Abstract
Cleavage of fibrinopeptide A is the first step in fibrin clot formation, and mutations at the fibrinopeptide A cleavage site are the most common cause of dysfibrinogenemia. We describe here the effect on clot structure of a mutant Aα R16C fibrinogen with defective fibrinopeptide A cleavage (designated fibrinogen Hershey III). The propositus, a young child with mild bleeding symptoms, was found to be heterozygous for the Aα R16C mutation. Fibrinogen was purified from Hershey III and control plasma via glycine precipitation. Hershey III fibrinogen was only 63 ± 10% clottable with thrombin (mean ± SEM), as compared to 96 ± 0.4% for normal fibrinogen. Since the propositus was heterozygous for the mutation, the unclottable portion likely consisted of mutant homodimers, but it was still possible that normal/mutant heterodimers existed. Because the cysteine in the mutant fibrinogen prevents thrombin-mediated fibrinopeptide A cleavage, we hypothesized that incorporation of uncleaved fibrinopeptide A, if present, would affect clot structure. Western blotting was used to evaluate the presence of fibrinopeptide A in clottable and unclottable fibrinogen. For fibrinogen Hershey III, both forms showed a substantial amount of fibrinopeptide A, suggesting that mutant fibrinogen was incorporated into the final clot. No fibrinopeptide A was seen in either the clottable or unclottable fibrinogen from the normal control. Next, fibrin clots were made with thrombin, critical-point dried, and visualized via scanning electron microscopy. Visco-elastic measurements were obtained with a torsion pendulum and clot permeability was compared to that of clots formed with normal fibrinogen. The relative proportions of normal vs. mutant fibrinogen in the clottable and unclottable fibrinogen were assessed by protein sequencing. Scanning electron microscopy showed that the Hershey III clots displayed abnormal architecture with many short fibrin fibrils, consistent with premature fibril termination. Hershey III clots also had thicker fibers, with an average fiber diameter of 182 nm compared to 151 nm for the normal control. A significant difference in clot stiffness (G′), energy dissipated by viscous processes (G″), and permeability (Ks) was seen when fibrinogen Hershey III was compared to a normal control (see table). Protein sequencing of the unclottable Hershey III fibrinogen showed only the homozygous mutant form, while the fibrin clot showed approximately 50% each of the wild-type and mutant fibrinogen chains. These results support the presence of both homodimers and heterodimers in fibrinogen Hershey III, and suggest that incorporation of Aα R16C heterodimers into the fibrin clot leads to defects in fiber formation and clot structure.
Mechanical Properties of Hershey III and Control Clots Hershey III Control P G′ (dyne/cm2) 10.8 37.9 0.03 G″ (dyne/cm2) 0.83 2.77 0.04 Tan δ (G″/G′) 0.077 0.076 0.79 Ks (10−7 cm2) 1.86 2.44 0.01
Fibrinogen variant BβD432A has normal polymerization but does not bind knob “B”