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Role of Engineered Proteins as Therapeutic Formulations
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Khushboo Gulati, Krishna Mohan Poluri
Blood clotting factors are being engineered to overcome the bleeding disorders such as hemophilia. Recombinant Factor VIII (rFVIII) was given first in March 1987 to the hemophilic patient and after five years it became available for clinical usage (White et al., 1989; Pipe, 2005). Recombinant Factor IX (rFIX) and recombinant factor VIIa (rVIIa) were later developed for the treatment of hemophilia A and B. Few of the clotting factors have been already approved, and some are under clinical trials (Pipe, 2005; Key and Negrier, 2007). Recombinant activated protein C (APC) has been developed and was reported to reduce the mortality in patients suffering from severe sepsis. This recombinant protein also showed high bleeding risk due to its high anticoagulant activity; hence, efforts were made to reduce its anticoagulant activity to overcome bleeding risk. Two variants of APC were designed by site directed mutagenesis. In one variant, the two arginine residues at 229th and 230th position were mutated to alanine and in other variant three adjacent lysines at positions 191, 192, and 193 were mutated to alanine. Both the variants held back their apoptotic activity and evidenced a decrease in anticoagulant activity. These two APC variants can serve as future engineered therapeutics (Mosnier et al., 2004).
Direct Oral Anticoagulants: New Options
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2020
The protein C system regulates coagulation by modulation of the activity of the cofactors FVIIIa and FVa. Protein C is activated by thrombomodulin-bound thrombin on the surface of endothelial cells. The resulting activated protein C (APC), which is supported by protein S, cleaves phospholipid membrane-bound FVa and FVIIIa and so leads to inhibition of coagulation. Consequently, thrombin has the capacity to express both procoagulant and anticoagulant functions.
Purification and characterization of Protein C activator from Agkistrodon acutus Venom
Published in Preparative Biochemistry & Biotechnology, 2020
Yao Sun, Peng-Ju Bao, Gen-Bao Zhang
Protein C (PC) is a vitamin K-dependent protein that circulates in the blood.[1] The protein C zymogen is activated upon binding to thrombin,[2] and its activation is induced by the presence of thrombomodulin (TM) and endothelial protein C receptors (EPCR).[3] PC is hydrolyzed from a peptide chain to become activated protein C (APC). APC plays important roles in the regulation of blood clotting,[4] inflammation,[5] cell death, and blood vessel wall permeability in humans and other animals[6] by proteolytically inactivating factors Va and VIIIa. Because of the crucial role of protein C as an anticoagulant, individuals with deficiencies in protein C or resistance to APC[7] are at increased risk of developing life-threatening blood clots. A decrease in the PC level generally associates with a decrease in PC activity. However, in some cases, the level of PC is normal but the activity of PC has been reduced by half.[8] Therefore, the detection of PC activity is more diagnostically significant for patients with cardiovascular diseases. Presently, the key methods to detect PC activity[9] are the chromogenic assay and APTT test.[10] In a previous study, it was reported that snake venom contains an enzyme that activates plasma PC independent of thrombomodulin–endothelial protein C receptor (TM/EPCR).[11] This protein has been designated as protein C activator (PCA). Other studies have demonstrated that PCA is mainly present in Agkistrodon halys halys snake venom.[12] PCA has a molecular mass of approximately 37 kD[13] and is a valuable commodity named PROTAC,[14] which is isolated from Agkistrodon contortrix contortrix venom (ACCV). However, the use of the chromogenic assay is limited because of the high price of PROTAC.