Recombinant human vimentin binds preferentially to P‐selectin through the rod domain to block leukocyte adhesion to platelets
Authors: Lam Fong , Da Qi , Cruz Miguel 
Published: June 2021

Background

Platelet‐leukocyte interactions play an important role in inflammation. Leukocyte adhesion to vascular endothelium is a step‐wise process that is initiated by leukocyte (WBC) P‐selectin glycoprotein ligand‐1 (PSGL‐1) to P‐selectin on adhered platelets and endothelium. These interactions then start a cascade of WBC firm adhesion and transmigration, which could lead to organ injury. Finding novel therapeutics to block specific aspects of inflammation may be useful in controlling unwanted inflammation. Recent reports suggest that vimentin may bind to PSGL‐1, although the function of this had not been previously described. We hypothesize that rhVim may block P‐selectin‐PSGL‐1 interactions and that this may be through the rod domain.

Methods

All research was approved by the Institutional Review Boards of the Michael E. DeBakey Veterans Affairs Medical Center and Baylor College of Medicine. Recombinant human vimentin (rhVim) and its rod domain (Rod) were expressed in E. coli and His‐purified using affinity chromatography. Blood was collected from healthy adult donors and mepacrine‐labelled whole blood or isolated neutrophils (PMN) were perfused in microfluidic channels at 2 dyn/cm2 over fibrinogen or isolated platelets in the presence of rhVim, Rod, or vehicle control. Blocking antibodies to P‐selectin (AK4) and PSGL‐1 (KPL‐1) were used in conjunction with rhVim to assess for additional decreases in PMN adhesion to platelets. Protein binding was assessed using ELISA and the equilibrium dissociation constant, KD, was measured using surface plasmon resonance (BIACore).

Results

rhVim significantly decreased WBC adhesion to fibrinogen coated surfaces by ~50% (p<0.05) and PMN adhesion to platelets in a dose dependent fashion (up to 40 ug/mL; p<0.05). In the presence of AK4 (10 ug/mL) and KPL‐1 (2.5 ug/mL) blocking antibodies, the addition of a submaximal dose of rhVim (5 ug/mL) did not further decrease PMN adhesion to platelets. Similar to rhVim, the Rod decreased WBC adhesion to fibrinogen‐coated channels by ~50% as compared to vehicle control (42 ± 13 vs 79 ± 20 WBC per field; p<0.05). There was no difference in WBC adhesion when comparing equimolar concentrations of rhVim and Rod. Likewise, the binding of rhVim and Rod to P‐sel/Fc were also similar (67 ± 1.2 and 125 ± 1.2 nM, respectively) and there was no binding of either to IgG or PSGL‐1.

Discussion

Our data suggests that rhVim blocks adhesion of WBC and PMN to platelets via binding to P‐selectin, and not PSGL‐1. This effect may be through binding within the rod domain. Further studies are warranted to determine the binding epitope of rhVim to P‐selectin as a potential therapy for unwanted inflammation.

Support or Funding Information

This work is supported by NIH/NHLBI HL‐116524, NIH/NIGMS GM‐112806 and GM‐123261, NIH/NINDS NS‐094280, and a Merit Review Award Number I01 BX002551 from the United States Department of Veterans Affairs Biomedical Laboratory Research and Development Service.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.