We activated human platelets with thrombin or Pam3
, and examined agonist-dependent effects on the phosphorylation kinetics of signaling proteins, on FXIIIa-protein interactions, and on α-granule release. Three major signaling proteins - Akt, ERK1/2, and p38 - were examined as to whether they are differentially affected by thrombin- vs.
-stimulation. Thrombin-induced signaling cascades in platelets have been intensively studied (9
), and PI3K is a known key player in these events (24
). However, the details of signaling pathways involving ERK1/2 or p38 remain controversial (16
). TLR2 signaling, on the other hand, culminates in NF-κB activation in various cell types (2
), and PI3K has been implicated as a regulator of TLR signaling (26
). Although anuclear platelets express NF-κB (27
), no reports describing mechanistic details of the TLR2 signaling pathway within platelets have been published. Recent data from our laboratory have shown that PI3K/Akt play a major role in Pam3
-induced platelet responses (8
). Here, we further demonstrate that Pam3
also induces phosphorylation of ERK1/2 and p38 in a time-dependent and agonist-dependent manner. This clearly suggests that other pathways beside PI3K/Akt are involved in TLR2 signaling and Pam3
-induced platelet responses.
The increased phosphorylation of Akt, ERK1/2, and p38 after activation with thrombin results in the formation of a more stable clot, as seen in the platelet aggregation and fibrin clot retraction assays, compared to Pam3
-stimulation. Furthermore, the phosphorylation kinetics of Akt is faster with thrombin treatment compared to Pam3
. PI3K/Akt pathways have been implicated in the outside-in signaling that occurs when fibrinogen binds to αIIb
and activates signaling pathways that stabilize platelet aggregates (28
). Important to clot stabilization is the greater extent of phosphorylation of both ERK1/2 and p38 with thrombin compared to Pam3
. Both kinases are phosphorylated in more rapid manner, suggestive of their role in inside-out signaling, when signaling events activated by an agonist lead to conformational changes in αIIb
). Therefore, the more stable clot formation occurring with thrombin is a result of the rapid phosphorylation of ERK1/2 and p38 causing conformational change in αIIb
to increase fibrinogen binding, which results in an increased phosphorylation of Akt to further stabilize the aggregate.
The second part of our study focused on protein-protein interactions with particular emphasis on FXIIIa. For anuclear platelets, MS-based proteomic methods have proven to be an important tool for characterizing protein expression, post-translational modifications, and protein release (13
). Eighteen FXIIIa-binding proteins were identified and validated in resting and activated platelets. Only four of these proteins were found to bind to FXIIIa specifically. Known FXIIIa-substrates have been classified into three groups: coagulatory/fibrinolytic proteins, adhesive proteins, and cytoskeletal proteins (20
). The four FXIIIa-binding proteins we identified in this study were: FAK, gelsolin, myosin IIA and thrombospondin. FAK and gelsolin are novel FXIIIa-interacting partners, while myosin IIA and thrombospondin had been reported to function as substrates for FXIIIa. FAK is a tyrosine kinase that mediates signaling through integrins and translocates to the cytoskeleton in platelets in the second wave of cytoskeletal rearrangements (36
). The FAK-FXIIIa interaction could lead to crosslinking of FAK to other proteins, or phosphorylation of FXIIIa by FAK. Gelsolin, a cytoskeletal protein that regulates actin assembly (39
), has also been found in α-granules (35
), where its interaction with FXIIIa might.
It was examined whether these FXIIIa-protein interactions are differentially altered upon thrombotic vs.
immune stimulation in comparison to resting platelets. We found differences in FXIIIa-binding to FAK, thrombospondin, and HSP27. The FXIIIa-FAK interaction changed in a statistically significant, agonist-dependent manner. The intensity of the interaction was not altered significantly after thrombin-stimulation, indicative of their translocation to the cytoskeleton where both proteins are thought to function after activation. In Pam3
stimulated platelets, the FAK-FXIIIa interaction decreased significantly compared to thrombin-activated platelets, suggesting a decrease in binding due to differences in signaling. The FXIIIathrombospondin interaction under resting conditions stems from α-granules, since thrombospondin is located only in these granules. After thrombin- or Pam3
-activation, the intensity of the FXIIIa-thrombospondin interaction decreased significantly indicating that less thrombospondin was bound to FXIIIa after activation with either agonist. The FXIIIa-HSP27 binding for thrombin-activated platelets decreased compared to resting but not to the extent as that seen with Pam3
. HSP27, like FAK, is thought to translocate to the cytoskeleton upon platelet stimulation (40
). The observed differences in signaling might have led to the differences in FXIIIa binding. Additional experiments showed the inhibition of PI3K did not affect the observed differences in these FXIIIa-interactions, suggesting that the PI3K/Akt-pathway is not involved in regulating FXIIIa-protein interactions.
Platelet activation with thrombin, Pam3
or ADP was examined whether it causes a differential release of various α-granule proteins. Platelet α-granules are the major storage compartment for adhesive proteins, chemokines, receptors, coagulatory and fibrinolytic proteins (35
). Granules are distributed randomly within platelets, but, upon stimulation, fuse with the plasma membrane or open canalicular systems to release their contents. Our data suggest a differential, agonist-dependent release for selected proteins upon platelet activation with thrombin, Pam3
, or ADP. The releasate of ADP-stimulated platelets lacked thrombospondin, while the releasate of thrombin-activated platelets lacked fibrinogen β, likely because it had been converted into fibrin (22
). For FXIIIa and gelsolin, differences occurred in protein spots representing multiple isoforms and in spot intensity. For PBP and PF4, the most abundant chemokines released from platelets (44
) were differently abundant in the respective releasates. One might expect that Pam3
-stimulation could lead to an excessive release of these chemokines, however, we did not observe this. Pam3
-stimulation of platelets that had been treated with increasing amounts of PI3K-inhibitor, caused a decrease in the release of PBP and thrombospondin. The thrombin-stimulated release of these two proteins was not affected by the inhibitor, suggesting that the PI3K/Akt signaling pathway is significantly involved in Pam3
-induced, but not in thrombin-induced, α-granule release.
Finally, these data demonstrate the functional differences that occur when platelets are stimulated in the context of inflammation and thrombosis. Using thrombin as the agonist, platelets release fibrinogen, which is converted into fibrin by thrombin. FXIIIa crosslinks fibrin to form a stable, platelet clot. In normal hemostasis, this stable clot prevents hemorrhage and allows for proper wound healing. However, in a pathologic setting, the same clot can also prevent blood flow and cause an ischemic event to occur, such as in myocardial infarction. Stimulation with Pam3CSK4, which binds to TLR2/1, leads to the release of fibrinogen, which not only binds to platelets, but also to immune cells such as neutrophils and monocytes. Platelets will bind these cells and each other to localize the immune response to the site of injury.
In conclusion, our data demonstrate for the first time that platelet stimulation with thrombin or Pam3CSK4 leads to differential activation of signaling proteins, changes in protein-protein interactions, as well as α-granule release. These results highlight the differences in the platelet's immune vs. thrombotic responses and form the basis for further functional and mechanistic studies to better understand the platelet's role in innate immunity and inflammation.