Bikunin in Rat Plasma, Lymph and Bile

Abstract

Bikunin is a protease inhibitor consisting of a 16 kDa polypeptide and an 8 kDa chondroitin sulphate chain which has an apparent molecular mass of 60-70 kDa upon gel filtration. It is synthesized by hepatocytes and occurs in plasma, both in free form, and in complex with other polypeptides--mainly as the 180 kDa protein inter-alpha-inhibitor. Bikunin binds to proteases less avidly than other plasma inhibitors, making its role in the blood unclear. However, some observations indicate that bikunin has important functions outside the blood system. To assess its capacity to reach extravascular spaces, we have determined the total concentration of bikunin in plasma (0.17 mg/ml), lymph (31 micrograms/ml) and bile (0.2 microgram/ml). Quantitation after removal of complexed bikunin (inter-alpha-inhibitor) by acid precipitation showed that the concentration of free bikunin in those fluids was 3, 1.4 and 0.05 micrograms/ml, respectively. These values yield a lymph/plasma ratio of free bikunin of 0.5, which is higher than expected for a protein of the hydrodynamic size and charge of bikunin. The bile/plasma ratio (0.02), however, is similar to that of other proteins of comparable size. The corresponding values for inter-alpha-inhibitor, 0.16 and 0.001, respectively, indicate that its capacity to pass through the vascular endothelium is relatively high whereas transfer to bile is restricted. Furthermore, we have found that in a perfusate of an isolated rat liver, the ratio of free to complexed bikunin was 30-40 times higher than in plasma, consistent with previous observations showing that free bikunin is cleared from the blood stream much more rapidly than inter-alpha-inhibitor.

Urinary Bikunin Determination Provides Insight into Proteinase/Proteinase Inhibitor Imbalance in Patients with Inflammatory Diseases

“…In a perfusate of rat liver, free BK is seen to be the major form present (40). However, in humans BK excretion remains low in urine from healthy subjects.…”

Generation of Catalytically Active Granzyme K from Escherichia coli Inclusion Bodies and Identification of Efficient Granzyme K Inhibitors in Human Plasma

“…Nevertheless the K i value of I␣I for human GzmK is lower than that determined previously for human plasmin (130 nM) and neutrophil elastase (150 nM) (36) and lower than that determined for the inhibition of factor Xa (450 nM) and plasma kallikrein (410 nM) by bikunin D2 (37). When we mixed purified I␣I with bikunin D2 at the same molar ratio as present in 2.5% normal plasma (30,38) and at a total concentration of 26 nM, GzmK activity was inhibited to the same degree (Fig. 5, 5th column).…”

“…The major Kunitz-type inhibitor of human plasma, known as inter-␣-trypsin inhibitor (I␣I) according to its electrophoretic mobility between ␣ 1 and ␣ 2 plasma proteins, however, is a mixture of various complexes between bikunin (which consists of two Kunitz-type inhibitor domains) and H2 or H3 or H1 and H2 of the three structurally related polypeptide chains H1 to H3 (29). I␣I occurs in concentrations between 0.4 and 0.7 mg/ml plasma, whereas free bikunin represents only about 2% of total plasma bikunin (30,31). Here, we report that I␣I is the most important physiological GzmK inhibitor of human plasma and that its inhibitory activity is mediated by the second carboxyl-terminal domain of bikunin.…”

Inter-α-trypsin Inhibitor, a Covalent Protein-Glycosaminoglycan-Protein Complex

“…However, more than 90% of circulating bikunin carries HCs, whereas urinary bikunin is predominantly free of HCs (46). Furthermore, free bikunin has a much shorter half-life in circulation (4 min in mice) than that of HC-bikunin complexes (several hours in mice) (47,48).…”