key: cord-0933970-w4bnbx8c authors: Smith, Christopher W.; Montague, Samantha J.; Kardeby, Caroline; Di, Ying; Lowe, Gillian C.; Lester, William A.; Watson, Steve P.; Nicolson, Phillip L.R. title: Antiplatelet drugs block platelet activation by VITT patient serum date: 2021-12-23 journal: Blood DOI: 10.1182/blood.2021012277 sha: f829cd3259d217d44e9e4d0b520510189e89fd53 doc_id: 933970 cord_uid: w4bnbx8c nan Vaccines are an important part of the response to the SARS-COV-2 global pandemic. Although rare, aggressive thrombotic events at unusual sites, with accompanying thrombocytopenia and bleeding with high mortality, have increasingly been reported in young, healthy individuals at 4 to 30 days after vaccination with the Oxford-AstraZeneca chimpanzee adenovirusvectored ChAdOx1 nCoV-19 (AZD1222). 1, 2 This syndrome of vaccine-induced immune thrombocytopenia and thrombosis (VITT) clinically resembles autoimmune heparin-induced thrombocytopenia (HIT), in which antibodies against platelet factor 4 (PF4) bind and cross-link to the platelet surface receptor FcgRIIA (CD32a), inducing platelet activation. [1] [2] [3] VITT after the first AZD1222 vaccination has a reported incidence of between 1 in 25 000 and 1 in 100 000. 2, 4, 5 In this study, we investigated the effect of serum from patients with VITT on platelet activation monitored by light transmission aggregometry (LTA), assessing the ability of clinically available antiplatelet drugs and kinase inhibitors to prevent platelet aggregation in vitro. Blood collection from patients, healthy individuals after AZD1222 vaccination, and nonvaccinated healthy donors were authorized under research ethics approvals 15/NW/0079 and 20/HRA/1817 and Birmingham University Internal Ethical Review approval ERN_11-0175, respectively. Experimental procedures are detailed in the supplemental Information (available on the Blood Web site). Patients (or their next of kin in the case of those patients who lacked capacity) gave informed consent for collection of their blood in line with ethical principles laid out in the Declaration of Helsinki. The presentations of 7 patients with VITT are summarized in Table 1 . All patients were Caucasian and under the age of 50 with no previous symptomatic COVID- 19 . Patients presented with thrombosis (6 patients with cerebral venous sinus thrombosis [CVST] and 1 patient with ischemic stroke) and thrombocytopenia 9 to 14 days after the first AZD1222 vaccination. Clinical investigation at the time of presentation revealed all patients had thrombocytopenia (range, 7-113 3 10 9 platelets per L), with massively elevated D-dimer (range, 6574-62 342 ng/mL) and low fibrinogen (range, ,0.35-2.36 g/L) levels. Despite no prior heparin exposure, HIT screening (anti-PF4 IgG Immucor enzymelinked immunosorbent assay) showed strong reactivity in all patients. Heparin-induced platelet activation (HIPA) assays in the 4 patients tested showed activation in response to patient serum that was reduced by low heparin concentrations and blocked by high ones. Similar findings are reported in other patients with VITT. 1,2 All patients received IVIg and the steroid dexamethasone, as recommended by VITT treatment guidelines, 6 and 2 patients received plasma exchange. Platelet counts improved over 1 to 4 days in all patients except 1 who died 24 hours after presentation. At the time of this writing, 3 patients had recovered and been discharged from the hospital with ongoing normal platelet counts, 1 patient remained in hospital, and 2 patients had died because of the sequelae of CVST and secondary intracerebral hemorrhage. In addition, 1 discharged patient, LETTERS TO BLOOD who was taking dabigatran, relapsed with thrombocytopenia and headaches but without thrombosis or raised D-dimer ,8 weeks after discharge and required repeat treatment with IVIg and corticosteroids. Serum from patients with VITT, but not age-matched AZD1222vaccinated or non-vaccinated healthy donors, induced platelet aggregation (Figure 1Ai -ii and data not shown). Variable degrees of platelet aggregation, depending on patient serum and platelet donor, were observed (Figure 1Ai-ii) , which is similar to results in HIT and other VITT studies, with platelets from some healthy donors not responding. 1, 7 Low-titer anti-PF4 antibodies have been shown to develop after vaccination in a small percentage of healthy individuals; however, they do not cause platelet activation. 