R1334 stabilizes the force-bearing von Willebrand factor A1-GPIbα interface.

Abstract

The VWF A1-GPIbα interaction mediates platelet tethering under flow and exhibits a force-dependent mechanical response. Previous simulations identified R1334 within a multi-residue electrostatic region of the interface, but its specific contribution to equilibrium interfacial stability and tensile resistance has been less directly examined through matched WT and R1334A comparisons. Here, replicated equilibrium and multi-rate steered molecular dynamics simulations were used to further examine how R1334A alters these previously reported interactions and the mechanical response of the complex. R1334A decreased the mean interfacial heavy-atom contact count from 306.9±15.5 to 244.3±40.1 and left only sparse residual contacts at residue 1334. Under tensile loading, the mutation lowered the maximum observed resistance by 423-482 pN across all tested rates. Residue-resolved analysis showed that WT R1334 repeatedly engaged GPIbα D18, H37, S39 and N61 during maintenance and tensile separation of the bound interface, whereas these coordinated interactions were largely absent after mutation. Notably, R1334A could reach sustained interfacial contact loss at greater extension despite its lower tensile resistance, showing that delayed contact loss does not necessarily indicate stronger binding but can instead arise from the persistence of weaker residual interactions. These results refine the established role of R1334 by directly linking its interactions with neighbouring GPIbα residues to equilibrium interfacial stability and tensile resistance across multiple loading rates.