Abstract
The conversion of the blood coagulation zymogen prothrombin to thrombin is associated with the production of several cleavage intermediates and products. In contrast to earlier studies of prothrombin cleavage in chemically defined systems, the current investigation examines the fragmentation of human prothrombin in normal plasma. Radiolabeled prothrombin was added to platelet-poor relipidated normal human plasma, and clotting was initiated with the addition of Ca(II) and kaolin. Analysis of the radiolabeled prothrombin cleavage products by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate and beta-mercaptoethanol identified a heretofore unobserved product of prothrombin activation with an apparent molecular weight of 45,000. This product was identified as fragment 1 X 2 X 3, the NH2-terminal 286 amino acids of prothrombin. The product was isolated from a prothrombin digest by immunoaffinity chromatography using anti-prothrombin:Ca(II) antibodies and by preparative gel electrophoresis. Its amino-terminal sequence is identical to that of prothrombin. Digestion of this product with either Factor Xa or thrombin yields, at a minimum, fragment 1 X 2 and fragment 1. Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2, and fragment 3. To unambiguously identify the COOH-terminal amino acid sequence of the product, its factor Xa digestion products were separated by reverse-phase high performance liquid chromatography. Edman degradation of one peptide revealed the complete sequence of fragment 3. On this basis, we identify the Mr 45,000 polypeptide as fragment 1 X 2 X 3 and indicate that it is a prominent product of prothrombin conversion to thrombin when activation occurs in plasma.
Highlights
The conversion of the blood coagulation zymogen leading to in uitro activation of both bovine andhuman prothrombin to thrombin is associated with the pro- prothrombin have been well characterized [3,4,5,6,7]
Amino-terminal sequence analysis of the products obtained by digestion with Factor Xa of the unknown activation product indicated 3 amino acid residues at each cycle consistent with the presence of fragment 1, fragment 2,and fragment3.To unambiguously identifythe COOH-terminal amino acid sequence of the product,its factorXa digestion products were separated by reverse-phasehighperformance liquid chromatography
O n this basis, we identify the M, 45,000 polypeptide as fragment 1-2-3and indicate that it isa prominent product of prothrombin conversion to thrombin when activation occurs in plasma
Summary
Prethrombin 1 and fragment 1 were obtained by digestion of 0.05 M Tris-HC1, pH 7.5, 0.15 M NaC1, 16 mM EDTA, 1.2 X 10" M human prothrombin by bovine thrombin and separated according to DAPA. Prethrombin 2 was prepared by digestion of human prethrom- and a resolving gel (1.5 X 6 cm) with an acrylamide concentration of bin 1 with bovine Factor Xa [15]. Reaction mixture was thoroughly mixed.At the specified time the Peptide fragmentsof peak B were prepared for automated sequence reaction was quenched by addition of sodium dodecyl sulfate polyac- analysis by digestion of the polypeptide by human Factor Xa. Peak rylamide gel incubation buffer (3.5% SDS, 10% fl-mercaptoethanol, B and Factor Xa (1000:1, w/w) were incubated in 0.05 M NH4HC03. Phenylhydantoins of the hydrolyzed and isolated amino acids were identified by HPLC
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