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Aprotinin

Created by Morgan Savoia

   Isolated from bovine pancreas, aprotinin (PDB ID: 3LDI) is a hydrolase inhibitor protein. Aprotinin is responsible for the inhibition of serine proteases, specifically those in the blood clotting enzyme cascade (kallikrein, XIIa, XIa, IXa, VIIa, Xa, and thrombin)(2). The blood clotting cascade consists of two pathways, intrinsic and extrinsic(2). Aprotinin and its action on kallikrein leads to inhibition of the formation of factor Xlla(4). This inhibition stops the intrinisic pathway so that Factor Xa cannot be made and the final pathway of coagulation, which is the activation of thrombin and the conversion of fibrinogen to fibrin(2), is hindered. In this way, aprotinin hinders the intrinsic pathway(1), which is "instigated when blood comes into physical contact with abnormal surfaces caused by injury"(2). Because of the anticoagulation properties, aprotinin has been used as an antifibrinolytic medicine (Trasylol, sold by Bayer), most appropriate for use during cardiopulmonary bypass (CPB)(1). Recently, aprotinin was taken off the market due to its side affects such as renal failure(1).

   Aprotinin belongs to a family of proteins called "bovine pancreatic trypsin inhibitor/kunitz-type inhibitor superfamily"(1). This family, and specifically aprotinin, inhibits serine proteases such as "plasmin, tissue plasminogen activator, kallikrien and thrombin"(1). The molecular weight is 6517.54 and the isoelectric point is 9.24 according to ExPASy. Herapin, which is a natural anticoagulant(2), binds strongly to aprotinin. The binding of aprotinin to the herapin molecule prevents herapin from binding to platelets. The exact mechanism by which aprotinin acts as an aggregation inhibitor is still in question(1).

   A mutant protein, G37A (PDB ID: 1JV8) has a similar primary structure to aprotinin according to BLAST. The E score found by BLAST is 9e-26. The mutant replaces a Gly residue with an Ala residue in the active site loop of the bovine pancreatic trypsin inhibitor. This one change in sequence causes a change in the stability of the aprotinin by about 5 kcal/mol of destabilization(4). The destabilization is accounted for in the "strain in the backbone configuration of residues 36 and 37 of G37A"(4). Replacing the Gly residue with an Ala residue at residue 37 causes increased flexibility of the protein(4). This observation indicates that residue 37, and its contribution to the "NH-aromatic-NH"(4) structure of the backbone of aprotinin is critical in the stabilization and function of aprotinin.

    Textilinin-1 (PDB ID: 3BYB) has similar tertiary structure to aprotinin according to the Dali Server(5)(red is textilinin and blue is aprotinin). The Z score is 12.2 and the root mean square deviation is 1.0. Textilinin-1 is from the venom of the common brown snake from Australia(3). Similar to aprotinin, textilinin-1 is a Kunitz-type serine protease inhibitor. The sequence of aprotinin and textilinin-1 is 45% similar(3). The most significant difference in sequence between the two proteins is the surface residues. In multiple cases aprotinin has more positively charged residues where textilinin-1 is either negatively charged or is missing a particular residue(3). The affinity of aprotinin to bind plasmin is higher than that of textilinin-1, making for a more efficient anticoagulant(3). Although textilinin-1 has a lower affinity for binding plasmin than aprotinin, this may be better for medical purposes due to "faster clearance of the drug"(3). Because of the similarities in function, textilinin-1 has been thought to be able to replace aprotinin (Trasylol) for use as an antifibrinolysis in cardiopulmonary bypass surgery(3).
   The primary structure of aprotinin consists of 58 amino acid residues per subunit(8). Aprotinin has three ligands, sulfate ion (red), mercury ion(green) and glyercol(blue). Residues Ala-16 to Gly-36 comprise the right-hand twisted double-stranded antiparallel beta sheet. Both the N-terminal and the C-terminal end's secondary structure of aprotinin are alpha helices (8). The residues from Pro-2 to Glu-7 form the N-terminus end of the protein and Ser-47 to Gly-56 form the C-terminus end(8). Arg-20 and Glu-49 form a salt bridge(1). There are twelve total hydrogen bonds, but important sites include the hydrogen bond between Ser-47 and Glu-49 with the amide nitrogen at the start of the helix and the two hydrogen bonds between the carbonyl oxygen of Asn-24 to the amide nitrogens of Ala-27 and Gly-287. Three disulfide bridges that occur at Cys30-Cys51, Cys5-Cys55 and Cys14-Cys-38 stabilize the secondary structure of aprotinin(7). A "specificity pocket"(8) is formed by the disulfide bond between Cys14-Cys38. The particular function of aprotinin, which is to inhibit the action of various serine proteases, takes place in this "specificity pocket"(8). Lys-15 is the active center of the aprotinin molecule. It is the residue that interacts with the active site of a serine protease, such as trypsin, to form an aprotinin-serine protease complex (4). Formation of this complex is the action that inhibits the serine protease(4). Inside the loop Arg-17 and Arg-39 are found. These residues are responsible for the strong inhibition properties of aprotinin due to their basicity(8).

   Aprotinin is a globular protein. The hydrophobic core consists of Phe-4, Cys5-Cys55, Phe22, Ty-r23, Cys-30-51, Phe-33, Tyr-35, and Phe-45(7). The compactness of the protein due to the burying of these hydrophobic residues, "results in a very compact tertiary structure and is mainly responsible for the remarkable stability of aprotinin against denaturation by high temperature, acids, alkalies and organic solvents or proteolytic degradation"(4). Five subunits form the aprotinin structure. Each subunit forms a pentamer that in turn oligomerizes to form a decamer. This structure creates a channel like center where serine proteases are inhibited by aprotinin(1). Along this channel, sulfate ions appear to make electrostatic interactions with the positively charged residues of Arg-20 and Lys-46(1). Aprotinin's ligand, SO4, is in part, also responsible for the stability of the pentameric and decameric structure of aprotinin(1).

   With increasing salt concentration aprotinin oligomerizes to a decamer(6). It is thought that the decamer is composed of two pentamers that are stacked on top of one another. This hamburger-like structure forms a basic channel, the "specificity pocket", that is thought to bind to herapin and other serine proteases(1). Specific residues that are thought to be important for the formation of oligomers include, Arg-42, Lys-46, and Ala-16(3).
   Aprotinin's structure allows for its function as a serine protease, and has been utilized as a drug to prevent blood clotting. The specific structure of aprotinin has proven to not be ideal for its use in medicine. Its affinity for binding serine proteases is too strong due to the specificity pocket of Pro-13, Cys-14, Lys-15, Ala-16, Arg-17, and Ile-18(3). The strong reaction of aprotinin with serine proteases in this specificity pocket "is due to the basic character of the residues"(3). Therefore the medicine that utilizes aprotinin (Traysol made by Bayer) does prevent blood clotting but for too long which leads to "increased risks of renal failure, myocardial infarction and stroke"(1).