Both platelet factor 4 (PF4) and b2-glycoprotein I (b2GPI) bind to von Willebrand factor (VWF) and inhibit ADAMTS13 cleavage: Mechanistic and clinical implications in thrombotic thrombocytopenic purpura (TTP)

Abstract Background: PF4 is a platelet-specific chemokine released in large amounts from activated platelets that binds to the A2 domain of VWF. β2-glycoprotein I (β2GPI) is an abundant circulating protein known to bind to the A1 domain of VWF. PF4 has also been shown to complex with b2GPI, an observation we confirmed in studies using dynamic light scattering (DLS). Whether PF4:VWF or b2GPI:VWF or PF4:b2GPI:VWF alter proteolytic cleavage of VWF by ADAMTS13 has not been reported. Aim: To test whether PF4 and b2GPI form complexes with VWF and whether these complexes inhibit ADAMTS13 proteolytic activity, potentially exacerbating the prothrombotic risk of TTP. Methods: Particle sizes were analyzed by DLS on a fixed scattering angle Zetasizer Nano-ZS system (Malvern Instruments Ltd). The Z-average size distribution (i.e., hydrodynamic diameters) of particles based on volume, were measured at RT with light backscattering of 173°. At least 25 repetitive measurements were made at the studied timepoints. Data analysis was performed using the Zetasizer software, version 7.03 (Malvern Inst Ltd). Recombinant human D'D3-A1-A2 VWF and PF4, and plasma-derived b2GPI were studied. ADAMTS13 activity was tested using plasma-derived, full-length VWF (Hematologic Technologies) denatured using 1.5 M urea in cleavage buffer to expose the A2 domain. PF4 (0-100 µg/mL) and β2GPI (0-200 µg/mL) were added to the VWF (Haematologic Technologies) alone or in combination, prior to ADAMTS13 (5nM final conc, rh-ADAMTS13, R&D) exposure. Cleavage products were analyzed by gel electrophoresis and western blot with a polyclonal anti-VWF-HRP antibody (Dako). In some studies, 100 µg/mL of the heparin-induced thrombocytopenia (HIT)-like anti-PF4 monoclonal antibody KKO or an isotype control TRA to further support role for PF4complexed to VWF on ADAMTS13 protease activity. A previously described hematoporphyrin photochemically injured human umbilical vein endothelial cell (HUVEC)-lined microfluidic channels system (PMC7146020) was perfused with washed human platelets with and without added ADAMTS13 (0.5 or 0.7µg/mL) with PF4, b2GPI and the above moAbs. VWF strand persistence on the HUVECs and platelet adhesion were visualized and quantified using confocal and epifluorescence microscopy. Results: DLS studies showed that both PF4 and b2GPI bind to the D'D3-A1-A2 fragment individually and as a triplex. PF4 inhibited ADAMTS13 activity in a dose-dependent manner, reducing cleavage by 38% at 10 µg/mL and 70% at 25 µg/mL. β2GPI alone reduced cleavage by 42% at 20 µg/mL and 91% at 50 µg/mL. When 20 µg/mL of b2GPI was added in addition to 10 µg/mL of PF4, cleavage was reduced by nearly 90%. The addition of KKO (100µg/mL), but not control TRA, fully prevented ADAMTS13-induced proteolysis whenever PF4 was present, independent of the presence of b2GPI. In the HUVEC-based microfluidic model, PF4 (25µg/mL) promoted VWF strand retention on injured endothelium, with further enhancement when combined with β2GPI (200µg/mL). VWF retention correlated with increased platelet adhesion. The addition of ADAMTS13 (0.7µg/mL) reduced both VWF retention and platelet binding, unless PF4 and/or β2GPI were present, especially, when both were present. The addition of KKO further enhanced PF4:VWF retention in the microfluidic channel as well as platelet binding. Conclusion: Our data suggest that PF4 and β2GPI each inhibit ADAMTS13 cleavage of VWF. PF4 reportedly binds directly to the A2 domain, whereas b2GPI reportedly binds to the A1 domain but still inhibits PF4 and ADAMTS13 cleavage within the A2 domain. Inhibition of ADAMTS13 is enhanced when both PF4 and b2GP1 are present, perhaps by forming generating tripartite complexes with VWF. At least one anti-PF4 monoclonal antibody KKO blocks proteolysis of PF4:VWF by ADAMTS13 as well. Our observations suggest that platelet activation with release of PF4 may contribute to the acute onset and unexplained clinical exacerbations in patients with TTP at varied and sometimes relatively stable levels of ADAMTS13 activity. Additionally, our data suggests that clinical assays measuring ADAMTS13 activity relying on short VWF A2 fragments in the absence of PF4 and b2GP1 may not fully characterize the risk of thrombosis. Inhibition of ADAMTS13 proteolysis of PF4:VWF by the HIT-like monoclonal antibody KKO also suggests that a similar process may contribute to the prothrombotic nature of HIT associated with increased levels of high molecular weight VWF multimers.