Activation of Human Platelets by the Membrane-Expressed A1 Domain of von Willebrand Factor

Abstract

Platelet activation and microthrombus formation are invariable features of xenograft rejection and the vascular injury observed when porcine organs are transplanted into primates. This pathological process could be mediated, at least in part, by aberrant interactions of von Willebrand Factor (vWF) associated with the donor vasculature with host platelets. Unlike human vWF, native porcine vWF (pvWF) interacts with human GPIb independently of shear stress or nonphysiological stimuli, eg, ristocetin. We therefore contrasted the potential of isolated human and porcine vWF–A1-domains to interact with human platelets in vitro. Both human and porcine vWF–A1-domains expressed as glycosyl phosphatidylinositol–linked FLAG fusion proteins on COS-7 cells induced GPIb-dependent aggregation and intracellular Ca++ uptake of platelets, independent of both the remainder of the vWF protein and additional modifying factors. Porcine A1-domains were more potent than human homologues, and in addition ristocetin could boost platelet aggregation only with the human A1-domain. Putative conformational changes in the porcine A1-domain could result in the heightened, ristocetin-independent interactions observed with human platelets and may be of importance for xenograft survival.

Impact of O‐linked glycosylation of the VWF‐A1‐domain flanking regions on platelet interaction

“…All experiments were performed in the presence of MgCl 2 (1AE0 mmol/l), CaCl 2 (1AE0 mmol/l) and fibrinogen (final concentration: 800 lg/ml). Co-incubation of washed platelets with ristocetin alone produced no aggragatory responses (data not shown), ruling out effective remnants of blood-donor-plasma VWF, as demonstrated previously (Schulte am Esch et al, 1997, 2003. Aggregation was determined as percentage of maximal light transmission at 8 min.…”

“…The purpose of this study was to obtain insights into the role of post-translational modifications of VWF-A1-domains and flanking regions for interaction with platelet GPIb. To enable functional studies of isolated, fully glycosylated VWF-A1domains, we utilized a previously developed model of the FLAG-tagged, via GPI-linker, COS-7-cell-membrane anchored VWF-A1-domain (Schulte am Esch et al, 1997, 2003Berndt et al, 2002) spanning amino acids 473-713 of the mature protein. As this fragment does not incorporate N-linked glycosylation sites, we focused on the role of O-linked glycosylation.…”

“…The pcDNA 3-based vector constructs coding for human (hu) VWF-A1 residues 473-713 of the mature protein as a FLAG-tagged, cell membrane GPI-anchored fusion protein have been previously described and were utilized as native forms in this study (Schulte am Esch et al, 1997, 2003Berndt et al, 2002).…”

“…To monitor levels of expression, all VWF-A1-expressing COS-7-cell-batches were analysed by cytofluometric analysis, as described (Schulte am Esch et al, 1997, 2003. Cells were stained in a single-step procedure, utilizing a FITC-conjugated anti-FLAG antibody analysed on a FACScalibur (Becton-Dickinson, Heidelburg, Germany).…”

“…Solutions of human washed platelets (HWP) were prepared and aggregation profiles following incubation with transfected COS-7 cells were evaluated as described (Schulte am Esch et al, 1997, 2003. Briefly, platelet rich plasma was adjusted to a pH of 6AE5to prevent fibrinogen binding to its receptor on platelets GPIIbIIIa -and co-incubated with two units of apyrase per millilitre to prevent low-level ADP-stimulation during preparation.…”