8 Aggregation was blocked after IVIg treatment, except in the 2 patients who did not clinically respond to IVIg and required plasma exchange (Figure 1Aii -iii). In these 2 patients, aggregation responses were blocked after plasma exchange (Figure 1Aii-iii) . Eptifibatide treatment confirmed that responses were aggregation not agglutination (data not shown). Platelet activation by patient serum was abolished by IV.3 F(ab) blockade of FcgRIIA (Figure 1Ai-iii) . This result is similar to those in another report 1 and implies that activation is most likely mediated by clustering of the receptor by IgG and immune complexes, 9 demonstrating that platelet activation in VITT is mediated by FcgRIIA. Low concentrations of heparin are known to enhance platelet responses in HIT assays, whereas high concentrations are inhibitory. 10, 11 In contrast, low (0.2 U/mL) concentrations of heparin prevented (5 of 7 patients) or delayed (2 of 7 patients) aggregation (Figure 1Ai-iii) . High heparin concentration (100 U/mL) blocked aggregation (data not shown). Immune complexes that activate platelets via FcgRIIA have been reported in patients critically ill with COVID-19. 12 In these patients, who had been exposed to heparin and displayed thrombocytopenia and thrombosis, HIT was ruled out, because of the lack of anti-PF4 antibodies and platelet activation independent of heparin. 12 Analogous to our findings, platelet activation by these immune complexes was blocked by both low and high concentrations of heparin. 12 Our observation that heparin blocks platelet aggregation, which is consistent with HIPA results and other reports, 1, 13, 14 implies that the decision to withhold heparin use in patients with VITT perhaps should be revisited. Unfractionated heparin treatment has been reported in 1 patient with VITT without deleterious effect. 14 Anti-SARS-CoV-2 spike protein IgG antibodies from patients with severe COVID-19 have been shown to induce apoptosis and increase phosphatidylserine externalization in platelets mediated by FcgRIIA, although IgG aggregates or immune complexes could not be isolated from patient sera. 15 It is possible that a similar mechanism is occurring in patients with VITT. Activation of FcgRIIA could give rise to phosphatidylserine exposure and procoagulant platelets, which may lead to the extensive thrombosis and thrombocytopenia observed in patients with VITT. 13 A role for complement has been proposed in VITT. Heat treatment of sera, which inactivates complement (56 C, 45 minutes), blocked aggregation in 3 of 7 patients (Figure 1Ai-iii) , whereas minor effects on aggregation were observed with compstatin (a C3a inhibitor) and FUT-175 (a C3, C4, and C5 inhibitor; Figure 1B ). These findings indicate that, although complement is not critical, it may reinforce platelet activation. Eculizumab (anti-C5 monoclonal antibody) treatment has been reported in 2 patients with VITT, in whom anticoagulation and IVIg or plasma exchange failed. 14 Both patients rapidly improved. The involvement of complement, which mediates a broad range of thromboinflammatory reactions involving endothelium, monocytes, and neutrophils, as well as platelets, in VITT pathology should be considered. 16 Normal serum complement levels in patients with VITT have been reported. 2 We tested a variety of clinically used antiplatelet drugs and inhibitors of kinases downstream of FcgRIIA to determine whether they could prevent platelet aggregation in response to patient sera. 17 The COX inhibitor indomethacin, which works via the same mechanism as aspirin, and the P2Y 12 inhibitor ticagrelor prevented aggregation in response to patient serum, as did the Src inhibitor dasatinib and the Btk inhibitors ibrutinib and rilzabrutinib, with a significant reduction observed in response to the Syk inhibitor entospletinib ( Figure 1C ). This inhibition occurred irrespective of heterogeneity in samples from patients with VITT. All inhibitors were used at a concentration that fully inhibited aggregation in response to 3 mg/mL collagen (results not shown). Although these antiplatelet and kinase inhibitors prevent aggregation in healthy donor platelets in vitro, further study in more physiological and clinically relevant assays assessing multiple additional readouts is needed before their use in treating patients with VITT can be considered. The potential clinical utility of some of these agents may be limited, however, by their associated bleeding risk. The risk of major bleeding with population-wide use of the COX inhibitor aspirin outweighs any theoretical benefit for this rare syndrome. 