Strategy for Recombinant Multichain Protein Synthesis: Fibrinogen Bβ-Chain Variants as Thrombin Substrates

Publisher Website
Google Scholar
Abstract
Thrombotic disease has been found in patients with congenital dysfibrinogens that have abnormalities in the amino terminal domain of the fibrinogen Bβ-chain. Surprisingly, these fibrinogens are poor substrates for thrombin. In order to examine the molecular basis for this impaired thrombin−fibrinogen interaction, we synthesized three fibrinogens with single amino acid substitutions in this domain: Bβ A68T, Bβ P70S, and Bβ L72S. Bβ-chain expression vectors were altered by PCR-directed mutagenesis of the Bβ cDNA. The altered vectors were transfected into a Chinese hamster ovary (CHO) cell line that was constructed as a first step in recombinant fibrinogen synthesis; this CHO line synthesizes fibrinogen Aα- and γ-chains. More than 86% of the stably selected clones expressed significant levels of fibrinogen, confirming that a two-step strategy permitted efficient synthesis of variant fibrinogens. In large-scale cultures variant fibrinogen accumulation in serum-free medium fluctuated between 1 and 15 μg/mL. Normal and variant recombinant fibrinogens were compared to plasma fibrinogen by following the time course of thrombin-catalyzed release of fibrinopeptides. Only the variant Bβ A68T, a change identified in a congenital dysfibrinogen, showed significantly impaired kinetics. The rate of fibrinopeptide A release was decreased 27-fold, and the rate of fibrinopeptide B release was decreased 45-fold relative to normal fibrinogen. Fibrinopeptide release was not significantly altered by the substitutions Bβ P70S or Bβ L72S. These results suggest that Bβ residue Ala68 has a novel and critical role in the interaction between thrombin and fibrinogen.