The Impact of Shear Stress On the Interaction of Human Platelet Integrin αIIbβ3 with Fibrinogen.
Authors: Gyenes Marianna , Stoldt Volker R. , Huynh Khon C. , Scharf Rüdiger E. 
Published: October 2019

Abstract
Abstract 2167

Objectives:
Shear stress can activate platelets resulting in subsequent platelet aggregation. The so-called “shear-induced platelet aggregation” (SIPA) contributes to various vascular diseases (Speich et al., Am J Physiol Cell Physiol 2008). Several signaling pathways were proposed to be involved in this process, e.g., αIIbβ3-mediated signaling (Feng et al., Am J Physiol Cell Physiol 2006). We investigated the impact of shear stress on the αIIbβ3–ligand interaction in human platelets adherent onto fibrinogen. Platelets on immobilized fibrinogen were exposed to various shear rates and signaling of Src and FAK tyrosine kinases, both essential in the integrin downstream signaling pathways, were examined. Specifically, we analyzed the role of αIIbβ3 in shear-induced platelet signaling (i) by comparing the Src Y418 and FAK Y397 phosphorylation activities between platelets on immobilized fibrinogen and platelets on BSA matrix in response to shear stress, and (ii) by performing experiments in the presence of the αIIbβ3 antagonist abciximab.

Methods:
Human washed platelets were incubated on immobilized fibrinogen 100 μg/ml or 1% BSA either under static conditions or exposed to shear rates of 500 s−1 or 5000 s−1, respectively. Specific phosphorylation of Src (pY418) and FAK (pY397) was determined by Western blot and quantified densitometrically. Experiments under flow conditions were performed in a cone-plate viscometer.

Results:
Both Src and FAK exhibited phosphorylation under static conditions on immobilized fibrinogen after 2 min of adhesion. A shear rates of 500 s−1 did not increase the phosphorylation activities. By contrast, high shear rates (5000 s−1) significantly enhanced both Src and FAK phosphorylations in fibrinogen-adherent platelets (3-fold increase each, p<0.05). In the absence of immobilized fibrinogen, platelets incubated with BSA matrix did not show any Src activation under static conditions and only a very low Y418 phosphorylation activity in response to a shear rate of 500 s−1. A shear rate of 5000 s−1 considerably induced Src pY418 activity compared to platelets exposed to physiological shear stress (10-fold increase, p< 0.01). In response to shear rates of 500 s−1 or 5000 s−1, we detected a significantly higher Src activation in platelets adherent onto fibrinogen (500 s−1: 10-fold higher, p<0.01; 5000 s−1: 2-fold higher, p<0.05) than in platelets incubated over a BSA matrix indicating a ligand-dependent signaling. When platelets over BSA were exposed to a shear rate of 5000 s−1, FAK also exhibited a significant elevation of pY397 activity (9-fold increase, p<0.05). By contrast to Src, in platelets exposed to a shear rate of 500 s−1 or 5000 s−1, we observed approximately equal FAK pY397 activation, independent of the presence or absence of immobilized fibrinogen. In platelets incubated for 10 min on a fibrinogen matrix under static conditions, we did not detect any change in the Src activation compared to 2 min incubation. The activity of FAK pY397, however, was time-dependent and showed a 3-fold higher phosphorylation extent after 10 min than after 2 min adhesion (p<0.05). In response to a shear rate of 500 s−1 both Src Y418 and FAK Y397 phosphorylations exhibited a considerable time-dependent enhancement (comparing the phosphorylation activities after incubation for 2 or 10 min). This enhancement could be seen both in platelets adherent onto fibrinogen and in platelets over BSA (3 to 6-fold increase, p<0.05). In platelets exposed to a shear rate of 5000 s−1 for 10 min, the Src and FAK phosphorylation activities were similar to platelets after 2 min. Abciximab inhibited the Src and FAK signaling in platelets exposed to 5000 s−1 on immobilized fibrinogen. The same inhibition was seen in platelets exposed to 5000 s−1 over BSA (p<0.05).

Conclusions:
Exposure of platelets to high shear rates induces a significant increase of both Src and FAK signaling compared to platelets under static conditions. Whereas Src activation remains predominantly ligand-dependent in fibrinogen-adherent platelets even under shear stress, FAK signaling appears to be shear-induced. The finding that, abciximab inhibits the activation of both Src and FAK in the absence of fibrinogen, emphasizes the role of integrin αIIβ3 in the shear-induced platelet signaling.

Disclosures:
No relevant conflicts of interest to declare.