Platelet microparticle formation and thrombin generation under high shear are effectively suppressed by a monoclonal antibody against GPIbα

Authors:
  • LPLuca Pontiggia
    Luca Pontiggia
  • BSBeat Steiner
    Beat Steiner
  • HUHans Ulrichts
    Hans Ulrichts
  • HDHans Deckmyn
    Hans Deckmyn
  • MFMarc Forestier
    Marc Forestier
  • JBJürg Beer
    Jürg Beer
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Abstract
Summary: We studied the inhibition of platelet microparticle (MP) formation and thrombin generation under high shear forces. We hypothesized that an inhibitor of the GPIbα-von Willebrand factor (vWF) interaction would be more effective in suppressing MP formation and thrombin generation than GPIIb/IIIa inhibitors. Platelet-rich plasma (PRP) anticoagulated with PPACK (D-Phe-Pro-Arg chloromethyl ketone) was exposed in a coneand-plate viscometer (shear: 5,000 s−1 for5 min) in the presence of antagonists to GPIbα (the monoclonal antibody [Mab] Ib-23) or to GPIIb/IIIa (abciximab, tirofiban, eptifibatide) at their IC90 determined in platelet aggregometry with ristocetin or ADP, respectively. We used double labeling (CD41-PE and annexin-V-FITC) for flow cytometric detection of MP and their aminophospholipid exposure. Thrombin generation was measured using PRP prepared fromACD anticoagulated blood. About 40% of the thrombin generation was found to be mediated by the MP fraction of the PRP. Blockade of GPIbα with Mab Ib-23 reduced MP formation and thrombin generation by 50%, and was more effective than any GPIIb/IIIa antagonist. The combination of Mab Ib-23 with one of the GPIIb/IIIa inhibitors further reduced the MP formation to ∼30%. The antibody also partially inhibited thrombin induced platelet aggregation. Epitope mapping suggested that Mab Ib-23 binds between the amino acids 201 and 268 of GPIbα, explaining the interference with vWF and thrombin interaction. In contrast to the commonly used GPIIb/IIIa antagonists, the blockade of GPIbα with Mab Ib-23 effectively reduces the prothrombotic MP generation and thrombin formation at shear rates typically found in arterial stenoses.
Funding Information
  • Swiss National Science Foundation (32–59449.99, 320080–104202)
  • Swiss Heart Foundation