Proteomics of fibrin amyloid microclots in long COVID/post-acute sequelae of COVID-19 (PASC) shows many entrapped pro-inflammatory molecules that may also contribute to a failed fibrinolytic system

Abstract

Background Post-acute sequelae of COVID-19 (PASC), also now known as long COVID, has become a major global health and economic burden. Previously, we provided evidence that there is a significant insoluble fibrin amyloid microclot load in the circulation of individuals with long COVID, and that these microclots entrap a substantial number of inflammatory molecules, including those that might prevent clot breakdown. Scientifically, the most challenging aspect of this debilitating condition is that traditional pathology tests such as a serum CRP (C-reactive protein) may not show any significant abnormal inflammatory markers, albeit these tests measure only the soluble inflammatory molecules. Elevated, or abnormal soluble biomarkers such as IL-6, D-Dimer or fibrinogen indicate an increased risk for thrombosis or a host immune response in COVID-19. The absence of biomarkers in standard pathology tests, result in a significant amount of confusion for patients and clinicians, as patients are extremely sick or even bed-ridden but with no regular identifiable reason for their disease. Biomarkers that are currently available cannot detect the molecules present in the microclots we identified and are therefore unable to confirm their presence or the mechanisms that drive their formation. Methods Here we analysed the protein content of double-digested microclots of 99 long COVID patients and 29 healthy controls. The patients suffering from long COVID reported their symptoms through a questionnaire completed by themselves or their attending physician. Results Our long COVID cohort’s symptoms were found to be in line with global findings, where the most prevalent symptoms were constant fatigue (74%,) cognitive impairment (71%) and depression and anxiety (30%). Our most noteworthy findings were a reduced level of plasma Kallikrein compared to our controls, an increased level of platelet factor 4 (PF4) von Willebrand factor (VWF), and a marginally increased level of α-2 antiplasmin (α-2-AP). We also found a significant presence of antibodies entrapped inside these microclots. Conclusion Our results confirm the presence of pro-inflammatory molecules that may also contribute to a failed fibrinolysis phenomenon, which could possibly explain why individuals with long COVID suffer from chronic fatigue, dyspnoea, or cognitive impairment. In addition, significant platelet hyperactivation was noted. Hyperactivation will result in the granular content of platelets being shed into the circulation, including PF4. Overall, our results provide further evidence of both a failed fibrinolytic system in long COVID/PASC and the entrapment of many proteins whose presence might otherwise go unrecorded. These findings might have significant implications for individuals with pre-existing comorbidities, including cardiovascular disease and type 2 diabetes.

Increased levels of inflammatory molecules in blood of Long COVID patients point to thrombotic endotheliitis

“…We have found that α-2AP is trapped inside microclots (10,19). This molecule prevents the fibrinolytic system from functioning optimally and impedes the digestion of pathological thrombi that cause microvascular thrombosis, thereby increasing the risk of thromboembolic events (10,19).…”

“…Participant identification for ELISA analysis For the enzyme-linked immunosorbent assay (ELISA) analysis, we randomly selected a subset of stored Long COVID and healthy samples, where we previously confirmed entrapped inflammatory molecules using proteomics analysis (10) (see Table 1). The sample included blood collected from 15 healthy individuals to serve as controls (1 male; 14 females; median age 42 ) and 25 Long COVID patients (9 males; 16 females; median age 52 ).…”

“…VWF is well-known inflammatory molecule that is widely used as a marker of inflammation and vascular damage (25), and is commonly available in pathology laboratories. We have found VWF to be trapped inside microclots and also upregulated in the soluble part of the plasma (10,19). When in circulation, it binds to two main receptors on the platelet membrane, including, GPIbα (in the GPIb-IX-V complex) and integrin αIIbβ3 (in the GPIIb-IIIa complex) (60) (see Figure 5).…”

“…There is also increasing evidence that autoantibodies may be involved in the lingering symptoms of individuals with Long COVID ( 15, 16 ). We have demonstrated that individuals with Long COVID have a significant fibrin amyloid microclot load in their circulation ( 2, 10, 17 ). These clots can be induced in normal plasma by (recombinant) SARS-CoV-2 spike protein ( 18 ), and are resistant to fibrinolysis ( 10, 19 ).…”

“…Post-Acute Sequelae of COVID-19 (PASC) or Long COVID is a major global health burden with its symptoms significantly impacting physical and cognitive function, health-related quality of life, and participation in society (1-9). The mechanisms for the persistent symptoms can be narrowed down to 1) viral persistence, 2) microclotting and platelet hyperactivation, 3) autoantibodies, 4) immune dysregulation, 5) widespread organ damage, and the 6) reactivation of dormant viruses (9)(10)(11)(12). SARS-CoV-2 (COVID-19) viral remnants including RNA and spike protein have been identified in patients with Long COVID.…”