COVID-19 microthrombosis: unusually large VWF multimers are a platform for activation of the alternative complement pathway under cytokine storm

Abstract

ADAMTS13, a metalloproteinase, specifically cleaves unusually large multimers of von Willebrand factor (VWF), newly released from vascular endothelial cells. The ratio of ADAMTS13 activity to VWF antigen (ADAMTS13/VWF) and indicators of the alternative complement pathway (C3a and sC5b-9) are both related to the severity of COVID-19. The ADAMTS13/VWF ratio is generally moderately decreased (0.18-0.35) in patients with severe COVID-19. When these patients experience cytokine storms, both interleukin-8 and TNFα stimulate VWF release from vascular endothelial cells, while interleukin-6 inhibits both production of ADAMTS13 and its interaction with VWF, resulting in localized severe deficiency of ADAMTS13 activity. Platelet factor 4 and thrombospondin-1, both released upon platelet activation, bind to the VWF-A2 domain and enhance the blockade of ADAMTS13 function. Thus, the released unusually-large VWF multimers remain associated with the vascular endothelial cell surface, via anchoring with syndecan-1 in the glycocalyx. Unfolding of the VWF-A2 domain, which has high sequence homology with complement factor B, allows the domain to bind to activated complement C3b, providing a platform for complement activation of the alternative pathway. The resultant C3a and C5a generate tissue factor-rich neutrophil extracellular traps (NETs), which induce the mixed immunothrombosis, fibrin clots and platelet aggregates typically seen in patients with severe COVID-19.

Keywords: ADAMTS13; COVID-19; Complement activation; Endotheliopathy; Microthrombosis; VWF.

Fig. 1

Structural domains of human von Willebrand factor (VWF) and its binding ligands. The cDNA of mature human VWF subunit includes the following structural domains: D’-D3-A1-A2-A3-D4-C1-C2-C3-C4-C5-C6-CK. The monomeric mature VWF subunit begins with amino acid residue number 763 and ends with 2813 (2050 amino acid residues). As shown in the upper panel, each A domain has a loop structure linked by an intramolecular disulfide bond. ADAMTS13 cleaves at the peptide bond of Y1605-M1606. LLG = Leucine-Leucine-Glycine motif, RGD = Arginine-Glycine-Aspartate motif. The lower panel lists the natural ligands that bind to each VWF domain involved in thrombosis/hemostasis, complement activation/inhibition, and others. TSP-1 = thrombospondin-1, PF4 = platelet factor 4, PSGL-1 = P-selectin glycoprotein ligand-1, NETs = neutrophil extracellular traps, SA-Protein A = Staphylococcus aureus Protein A. SA-VWFbp = Staphylococcus aureus VWF binding protein. Note that the ligands shown by the red bars bind to VWF domains in a shear-dependent manner. The binding of platelet GPIb to the A1-domain initiates platelet activation, alongside the enhanced proteolysis by ADAMTS13 [48], as does factor H [40]. Notably, both TSP-1 and PF4, released from α-granules of platelets upon activation, bind to the A2-domain, preventing cleavage by ADAMTS13. (See text in detail.)

Fig. 2

Activation and amplification of the complement alternative pathway (AP), according to Pangburn and Müller-Eberhard [83]. In this pathway, plasma C3, which has a thioester domain, is spontaneously hydrolyzed by H₂0 to form C3(H₂0), to which binds factor B, leading to proteolysis by factor D releasing a 32 kDa-Ba protein and thereby generating C3(H₂0)Bb, which is the initiation C3-convertase. This convertase then cleaves C3 into 2 fragments–C3a and metastable C3b. The latter further changes either to the fluid-phase C3b by hydrolysis or to the surface-bound C3b through covalent binding of a thioester bond to the cell surface. The surface-bound C3b has been well characterized, but the fluid-phase ‘soluble’ C3b, particularly its function, has been little studied in vitro, presumably due to its instability in vivo (See text in detail.)

Fig. 3

The role of UL-VWFMs in activation of the alternative complement pathway (AP) in the microvasculatures during COVID-19 thrombosis. SARS-CoV-2 invades lung alveoli from the respiratory tract and infects type 2 alveolar pneumocytes and macrophages. This causes release of cytokines from resident cells, such as macrophages, CD4-T lymphocytes and neutrophils. Inflammatory cytokines further stimulate release of cytokines from blood cells and vascular ECs, generating a cytokine storm. Consequently, IL-8, TNFα, and a complex of IL-6 and its soluble receptor (sIL-6R) stimulate vascular ECs, and induce exocytosis of UL-VWFMs from Weibel-Palade Bodies (WPBs). Under a high shear flow, UL-VWFMs undergo a conformational change, allowing ADAMTS13 more accessibility; however, IL-6 interferes with this interaction, resulting in inhibition of ADAMTS13 activity. In such microenvironments, the A1-loop domain of VWF binds platelets, forming platelet aggregates with or without involving resident megakaryocytes. The activated platelets release PF4 and TSP-1 from the α-granules, both of which bind to the A2-domain of VWF and block cleavage by ADAMTS13. The A1-loop domain of VWF binds to the heparan sulfate of syndecan-1 on the vascular EC surface, while the A2 domain binds to C3b generated by the AP activation. C3b bound to UL-VWFM anchored on the EC surface binds factor B, which is proteolyzed by factor D. Then binding of properdin to the C3b moiety as a stabilizer results in the formation of C3-convertase in the AP. Subsequent activation through the AP (C5-convertase) (C3bBbC3b) produces C5b, to which C6 ~ C9 bind, finally forming C5b-9 (MAC), which in turn activates endothelial cells (EC) together with endotheliopathy, UL-VWFM is a major constituent of WPBs, which also contain IL-8, Ang-2, t-PA, etc. The secretion of IL-8 into the circulation enhances UL-VWFM release and accelerates C3b binding to UL-VWFM on the vascular EC surface, promoting platelet thrombi formation. The released t-PA generates plasmin under microenvironments in which thrombomodulin (TM) on the vascular EC surface undergoes shedding. TM binds to thrombin to form a complex that inhibits thrombin activity, but activates protein C and thrombin-activatable fibrinolysis inhibitor (TAFI) to TAFIa (carboxypeptidase), which can proteolytically inactivate both C3a and C5a. Shedding of TM loses the antithrombotic function of vascular ECs, turning them into the thrombogenic surface