, a type of autoimmune HIT seen in pro-inflammatory milieus such as orthopedic surgery and infectious prodromes also demonstrated monoclonal anti-PF4 antibodies (Fig 1D). Eluates from control heparin-sepharose beads did not reveal any immunoglobulins (data not shown). Conclusion: Although development of platelet-activating anti-PF4 antibodies and the thrombotic thrombocytopenia syndrome seen after ChAdOx1 nCoV-19 and Ad26.COV2.S vaccination resembles HIT, these findings demonstrate that clonally restricted anti-PF4 antibodies mediate VITT while polyclonal anti-PF4 antibodies mediate HIT. In addition, we noted clonally-restricted anti-PF4 antibodies in another condition that does not require proximate heparin exposure, spontaneous ("autoimmune") HIT. In VITT, the strong immune response after vaccine administration may result in the activation of a single or few pre-existing anti-PF4 reactive clones, and development of clonally restricted anti-PF4 antibodies with a similar pathophysiology to Spontaneous HIT. It is also likely that high levels of monoclonal/oligoclonal anti-PF4 antibodies cause the severe thrombotic phenotypes seen in VITT and Spontaneous HIT. The high mortality rate and reports of disease refractoriness to therapy in VITT may warrant consideration of additional therapeutic modalities like rituximab and therapeutic plasma exchange in select cases. Figure Legends: (A): VITT (Patient 1-ChAdOx1 nCoV-19; Patients 2-5, Ad26.COV2.S) patient samples were tested in ELISA against uncomplexed PF4 (white), and PF4 in complex with polyvinyl sulfonate (light grey), or unfractionated heparin (dark gray). (B-D) Mass spectrometric evaluation of anti-PF4 antibodies isolated from VITT (B), HIT (C) and spontaneous HIT patient sera (D). "Relative Intensity" refers to abundance of the Ig light chain relative to the polyclonal background. Numbers above Ig light chain peaks depict mass/charge ratios. NC- Normal control. Figure 1 Figure 1. Disclosures Murray: Mayo Clinic: Other: Has received patents for the Mass-Fix technology which has been licensed to the Binding Site with potential royalties.. Padmanabhan: Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees.

Spontaneous heparin-induced thrombocytopaenia with adrenal haemorrhage following orthopaedic surgery: a case report and literature review

A 68-year-old woman was admitted to the hospital for elective total knee arthroplasty in both knees without preceding heparin exposure. She developed adrenal haemorrhage and thrombocytopaenia on postoperative day 12, followed by right leg arterial occlusion and multiple venous intra-abdominal sites thrombosis. After given unfractionated heparin to treat arterial occlusion, platelet count was gradually declined. Spontaneous heparin-induced thrombocytopaenia was diagnosed by heparin-induced platelet activation test with light transmission aggregometry. The patient was successfully treated with fondaparinux and intravenous immunoglobulin. Apixaban was given after recovery of platelet count. Resolution of both thrombus along aorta and adrenal haemorrhage were shown by CT of whole abdomen after 2 months of treatment. Our case demonstrates that this serious complication is important but seldom recognised early.

Autoimmune heparin-induced thrombocytopenia: a rare manifestation of COVID-19

We describe the case of a 65-year-old male who presented to an outside hospital for shortness of breath, nausea and vomiting 8 days after testing positive for COVID-19. Initial workup revealed massive bilateral pulmonary emboli and thrombocytopenia. The patient was then admitted to our hospital, received an inferior vena cava filter and initially started on argatroban for autoimmune heparin-induced thrombocytopenia (HIT) prophylaxis. On hospital stay day 6, labs revealed a diagnosis of HIT in the setting of COVID-19. This case highlights the rare occurrence of a patient developing HIT without heparin exposure and in the setting of a novel infectious agent, COVID-19.

SARS-CoV-2 receptor binding domain-specific antibodies activate platelets with features resembling the pathogenic antibodies in heparin-induced thrombocytopenia

Severe COVID-19 is associated with unprecedented thromboembolic complications. We found that hospitalized COVID-19 patients develop immunoglobulin Gs (IgGs) that recognize a complex consisting of platelet factor 4 and heparin similar to those developed in heparin-induced thrombocytopenia and thrombosis (HIT), however, independent of heparin exposure. These antibodies activate platelets in the presence of TLR9 stimuli, stimuli that are prominent in COVID-19. Strikingly, 4 out of 42 antibodies cloned from IgG1+ RBD-binding B cells could activate platelets. These antibodies possessed, in the heavy-chain complementarity-determining region 3, an RKH or Y5 motif that we recently described among platelet-activating antibodies cloned from HIT patients. RKH and Y5 motifs were prevalent among published RBD-specific antibodies, and 3 out of 6 such antibodies tested could activate platelets. Features of platelet activation by these antibodies resemble those by pathogenic HIT antibodies. B cells with an RKH or Y5 motif were robustly expanded in COVID-19 patients. Our study demonstrates that SARS-CoV-2 infection drives the development of a subset of RBD-specific antibodies that can activate platelets and have activation properties and structural features similar to those of the pathogenic HIT antibodies.

