Cdc42 is a member of the Rho family of small GTPases and has been known to regulate actin organization and associated morphological changes such as filopodia formation in a variety of cell types. However, its role in platelet activation, particularly filopodia formation, granular secretion and aggregation, is controversial at this time due to recent contradictory reports 
. This study was undertaken to more clearly define the role of Cdc42 in regulation of platelet activation using an inducible Cdc42 knockout mouse model. The data in show that gene targeting of Cdc42 resulted in a lack of expression of Cdc42 but did not affect expression of related Rac1 and RhoA. Our findings that inducible gene targeting of Cdc42 results in a significant thrombocytopenia () and prolonged tail bleeding times in Cdc42-deficient mice () demonstrate that Cdc42 is essential for normal platelet function. These observations are in agreement with a recent report that megakaryocyte/platelet specific constitutive deletion of Cdc42 results in thrombocytopenia as well as prolonged tail bleeding times 
We have previously reported that deletion of Cdc42 leads to hematopoietic stem/progenitor cell defects in differentiation, cell cycle progression, and homing/lodging in the bone marrow 
. Thus, the observed partial reduction in platelet production in Cdc42 deficient mice may be due to early progenitor defects as well as defects in megakaryocyte differentiation. The mechanisms by which Cdc42 regulates platelet production remain unclear at this time. The interferon inducible deletion of Cdc42 in Mx-cre;Cdc42lox/lox
mice affects all blood lineages including the stem/early progenitor compartments and therefore makes this particular mouse model unsuitable for studying the developmental mechanism of a terminal lineage like platelet.
Wiskott-Aldridge Syndrome (WAS) patients exhibit thrombocytopenia 
. Since Wiskott-Aldridge Syndrome Protein (WASP) is regulated by Cdc42, deficiency of Cdc42 may be involved in thrombocytopenia. However, Cdc42 engages multiple effector pathways including PAK, WASP, IQGAP, Par6 
, making it difficult to sort out the specific contribution of WASP to the diminished platelet number in Cdc42-deficient mice. Technical difficulties in reintroducing Cdc42 mutants (e.g. defective in WASP binding) to platelets pose another challenge for demonstrating a causal relationship between Cdc42 deficiency, defective WASP activation and thrombocytopenia. More recently WASP has been shown to play an important role in regulation of integrin αIIb
mediated platelet activation 
and it is possible that this pathway is involved in Cdc42 mediated αIIb
The p-21activated kinases (Paks) are known effector molecules of Cdc42 and have been linked with actin polymerization and lamellipodia formation. The Paks have been shown to be phosphorylated in platelets by thrombin, TRAP as well as convulxin, a GPVI agonist 
. Our finding that CRP, a GPVI specific agonist, as well as thrombin induced phosphorylation of PAK1/2 is inhibited in Cdc42-deficient platelets () confirms not only deletion of Cdc42 in the gene targeted mice but also demonstrates that signaling downstream of Cdc42 has been effectively blocked in Cdc42−/−
platelets. This is a first report to show that the Cdc42 is involved in GPVI-specific signaling leading to phosphorylation of PAK1/2 in murine platelets. We have reported earlier that gene targeting or pharmacologic inhibition of Rac1 also inhibits activation of PAK1/2 
. Thus, PAK1/2 appears to be subjected to a dual regulation by Cdc42 and Rac1 in platelets. This suggests that Cdc42/Rac1 either function sequentially or in parallel on this “shared” effector. How PAK1/2 is regulated by both Cdc42 and Rac1 in platelets is not clear at this point. One possibility is that the regulation of PAK1/2 by Cdc42/Rac1 may involve heterodimerization of PAK1/2 and binding of the PAK1/2 dimer to Cdc42/Rac1 simultaneously. Alternatively, Cdc42 and Rac1 may act sequentially in a signaling cascade leading to PAK regulation. Investigations of these hypotheses to resolve the functional relationship between Cdc42 and Rac1 would require multiple gene targeting and transgenic approaches as most standard gene transfer techniques are not applicable in platelets.
Agonists that induce platelet activation also activate Cdc42 
and active Cdc42 can initiate platelet actin cytoskeleton reorganization 
leading to lamellipodia 
and filopodia formation 
. Ligand density has been shown to affect integrin αIIb
-mediated platelet signaling and spreading 
and increasing agonist concentrations, at least partially, reverse the diminished aggregation responses observed in Cdc42 deficient platelets (). We therefore utilized lower concentrations of immobilized fibrinogen (3 µg/ml) and CRP ((0.5 µg/ml) to investigate the role of Cdc42 in platelet morphological changes. Our findings that Cdc42−/−
platelets failed to form filopodia or lamellipodia and exhibited diminished spreading on immobilized fibrinogen or CRP (, , and ) clearly demonstrate that Cdc42 is involved in reorganization of actin cytoskeleton. The inability of Cdc42−/−
platelets to form filopodia on immobilized fibrinogen clearly demonstrate that Cdc42 is essential for integrin αIIb
-depedent outside-in signaling involved in reorganization of actin cytoskeleton. The findings that murine platelets were able to form filopodia on immobilized CRP show that GPVI is involved in filopodia formation. Moreover, our observations that Cdc42−/−
platelets were unable to form filopodia on CRP clearly demonstrate that Cdc42 is critical for the GPVI mediated actin reorganization. Using secramine A, a non-specific inhibitor, Pula and Poole 
have suggested that inhibition of Cdc42 partially blocks spreading of platelets on collagen, CRP and fibrinogen. However, our findings disagree with a recent report showing that megakaryocytes/platelets specific deletion of Cdc42 had no effect on filopodia formation on immobilized fibrinogen or CRP 
. The reason for this discrepancy is not clear at this time. These investigators have also suggested that Cdc42-deficient platelets exhibit specific defect in GPIb-specific signal as evidenced by the reduced extension of filopodia on immobilized vWf. However, they used a significantly lower concentration of vWf (10 µg/ml) as compared to the ten-fold excessive amounts of fibrinogen or CRP. It is possible that platelets from their Cdc42−/−
mice would have exhibited defective filopodia formation if significantly lower concentrations of fibrinogen or CRP were used. Another possible explanation for the discrepancy may be that constitutive, but not transient inducible deletion of Cdc42, in megakaryocytes/platelets results in compensatory expression and/or activation of Cdc42-related Rho GTPases such as TC10, TCL or Rif. Such differences between the two knockout systems are difficult to confirm at this time due to the lack of specific antibodies and probes.
