Csk/CD148 and platelet SFK activation: a balancing act!

Abstract

In this issue of Blood, Mori et al provide important insights into the regulation of the activity of Src family kinases (SFKs), a family of enzymes that play a critical role in the ability of platelets to regulate both hemostasis and thrombosis.¹

SFKs in platelets, which include Src, Lyn, and Fyn, are downstream effectors of many cell surface receptors, including integrins, GPVI, GPIb-IX-V, CLEC-2, and G protein–coupled receptors.² SFKs have wide-ranging roles in platelet function, including such key processes as adhesion, cell spreading, and aggregation. Following blood vessel injury, platelet activation is initiated and propagated by a series of agonists, many of which result in the changes in the phosphorylation state of key tyrosine residues that regulate SFK activity. Interestingly, although activation of some SFKs leads to platelet activation, some of the same SFKs can limit platelet function and reduce the likelihood of thrombosis. Regulation of SFK activity in such a way as to enable normal hemostasis while avoiding unwanted thrombosis is poorly understood. Studies by Mori et al in this issue of Blood provide a clue in understanding this important balance.

The activity of SFKs is regulated by the phosphorylation of 2 conserved regulatory tyrosines, one located in the C-terminal tail and the other in the kinase domain activation loop.³ In platelets, it is known that phosphorylation of the C-terminal tyrosine, mainly by the C-terminal Src kinase (Csk), inhibits SFK activity, and that dephosphorylation of this tyrosine, mainly by the receptor-like tyrosine phosphatase CD148 (also known as Dep-1 or PTPRJ), activates the SFKs. Full activation of SFKs, however, requires transautophosphorylation of the activation loop tyrosine. To investigate how the activation of SFK is balanced by Csk and CD148, Mori et al generated mice with platelet-specific deletions of Csk, CD148, and Csk plus CD148. As was expected, Csk knockout resulted in elevated SFK activity, whereas CD148 knockout led to greatly decreased SFK activation. Unexpectedly, however, SFK activity was significantly greater in the Csk/CD148 double-knockout mice than in mice lacking only Csk. This intriguing observation suggests that CD148 is also capable of dephosphorylating the activation loop tyrosine, thus negatively regulating SFK activity. Negative regulation of SFKs by CD148 was also detected previously in endothelial cells.⁴

A second surprising observation made by Mori et al is that elevated SFK activity in the double-knockout mice was associated with dampened rather than enhanced platelet function in both hemostasis and thrombosis. This was at least in part due to the reduced expression of the activating receptors GPVI and CLEC-2 and to the increased expression of the inhibitory receptor G6b-B. In addition, the elevated Lyn activity may exert an inhibitory function on platelet activation. It is tempting to speculate that sustained, high-level SFK activity acts as a “danger signal” for a prothrombotic state, somehow modifying the expression level of these receptors. However, one of the limitations of gene-knockout technology is that the resulting protein modifications sometimes are due to a compensatory mechanism that may not operate under normal conditions. To circumvent this issue, Mori et al used a novel mouse model expressing an inhibitor-sensitive form of Csk, enabling rapid and specific inhibition of Csk activity in mature platelets. Inhibition of Csk in these platelets demonstrated that a sudden increase in SFK activity is able to initiate inhibitory signaling from G6b-B.

Studies by Mori et al demonstrate a novel mechanism by which platelet homeostasis is maintained through the regulation of the activities of SFKs. On one hand, SFK activities are balanced by the kinase and phosphatase pairing of Csk and CD148. On the other hand, high SFK activity induces negative feedback that restrains hyperactivation of platelets. It remains to be determined how the threshold of activation of SFKs is set and checked in platelets so as to satisfy the different requirements of hemostasis versus thrombosis. Moreover, because SFKs are involved in multiple receptor signaling pathways, an intriguing question is whether the SFKs associated with different receptors are regulated in the same or different ways.

An emerging understanding of the role played by CD148 in regulating platelet activation makes it an attractive drug target for antithrombotic therapy. Intriguingly, CD148 knockout mice exhibit normal hemostasis but reduced thrombosis. In addition to the previously defined positive regulatory function of CD148 in SFK activation,⁵ this study reveals a negative regulatory effect of CD148 on SFKs. A remaining question is how the phosphatase activity of CD148 is regulated to fulfill its dual activating and inhibitory functions. CD148 also exerts both activating and inhibitory effects on immune cell signaling such as in T-cell receptor (TCR) triggering.⁶ It has been established that the inhibitory effect of CD148 on TCR triggering is regulated by its large extracellular domain, which passively segregates the phosphatase from the close-contact area containing engaged TCRs.⁷ Whether a similar mechanism operates in platelets is unknown. Nor is it known whether the activity of CD148 in platelets can be regulated by binding of recently identified extracellular ligands for CD148 such as thrombospondin-1.⁸ Answering these questions is critical to fully understanding the regulatory function of CD148 in platelets and for the development of novel antagonists as antithrombotic reagents that target the extracellular or phosphatase domain of CD148.