The Binding of the SARS-CoV-2 Spike Protein to Platelet Factor 4: A Proposed Mechanism for the Generation of Pathogenic Antibodies.

Pathogenic platelet factor 4 (PF4) antibodies contributed to the abnormal coagulation profiles in COVID-19 and vaccinated patients. However, the mechanism of what triggers the body to produce these antibodies has not yet been clarified. Similar patterns and many comparable features between the COVID-19 virus and heparin-induced thrombocytopenia (HIT) have been reported. Previously, we identified a new mechanism of autoimmunity in HIT in which PF4-antibodies self-clustered PF4 and exposed binding epitopes for other pathogenic PF4/eparin antibodies. Here, we first proved that the SARS-CoV-2 spike protein (SP) also binds to PF4. The binding was evidenced by the increase in mass and optical intensity as observed through quartz crystal microbalance and immunosorbent assay, while the switching of the surface zeta potential caused by protein interactions and binding affinity of PF4-SP were evaluated by dynamic light scattering and isothermal spectral shift analysis. Based on our results, we proposed a mechanism for the generation of PF4 antibodies in COVID-19 patients. We further validated the changes in zeta potential and interaction affinity between PF4 and SP and found that their binding mechanism differs from ACE2-SP binding. Importantly, the PF4/SP complexes facilitate the binding of anti-PF4/Heparin antibodies. Our findings offer a fresh perspective on PF4 engagement with the SARS-CoV-2 SP, illuminating the role of PF4/SP complexes in severe thrombotic events.

High-frequency Contactless Sensor for the Detection of Heparin-Induced Thrombocytopenia Antibodies via Platelet Aggregation.

Heparin-induced thrombocytopenia (HIT), a severe autoimmune disorder, occurs in patients undergoing heparin therapy. The presence of platelet-activating antibodies against platelet factor 4/Heparin in the blood confirms patients suffering from HIT. The most widely used methods for HIT diagnosis are immunoassays but the results only suit to rule out HIT as the assays provide only around 50% specificity. To confirm HIT, samples with positive results in immunoassays are retested in functional assays (>98% specificity) that track platelet-activating antibodies via platelet aggregation. However, the protocols in functional assays are either time-consuming (due to the requirement of the detection of serotonin release) or require highly trained staff for the visualization of platelets. Here, we applied a cheap and easy-to-use contactless sensor, which employs high-frequency microwaves to detect the changes in the resonant frequency caused by platelet aggregation/activation. Analysis of change in conductivity and permittivity allowed us to distinguish between HIT-like (KKO) and non-HIT-like (RTO) antibodies. KKO caused a stronger reduction of conductivity of platelet samples than RTO. Our results imply that the high-frequency contactless sensor can be a promising approach for the development of a better and easier method for the detection of HIT.

Label-Free Detection and Characterization of Heparin-Induced Thrombocytopenia (HIT)-like Antibodies.

Heparin-induced thrombocytopenia (HIT) antibodies (Abs) can mediate and activate blood cells, forming blood clots. To detect HIT Abs, immunological assays with high sensitivity (≥95%) and fast response are widely used, but only about 50% of these tests are accurate as non-HIT Abs also bind to the same antigens. We aim to develop biosensor-based electrical detection to better differentiate HIT-like from non-HIT-like Abs. As a proof of principle, we tested with two types of commercially available monoclonal Abs including KKO (inducing HIT) and RTO (noninducing HIT). Platelet factor 4/Heparin antigens were immobilized on gold electrodes, and binding of antibodies on the chips was detected based on the change in the charge transfer resistance (R ct). Binding of KKO on sensors yielded a significantly lower charge transfer resistance than that of RTO. Bound antibodies and their binding characteristics on the sensors were confirmed and characterized by complementary techniques. Analysis of thermal kinetics showed that RTO bonds are more stable than those of KKO, whereas KKO exhibited a higher negative ζ potential than RTO. These different characteristics made it possible to electrically differentiate these two types of antibodies. Our study opens a new avenue for the development of sensors for better detection of pathogenic Abs in HIT patients.