TO THE EDITOR:
In the article entitled “SARS-CoV-2 spike protein can bind fibrin(ogen), but does not alter plasma fibrin formation, clot structure, or lysis,”1 we reproduced the reported finding that soluble spike protein (SP) binds immobilized fibrinogen2 and found that fibrinogen in solution binds immobilized SP. However, an important additional new result was that no binding was detected between these proteins in solution. Moreover, despite carefully replicating (and building on) the experimental conditions of Ryu et al,2 we observed no impact of SP on fibrin clot properties using human plasma.
In their letter, Pretorius and Kell3 take issue with the overarching conclusion of our work. Although SP or SP fragments have been identified within the thrombi of infected patients,4 a key question is whether SP alters plasma fibrin formation and breakdown. It is suggested, without evidence, that once in a plasma milieu the small amount of SP relative to fibrinogen forms a nonfunctional complex. These authors appear to fail to recognize the core tenet of their stated hypothesis. If SP associates with fibrinogen and the trace amounts of these complexes are partitioned from the bulk functional fibrinogen, they would be incapable of influencing clot dynamics. This is exactly what our data reveal using the turbidimetric assays of formation and lysis and confocal microscopy-based analyses of structure. Thus, their hypothesis, even if true, supports our conclusion that SP does not alter plasma fibrin formation, clot structure, or lysis.
Our data question whether thrombosis in COVID-19 is affected through mechanisms coupled to SP-fibrinogen binding. The original Wuhan strain of the virus was linked to increased rates of venous thromboembolism (VTE).5, 6, 7 However, assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants indicates that the VTE risk significantly diminished later in the pandemic and in the contemporary period (May 2022 to November 2024) is no greater than for other acute respiratory infections.8 These data are notable when considering amino acid (AA) changes in SP that define the variant SARS-CoV-2 strains. Five putative fibrinogen-binding domains in SP have been suggested that span AA 37 to 103, AA 229 to 251, AA 305 to 319, AA 341 to 355, and AA 1049 to 1063.2 Despite significant AA changes in other regions of the SP across the variants, including α, β, γ, and δ, through numerous omicron sublineages, few (if any) changes occur within the putative fibrinogen-binding domains (Table 1), suggesting fibrin(ogen) binding potential is likely maintained. Thus, it is unlikely that SP binding directly to fibrinogen, if it even occurs in circulation, contributes to COVID-19–induced VTE.
Table 1.
Alignment of the proposed SP-fibrinogen–binding regions across SARS-CoV-2 variants with differences highlighted in red text
| WHO label | Pango lineage | AA 37-103 | AA 229-251 | AA 305-319 | AA 341-355 | AA 1049-1063 |
|---|---|---|---|---|---|---|
| A.23.1 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL | |
| B.1.1.318 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL | |
| B.1.177 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL | |
| B.1.214.2 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL | |
| B.1.375 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI--SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL | |
| B.1.616 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFDAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL | |
| α | B.1.1.7 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI--SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| β | B.1.351 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNAGVYFASTEKSNIIRG | LPIGINITRFQTL---HISYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| δ | B.1.617.2 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ε | B.1.427 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| η | B.1.525 | YYPDKVFRSSVLHSTRDLFLPFFSNVTWFHVI--SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| γ | P1, B.1.1.28.1 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ι | B.1.526 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| κ | B.1.617.1 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| λ | C.37, B.1.1.1.C37 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNVIKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALH------ | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| μ | B.1.621 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATKFASVYAWNR | LMSFPQSAPHGVVFL |
| θ | P.3, B.1.1.28.3 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ζ | P2, B1.1.28.2 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ο | BA.1 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHVI--SGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ο | BA.2 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ο | BA.2.12.1 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ο | BA.2.75 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ο | BA.2.86 | YYPDKVFRSSVLHLTQDLFLPFFSNVTWFHAI--SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALNRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ο | BA.4 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI--SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ο | BA.5 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI--SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATRFASVYAWNR | LMSFPQSAPHGVVFL |
| ο | BQ.11 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAI--SGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATTFASVYAWNR | LMSFPQSAPHGVVFL |
| ο | EG.5.1 | YYPDKVFRSSVLHSTHDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATTFASVYAWNR | LMSFPQSAPHGVVFL |
| ο | XBB.1.5 | YYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRG | LPIGINITRFQTLLALHRSYLTP | SFTVEKGIYQTSNFR | VFNATTFASVYAWNR | LMSFPQSAPHGVVFL |
WHO, World Health Organization.
In a second Letter to the Editor, Hammarström and Nyström9 agree with our conclusion that intact SP is “inert in fibrin(ogen) clot formation.” Rather, they suggest the intriguing hypothesis that SP proteolytic fragments with amyloidogenic activity form pathogenic complexes with fibrin(ogen). This mechanism is akin to Aβ-peptide–forming amyloids with fibrin(ogen) to provoke Alzheimer’s disease and cerebral amyloid angiopathy.10, 11, 12 Degradation of SP by inflammatory cell–derived proteases (eg, neutrophil elastase) has been found, and SP peptides were documented to form amyloid fibrils with fibrinogen.13 Importantly, the amyloidogenic sequences analyzed do not overlap with the fibrinogen-binding regions identified by Ryu et al,2 indicating that fibrin(ogen) effects arise from amyloid-driven interactions rather than the canonical binding motifs. Two important remaining questions are whether sufficient quantities of SP peptides are formed in vivo and in what microenvironment the complexes would accumulate to influence COVID-19 disease. We postulate that SP-peptide/fibrinogen amyloids could influence disease within extravascular inflammatory foci in tissues (eg, lung, brain) where accumulation and persistence of inflammatory cells and fibrin(ogen) could allow sufficient accumulation of amyloid SP-fibrinogen fibrils to promote COVID-19 pathologies. This mechanism would be consistent with the findings of Ryu et al,2 but definitive documentation of such a pathogenic pathway remains to be found.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Acknowledgments
Contribution: K.K., A.S.W., and M.J.F. wrote and edited the manuscript.
References
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