Enhanced Activation of Integrin αIIbβ3-Dependent Signaling in the Pro33 (HPA-1b) Variant

Abstract

The major platelet integrin αIIbβ3 is polymorphic at residue 33, and the Pro33 isoform exhibits prothrombotic character. We explored via which pathways the Leu33/Pro33 polymorphism modulates integrin αIIbβ3 mediated outside-in signaling. We have investigated Src Y418 phosphorylation in human platelets adherent on fibrinogen in the presence or absence of divalent cation Mn2+ and of the soluble form of the CD40 ligand (sCD40L). Whereas HPA-1a/1a platelets did not show Src activation on 10 µg/ml immobilized fibrinogen even after 40 min, we observed a significant Src phosphorylation increase in the HPA-1b/1b isoform. Mn2+ alone also increased the Src activity in the platelets and addition of Mn2+ to platelets adherent on fibrinogen resulted in a synergism of Src Y418 phosphorylation in both isoforms, whereas after 40 min adhesion HPA-1b/1b platelets demonstrated a substantially (sixfold) higher Src kinase activation than HPA-1a/1a. Our observation that in the presence of Mn2+ the Src pY418 phosphorylation increases in both isoforms was independent of the fibrinogen concentration suggesting a dominant role of the Mn2+. Whereas sCD40L, another αIIbβ3 ligand, did not enhance the Src activity in HPA-1a/1a platelets adherent on fibrinogen, we observed a threefold Src kinase activation in HPA-1b/1b platelets in the presence of sCD40L.