Cdc42 has been shown to be critical in exocytosis in a number of cell types 
. Our findings that platelets from mice with inducible deletion of Cdc42 exhibit diminished secretion from platelet granules () suggest that Cdc42 plays a critical role in platelet secretory responses. However, Pleines et al. 
have shown that platelets from mice with constitutive deletion of Cdc42 exhibit increased secretory responses. There is no apparent mechanistic explanation for the decreased secretion observed by us () and the enhanced secretion reported by them. We quantified secretion in washed murine platelets using a luciferin/luciferase chemi-luminescence method. They quantified ATP in supernatants of PRP from heparinized blood after terminating the reactions with EDTA and fixing platelets with formaldehyde. The differences in methodology for assessing secretion, in addition to above discussed genetic models, may account for the differences in these observations.
The functional consequence of platelet activation reactions is aggregation or bridging of platelets that occurs due to binding of fibrinogen to integrin αIIb
on the adjoining platelets. Diverse agonists bind to their specific receptors on platelets and initiate signaling cascades leading to the so-called inside-out signaling that culminates in secretion from platelet granules and a many-fold increase in exposure of integrin αIIb
on platelet surface and consequently a much greater aggregation response. If secretion from platelet granules is essential for maximal, or the so-called irreversible, platelet aggregation response then diminished secretion responses observed in Cdc42−/−
platelets () would result in diminished platelet aggregation response. Indeed our findings that platelet aggregation responses induced by thrombin, collagen or CRP are either minimal or diminished in Cdc42−/−
platelets () suggest that diminished secretion, at least in part, is responsible for impaired aggregation responses in Cdc42−/−
platelets. Impaired aggregation responses induced by diverse agonists such as thrombin, collagen or CRP imply that a common rather than an agonist-specific signaling mechanism is responsible for defective platelet aggregation in Cdc42−/−
mice. Agonist induced activation of Akt is one such common signaling mechanism. Secondary or irreversible platelet aggregation has been linked to phosphorylation of Akt 
. Secreted ADP, regardless of the nature of the agonist employed, induces phosphorylation of Akt via activation of Gαi 
and GPVI specific agonists such as convulxin induce phosphorylation of Akt in part due to secreted ADP and in part due to activation of PI3Kβ 
. Our findings that CRP, a GPVI specific agonist, and thrombin, that activates murine platelets via PAR3/4 receptors, both induced minimal or only reversible aggregation () as well as induced only nominal phosphorylation of Akt in Cdc42−/−
, as compared to the Cd42+/+
, platelets () suggest that deficiency of Cdc42 results in defective platelet aggregation, at least in part, due to inhibition of phosphorylation of Akt.
Our observation that Cdc42−/−
platelets exhibit impaired platelet aggregation in response to CRP, collagen or thrombin differ from the report showing that platelets from constitutively deleted Cdc42 mice exhibit increased aggregation responses upon stimulation with collagen or CRP and a similar aggregation response upon stimulation with thrombin 
. These investigators have suggested that increased aggregation induced by collagen and CRP is due to enhanced secretion from platelet granules. There is no obvious explanation for the different observations made by them and us. Again, methodological differences may be attributed to the different findings. However, our data () and their observation of a prolonged bleeding time in Cdc42−/−
mice are hard to reconcile with enhanced secretion/aggregation responses in Cdc42−/−
Pula and Poole 
have reported that secramine A blocks collagen but not CRP induced platelet aggregation. However, the non-selective nature of the inhibitor as well as the excessively high concentration of collagen (30 µg/ml) and CRP (5 µg/ml) used in this study make it difficult to compare their data with our data in Cdc42−/−
platelets where we used 10-times lower concentration of collagen and CRP. It is possible that secramine A might have inhibited normal i.e. typical aggregation response induced by a lower concentration of CRP.
Platelet aggregation at the site of injury forms the primary hemostatic plug leading to an arrest of bleeding. Standardized bleeding times are considered a measurement of platelets functional integrity. Our findings of prolonged tail bleeding times in Cdc42−/−
mice (), consistent with observation by Pleines et al. 
, along with the observed impaired secretion and aggregation in Cdc42−/−
platelets (,) clearly demonstrate that Cdc42 plays an essential role in regulation of platelet function and thus is critical for platelet mediated primary hemostasis.
In summary, our data implicate Cdc42 not only in reorganization of actin cytoskeleton including filopodia formation induced by fibrinogen, an integrin αIIbβ3-dependent and CRP, a GPVI-dependent, pathway, but also in platelet aggregation. Regulation of granular secretion and Akt phosphorylation by Cdc42 appears to determine, at least in part, the extent of platelet aggregation induced by agonists that employ the GPVI and non-GPVI signaling pathways.