ThrombinJanuary 2002 Molecule of the Month by David Goodsell
doi: 10.2210/rcsb_pdb/mom_2002_1 (PDF Version, ePub Version )
Oxygen and nutrients are delivered throughout our bodies through the watery transport system of the blood. Using a liquid delivery method poses two challenges. First, it leaves the entire body open to infection, since bacteria and viruses will be quickly distributed everywhere that the blood goes. The immune system, with antibodies as the first line of defense, fights this danger. Second, there is the constant danger of damage to the blood circulatory system. Blood is pumped throughout the body under pressure, and any small leak could lead to a rapid emptying of the entire system. Fortunately, the blood carries an emergency repair system: the blood clotting system. When we are cut or wounded, our blood builds a temporary dam to block the damage, giving the surrounding tissues time to build a more permanent repair.
A Molecular Pyramid Scheme
Thrombin is at the center of this process of blood clotting. Blood clotting starts with molecules that sense that something is wrong. For instance, the protein tissue factor is found on the surfaces of cells that are not normally in contact with the blood. If tissue is cut, the blood flows out of the blood vessels and encounter tissue factor. Then, a cascade of signaling starts, beginning with a few tissue factor molecules and amplifying, like a pyramid scheme, into a response large enough to cure the entire problem. Tissue factor activates a few molecules of Factor VII. These then activate a lot of Factor X. And finally, these activate even more thrombin. Thrombin, when activated, then translates this signal into action. It clips a little piece off of the large protein fibrinogen, causing it to assemble into large stringy networks. These networks then trap lots of blood cells, forming the dark red scab that blocks the damage.
Thrombin is a serine protease: a protein-cutting enzyme that uses a serine amino acid to perform the cleavage. Other examples of serine proteases are trypsin and chymotrypsin, enzymes involved in digestion. Thrombin, however, is more specific than these gastrointestinal cleavage machines. It is designed to perform the specific cleavage needed to activate fibrinogen, without digesting all the other important proteins in the blood. The active site is seen here, in the structure of activated thrombin from PDB entry 1ppb, at the base of a deep groove. The oxygen atom of the key serine amino acid is shown in bright red, and the two bright blue nitrogen atoms are part of a histidine that activates the serine. An aspartate, to the left of the histidine and hidden under another amino acid, also helps in the activation.
In the Right Place at the Right Time
Of course, blood clotting must be carefully regulated, otherwise the blood would be clotting in all of the wrong places. Errors in blood clotting have disastrous effects: improper blood clots in the heart can cause heart attacks and misplaced blood clots in the brain cause strokes. Thrombin is controlled in two ways. First, it is built as an inactive precursor, shown here at the bottom (two PDB files are used, 1a0h on the left and 2pf2--the little dotted line shows the piece that is missing). The inactive form has several extra domains, colored light blue here, that are clipped off when the protein is activated. The purple atoms at right-bottom are calcium ions, bound to specially modified glutamate amino acids. The strong positive charge on these ions tether the protein to the surfaces of blood vessels, so that thrombin stays put. Since thrombin is not free to spread, blood clots, once they start, will not spread everywhere. Only the thrombin right next to damage will be activated. Second, once thrombin is activated, as in the upper structure shown here (PDB entry 1ppb), it lasts only seconds, also limiting the clot to area of damage.
Blood clots are not always wanted. For instance, many people take small doses of aspirin, under the direction of their doctors, to reduce the chance of the blood clots that cause heart attacks. Aspirin acts on the protein cyclooxygenase, which is important in another aspect of clot formation that uses small cell fragments called platelets. The rat poison warfarin, not commonly used these days, blocks the formation of the modified glutamate amino acids that hold calcium ions, shown on the last page. The unfortunate rats then die because of uncontrolled blood clotting. Leeches, as you might expect, also detest blood clots, because it means the end to their meal. They make special proteins that block thrombin (or other enzymes), stopping the formation of the clot. One example, a protein called hirudin, is shown here on the left (from PDB entry 2hgt). The leech protein is shown in blue-- notice how it blocks the active site of thrombin perfectly.
Exploring the Structure
PDB entry 1mkx is a perfect structure for exploring thrombin. It contains two molecules of the protein, one in inactive form (chain K) and one activated (chain H and L). The inactive form is shown on the left. In order to activate the protein, the protein strand must be cleaved between the yellow and red segments on the left side. Then, the two new ends separate and the whole protein relaxes into the active form, shown on the right. Notice how, in the active form, the key catalytic serine amino acid, with oxygen atom in bright red, changes position and points straight out into the active site, ready to perform the cleavage.
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Further information on thrombin
Milton T. Stubbs and Wolfram Bode (1993): A Player of Many Parts: the Spotlight Falls on Thrombin¹s Structure. Thrombosis Research 69, pp. 1-58.
Earl W. Davie, Kazuo Fujikawa and Walter Kisiel (1991): The Coagulation Cascade: Initiation, Maintenance and Regulation. Biochemistry 30, pp. 10363- 10370.
© 2013 David Goodsell & RCSB Protein Data Bank