Abstract

Microparticles (MP) are circulating submicroscopic cell fragments expressing procoagulant phospholipids and haemostatic proteins such as tissue factor. Several evidences support a pro-haemostatic role of MP and suggest that the axis VWF-platelet GpIb might be critical in their generation. The aim of this study was to measure the levels of platelet-derived MP (PMP) and tissue factor-expressing MP (TFMP) in relationship with VWF levels and multimeric composition as well as shear stress exploiting ex vivo and in vitro natural models. PMP and TFMP were analyzed by flow cytometry and defined as Annexin V and GpIIb/IIIa or Tissue Factor positive events falling in a gate defined by 1 μm beads. MP were enumerated adding a known number of 7 μm beads to each test tube. In the first set of experiments, levels of PMP and TFMP were measured before and after DDAVP administration in 9 patients with mild hemophilia A. FVIII:C, VWF:Ag, PMP and TFMP were measured in plasma at baseline and 30 min, 1, 2, 4, 8 and 24 h after DDAVP 0.3 μg/kg, subcutaneously. A significant increase in the levels of PMP and TFMP (p<0.05 at all time points vs baseline) was observed after DDAVP; peak levels were achieved after 2–8 h. Correlation was found between the increase of VWF:Ag and FVIII:C levels and the peak levels of MP (r=0.62 and r=0.7, respectively). We then studied 27 patients with different types of von Willebrand disease (VWD) and 15 normal controls: citrated platelet rich plasma was exposed to increasing shear stress (from 0 to 90 dyne) for 45 seconds in a cone and plate viscometer and generated PMP were then enumerated by flow cytometry. In normal controls the number of MP at 25 dyne was significantly higher than the number at 0 dyne (p<0.05). In patients with type 1 VWD and type 2B PMP significantly increased as in normal controls (p<0.05 at 25 dyne), while in type 3 patients no PMP increase was observed (p n.s. till 90 dyne). Interestingly, in type 2M patients PMP started to increased significantly at 50 dyne (p<0.05), while type 2A patients had the same behaviour of type 3 patients. This study support the hypothesis that ultralarge VWF multimers released by the endothelium interact with platelets and trigger MP generation, both in vivo and in vitro.

Disclosures: No relevant conflicts of interest to declare.

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