Key witnesses in the Alexander Litvinenko investigation are missing, with their families claiming that they fear for their lives.

The sudden disappearance of a number of leading figures linked to the affair will make it even harder for British detectives, whose inquiry has now spread across five countries.

Interpol joined the hunt for the murderer yesterday, saying that it hoped to exchange information coming from Britain, France, Italy, Germany and Russia.

Scotland Yard was struggling to gain access to vital witnesses with former associates of Litvinenko claiming that they are too scared to come forward.

Evgeny Limarev, who told the former KGB officer that he was on a death list just hours before he was poisoned, was reported to have fled his home in the French Alps, where he was under police protection. Before his disappearance, he said that British detectives wanted to question him about the origins of a hitlist that included Litvinenko’s name among the targets being hunted by a team of former Russian agents working across Europe.

He sent this list to the Italian security expert Mario Scaramella, who showed it to Litvinenko in a Piccadilly sushi bar on the day that he fell ill.

Mr Limarev, who used to work with Russia’s Federal Security Bureau (FSB) before he fled Moscow, did not expect to be identified publicly. “Now my name has been linked to this case I really fear something might happen to me,” he said last week. He claims that sources in Russia who passed him the hitlist are being hunted by the FSB and have had to go to ground after armed agents searched their homes and other locations.

In Moscow, nine detectives from Scotland Yard’s counter-terror squad are seeking a further interview with the two Russian businessmen who met Litvinenko in a London hotel where police now believe that he was poisoned.

Both men are still being kept at a clinic run by the Federal Medico-Biological Agency of Russia, which is sealed off.

Russian authorities describe Dmitri Kovtun as a target for the assassin who killed Litvinenko. British police still call him a significant witness, while prosecutors in Hamburg say they are investigating him for allegedly illegally handling the radioactive polonium-210, which they believe was smuggled from Russia through Germany to Britain. Mr Kovtun denies any role in the assassination.

His business partner and a former KGB bodyguard, Andrei Lugovoy, refused to divulge details of his questioning by Russian and British investigators, saying that Russian officials had made him sign a gagging order.

Doctors say that the results of medical tests on whether the two men are contaminated will be revealed on Friday.

With police forces from five countries now involved, Russian prosecutors made clear yesterday that they intend to use their visit to London later this week to raise the issue of a number of the Kremlin’s enemies who have been given asylum in Britain, as well as the Litvinenko affair.

Russian officials have suggested that the poisoning may have been an attempt to discredit Moscow and have thrown suspicion on the London-based dissidents Boris Berezovsky, a wealthy businessman, and Akhmed Zakayev, a spokesman for the rebel government in Chechnya.

Both men were close friends of Litvinenko and have denied any role in his murder. They have accused the Kremlin of orchestrating the killing.

An unnamed Russian businessman who flew from Moscow to Hamburg on October 28 with Mr Kovtun is also being sought. Police believe this flight was used to transport polonium-210 into Europe.

• The Moscow headquarters of Garry Kasparov’s United Civil Front movement were raided yesterday. Police took away literature produced by the former world chess champion’s group, which is highly critical of President Putin.