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N. Eaton et al.
   ishes both GPVI signaling and internalization.44 Besides its role in membrane fission during RME, DNM2 can also serve as a scaffolding protein for signaling intermediates.2 In T cells, DNM2 directly interacts with the guanine nucleotide exchange factor Vav1 to regulate activation of PLC-γ1 and the accumulation of cortical actin at sites of T-cell receptor activation, thereby regulating T-cell recep- tor signaling.45 Vav1 and Vav3 play critical but redundant roles in the activation of PLC-γ2 downstream of GPVI,46 and clustering of GPVI dimers, the mechanism of which depends on a dynamic actin cytoskeleton, contributes to GPVI signaling.47 It is, therefore, tempting to speculate that platelet DNM2 serves as a scaffolding protein for signaling intermediates and/or actin-regulatory proteins down- stream of GPVI.
Dnm2Plt–/– mice had a severe bleeding diathesis, which was intrinsic to platelets as Dnm2 deletion under control of the Pf4 promoter is specific to the platelet lineage.9 Consistently, Dnm2Plt–/– platelets adhered poorly to colla- gen under arterial shear rates and were depleted of fibrino- gen. Further, α-granule secretion, integrin αIIbβ3 activa- tion, and spreading onto fibrinogen were markedly reduced in Dnm2Plt–/– platelets stimulated through GPVI or with thrombin. The lack of GPVI signaling is likely explained by the profound deficit in GPVI expression.9 Whether this deficit is due to decreased GPVI synthesis in Dnm2Plt–/– MK, increased internalization and degradation, or ADAM10-mediated extracellular domain shedding is unclear. The differences between the pharmacological and genetic approaches indicate that either dynasore treat- ment does not completely inhibit platelet DNM2 GTPase activity or Dnm2Plt–/– platelets acquire defects during pro- duction affecting their functional responses. Because RME is a critical component of cellular cholesterol homeostasis,1 it is possible that Dnm2 genetic deletion impacts cell membrane composition and lipid raft organi- zation, thereby affecting signaling in cholesterol-rich membrane domains. While the tail bleeding time in mice is largely unaffected by severe reduction of platelet count or lack of GPVI,48,49 it cannot be excluded that the macrothrombocytopenia of Dnm2Plt–/– mice combined with the profound deficit in GPVI expression contributes to the bleeding diathesis.
The severe reduction in fibrinogen content of Dnm2Plt–/– platelets is consistent with impaired RME and a role for DNM2 downstream of integrin αIIbβ3 function as plasma fibrinogen is taken up by platelets and MK in an integrin αIIbβ3-mediated manner.31,32 Beside fibrinogen, platelets and MK take up and store in their α-granules a long list of
plasma proteins such as coagulation factor V and regula- tors of angiogenesis.50,51 While the hypothesis was not tested here, endocytosed proteins are expected to be absent in Dnm2Plt–/– platelets due to defective RME, affect- ing other biological processes aside from hemostasis.
Platelets contained major endocytic and endosomal pro- teins such as clathrin and Rab GTPases.33,39 The increased expression of clathrin heavy chain in Dnm2Plt–/– platelets likely reflects the accumulation of clathrin-coated vesicles observed in the demarcation membrane system of Dnm2Plt–/– MK.9 Caveolin 1 was not detected in platelets, consistent with mRNA and protein profiling,14,15,17 and cavin 2, a detergent-insoluble caveolae marker in cells expressing caveolin 1,38 remained soluble following sucrose gradient of human platelet lysates. While cavin 2 is abundantly expressed in platelets,14,15,17 where it was originally characterized,52 its role in the absence of cave- olin 1 is unclear. Taken together, the observations show that CME is the primary mechanism for RME in platelets. Consistently, fibrinogen uptake involves the recruitment of clathrin-associated adaptor protein 2 complexes to the β3 subunit of the integrin αIIbβ3 via clathrin adaptor pro- teins such as Dab2 and Numb.53,54 After its uptake by CME and transport via the early (Rab5) and late (Rab7) endo- some, fibrinogen is retained within α-granules by mecha- nisms dependent on NBEAL2, mutated in gray platelet syndrome.33 The remaining fibrinogen content of Dnm2Plt–/– platelets is likely associated with the plasma membrane or trapped in the open canalicular system.
In conclusion, our work provides pharmacological and genetic evidence for a role of DNM2 in GPVI signaling and platelet hemostatic function. DNM2 regulates signaling events downstream of the collagen receptor GPVI, includ- ing the activation of the proximal tyrosine kinase Lyn, and is required for the integrin αIIbβ3-mediated accumulation of plasma fibrinogen into α-granules. Our data also show that DNM2 does not contribute to GPIbα downregula- tion, as dynasore-treated platelets and Dnm2Plt–/– platelets down-regulated GPIbα normally in response to thrombin. Whether DNM2 is a reasonable target for antithrombotic therapies remains to be determined.
Acknowledgments
The authors would like to thank Emily Legan and Scott Wood for technical assistance and Drs. Harry Heijnen, Karin Hoffmeister, and Walter Kahr for helpful discussions. This work was supported by the American Society of Hematology Foundation and National Institutes of Health R01 grant HL126743 (HF).
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