Endothelial Microparticles Improve Primary and Secondary Hemostasis in Patients with Von-Willebrand Disease

Abstract 2287

The bleeding phenotype in patients with von Willebrand disease (vWD) type 1 and 3 can usually be correlated to the amount and function of von Willebrand factor (vWF:Ag, vWF:RCo) but also to the impairment of functional coagulation assays: the ristocetin-induced platelet aggregation (RIPA) for primary hemostasis and the parameters of thrombin generation in a calibrated automated thrombogram (CAT, Thrombinoscope®) for secondary hemostasis. We therefore evaluated whether addition and depletion of endothelial microparticles (EMP) might influence these assays for samples of vWD patients.

EMP were obtained from stimulated endothelial cell lines, isolated either by ultracentrifugation or magnetic beads selection for Annexin V and detected and quantified by flow cytometry after labelling for Annexin V and vWF. For CAT, EMP concentration was adjusted to 3×10³/μl, for RIPA to 2×10⁴/μl. vWD patient samples were diluted 3:1 or 2:1 with control buffer (HEPES/CaCl) or EMP solution.

For thrombin generation, there was a significant shortening of lag time (6.8min vs 16.5min, p=.029) and time-to-peak (13.6min vs. 23.0min, p=.049) in vWD type 3 plasma (n=4) after addition of EMP using no reagent or MP reagent while there was no significant effect on these parameters in normal plasma. After addition of a tissue factor blocking antibody, the EMP effect could be largely reversed. For RIPA, we found a median maximum aggregation of 4% in vWD patients (n=6, vWF-Ag <40%). After addition of EMP, aggregation levels increased to 17.3%, which was significantly higher compared to control buffer (9.8%, p=.028), but lower compared to 250 IU/ml of a plasma-derived vWF product (Humate P®, 88.7%). To evaluate if significant amounts of vWF are bound to circulating microparticles in human plasma, we used magnetic bead selection to reduce MP counts in plasma of DDAVP stimulated vWD patients (n=10), because depletion of microparticles by 0.2μm filtration showed significant disruption of large vWF multimers. After MP reduction (1464/μl vs. 1863/μl, p=.036), both vWF:Ag (134.7% vs 161.1%, p=.005) and vWF:RCo (131.8% vs. 167.2%, p=.017) were significantly decreased.

In summary, we could show that in vitro generated endothelial microparticles are able to improve thrombin generation and platelet aggregation in vWD patients and that significant amounts of vWF are bound to circulating microparticles in human plasma after DDAVP stimulation. Thus, in vitro generated EMP might have the potential to improve replacement therapy for vWD patients.