C3G transgenic mouse models with specific expression in platelets reveal a new role for C3G in platelet clotting through its GEF activity.
We have generated mouse transgenic lineages for C3G (tgC3G) and C3GΔCat (tgC3GΔCat, C3G mutant lacking the GEF domain), where the transgenes are expressed under the control of the megakaryocyte and platelet specific PF4 (platelet factor 4) gene promoter. Transgenic platelet activity has been analyzed through in vivo and in vitro approaches, including bleeding time, aggregation assays and flow cytometry. Both transgenes are expressed (RNA and protein) in purified platelets and megakaryocytes and do not modify the number of platelets in peripheral blood. Transgenic C3G animals showed bleeding times significantly shorter than control animals, while tgC3GΔCat mice presented a remarkable bleeding diathesis as compared to their control siblings. Accordingly, platelets from tgC3G mice showed stronger activation in response to platelet agonists such as thrombin, PMA, ADP or collagen than control platelets, while those from tgC3GΔCat animals had a lower response. In addition, we present data indicating that C3G is a mediator in the PKC pathway leading to Rap1 activation. Remarkably, a significant percentage of tgC3G mice presented a higher level of neutrophils than their control siblings. These results indicate that C3G plays an important role in platelet clotting through a mechanism involving its GEF activity and suggest that it might be also involved in neutrophil development.
Centro de Investigación del Cáncer, Universidad de Salamanca, Salamanca, Spain.
Sara
S
Gutiérrez-Herrero
Vera
V
Maia
Javier
J
Gutiérrez-Berzal
Nuria
N
Calzada
María
M
Sanz
Consuelo
C
González-Manchón
Miguel
M
Pericacho
Sara
S
Ortiz-Rivero
José R
JR
González-Porras
María
M
Arechederra
Almudena
A
Porras
Carmen
C
Guerrero
D000818:Animals; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D002478:Cells, Cultured; D004789:Enzyme Activation; D005260:Female; D005818:Genetic Engineering; D020735:Guanine Nucleotide-Releasing Factor 2 / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007958:Leukocyte Count; D008297:Male; D008533:Megakaryocytes / Q000378:metabolism / Q000502:physiology; D051379:Mice; D008822:Mice, Transgenic; D009504:Neutrophils / Q000502:physiology; D015539:Platelet Activation; D010976:Platelet Count; D010978:Platelet Factor 4 / Q000235:genetics; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020827:rap1 GTP-Binding Proteins / Q000378:metabolism
D020735:Guanine Nucleotide-Releasing Factor 2; D010978:Platelet Factor 4; D011493:Protein Kinase C; D020827:rap1 GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate