Created by: Javier Mery

          Blood coagulation factor VIIa light chain (pdb ID: 1DAN) from Homo sapiens is one of two chains that compose the blood coagulation factor VIIa. Coagulation factor VIIa heavy chain is the other chain. Blood coagulation factor VIIa is a trypsin-like serine protease that cleaves peptides bond on the carbonyl side of lysine and arginine residues. Vascular injury exposes blood coagulation factor VII to tissue factor expressing cells and forms a complex with tissue factor. Because blood transports nutrients and oxygen, protects cells from infection, and trasnports messengers to different locations in the body, disruption of the blood vessel endothelium would form a leak and disrupt the circulatory system. Therefore, trypsin-like serine protease enzymes, like the complex of tissue factor and coagulation factor VIIa, are necessary in propagating the coagulation cascade and resulting in fibrin deposition and clot formation. Coagulation factor VIIa circulates in blood plasma at a concentration of 10 nM with a half-life of 90 minutes (1). Blood coagulation factor VIIa is thus implicated in bleeding episodes of patients with haemophilia A and B with inhibitors, congenital FVIIa deficiency, acquired hemophilia, and Glanzmann's thrombasthenia (2). Because of these associations, blood coagulation factor VIIa light chain is an important drug target. 

          A complex of tissue factor and coagulation factor VIIa was crystalized using microseeding techniques. For this, the coagulation factor VIIa was mixed with a mixtre of N- and C-terminal soluble tissue factor fragments at a 1:1 molar ratio and incubated for 5 min at 37?C. The complex was concentrated to 10 mg/mL in Microcon micro concentrators with a molecular weight cut-off of 30000. A volume of 3 µL of the complex was mixed with 3 µL of 12% polyethylene glycol 8000, 100 mM cacodylate at pH 5.0 and the drops were sealed over a reservoir. After 24 hours the drops were seeded with microcrystals, which were prepared by crushing the initially obtained crystals (3). 

          The activity of blood coagulation factor VIIa light chain is dependent on its structure. Blood coagulation factor VIIa has three domains: the N-terminal γ-carboxyglutamate-rich (Gla) domain, composed of residues 1 through 35, epidermal growth factor-like 1 domain, composed of residues 49-84, and epidermal growth factor-like 2 domain, composed of residues 91 through 142 (4). The Gla domain is composed of two alpha helices and one random coil. The Gla domain contains 10 vitamin K-dependent, posttranslational modified γ-carboxyglutamate residues and coordinates with seven Ca+2 ions to induce a 'W-like' loop that exposes the hydrophobic residues, Phe-4, Leu-5, and Leu-8. The hydrophobic residues protrude toward the cell membrane (5). Blood coagulation factor VIIa binds to the phospholipid membrane through coagulation factor VIIa light chain's Gla domain. The metal ions induce conformational rearrangements to increase the affinity with cell membrane surfaces. The epidermal growth factor-like 1 domain contains one anti-parallel sheet. The epidermal growth factor-like 1 domain binds to a single Ca²⁺ ion, which increases blood coagulation factor VIIa amidolytic activity and tissue factor association (1). The epidermal growth factor-like 1 domain interacts with the tissue factor through Lys-62, Gln-64, Ile-69, Phe-71, and Arg-79. These four amino acids are important binding contacts at the interface of tissue factor. The epidermal growth factor-like 2 domain has two anti-parallel beta sheets in close proximity. The epidermal growth factor-like 2 domain on the light chain and the catalytic domain on the heavy chain form a coherent structural unit that interacts with the tissue factor. Blood coagulation factor VIIa light chain has three instances of random coil. Overall, the secondary structure of coagulation factor VIIa light chain has multiple alpha helices, beta sheets, and random coils. 

          When a vascular injury occurs, the blood coagulation factor VIIa, composed of both heavy and light chains, complexes with tissue factor. The heavy chain is composed of multiple  antiparallel beta sheets, three alpha helices, and multiple random coils. The heavy chain contains the trypsin-like protease domain with a catalytic triad comprised of His-193, Asp-242, and Ser-344. Blood coagulation factor VIIa heavy chain binds to a single Ca2+ ion(6). The formation of the tissue factor and blood coagulation factor VIIa complex increases the enzymatic activity of coagulation factor VIIa. The active enzyme can hydrolyze small, tripeptidyl-amide substrates. Coagulation factor VIIa's amidolytic activity increases 50 fold upon binding of tissue factor (7). 

