Fibrinogen Petoskey, a dysfibrinogenemia characterized by replacement of Arg-A alpha 16 by a histidyl residue. Evidence for thrombin-catalyzed hydrolysis at a histidyl residue.

D.L Higgins,J.A Shafer

doi:10.1016/s0021-9258(18)43225-5

Abstract

Inherited abnormal fibrinogens that exhibit altered behavior in one or more of the steps in the conversion of fibrinogen to fibrin should be useful for determining structure-function relationships important in the final stages of blood coagulation. In this work, we show that the inherited dysfibrinogenemia, fibrinogen Petoskey, is due to an Arg --* His replacement at residue 16 in the Aa chain of fibrinogen. This mutation involves the aminoacyl residue that provides the carbonyl portion of the peptide bond cleaved in the thrombin-catalyzed release of fibrinopeptide A from fibrinogen in the conversion of fibrinogen to fibrin. Thrombin treatment of fibrinogen from patients with fibrinogen Petoskey, followed by high performance liquid chromatography, indicated that equal amounts of normal and an abnormal fibrinopeptide A were liberated from the patients’ fibrinogen. This result indicates that the patients studied are heterozygotes with respect to their fibrinogen, which is consistent with the autosomal dominant mode of inheritance observed for their phenotypic prolonged thrombin clotting time. Amino acid analysis and carboxypeptidase Y digestion of the fibrinopeptides established the Arg + His interconversion for fibrinogen Petoskey. Our observation that thrombin catalyzes the hydrolysis of a histidyl peptide bond in the abnormal hrinogen is the first report of a thrombin-catalyzed cleavage of a peptide bond other than at an arginyl or lysyl residue, and suggests that the arginyl residue in fibrinogen can bind to the active site of thrombin with its side chain extended in a conformation similar to that of a histidyl side chain.

Highlights

Inherited abnormal fibrinogens that exhibit altered rates of the thrombin-catalyzed hydrolyses of the Arg-Gly behavior in one or more of the steps in theconversion bonds near the NH2 termini of the A a and BP chains of a of fibrinogen to fibrin should be useful for determining fibrinogen molecule to release 2 molecules of fibrinopeptide A structure-function relationships important in thefinal and 2 molecules of fibrinopeptide B
We show that the inherited dysfibrinogenemia, fibrinogen Petoskey, is due to an Arg --* His replacement at residue 16 in the Aa chain of fibrinogen
We show that an abnormal fibrinopeptide A is released from fibrinogen Petoskey upon treatment with thrombin, and thatfibrinogen Petoskey is the result of an Arg-16 -+ His replacement in the Aa chain of fibrinogen

Summary

FACTOR XlllA

Fibrinopeptides-These were obtained by treating human fibrinogen (1.5to 3.0 mg/ml in 0.2 M NH4HCOs,pH 7.8) with approximately 7 units/ml of thrombin. Less concentrated solutions of fibrinopeptides (-9 p ~ w)hich were stable for several days at 4 "C wereprepared by clotting fibrinogen (-1.5 mg/ml) in 0.3 M NaC1, 0.025 M sodium phosphate, pH 6.4, with either human thrombin or bovine thrombin (from Sigma, 2000 units/mg). The relationship between the area under the peak of fibrinopeptide A and fibrinopeptide B was obtained by subjecting an aliquot of a solution containing the pure fibrinopeptide to amino acid analysis (for determination of molar concentration) and subjecting another aliquot of the solution to HPLC (fordetermination of peak area). Digestion of Fibrinopeptides with Carboxypeptidase Y-A fraction from the HPLC column containing a known amount of fibrinopeptide A, as judged by amino acid analysis of a sample of the fraction, was adjusted to pH 6.1 by slow addition (with stirring) of 1 M NaOH. Losses of material during this procedure, were corrected for using the recovery (>50%)of a standard solution containing 5 p~ histidine and 5 p~ valine which wassubjected to the HPLC procedure and all subsequent steps

RESULTS

Fibrinopeptide A Petoskey

Glycine Alanine Valine Leucine

DISCUSSION