Incidence, Outcomes and Risk Factors of Heparin-Induced Thrombocytopenia After Total Joint Arthroplasty: A National Inpatient Sample Database Study

Backgrounds: Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated complication of heparin exposure, leading to negative consequences after total hip (THA) and knee arthroplasty (TKA). Materials and Methods: A retrospective study was conducted using the National Inpatient Sample (NIS) database from 2005 to 2014. The incidence and outcomes of HIT after THA or TKA were documented. Logistic regression analysis was performed to identify the postoperative HIT risk factors. Results: A total of 59 3045 patients who underwent THA and 1228 707 patients who underwent TKA were identified. The cumulative incidences were 0.02% and 0.01%, respectively. The HIT group presented significantly higher Charlson Comorbidity Index and Elixhauser Comorbidity Index scores, longer hospital stays (LOS), and higher medical costs. HIT led to a significantly higher mortality rate after THA (2.17% vs 0.16%, P = .0091). In THA, the HIT risk factors were racial minority, AIDS, pulmonary circulation disorders (PCD), psychoses, and hypertension. In TKA, the HIT risk factors were racial minority, PCD, and weight loss. Conclusion: The incidence of HIT after THA and TKA is relatively low; however, HIT significantly increases inpatient mortality, LOS, and medical cost.

Heparin-induced thrombocytopenia (HIT): Review of incidence, diagnosis, and management

Heparin-induced thrombocytopenia (HIT) is a life and limb-threatening complication of heparin exposure. Here, we review the pathogenesis, incidence, diagnosis, and management of HIT. The first step in thwarting devastating complications from this entity is to maintain a high index of clinical suspicion, followed by an accurate clinical scoring assessment using the 4Ts. Next, appropriate stepwise laboratory testing must be undertaken in order to rule out HIT or establish the diagnosis. In the interim, all heparin must be stopped immediately, and the patient administered alternative anticoagulation. Here we review alternative anticoagulation choice, therapy alternatives in the difficult-to-manage patient with HIT, and the problem of overdiagnosis.

Heparin ‘flush’ induced thrombocytopenia triggered by total hip replacement: a case report

Background Heparin-induced thrombocytopenia (HIT) typically responds to heparin termination. Some types of HIT can persist after heparin discontinuation. Case summary A 95-year-old woman was referred to the cardiology from orthopaedics because of acute limb ischaemia (ALI) 1 day after surgery of a femoral neck fracture. Despite thrombectomy, ALI relapsed the next day. She had been treated with intravenous antibiotics with a diagnosis of aspiration pneumonia for 1 week until 3 days before surgery, together with heparin flush twice a day. Of note, no intra-/post-operative heparin was administered, no cell salvage device, central venous, nor arterial catheters were used before development of ALI. The patient and her family refused reattempting invasive therapies; consequently, the patient continued to worsen and died on post-operative day 3. Diagnosis of autoimmune HIT, which was prompted by surgery without re-exposure to heparin, was confirmed posthumously. Discussion This case emphasizes the significance of suspecting autoimmune HIT in any patient presenting with thrombosis, even if the heparin exposure dates back more than a few days or even without heparin exposure.

A practical approach to evaluating postoperative thrombocytopenia

Identifying the cause(s) of postoperative thrombocytopenia is challenging. The postoperative period includes numerous interventions, including fluid administration and transfusion of blood products, medication use (including heparin), and increased risk of organ dysfunction and infection. Understanding normal thrombopoietin physiology and the associated expected postoperative platelet count changes is the crucial first step in evaluation. Timing of thrombocytopenia is the most important feature when differentiating causes of postoperative thrombocytopenia. Thrombocytopenia within 4 days of surgery is commonly caused by hemodilution and increased perioperative platelet consumption prior to thrombopoietin-induced platelet count recovery and transient platelet count overshoot. A much broader list of possible conditions that can cause late-onset thrombocytopenia (postoperative day 5 [POD5] or later) is generally divided into consumptive and destructive causes. The former includes common (eg, infection-associated disseminated intravascular coagulation) and rare (eg, postoperative thrombotic thrombocytopenic purpura) conditions, whereas the latter includes such entities as drug-induced immune thrombocytopenia or posttransfusion purpura. Heparin-induced thrombocytopenia is a unique entity associated with thrombosis that is typically related to intraoperative/perioperative heparin exposure, although it can develop following knee replacement surgery even in the absence of heparin exposure. Very late onset (POD10 or later) of thrombocytopenia can indicate bacterial or fungal infection. Lastly, thrombocytopenia after mechanical device implantation requires unique considerations. Understanding the timing and severity of postoperative thrombocytopenia provides a practical approach to a common and challenging consultation.

Spontaneous heparin-induced thrombocytopenia syndrome presenting as bilateral adrenal infarction after knee arthroplasty

Adrenal gland infarction resulting from adrenal vein thrombosis is an infrequently recognised entity with a limited differential diagnosis. When bilateral, it can result in acute life-threatening adrenal failure. Heparin-induced thrombocytopenia (HIT) is an antibody-mediated, prothrombotic state that represents an important cause of adrenal vein thrombosis leading to associated infarction. Sometimes, the clinical picture of HIT—including the presence of HIT antibodies—occurs despite absence of proximate heparin exposure (‘spontaneous HIT syndrome’). We report a case of nearly missed adrenal failure secondary to bilateral adrenal infarction that evolved during the second week following knee arthroplasty (a known trigger of spontaneous HIT syndrome). The combination of bilateral adrenal infarction, thrombocytopenia and presence of platelet-activating HIT antibodies not explainable by preceding heparin exposure led to a diagnosis of postknee arthroplasty spontaneous HIT syndrome. The case also highlights the clinical and laboratory findings associated with rapidly progressive acute adrenal failure.