Signaling by EphrinB1 and Eph Kinases in Platelets Promotes Rap1 Activation, Platelet Adhesion, and Aggregation via Effector Pathways that Do Not Require Phosphorylation of EphrinB1

We have previously shown that platelets express 2 receptor tyrosine kinases, EphA4 and EphB1, and the Eph kinase ligand, ephrinB1m and proposed that transcellular Eph/ephrin interactions made possible by the onset of platelet aggregation promote the further growth and stability of the hemostatic plug. The present study examines how this might occur. The results show that clustering of either ephrinB1 or EphA4 causes platelets to adhere to immobilized firinogen via αIIbβ3. Adhesion occurs more slowly than with adenosine diphosphate (ADP) abd requires phosphatidylinositol 3 (PI3)—kinase and protein kinase C activity but not ephrinB1 phosphorylation. By itself, Eph and ephrin signaling is insufficient to cause aggregation or the binding of soluble fibrinogen, but it can potentiate aggregation initiated by a Ca++ ionophore or by agonists for thrombin and thromboxane receptors. It also enhances Rap1 activation without requiring ADP secretion, ephrinB1 phosphorylation, or the activation of PI3-kinase and Src. From this we conclude that (1) Eph/ephrin signaling enhances the ability of platelet agonists to cause aggregation provided that those agonists can increase cytosolic Ca++; (2) this is accomplished in part by activating Rap1; and (3) these effects require not phosphotyrosine-based interactions with the ephrinB1 cytoplasmic domain

Thrombin responses in human endothelial cells. Contributions from receptors other than PAR1 include the transactivation of PAR2 by thrombin-cleaved PAR1.

The recent identification of two new thrombin receptors, PAR3 and PAR4, led us to re-examine the basis for endothelial cell responses to thrombin. Human umbilical vein endothelial cells (HUVEC) are known to express PAR1 and the trypsin/tryptase receptor, PAR2. Northern blots detected both of those receptors and, to a lesser extent, PAR3, but PAR4 message was undetectable and there was no response to PAR4 agonist peptides. To determine whether PAR3 or any other receptor contributes to thrombin signaling in HUVEC, PAR1 cleavage was blocked with two selective antibodies and PAR1 activation was inhibited with the antagonist, BMS200261. The antibodies completely inhibited HUVEC responses to thrombin, but BMS200261 was only partly effective, even though separate studies established that the antagonist completely inhibits PAR1 signaling at the concentrations used. Since peptides mimicking the PAR1 tethered ligand domain can also activate PAR2, we asked whether the remaining thrombin response in the presence of the antagonist could be due in part to the intermolecular transactivation of PAR2 by cleaved PAR1. Evidence that transactivation can occur was obtained in COS-7 cells co-expressing PAR2 and a variant of PAR1 that can be cleaved, but not signal. There was a substantial response to thrombin only in cells expressing both receptors. Conversely, in HUVEC, complete blockade of the thrombin response by the PAR1 antagonist occurred only when signaling through PAR2 was also blocked. From these observations we conclude that 1) PAR1 is the predominant thrombin receptor expressed in HUVEC and cleavage of PAR1 is required for endothelial cell responses to thrombin; 2) although PAR3 may be expressed, there is still no evidence that it mediates thrombin responses; 3) PAR4 is not expressed on HUVEC; and 4) transactivation of PAR2 by cleaved PAR1 can contribute to endothelial cell responses to thrombin, particularly when signaling through PAR1 is blocked. Such transactivation may limit the effectiveness of PAR1 antagonists, which compete with the tethered ligand domain rather than preventing PAR1 cleavage

GSK3β is a negative regulator of platelet function and thrombosis

Glycogen synthase kinase (GSK)3β is a ser-thr kinase that is phosphorylated by the kinase Akt. Although Akt has been shown to regulate platelet function and arterial thrombosis, its effectors in platelets remain unknown. We show here that agonist-dependent phosphorylation of GSK3β in platelets is Akt dependent. To determine whether GSK3β regulates platelet function, platelets from mice lacking a single allele of GSK3β were compared with those of wild-type (WT) controls. GSK3β+/− platelets demonstrated enhanced agonist-dependent aggregation, dense granule secretion, and fibrinogen binding, compared with WT platelets. Treatment of human platelets with GSK3 inhibitors renders them more sensitive to agonist-induced aggregation, suggesting that GSK3 suppresses platelet function in vitro. Finally, the effect of GSK3β on platelet function in vivo was evaluated using 2 thrombosis models in mice. In the first, 80% of GSK3β+/− mice (n = 10) formed stable occlusive thrombi after ferric chloride carotid artery injury, whereas the majority of wild-type mice (67%) formed no thrombi (n = 15). In a disseminated thrombosis model, deletion of a single allele of GSK3β in mice conferred enhanced sensitivity to thrombotic insult. Taken together, these results suggest that GSK3β acts as a negative regulator of platelet function in vitro and in vivo