The results of this study demonstrate that Rap1b-null mice have a bleeding defect due to abnormal platelet function. In vitro aggregation responses of Rap1b-null platelets are impaired, and this impairment appears to be due to impaired activity of integrin. Moreover, in an in vivo, platelet-dependent arterial thrombosis model, in which integrin αIIbβ3 is a central regulator, Rap1b-null platelets are protected from thrombosis.
In contrast to previous studies that have placed Rap1b in the signaling pathway downstream from the Gi
-coupled receptors (16
), the data presented in this paper suggest that Rap1b is involved in a common critical step required for platelet activation. Consistent with the former model and similar to the Gαi2
-null phenotype (28
), ADP and epinephrine-induced aggregation is attenuated in Rap1b-null platelets. Rap1b-null platelet aggregation is also dramatically reduced at lower collagen concentrations, where normal platelet responses are dependent on secreted ADP and integrin αIIb
). However, ADP-induced aggregation of Rap1b-null platelets is further inhibited when signaling from either of the purinergic receptors is blocked with receptor-specific antagonists, which argues against Rap1b functioning exclusively in one of those pathways. Moreover, early signaling events from the P2Y1
receptors (the increase in cytosolic calcium and the inhibition of adenylyl cyclase, respectively) are not affected in Rap1b-null platelets (data not shown), indicating that Rap1b lies further downstream from these events.
Strikingly, aggregation of Rap1b-null platelets is impaired in response to all agonists tested, not just those coupled to GPCRs. Dose-response curves to the Gαq
-coupled PAR4 agonist peptide or non-GPCR-coupled GPVI receptor agonists (convulxin and high concentration of collagen, which is ADP-independent; ref. 30
) are all right-shifted in Rap1b-null platelets relative to normal platelets. Moreover, inhibition of the effects of released ADP by the use of purinergic receptor antagonists further reduces aggregation of Rap1-null platelets. The abnormalities in Rap1b-null platelets are not overcome by higher doses of agonist: the slower shape change and reduced rate of aggregation persist. Additionally, Rap1b-null platelets manifest similar defects in response to calcium ionophore, indicating that Rap1b acts downstream from calcium release.
One critical step in platelet activation regulated by Rap1b is integrin αIIbβ3 activation. We show that Rap1b deficiency impairs both weak and strong agonist-induced soluble fibrinogen binding, indicating that Rap1b is involved upstream from integrin αIIbβ3 activation. In addition, platelet spreading on fibrinogen, a complex phenomenon dependent on the signaling downstream from integrin engagement, is decreased in Rap1b-null platelets. Our thrombosis model results support the idea that Rap1b is a critical regulator of integrin function in vivo.
While the exact mechanism through which Rap1b regulates integrin activity remains unknown, several of our results and previous reports suggest that Rap1b may be acting between integrin αIIb
and the cytoskeleton. A universal characteristic in all Rap1b-null platelet aggregations is slower shape change. This could result from slower integrin activation and/or decreased actin microfilament dynamics. This phenotype is opposite to that seen in another knockout model of a platelet-abundant protein, vasodilator-stimulated phosphoprotein (VASP), which is believed to negatively regulate actin polymerization. In VASP-null platelets, collagen induces faster shape change (31
) and increased fibrinogen binding compared with normal platelets (31
). Moreover, VASP, which, like Rap1b, is phosphorylated by PKA upon elevation of cellular cAMP level, has been shown to be a mediator of that inhibition (31
). Unlike VASP-null platelets, aggregation of Rap1b-null platelets is susceptible to inhibition by a full range of cAMP concentrations, indicating that Rap1b is not a critical effector of cAMP inhibition.
Another indication that Rap1b may be involved in regulating cytoskeleton-integrin interaction comes from localization studies. Upon stimulation and aggregation of platelets, Rap1b translocates to the cytoskeleton (33
) in a process that is largely integrin-independent, as it occurs in platelets from Glanzmann thrombesthenia patients (35
). Lastly, studies in megakaryocytes have shown that Rap1b-induced upregulation of integrin is blocked when actin polymerization is blocked by cytochalasin D (20
This study shows that Rap1b is involved in regulation of platelet function and that its deficiency leads to a mild bleeding defect in otherwise apparently normal mice. However, the mildness of the defect in surviving adults contrasts with a severe bleeding phenotype and lethality of Rap1b-null embryos. While the surviving Rap1b-null mice may have a genotypic or phenotypic compensation that ameliorates the consequences of rap1b
deletion, we believe that the defective function of Rap1b-null platelets is unlikely to be the cause of increased embryonic lethality. In fact, neither platelets nor fibrinogen are required for embryonic hemostasis (36
). Instead, embryonic bleeding is often caused by a defective vascular component of hemostasis. Whether Rap1b is involved in the embryonic vasculogenesis is an intriguing question.
Interestingly, Rap1a, which is highly homologous to Rap1b and is also expressed in platelets, albeit at a lower level (12
), does not appear to be required for platelet function, as Rap1a-null platelets have normal aggregation responses (M. Chrzanowska-Wodnicka and L.A. Quilliam, unpublished data). Moreover, the activity of Rap1a is undetectable in Rap1b-deficient platelets (M.K. Larson, M. Chrzanowska-Wodnicka, G.C. White II, and L.V. Parise, unpublished observations), and thus, Rap1a does not appear to compensate for Rap1b deficiency in platelets.
This report provides the first genetic evidence to our knowledge that Rap1b is required for normal integrin αIIb
signaling in platelets. Impaired integrin signaling in Rap1b-null platelets is likely to be a major factor responsible for protection from thrombosis in Rap1b-null mice in the in vivo arterial thrombosis model, where integrin αIIb
is key (27
). Our study shows that Rap1b is required for normal hemostasis in vivo and, because its deficiency confers protection against thrombosis without spontaneous bleeding, Rap1b emerges as a novel antithrombotic therapy target. The next challenge will be to elucidate the exact mechanisms by which Rap1b regulates signaling from the receptor agonists to integrin αIIb
, affecting platelet function. The Rap1b-knockout mice should provide a useful tool to accomplish that goal.