Enhanced Outside-In Signaling Through the Prothrombotic Pro33 Variant of Integrin αIIbβ3 in Adherent Platelets Under Static and Flow Dynamic Conditions

Platelet integrin αIIbβ3 is polymorphic at residue 33 (Leu33/HPA-1a or Pro33/HPA-1b). In patients with coronary artery disease, the Pro33 isoform is associated with premature manifestation of myocardial infarction (Zotz et al., JTH 2005). Based on this finding, it has been postulated that the Pro33 variant of αIIbβ3 has a prothrombotic phenotype. Moreover, several studies have shown that elevated shear stress can activate platelets leading to shear-induced platelet aggregation contributing to acute myocardial infarction. Thus, it has been reported that pathological shear stress directly regulates αIIbβ3 (Feng et al., Am J Physiol Cell Physiol 2006). We have shown that adherent Pro33 platelets have a higher resistance than Leu33 platelets upon exposure to high shear rates (Loncar et al, Thromb J 2007). We now studied the impact of the Leu33/Pro33 polymorphism on αIIbβ3-mediated outside-in signaling under static and flow dynamic conditions in the presence or absence of Mn²⁺, analyzing the Src pY418 and FAK pY397 activities.

Adhesion assays were performed with 10 or 100 μg/ml of immobilized fibrinogen for different incubation times with washed human platelets in the presence or absence of Mn²⁺ (0.5 mM). Mn²⁺ induces the active conformation of αIIbβ3. Src pY418 and FAK pY397 activities were determined by Western blotting and quantified densitometrically. Control experiments were performed with 1% BSA. Adhesion experiments under flow conditions were carried out with a cone-plate viscometer.

Under static conditions, Pro33 platelets adherent onto fibrinogen exhibited a 2.5-fold higher Src pY418 activity than Leu33 platelets after incubation for 20 min (p<0.01). Presence of Mn²⁺ (0.5 mM) in suspended Leu33 platelets stimulated their Src pY418 activity in an extent comparable to that of platelets adherent onto fibrinogen, while addition of Mn²⁺ (0.5 mM) to platelets adherent onto fibrinogen yielded a 3.5-fold increase in Src pY418 activity (p<0.05). Increase of the Mn²⁺ concentration (2 mM) raised the Src pY418 activity 1.5-fold, as compared to 0.5 mM Mn²⁺ (p<0.05). In parallel experiments with both HPA-1 isoforms of αIIbβ3, Pro33 platelets adherent onto fibrinogen in the presence of 0.5 mM Mn²⁺ revealed a 2.5-fold higher Src pY418 activity than Leu33 platelets after 5 min (p<0.05). This difference further increased upon prolonged adhesion for 10 or 20 min with a 5-fold increase after 40 min (p<0.01). The concentration of immobilized fibrinogen (10 or 100 μg/ml) had no influence on this effect. Addition of abciximab completely abolished the Src pY418 activation. Upon exposure to abnormal (5000 s⁻¹) but not physiological (500 s⁻¹) shear rates for 2 or 5 min, Pro33 platelets adherent onto 100 μg/ml fibrinogen exhibited a 2- or 2.5-fold higher Src pY418 activity than under static conditions (p<0.05 or 0.02, respectively). Under the same conditions, the Src pY418 activity of Pro33 platelets was 2-fold higher than that of Leu33 platelets (p<0.05). Again, different concentrations of fibrinogen (10 μg/ml and 100 μg/ml) did not affect these results. In comparison to Src (pY418), phosphorylation of FAK (pY397) increased slower both in Leu33 and Pro33 platelets adherent onto fibrinogen, when exposed to a shear rate of 5000 s⁻¹ for 10min. Both isoforms (Leu33 or Pro33) exhibited a significantly higher FAK pY397 activity than under static conditions (3-fold increase, p<0.05). The Pro33 variant of αIIbβ3 showed a higher FAK pY397 activation than the Leu33 isoform (p<0.05). Again, abciximab completely blocked both the Src and FAK activation not only under static conditions but also upon exposure to shear.

Our observations indicate that the HPA-1 polymorphism of αIIbβ3 has a considerable impact on the integrin-mediated outside-in signaling. The significantly higher Src (pY418) and FAK (pY397) activities of Pro33 platelets adherent onto fibrinogen under static and flow conditions are in agreement with the contention that the Pro33 (HPA-1b) isoform of αIIbβ3 is indeed a prothrombotic integrin variant.

No relevant conflicts of interest to declare.