By Dean A. Haycock

Special to BioWorld Today

You don't need a degree in biology or medicine to recognize the word "heparin." It is such a commonly used drug that many people immediately identify it as something that prevents blood clots.

Biologists, however, know that this is far from a complete description of heparin's functions in the body. They know that heparin is not normally found circulating in the bloodstream. They know it is contained in resting mast cells, connective tissue cells containing granules filled with substances such as histamine and enzymes that break down proteins.

What they didn't know, until now, was heparin's physiological role in the body - it's "real job," so to speak - before its anticoagulant properties were discovered accidentally in 1916 by a medical student.

With the publication today of two papers in Nature, submitted by competing labs in the U.S. and Sweden, the longstanding question of what heparin really does is answered in large part. The molecule can be assigned an important physiological function independent of its serendipitously discovered property as an effective anticoagulant. The discovery is the result of years of basic research directed at uncovering the way mast cells work.

Both groups studied mice in which a gene necessary for the production of fully functional heparin was disrupted or knocked out. The gene encodes glucosaminyl N-deacetylase/N-sulphotransferase-2, an enzyme that adds sulfur to heparin. Loss of this enzyme results in improper packaging of key components of the storage granules in mast cells, which serve as the first line of defense for the immune system. The components affected are several enzymes called mouse mast-cell proteases. These proteases are used by the immune system to destroy foreign proteins. The transgenic, knockout mice thus indicate a physiologic function for heparin unrelated to its ability to prevent blood clots.

"Last year there were more than 2,000 papers published according to a Medline search that either were related to heparin or used heparin in some particular study. So, it is one of the most thoroughly studied molecules in the body," said Richard Stevens, associate professor at Brigham and Women's Hospital and Harvard Medical School. Stevens is a co-author of "Heparin is essential for the storage of specific granule proteases in mast cells" in the Aug. 19 issue of Nature.

A report by Lena Kjellin, associate professor at Swedish University of Agricultural Sciences in Uppsala and her co-authors, "Abnormal mast cells in mice deficient in a heparin-synthesizing enzyme," appears in the same issue.

"Mast cells are basically protease factories," Stevens said. They reside in connective tissue and release proteases, histamine and other substances to help the body fight off invaders in damaged tissue. Heparin is a proteoglycan, a molecule with a peptide core and a carbohydrate side chain. Other proteoglycans have different carbohydrate side chains. Heparin is the major chain found in mast cells. To work, it must be fully sulfated.

An important question in immunology is how is it possible to store high concentrations of proteases in granules in such a way that they are activated only after they are safely stored.

"You want to ensure that the proteases are activated only when they get in the granules. You don't want them activated early in the pathway," Stevens explained. "Then you want to package them in specific molar ratios in such a way that the proteases don't eat themselves and don't eat the other proteases in that granule. And you want to package them in the most concentrated form that you can."

The papers in Nature indicate that heparin plays a key role in this process.

Stevens said, "The carbohydrate chain heparin is very important in packaging the proteases - but not all proteases - in the mast cell granules. So the nature of the carbohydrate chain is very important in dictating what type of molecule you want to package in the granule."

When the immune system unleashes mast cells, it must depend on enzymes with restricted specificity. The protein-destroying proteases the cells release must attack foreign proteins while limiting damage to the body they are acting to protect.

"The heparin chain plays a role in restricting enzyme specificity. In terms of the biotech field, the concept of cofactors in enzyme specificity needs to be addressed more," Stevens told BioWorld Today.

He added that although 2,000 papers were published last year on heparin, there was almost nothing on another mast cell proteoglycan, chondroitin sulphate E. "There is a whole area of attack there," Stevens said. "This one carbohydrate chain [heparin] does this. What about the other chain?"

He provided the example of human lung mast cells in which half of the carbohydrate chains attached to a common peptide sequence are made up of chondroitin sulphate E. "There is a whole area of investigation that will open up" he said.

Kjellin told BioWorld Today, "The fact that the absence of functional heparin resulted in decreased amounts of pro-inflammatory substances like histamine and tissue-degrading proteases, may be possible to exploit for drug developers. If we can find a way to lower the endogenous production of heparin this may result in less aggressive mast cells."

The Swedish researchers are now conducting a similar study with mice that lack an enzyme that is more active in heparan sulfate biosynthesis. "We are presently studying the phenotype of these mice," Kjellin said. "We have also been involved in European Union-supported network aiming at finding new ways of designing and producing heparin/heparan sulfate oligosaccharides of defined structure and function."

The National Institutes of Health supported the work by the Boston researchers. The Swedish research was supported by grants from the Swedish Natural Science Research Council, the Swedish Medical Research Council, the European Commission, private companies and others.