Page 114 - 2022_03-Haematologica-web

S.E. Ward et al.
ysuccinimide-biotin (10 mg/kg; Thermo-Scientific), residual biotinylated murine VWF was quantified using a modified VWF ELISA. All clearance data were fitted to monoexponential equa- tions, based on analysis of the Akaike information criterion. The slope and intercept of the equation of the line were used to calcu- late pharmacokinetic parameters including mean residence time (MRT) and half-life (t1/2).
Data presentation and statistical analysis
Experimental data were analyzed with GraphPad Prism version 8.0 (GraphPad Software, San Diego, CA, USA). Data were expressed as mean values ± standard error of the mean. Data were analyzed with the Student unpaired two-tailed t-test and P-values <0.05 were considered to be statistically significant.
Results
Physiological importance of MGL in regulating von Willebrand factor clearance in vivo
Mice have two distinct MGL homologs - murine MGL1 (mMGL1) and murine MGL2 (mMGL2).45 To gain insight into the biological importance of MGL, we first investigat- ed murine MGL1 and MGL2 binding to VWF in vitro.
AB
Similar to human MGL, dose-dependent binding of both mMGL1 and mMGL2 to VWF was observed (Figure 1A). VWF binding to MGL2 was significantly greater than to mMGL1 (P<0.001). Murine plasma VWF:Ag levels are sig- nificantly elevated (~ 1.5 fold) in MGL1-/- mice. Since both mMGL1 and mMGL2 bind VWF, we hypothesized that knocking down mMGL1 alone may underestimate the bio- logical importance of MGL-mediated clearance. Dual mMGL1-/-/mMGL2-/- mice are not commercially available; thus, to address this hypothesis, in vivo clearance studies were repeated in mMGL1-/- mice in the presence or absence of dual anti-MGL1/2 inhibitory antibodies. Following treatment with anti-MGL2, murine VWF:Ag levels were significantly increased compared to those in mMGL1-/- con- trols (2.78±04 U/mL vs. 1.5±0.5 U/mL respectively; P<0.05) (Figure 1B). Thus, complete murine MGL inhibition was associated with an almost 3-fold increase in endogenous plasma VWF:Ag levels compared to the levels in wild-type (mMGL1+/+mMGL2+/+) controls. In the presence of com- bined mMGL1 and mMGL2 inhibition, endogenous VWF clearance was significantly attenuated compared to that in controls (P<0.05) (Figure 1C) and murine VWF MRT was increased 2.4-fold (Figure 1D). These data confirm that the observed increase in murine VWF levels associated with
Figure 1. Physiological importance of MGL in regulating von Willebrand fac- tor clearance. (A) In vitro binding of purified human plasma-derived (pd) von Willebrand factor (VWF) to murine MGL1 and MGL2 (mMGL1 and mMGL2) receptors was assessed using a plate binding assay as detailed in the ‘Methods’ section. (B) Plasma VWF levels were measured using a VWF:antigen (Ag) enzyme-linked immunosorbent assay (ELISA) in wild- type mice, MGL1-/- mice, and MGL1-/- mice 24 h following infusion of anti- MGL2 antibody. (C) NHS-biotin (10 mg/kg) was infused at t=0 h. Subsequently, residual biotinylated VWF clearance was quantified by a modified VWF ELISA. Clearance experi- ments were performed in MGL1-/- mice in the presence or absence of anti- MGL1/2 antibody. (D) Mean residence time (MRT) for endogenous murine VWF was determined for wild-type mice, MGL1-/- mice and MGL1-/- mice following infusion of anti-MGL2 anti- body. Three to five mice were studied per time point, and data are represent- ed as mean ± standard error of mean. *P<0.05, **P<0.01.
CD
670
haematologica | 2022; 107(3)