BioWorld International Correspondent

LONDON - One of the final players in the chain of events that leads to clot formation could prove a good target for a new generation of clot-busting drugs, a new study suggests. An analysis of minor variations in the gene encoding Factor XIII has shown this enzyme, which helps clots resist destruction, can influence the type of clot produced during thrombosis.

Peter Grant, professor of medicine at the academic unit of molecular vascular medicine of the University of Leeds in the UK told BioWorld International, "Our study shows that the genetics of Factor XIII and the activation of Factor XIII are very important in determining vascular risk."

The coagulation cascade - a series of enzymes, each of which activates and amplifies the next in the chain - culminates in the generation of the enzyme thrombin, which cleaves two peptides off fibrinogen. That allows fibrinogen to align itself into parallel sheets, forming "soluble fibrin." Simultaneously, thrombin activates Factor XIII, which cross-links the soluble sheets of fibrin to form "cross-linked fibrin," which comprises the final clot.

"This key role for Factor XIII in the coagulation cascade and the formation of fibrin explains why I believe fibrin is a fantastic target for new thrombolytic therapies. It is right at the business end of thrombus formation," Grant said.

The latest study by Grant and his colleagues, together with collaborators at the University of Pennsylvania School of Medicine, is published in the April 26, 2003, issue of The Lancet in a paper titled "Genetic regulation of fibrin structure and function: complex gene-environment interactions may modulate vascular risk."

Grant said the genetics of Factor XIII had been largely ignored by the scientific community until after he and his group began work on the subject. In their first paper, published in 1998, they showed that a common polymorphism in the gene encoding Factor XIII, which substituted a leucine for a valine at codon 34, was associated with a reduced risk of myocardial infarction. Yet, oddly, further investigations appeared to show that the type of clot made by people who had this polymorphism was more thrombotic - denser, stronger and less liable to be broken down.

Then Grant and his team realized they had done all their studies on the effects of mutations on the structure and function of fibrin at low fibrinogen concentrations. They began to wonder whether the polymorphisms they were studying were able to modulate the type of clot produced by the coagulation cascade in different ways according to the level of fibrinogen.

Their hunch proved correct. The leucine allele in the gene for Factor XIII - which in epidemiological studies protected those who had it from myocardial infarction, but did not seem to make the structure of the clot any more amenable to being broken down at low fibrinogen levels - turned out to be cardioprotective at high fibrinogen levels.

The study in The Lancet reports their experiments exploring this effect in more detail. They used plasma from patients who had had strokes, who were either homozygous for valine, homozygous for leucine or heterozygous at the codon 34 polymorphism. The researchers found that blood clots formed in samples from those homozygous for valine were less permeable and had a tighter structure than those who were homozygous for leucine, in the presence of high concentrations of fibrinogen. Those who were heterozygous showed intermediate results.

Grant and his colleagues confirmed those results with scanning electron micrographs of the fibrin clots, which showed thinner fibers with smaller pores (which are thus less permeable to enzymes that could break down the clot). They were also able to replicate their results using an in vitro system.

"The importance of this finding is that it demonstrates some of the complexities that can occur in this system," Grant said, "as well as providing a real mechanism for how a mutation can affect vascular risk. We know that hyperfibrinogenaemia is a risk factor for heart disease, and this mutation - rather beautifully and in a very coherent way - only becomes cardioprotective at high levels of fibrinogen."

The team will now continue its work on the genetic and environmental factors that alter fibrin structure and make it more thrombotic. That will include looking at the effects of glycosylation, and of glucose itself, on fibrin. "We will also be taking a proteomic approach," Grant said. "Ultimately, we hope to gain a better understanding of the mechanism of fibrin formation and how we can influence this process."