This is the first study of the functional role of CD148, the only RPTP expressed in platelets, in hemostasis and thrombosis in vivo.21
The present study shows the following: (1) CD148 is a global regulator of SFKs in platelets. (2) CD148 plays a critical role in signaling through GPVI and the major platelet integrin, αIIbβ3. (3) CD148 contributes to signaling by the G protein-coupled receptors for thrombin and TxA2
, PAR-4, and TP, respectively. (4) CD148 is required for megakaryocyte spreading and migration on a variety of surfaces. (5) CD148 plays a vital role in mediating platelet aggregation under flow and in hemostasis and thrombus formation in vivo, with the latter being shown using 3 distinct models. These findings, in addition to the absence of pathologic hemorrhaging in mice lacking cell surface expression of CD148, make this RPTP an attractive novel antithrombotic drug target.
Platelet functional responses are regulated by a diverse repertoire of tyrosine kinase–linked and G protein–coupled receptors. SFKs are essential for initiating and propagating signaling from several major platelet tyrosine kinase-linked receptors. They also contribute to signaling downstream of several stimulatory G protein–coupled receptors.48,49
SFK activity is tightly regulated by tyrosine phosphorylation and intramolecular interactions that constrain the kinase domain.10,11
Phosphorylation of the C-terminal inhibitory tyrosine maintains the SFK in an inactive conformation, whereas dephosphorylation of this site in parallel with trans
-autophosphorylation of the activation loop tyrosine renders it maximally active.15,50,51
One of the main findings of this study was that global SFK activity, as detected using phosphospecific antibodies, was significantly lower in both resting and activated CD148-deficient platelets compared with wild-type platelets, demonstrating for the first time that CD148 is required for maintaining a pool of active SFKs in resting platelets and increasing SFK activity upon platelet activation. We hypothesize that the pool of active SFKs in resting platelets is to maintain essential constitutive biologic processes and to initiate a rapid response to a stimulatory signal.
Until now, the earliest characterized signaling event downstream of GPVI is the activation of constitutively associated SFKs.5,6,37
It has yet to be elucidated, however, how SFKs become activated after GPVI-collagen engagement.6
Our biochemical data demonstrated a GPVI proximal signaling defect in the absence of CD148 as tyrosine phosphorylation of the FcR γ-chain, Syk, and PLCγ2 were all substantially reduced in response to CRP. Consistent with a major signaling defect, functional and biochemical defects exhibited by CD148-deficient platelets were substantially more severe than those observed in platelets from FcR γ-chain heterozygous-deficient mice, which express a similar reduction in the GPVI-FcR γ-chain complex.27,34
Additionally, SFKs exhibited significantly reduced phosphorylation of their activation loop tyrosines in parallel with increased phosphorylation of their C-terminal inhibitory tyrosines in the absence of CD148. These findings demonstrate that CD148 plays a critical role in activating SFKs downstream of GPVI. CD148 would therefore appear to play a similar role to that of the structurally distinct RPTP CD45 in B- and T-cell receptor signaling.16,20
The reduction in the signaling response to engagement of integrin αIIbβ3 in the absence of CD148 is similarly explained by the altered tyrosine phosphorylation of the SFKs. Interestingly, the group of Shattil has also demonstrated a role for the nontransmembrane tyrosine phosphatase, PTP-1B, in signaling by the major platelet integrin, although it does not play a role in platelet activation by the snake venom toxin, convulxin.9
Thus, CD148 and PTP-1B appear to work together to regulate αIIbβ3 integrin signaling.
Results from this study demonstrate a critical role of CD148 in regulating global SFK activity in platelets and suggest that SFKs may be direct physiologic substrates of CD148. In support of this hypothesis, CD148 has previously been shown to interact directly with Src and to dephosphorylate both of its regulatory phosphorylation sites in vitro.40
The interaction was also observed in transfected cells; however, CD148 only dephosphorylated the inhibitory site and not the activation site in transfected cells.40
Other potential physiologic substrates of CD148 in platelets and other cells include: the tyrosine kinase-linked receptors Met and PDGFβ, the adapter proteins LAT and Gab1, the adherens junction protein p120catenin, PLCγ1, and more recently the p85 subunit of PI 3-kinase, although the latter is not tyrosine phosphorylated in platelets.52–57
Interestingly, LAT, PLCγ1 and PI 3-kinase all lie downstream of SFKs in the GPVI signaling cascade; therefore, CD148 may be regulating multiple points of the GPVI signaling pathway.58–61
Residual GPVI signaling in CD148-deficient platelets raises the possibility that one or more other PTPs partially compensate for the absence of CD148. Because we were unable to identify another RPTP in platelets using a proteomic approach, we hypothesize that a nontransmembrane PTP may fulfill the role as has been shown to be the case for signaling by αIIbβ3.7–9,21
Possible candidates include the SH2 domain-containing PTPs, Shp1 and Shp2, as they have been previously shown to regulate ITAM receptor signaling in immune cells. Shp1 has also been shown to interact with Src in platelets and to positively regulate Src activation by preferentially dephosphorylating inhibitory Tyr-529.62
In support of this model, Shp1 was demonstrated to play a positive regulatory role in GPVI-mediated platelet activation.63
This was shown through the use of naturally occurring motheaten viable
mice, which have reduced Shp1 activity.63
A question that arises from our proposed mechanism is how approximately 2800 copies of CD148 molecules can regulate the pools of SFKs associated with approximately 4000 copies of GPVI and more than 80
000 copies of αIIbβ3.33,64
We hypothesize the explanation lies in the high catalytic activity of PTPs, which have kcat
values up to 3 orders of magnitude greater than those of protein tyrosine kinases.65
Regulation of CD148 activity and localization in the platelet plasma membrane are now critical to understanding how CD148 regulates both GPVI and αIIbβ3 signaling. This might be mediated by interaction with a ligand or through compartmentalization into membrane microdomains. The ligand for CD148 is presently not known. One report, however, suggests that it may be an extracellular matrix protein.66
Moreover, work done in Jurkat T cells has shown that CD148 can be regulated by membrane compartmentalization, as it is excluded from the immunologic synapse by mechanical forces.18,19
Both our ex vivo flow adhesion studies and in vivo analysis of hemostasis and thrombosis indicate that CD148 plays a novel physiologic role in preventing blood loss from sites of vascular injury. The lack of evidence for a severe bleeding diathesis in CD148 mutant mice makes it a potentially exciting antithrombotic drug target. Structural and functional features of CD148 also lend it to drug targeting, including its large extracellular domain that could be targeted by small molecule inhibitors without the need to cross the plasma membrane. These features, together with its unique function in platelets, make it an ideal target for development of a new class of antiplatelet agents.