Impact of the Leu33Pro Polymorphism of αIIbβ3 on Integrin – Ligand Interaction Under Abnormally High Shear Conditions
Authors: Gyenes Marianna , Stoldt Volker R. , Huynh Khon C. , Scharf Rudiger E. 
Published: October 2019

Abstract
Objectives: Several studies have shown that shear stress can activate platelets leading to shear-induced platelet aggregation. Moreover, it has been reported that pathological shear stress can directly regulate platelet αIIbβ3 (Feng et al. 2006). This integrin is polymorphic at residue 33 (Leu or Pro) of the β subunit. The Pro33 isoform of αIIbβ3 may be a prothrombotic integrin variant. For example, patients with coronary artery disease, who are carriers of Pro33 platelets, experience their myocardial infarction 5.2 years (median) earlier than those with Leu33 platelets (Zotz et al. 2005). In this work, we studied the impact of the Leu33Pro polymorphism on Src pY418 and FAK pY397 signaling in human platelets adherent onto fibrinogen and subsequently exposed to physiological (500 s-1) or abnormally high (5000 s-1) shear rates.
Methods: Washed platelets were placed onto immobilized fibrinogen (100 µg/ml) or BSA (2 %) either under static conditions or upon exposure to shear rates of 500 s-1 or 5000 s-1. Incubation times were 2, 5 or 10 min. Specific phosphorylations of Src (pY418) and FAK (pY397) were determined by Western blot and quantified densitometrically. Experiments under flow conditions were performed in a cone-plate viscometer.
Results: Adherent Pro33 positive platelets exhibited a significantly higher Src activity than Leu33 platelets throughout under static conditions (2-fold higher after 2 and 5 min, 3-fold after 10 min, p<0.01, each). A physiological shear rate of 500 s-1 did not have a further effect on the Src signaling in fibrinogen-adherent Pro33 or Leu33 platelets over an incubation time of 10 min. By contrast, in response to a shear rate of 5000 s-1, both genotypes exhibited a rapid, significant increase in Src (pY418) activation compared to platelets under static conditions (3-fold higher Src phosphorylation after 2 min, p <0.01). While Src activation in Pro33 platelets remained constantly elevated during 10 min of adhesion, a decrease in pY418 activity of Leu33 platelets was found, resulting in a 4-fold higher Src signaling of Pro33 platelets, as compared to Leu33 platelets (p<0.001). For FAK, we observed low Y397 phosphorylation both in adherent Leu33 and Pro33 platelets after 2 min under static conditions. Upon prolonged adhesion (> 5 min), Pro33 platelets exhibited a significantly higher FAK (pY397) activity than Leu33 platelets (5-fold higher after 5 min, 3-fold higher after 10 min, p<0.01, each). In contrast to Src activation, both shear rates (500 s-1 and 5000 s-1) affected specific FAK phosphorylation considerably in Pro33 platelets. This resulted in significantly higher FAK signaling of Pro33 than of Leu33 platelets (5-fold higher, p<0.01). In Leu33 platelets, we observed an enhanced pY397 activity only in response to 5000 s-1. Next, we examined the impact of the Leu33Pro polymorphism of αIIbβ3 on Src and FAK signaling without fibrinogen, exclusively in response to shear. Under this condition, we detected only a slight increase in Src activity in Pro33 but not in Leu33 platelets. By contrast, FAK signaling in response to shear rates of 500 s-1 and 5000 s-1 exhibited a significantly higher pY397 activity in Pro33 than in Leu33 platelets (p<0.01). In control experiments, abciximab completely inhibited Src and FAK signaling in fibrinogen-adherent platelets exposed to a shear rate of 5000 s-1 but also in non-adherent platelets exposed to 5000 s-1over BSA (p<0.01, each).
Conclusion: Exposure of human platelets to shear enhances specific phosphorylation of Src and FAK in an integrin-dependent manner. This effect is modulated by the Leu33Pro polymorphism of αIIbβ3. Pro33 platelets exhibit higher phosphorylation activities than Leu33 platelets both under static and flow dynamic conditions. The observation of higher phosphorylation activities of both tyrosine kinases, Src and FAK, in Pro33 than in Leu33 platelets in suspension upon exposure to shear (without concomitant adhesion) suggests that the Leu33Pro polymorphism of αIIbβ3 plays a role in shear-induced signaling. This contention is also supported by the strong inhibition of abciximab, abrogating shear-induced activation of both kinases not only in fibrinogen-adherent platelets but also in platelets in suspension. Overall, these observations suggest that platelet αIIbβ3 plays a relevant role in shear-induced integrin signaling.

Disclosures
No relevant conflicts of interest to declare.