COVID-19 (Coronavirus Disease 2019) is a now well-recognized pandemic caused by infection with the virus SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2). COVID-19 is a clearly prothrombotic disorder that involves multiple hemostasis pathways of interest, including platelet activation [19]. However, thrombosis in COVID-19 patients is multi-factorial, and platelets only play a part in a larger coagulopathic process. Unlike in HIT patients, platelet counts in COVID-19 patients are not usually very low, and so these patients are considered only mildly thrombocytopenic. In COVID-19, multiple hemostatic pathways can be affected, including primary hemostasis (platelets and von Willebrand factor [VWF]), secondary hemostasis (‘coagulation’), and fibrinolysis. Moreover, anti-PF4 antibodies do not arise in the majority of COVID-19 patients [20] and so cannot be considered a major driver of COVID-19-associated coagulopathy.

Nevertheless, of relevance to the current review, beside direct platelet activation resulting from direct interaction of SARS-CoV-2 spike protein with platelet receptors [15], ‘HIT-like’ events may occur in a small proportion of patients with COVID-19, and there have been several reports of anti-PF4 antibodies in COVID-19 patients, as recently reviewed by some of us [20]. In some cases, these were identified as involving heparin (i.e., anti-PF4/H antibodies were identified); however, in other cases, they did not involve prior heparin exposure (i.e., they were not anti-PF4/H antibodies, and so can be considered anti-PF4/X antibodies). Indeed, in some reports, the addition of therapeutic heparin levels in an assay can be shown to decrease antibody detection by immunological assays or inhibit platelet activation in functional assays, further confirming that these are not anti-PF4/H antibodies. Nevertheless, these events cannot really be considered as a form of ‘spontaneous HIT-like syndrome’, since SARS-CoV-2 is a likely trigger in at least some of these patients. Nevertheless, they do likely represent an analogous entity to the HIT-like syndromes mentioned in the preceding section, albeit associated with a particular viral infection, being SARS-CoV-2 [16]. Whether the anionic species associated with PF4 (i.e., the ‘X’ in PF4/X) is part of the virus, part of an associated co-bacterial infection, or simply arises due to platelet activation and consequent complex formation is still unknown.

In summary, a small proportion of COVID-19 patients may have anti-PF4/X antibodies, only a fraction of which can be identified as anti-PF4/H antibodies, with the remainder representing antibodies against PF4, potentially in complex with an as yet unknown ‘anionic species’ (‘X’) [20,21,22]. From the laboratory testing perspective, these will be anti-PF4 ELISA antibody-positive by immunological assessment, and SRA testing may show platelet activation in either presence or absence of added therapeutic heparin, depending on whether antibody development was due to heparin exposure (PF4/H complexes) or not (PF4/X complexes). In either case, a high dose (supra-therapeutic) of heparin should show inhibition of platelet activation/aggregation.

The COVID-19 pandemic has led to the rapid production and deployment of a large number of COVID-19 vaccines [23,24]. Notably, some minor adverse reactions may be anticipated for any vaccination program. Unfortunately, occasional rare and potentially fatal adverse events may also arise. One such event appears to arise in a small proportion of individuals vaccinated with COVID-19 adenovirus-based vaccines. Termed VITT (for vaccine-induced [immune] thrombotic thrombocytopenia) by the workers who first reported on the associated pathophysiological events [25,26,27], the term TTS (for thrombotic thrombocytopenia syndrome) may preferentially be used by government reporting agencies (for example, the FDA [Food and Drug Administration] in the USA, the EMA [European Medicines Agency] in Europe, and the TGA [Therapeutic Good Administration] in Australia) [23]. VITT and TTS after COVID-19 vaccine use essentially represent the ‘same’ condition, albeit that the specific case definition used to define VITT or TTS may result in the recognition of different patient cohorts. Even within the entity described as VITT, different patient cohorts may be identified, according to the diagnostic pathway used [28]. In a recent review, some half-dozen diagnostic pathways were identified as being recommended by various expert groups [28]. Whilst the pathways in general aimed to accurately identify VITT patients, differences in the approach could lead to inclusion or exclusion of some cases relative to a different diagnostic approach. For example, some diagnostic pathways restricted the inclusion of cases up to 28 days post-vaccine exposure, whereas others captured cases up to 42 days post-exposure [28]. Some diagnostic pathways placed stronger emphasis on D-dimer measurements than others, and some diagnostic pathways restricted case capture only to patients with thrombocytopenia, whereas some pathways included case capture for patients without thrombocytopenia but with a substantive fall in platelet counts, akin to 4Ts in HIT assessment.

VITT/TTS also has a recognized prevalence, albeit potentially different according to the vaccine with which it occurs. For example, for the AstraZeneca vaccine (also known as ChAdOx1 nCoV-19, AZD1222, Vaxzevria), the prevalence is around 1 in 80,000 doses or between 10 and 15 cases per million doses (Figure 1). For the Janssen (Johnson & Johnson) vaccine (alternatively known as Ad26.COV2.S or JNJ-78436735), the prevalence seems to be lower, perhaps 1 in 500,000 doses, or ~2 cases/million doses [23]. The prevalence of VITT/TTS will also differ according to the case definition and the diagnostic pathway chosen, as further outlined above, of which there are many [28]. For example, a total of only 58 cases of TTS worldwide were identified by one recent systematic review as of 23 August 2021, as based on WHO criteria for TTS identification [29]. In contrast, a separate review performed by one of us [30] identified at least 83 cases of VITT worldwide as of a much earlier date, i.e., 27 May 2021, and based on clinical presentations and results of laboratory tests. Thus, the systematic review would significantly underestimate the number of TTS cases worldwide, which currently stand at >150 for Australia alone, according to a recent TGA report (Figure 1).

Of major relevance to the current review, VITT/TTS is also characterized by the presence of anti-PF4 antibodies, or more likely anti-PF4/X antibodies, with X being an unknown anionic ‘species’ at present (perhaps, heparan sulfate proteoglycans or vaccine components such as adenovirus-derived hexon) [32,33]. Again, these anti-PF4 antibodies can be detected by immunological assays, but unlike in HIT, not all such assays can identify these antibodies with sufficient diagnostic sensitivity. Indeed, only the ELISA-based techniques can consistently identify anti-PF4 (or anti-PF4/X) antibodies in VITT/TTS, with all other immunological methods, including rapid assays, either not detecting the antibodies or only detecting these in a minor proportion of patients (i.e., typically below 30%) [21]. This tends to support the concept that the ‘X’ in anti-PF4/X for VITT/TTS is not heparin. Perhaps also interesting here is that the in vitro use of heparin in laboratory testing tends to reduce the level of detected antibodies in immunological assays for the majority of VITT patients and tends to inhibit platelet activation in functional assays, essentially proving that the species in complex with PF4 (at least, in the majority of patients) is not heparin. An example of this for two patients from the Westmead Hospital laboratory is provided in Figure 2. However, therapeutic heparin does not inhibit platelet activation in all VITT patients [34,35], and so this does not provide an infallible distinction from HITT.