Structural Examination of the Influence of Phosphorylation on the Binding of Fibrinopeptide A to Bovine Thrombin

Abstract

Upon addition of thrombin, fibrinopeptides A and B are cleaved off from the N-termini of four chains of fibrinogen (Aalpha Bbeta gamma)2, and sites of polymerization are exposed, resulting in formation of a fibrin clot. For the fibrinogen Aalpha chain, cleavage occurs most prevalently at the Arg16-Gly17 peptide bond. About 25-30% of the human fibrinogen Aalpha chains are phosphorylated in nature at the position of Ser3, but the function for this modification is not understood. Previous NMR studies indicated that the N-terminal portion (1ADSGE5) of unphosphorylated fibrinopeptide A does not interact with the surface of bovine thrombin. Kinetic and NMR studies have now been carried out to assess whether phosphorylation at Ser3 allows the N-terminal segment (1ADSGEGDFLAEGGGVR16) to become anchored on the thrombin surface, leading to formation of a catalytically more efficient enzyme-substrate complex. Kinetic results indicate that phosphorylation leads to an approximately 65% increase in substrate specificity (kcat/Km) toward hydrolysis of fibrinogen Aalpha(1-20). 31P NMR studies reveal that the phosphorylated group does interact with thrombin, and 1H line broadening studies suggest that phosphorylation does promote binding of amino acids 1-5. Two-dimensional transferred nuclear Overhauser effect spectroscopy studies of bound fibrinopeptide A(1-16 Ser3P) indicate that phosphorylation allows new through-space interactions involving amino acid residues 1ADSGE5 to be observed. Computational docking of the peptide onto the X-ray structure of thrombin suggests that the phosphate may interact with basic residues at the rim of the heparin binding site of thrombin. As a result, the phosphate may serve as an anionic linker between the fibrinopeptide and the enzyme thrombin.

Examining Thrombin Hydrolysis of the Factor XIII Activation Peptide Segment Leads to a Proposal for Explaining the Cardioprotective Effects Observed with the Factor XIII V34L Mutation

“…HPLC grade acetonitrile, trifluoroacetic acid, Trizma (Tris base), NaCl, PEG 8000, and phosphoric acid were purchased from Fisher and Sigma. Thrombin Preparation-Bovine thrombin was isolated and purified by methods similar to those of Ni et al (12) and Maurer et al (26). Briefly, bovine plasma barium citrate eluate containing prothrombin was activated with E. carinatus snake venom.…”

“…Kinetics-Experimental kinetic methods similar to those of Maurer et al (26) were employed. Unique features of the experimental design are summarized.…”

Three-dimensional Modeling of Thrombin-Fibrinogen Interaction

“…Analysis of the 31 P onedimensional NMR spectral changes caused by the synthetic phosphorylated peptide A␣-(20 -41) binding to thrombin suggests that this group contributed to binding (26 (28). A three-dimensional model of thrombin complexed with the A␣ chain carrying the F27Y mutation shows that the hydroxyl group of A␣-Tyr 27 H-bonds to the nitrogen atom of A␣-Val 34 , fixing the aromatic ring of A␣-Tyr 27 into a distorted position that pulls thrombin Trp 215 1.4 Å higher and out of the aryl binding site (Fig.…”

Bioactive Casein Phosphopeptides in Dairy Products as Nutraceuticals for Functional Foods

“…A similar set of phosphorylated peptides was previously obtained using a modified IMAC strategy coupled to iterative mass spectrometry-based scanning techniques [95], using the titanium ion-immobilized mesoporous silica particles and MALDI-TOF [96] and cerium ion-chelated magnetic silica microspheres [97]. (Table 4), is the segment anchored on the thrombin surface [98]. The other three phosphopeptides, (f1-15)1P, (f2-15)1P and (f2-16)1P (Table 4), are hydrolytic products of FPA.…”