By Dean A. Haycock

Special To BioWorld Today

It is not known as "coagulable lymph" anymore. That is what William Hewson, a British anatomist and physiologist, called the serum protein he isolated back in the eighteenth century. Today it is known as fibrinogen, an essential component of the blood clotting process.

The clots it helps form preserve life by limiting blood loss from damaged tissues. It also takes life when it forms unwanted clots that block blood flow to the heart or brain. For these reasons, understanding the molecular basis of clot formation is of particular interest to many in the biotechnology industry.

We know when damaged tissue bleeds, a complex series of biochemical steps are initiated. These culminate in the clotting enzyme thrombin converting soluble fibrinogen into insoluble fibrin. Fibrin forms long, tough threads that trap platelets, forming a mass that hardens into a clot.

In rare hereditary disorders, the failure of blood to clot may be traced to the inability of fibrinogen to form fibrin.

Protein Crystal Had To Be Made

To understand the clotting process on the molecular level, it is necessary to understand how fibrinogen is converted into fibrin. But to understand protein interactions on the molecular level, a protein must first be crystallized. Only the atomic regularity seen in a crystal allows scientists to determine a protein's structure to a high degree of resolution.

In the case of fibrinogen, getting a decent crystal is not easy. Scientists have been trying to get useful crystals of it for at least the last quarter century.

"Fibrinogen is big and floppy. Also it tends to be fragile. If you look at it the wrong way, it wants to precipitate instead of crystallize," said Russell Doolittle, professor at the Center for Molecular Genetics at the University of California, San Diego (UCSD).

Under the electron microscope, fibrinogen looks like a rod approximately 47.5 nanometers long. On the rod, two terminals and a central nodule can be seen.

The molecule has been divided into two core structures: fragment E and fragment D. The complete molecule consists of one fragment E with a fragment D at both ends. The latter account for one half of the molecule's mass.

Recently Doolittle and two colleagues at UCSD, postdoctoral fellows Glen Spraggon and Stephen Everse, succeeded in determining the crystal structure of fragment D. In their paper, "Crystal structures of fragment D from human fibrinogen and its cross linked counterpart from fibrin," in the Oct. 2 Nature, they describe its structure to a resolution of 2.9 angstroms.

In the same study, they describe, to the same resolution, the structure of the covalently bound dimer of fragment D, called "double-D."

These detailed descriptions will allow scientists to understand the molecular basis of blood clotting on a new level and perhaps shed light on pathological processes seen in cardiovascular disease and certain hereditary disorders.

It took the group four years to obtain a suitable crystal of fragment D from fibrinogen. Their sample came from human blood plasma obtained from a San Diego blood bank.

Using x-ray equipment at UCSD, Everse collected much of the early data in an attempt to build a detailed 3-D map of the crystal. Unfortunately, the double D crystal would not yield all its structural secrets under the x-rays' wavelength available to the researchers on campus.

"This is quite a big structure and it had a lot of difficult features associated with it. The quality of the crystal is a big factor. When we shined ordinary x-rays through, they just weren't sufficient. We just didn't have the kind of crystal that could be managed by the x-rays we had available," Doolittle explained.

The necessary data were obtained only after Everse and Spraggon, an Oxford University-trained crystallographer, gained access to a facility equipped with a subatomic particle accelerator that could provide x-rays with wavelengths not available locally. The x-rays the group needed were available at a synchrotron facility in the U.K. where William Hewson first described his "coagulable lymph." So Spraggon and Everse flew with the crystal across the Atlantic and subjected it to the tunable x-rays available in Daresbury, U.K.

Spraggon then applied an innovative averaging approach to the data obtained during the day-long analysis in England. The result was a clearer picture of the molecular structure that allowed the team to build their successful model of the fragment D structure.

Complete Picture Still To Be Developed

Future work will be directed toward determining the structure of the entire fibrinogen molecule.

"Everybody is still trying to make crystals. With these data as a starter, this will definitely help other people who have other (crystal) derivatives in hand," Doolittle said.

Already, the California researchers have sent the molecular coordinates they have determined to another group that has a low resolution structure of fibrinogen.

"If they are lucky, they will be able to use our information to go ahead and solve that structure which will be much bigger. Then, beyond that, everybody will want to try to get pieces of bigger complexes of fibrin welded together. So there is a lot more to be exploited here," Doolittle said. *

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