          The activation of blood coagulation factor VIIa results in the proteolytic cleavage of the Arg-152 residue from the light chain and Ile-153 from the heavy chain. The two independent chains then form a single disulfide bond at Cys-135 on the light chain and Cys-262 on the heavy chain (8). In addition, the new N-terminus at Ile-153 on the heavy chain can fold back and insert into the activation pocket. Ile-153 forms a salt bridge with the carboxylate of Asp-343 to generate a oxyanion hole. There is a partial helix in the heavy chain from residue 307-312. The tissue factor induces stabilization on the helix by causing the formation of a hydrogen bond between the amide of Arg-315 and the carbonyl of Gly-372 (4). This further stabilizes the active site. The complex is now activated and can catalyze the conversion of factor IX (pdb ID: 2WPI) and factor X (pdb ID: 1EZQ) into their active form. This leads to the formation of a fibrin clot. 

          Previous experiments have shown that alanine replacements of Lys-62, Gln-62, Ile-69, Phe-71, and Arg-79  in factor VIIa light chain compromised the binding of tissue factor. Mutations in the protease domain on the heavy chain reduced the proteolytic function without affecting tissue factor binding. In addition, loop 236-239 and basic residues flank the catalytic triad. Alanine replacement of Thr-239 and Asn-240 demonstrated that Thr-239 contributes to factor X activation and Asn-240 provides a conformation effect on Thr-239, Lys-341, and Arg-290 producing substrate specificity and are critical for proteolytic function of coagulation factor VIIa (9). 

         The catalytic triad is composed of His-193, Asp-242, and Ser-344 and cleaves peptides through an acylation phase and deacylation phase. The substrate enters the binding pocket of coagulation factor VIIa and triggers the formation of a hydrogen bond between Asp-242 and His-193 that orientates His-193 toward Ser-344. Afterwards, His-193 removes a proton from Ser-344, increasing Ser-344 nucleophilicity. Ser-344 attacks the carbonyl carbon of an arginine or lysine residue on factor X, which eventually cleaves the peptide (9). The hydrolysis of factor X advances blood coagulation. 

          Blood coagulation factor VIIa's role in blood coagulation has resulted in the development of recombinant activated factor VII in the treatment of bleeding episodes and prevention of bleeding during surgical procedures. Recombinant activated factor VII (NovoSeven®) was developed for the management of bleeding episodes in haemophilia A and B patients with inhibitors to factor VIII (pdb ID: 2R7E)  or factor IX. Recombinant activated factor VII binds to tissue factor and initiates coagulation, leading to the generation of thrombin (pdb ID: 3U69). The localized action of recombinant activated factor VII results in control of bleeding episodes without inducing a hypercoagulable state in blood from patients with haemophilia (10). Recombinant activated factor VII was first approved for use in Europe in 1966 (2). 

          Blood coagulation factor VIIa light chain is a crucial subunit of blood coagulation facotor VIIa. Coagulation factor VIIa is a member of the serine protease class with a molecular weight of 67,792 Da. The isoelectric point of blood coagulation factor VIIa is between 4.8 and 5.1 (11). Blood coagulation factor Xa is another serine protease with 33% primary structure similarity as shown by protein BLAST (E=2e^-59) (12). Protein-Blast provides a way of spotting distant relationships between proteins by analyzing protein query sequences and protein structure; a E score below 0.5 indicates high similarity. In addition, coagulation factor Xa has a similar tertiary structure as blood coagulation factor VIIa, as shown by the Dali server (Z=38.0) (13). The Dali server provides insight into the protein's function by comparing three-dimensional protein structures using a sum of pairs method; a Z score above 2 means the proteins have similar folds. Factor Xa is a key enzyme in the coagulation cascade and it converts prothrombin (pdb ID: 4NZQ) to thrombin by cleaving the Arg-271 bond and Arg-320 bond in prothrombin (14). Factor Xa is also activated by blood coagulation factor VIIa and factor XI. Coagulation factor Xa is composed of a light chain and a heavy chain. The light chain of factor Xa has two anti-parallel beta sheets. In addition, the light chain of factor Xa has no alpha helices, where as the light chain of coagulation factor VIIa has multiple alpha helices. Both of the heavy chains of factor Xa and factor VIIa have multiple beta sheets, alpha helices, and random coils. Both enzymes have a Gla domain in their light chain. The catalytic triad is lcoated on the heavy chain of coagulation factor Xa (15). The main difference in structure lies in the light chain of factor Xa. Compared to factor VIIa, the Gla domain of the light chain of factor Xa is 46 residues shorter. The secondary structures of factor Xa allow it to bind to factor Va (pdb ID: 1C7S) and the plasma membrane of an endothelial cell and cleave prothrombin to generate thrombin (16).