Brief Overview of Thrombin

Biological Function(s) of Thrombin: α-Thrombin is a trypsin-like serine protease involved in a multitude of processes in the human body.  Thrombin generation is the result of limited proteolysis of the vitamin K-dependent zymogen prothrombin.  Thrombin is the last enzyme in the clotting cascade functioning to cleave fibrinogen to fibrin which forms the fibrin gel of a hemostatic plug or a pathologic thrombus.  In addition, thrombin potentiates the procoagulant process by activating Factors V, VIII, XI and XIII.  Conversely, following its binding to thrombomodulin, thrombin aids the anticoagulant process by activation of protein C.  Activated protein C inhibits activated Factors V and VIII ultimately down regulating further thrombin production.  Thrombin is also involved with other activities including inflammation and wound healing.  In response to injury, thrombin is chemotactic for monocytes, fibroblasts and smooth muscle cells.  Thrombin can activate neutrophils and platelets which release a myriad of mediators including cytokines, chemotactic factors and growth factors, all of which influence inflammation and lead to resolution of an injury.  Thrombin has been shown to be mitogenic for chick embryo fibroblasts and smooth muscle cells.  In addition, thrombin has been shown to stimulate angiogenesis in a chick chorioallantoic membrane system.   Thrombin activates platelets through a tethered ligand receptor identified by Vu et al. in 1991.  Efficient cleavage of the receptor is dependent on binding of the amino terminal region, which has an acidic stretch of residues, to anion-binding exosite-1 (ABE-1) of thrombin.  This domain is very similar to the sequences of the amino terminal acidic domain of HCII and the carboxyl terminal region of hirudin.  The sequence LDPR/S, further upstream from the acidic region of this receptor, can then be cleaved by thrombin and the new amino terminus functions as the receptor’s ligand.  Activation of this receptor leads to an increase in cytosolic calcium and secretion from granules.  Much of our work is focused on better understanding the biological function of thrombin, dissecting out its structure-activity relationships, and using thrombin as a paradigm for protease action in both health and disease.

Structure of Thrombin:  Thrombin is the product of activation of the zymogen prothrombin.  Prothrombin is a vitamin K-dependent protein produced in the liver.  Vitamin K is necessary for proper synthesis of the prothrombin structure, which contains Gla residues (γ-carboxylated glutamate residues), during post-translational modification in the endoplasmic reticulum of the hepatocyte.  The Gla residues allow prothrombin to bind calcium and adhere to phospholipid surfaces.  Surface binding ability is necessary for prothrombin’s activation by factor Xa and factor Va which are also localized to phospholipid surfaces in what is known as the prothrombinase complex.

In 1989, Bode et al. published the X-ray structure of thrombin.  The same group reported a refined crystal in 1992.  Because of sequence homology, the numbering of thrombin is based on the sequence of chymotrypsinogen.  The thrombin molecule contains two chains.  The A chain is composed of 36 residues and is non-essential for proteolytic activities.  The B chain is composed of 259 amino acids and is derived from the carboxyl terminal sequence of prothrombin.  The B chain contains the three active site amino acids, His57, Asp102, and Ser195. Thrombin is a serine protease that possesses trypsin-like behavior in that it prefers to cleave its substrates after arginine residues.  Upon recognition, the hydroxyl group of Ser195 of thrombin’s active site attacks the reactive site P1 residue of the substrate.  Thrombin consists of two disulfide-linked polypeptide chains that are folded into a trypsin-like protease.  Like trypsin, the thrombin B chain contains the active site residues Ser, His, and Asp, but also contains insertion loops that extend around the active site cleft, making it deeper and more narrow than that of trypsin.  The  “60-insertion loop” to the “north” of the active site cleft forms a hydrophobic lid to the S2 subsite (the S1, S2, etc. subsites of the protease recognize the P1, P2, etc. residues of the substrate), which restricts the P2 site in substrates and inhibitors to mostly small hydrophobic residues, such as Val and Pro.  At the base of the S1 subsite is an Asp, which explains thrombin’s preference for Arg at the P1 position in substrates. 

Thrombin, however, is unlike trypsin in that it has extra surface structures that influence the interactions with macromolecular substrates and thus make it a more discriminating protease.  One of these subsites is called the anion-binding exosite-1 (ABE-1).  The ABE-1 is made up of residues Arg67 through Glu80 and also includes Arg35, Lys149e, Lys81, Lys110, Lys109, and Lys36.  ABE-1 of thrombin has been implicated in binding of fibrinogen, the leech anticoagulant hirudin, and heparin cofactor II.  Another surface structure of thrombin is called the anion-binding exosite-2 (ABE-2).  ABE-2 is primarily responsible for thrombin’s glycosaminoglycan binding abilities.  Another surface subsite is made up of the insertion from Leu144 to Gly150.  This region is reported to interact with hirudin and thrombomodulin.
   
In most serine proteases, the residues homologous to amino acid 192 of thrombin are usually a glutamine residue.  In thrombin this position is occupied by glutamate.  Based on Bode’s crystal structure, Glu192 protrudes into the active site region of thrombin.  This residue has been implicated in the binding of hirudin.  A mutation from Glu192→Gln has been shown to make thrombin a more effective activator of protein C in the absence of thrombomodulin.  The same mutation leads to increased inhibitory activity by the serine protease inhibitor α1-antitrypsin.  These studies have led researchers to conclude that amino acid 192 of thrombin is important for determining the specificity of the P3/P3′ residues of the substrate or inhibitor.

Yet another surface loop is called the 60-insertion loop.  The 60-insertion loop is an 11 amino acid insertion that starts with the very unique Tyr-Pro-Pro-Trp sequence and forms a lid-like structure over the catalytic triad.  This structure creates most of one side of a “canyon” leading to the active site and has been implicated as part of the extended binding groove that generates specificity toward macromolecular substrates and inhibitors.  This region is believed to participate in controlling access of certain substrates and inhibitors to the active site of thrombin, such as fibrinogen, protein C, antithrombin, and hirudin.  The 60-insertion loop has been proposed to be the region responsible for chemotactic activity towards monocytes.  In the past, the 60-insertion loop was thought of as a rigid structure.  However, it has been suggested that this region may be more flexible or subject to conformational changes following binding to alternate portions of the thrombin molecule such as ABE-1.  It has been shown that binding of thrombomodulin to ABE-1 of thrombin displaces residues near the active site, in particular residue Trp60d, which makes this region more accessible to substrates not recognized by thrombin in vivo.