Severely Impaired Polymerization of Recombinant Fibrinogen γ-364 Asp → His, the Substitution Discovered in a Heterozygous Individual

Nobuo Okumura,Oleg V Gorkun,Susan T Lord

doi:10.1074/jbc.272.47.29596

Nobuo Okumura, Oleg V Gorkun + Show 1 more

Open Access

https://doi.org/10.1074/jbc.272.47.29596

Copy DOI

Abstract

During blood coagulation, soluble fibrinogen is converted to fibrin monomers that polymerize to form an insoluble clot. Polymerization has been described as a two-step process: the formation of double-stranded protofibrils and the subsequent lateral aggregation of protofibrils into fibers. Previous studies have shown that gamma chain residues Tyr-363 and Asp-364 have a significant role in polymerization, most likely in protofibril formation. To better define the role of these residues, we synthesized three fibrinogens with single substitutions at these two positions: Tyr-363 --> Ala, Asp-364 --> Ala, and Asp-364 --> His. We found that the release of fibrinopeptides A and B was the same for these variants and normal recombinant fibrinogen, showing that all variants had normal fibrin formation. In contrast, we found that polymerization was significantly delayed for both Ala variants and was almost nonexistent for the His variant. Clottability for the Ala variants was only slightly reduced, and fibrin gels were formed. Surprisingly, clottability of the His variant was substantially reduced, and fibrin gels were not formed. Our data suggest that both protofibril formation and lateral aggregation were altered by these substitutions, indicating that the C-terminal domain of the gamma chain has a role in both polymerization steps.

Highlights

Fibrinogen is a plasma glycoprotein composed of a pair of three polypeptide chains, A␣, B␤, and ␥
The association of fibrin monomers into a fibrin clot has long been described as a two-step process, where the first step involves half-staggered, end-to-end interactions leading to doublestranded protofibrils and the second step, usually called lateral aggregation, involves the assembly of protofibrils into thick, multi-stranded fibers that branch to form a fibrin network
We measured turbidity, which with normal fibrin polymerization gauges the rate of protofibril formation from the lag period, and the rate of fiber formation from the slope of the turbidity change

Summary

EXPERIMENTAL PROCEDURES

Materials—The plasmid vectors, Chinese hamster ovary (CHO) cells, and culture medium have been previously described [15]. One cell line that synthesized high and approximately equivalent levels of both A␣ and B␤ chains was selected for further transfection This CHO-A␣1⁄7B␤ line has been described, as it was used to synthesize a ␥ chain variant fibrinogen that lacked the four C-terminal residues [16]. Fibrinogen (90 ␮l at 0.1 or 0.5 mg/ml) in 20 mM HEPES, pH 7.4, 0.12 M NaCl, and 10 mM CaCl2 was mixed with human ␣-thrombin (10 ␮l at 5 or 0.5 unit/ml), and changes in turbidity were monitored at ambient temperature. Fibrinogen Clottability—Clottability of the purified fibrinogens was determined essentially as described [23], mixing human ␣-thrombin (final concentration 0.05 unit/ml) and fibrinogen (final concentration 0.45 mg/ml) in 20 mM HEPES, 0.12 M NaCl, and 10 mM CaCl2. No correction was made for absorbance from the added thrombin

RESULTS

TABLE II Analysis of fibrin polymerization curves initiated by thrombin

Lag period

DISCUSSION