18 It should also be noted that VITT has been diagnosed in a patient already taking aspirin, 19 and our patient, who was initially treated with aspirin for a stroke, still developed progressive thrombocytopenia despite this intervention. Similarly, ticagrelor, dasatinib, and ibrutinib are associated with increased bleeding risk, so their use in patients with thrombocytopenia cannot be recommended. [20] [21] [22] Rilzabrutinib, currently in trials for immune thrombocytopenia (ITP) with no bleeding or thrombotic events reported, 23 appears to be a more promising treatment for further study, as does the Syk inhibitor fostamatinib, which is also an ITP treatment that lowers thrombosis without causing bleeding 24 ; however, its active metabolite R406, used in this study at its clinically relevant concentration, did not effectively block platelet activation in vitro. Entospletinib, although not associated with bleeding, is not yet routinely used outside of clinical trials and has not been used in patients with thrombocytopenia. 25 If ongoing treatment is required because of inadequate response to the scarce and expensive IVIg and plasma exchange, then these antiplatelet agents have a potential role and warrant further evaluation. The limitations of this study are the small sample size and the differing treatments received before collection of the patient samples. In addition, only a limited number of conditions were tested because of the volume of sera available, and aggregation was measured only over a period of 10 minutes, with current consensus for examining aggregation in response to serum from patients with VITT for 30 minutes. Overall, we have demonstrated that serum from patients with VITT, but not healthy AZ1222D-vaccinated donors, activates platelets via FcgRIIA, which can be blocked in vitro by antiplatelet therapies and tyrosine kinase inhibitors. Further assessment of these potential therapeutic interventions in physiological and clinically relevant models are needed before their use in patients with this rare syndrome can be considered. Thrombotic thrombocytopenia after ChAdOx1 nCov-19 vaccination Thrombosis and thrombocytopenia after ChAdOx1 nCoV-19 vaccination Autoimmune heparin-induced thrombocytopenia Arterial events, venous thromboembolism, thrombocytopenia, and bleeding after vaccination with Oxford-AstraZeneca ChAdOx1-S in Denmark and Norway: population based cohort study MHRA -Medicines & Healthcare products Regulatory Agency. Coronavirus vaccine -weekly summary of Yellow Card reporting Guidance Produced from the Expert Haematology Panel (EHP) Focussed on Covid-19 Vaccine Induced Thrombosis and Thrombocytopenia (VITT) Determinants of donor platelet variability when testing for heparininduced thrombocytopenia Frequency of positive anti-PF4/ polyanion antibody tests after COVID-19 vaccination with ChAdOx1 nCoV-19 and BNT162b2 Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia A comparative study of platelet factor 4-enhanced platelet activation assays for the diagnosis of heparin-induced thrombocytopenia Beneficial effect of exogenous platelet factor 4 for detecting pathogenic heparininduced thrombocytopenia antibodies Platelet-activating immune complexes identified in critically ill COVID-19 patients suspected of heparin-induced thrombocytopenia Antibody-mediated procoagulant platelets in SARS-CoV-2-vaccination associated immune thrombotic thrombocytopenia Prothrombotic immune thrombocytopenia after COVID-19 vaccine Antibody-induced procoagulant platelets in severe COVID-19 infection Is complement the culprit behind COVID-19 vaccine-related adverse reactions? Human platelet IgG Fc receptor FcgRIIA in immunity and thrombosis Association of aspirin use for primary prevention with cardiovascular events and bleeding events: a systematic review and meta-analysis Adjunct immune globulin for vaccine-induced thrombotic thrombocytopenia Bleeding complications with the P2Y12 receptor antagonists clopidogrel and ticagrelor in the PLATelet inhibition and patient Outcomes (PLATO) trial Ibrutinib-associated bleeding: pathogenesis, management and risk reduction strategies Bleeding diathesis in patients with chronic myelogenous leukemia receiving dasatinib therapy Phase I/II, open-label, adaptive study of oral bruton tyrosine kinase inhibitor prn1008 in patients with relapsed/refractory primary or secondary Immune thrombocytopenia Assessment of thrombotic risk during long-term treatment of immune thrombocytopenia with fostamatinib Entospletinib monotherapy in patients with relapsed or refractory chronic lymphocytic leukemia previously treated with B-cell receptor inhibitors: results of a phase 2 study © 2021 by The The online version of this article contains a data supplement.