The Supreme Court's electoral branch declared Luisa Ortega Diaz's request inadmissible on the same day anti- government demonstrators were marching toward the high court to protest its refusal to stop Maduro's special assembly.
Opposition leaders said pro-government armed groups known as "colectivos" clashed with protesters and journalists near the Supreme Court and witnesses' videos showed fistfights and people being shoved to the ground at the demonstration site.
The decision came four days after Ortega Diaz made an impassioned plea on the Supreme Court steps, grasping Venezuela's small blue constitution book in her hands and declaring the future of the nation's democracy was at stake.
Two months of anti-government protests have left at least 68 people dead as demonstrators demand new presidential elections in the face of triple-digit inflation that keeps rising, soaring crime and crippling food and medical shortages.
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Venezuelans in Caracas awoke yesterday to find their city paralyzed by a public transportation strike that union leaders said stretched through 90 percent of the capital.
Bus driver Santos Quevedo was charged with terrorism after allegedly transporting a group of opposition protesters, but local reports say the government opponents forced him to give them a ride.
As during previous protests, the government closed several metro stations.
Speaking outside the Supreme Court, union leaders said transit workers are the first to wake up in the morning and often exposed to dangerous conditions in a country with one of the highest homicide rates in the world.
"Every time we leave our homes we don't know if we'll return alive," said Pedro Jimenez, president of a local union called the Southwest Transporters Bloc. He demanded that the government take action to ensure drivers' safety.
The last time the flag is believed to have been raised was in April 2013 during the presidential election to replace the late President Hugo Chavez, which Maduro won by a narrow vote.
The Red Cross raised the flag again as a protective measure in light of recent protests in which authorities have used tear gas near the institution's hospital, said Jose Ramon Gonzalez, the group's national relief director.