Summary

The work detailed in this dissertation combines biochemical assays and X-ray crystallography to deepen the current understanding of the mechanism of fibrin polymerization. In Chapter 2, we solved the crystal structure of fragment D from γD364A to understand the molecular basis for the aberrant function of γ364Asp variants. Crystallographic studies revealed γ364Asp is part of hole “a” that interacts with knob “A” peptide mimic, GPRP. Biochemical studies have shown γ364Asp is critical to polymerization, based on the observation that the polymerization of variants γD364A, γD364H and γD364V is exceptionally impaired (1). Surprisingly, the structure (rfD- γD364A+GP) showed near normal “A:a” interactions with GPRP bound to hole “a” and no change in the overall structure of γD364A (2). Of note, inspection of the structure showed that the negative electrostatic potential inside hole “a” was diminished by this substitution. We examined GPRP binding to the γ364Asp variants in solution by plasmin protection assay and found no protection of either γD364H or γD364V but partial protection of γD364A, indicating the peptide does not bind detectably to either γD364H or γD364V and binds more weakly than normal to γD364A. We also examined protection by calcium and found all variants were indistinguishable from normal suggesting the global structures of the variants are not markedly different from normal. The prolonged lag time during thrombin-catalyzed polymerization suggests slow protofibril formation in γD364A which may be a consequence of the diminished electrostatic steering due to the loss of the acidic γ364Asp; that the local negative charge on holes “a” reinforce electrostatic steering so that knobs “A” at the center of one molecule are able to find the holes at the terminal ends of another molecule during

protofibril formation. Our data imply that γ364Asp per se is not required for knob “A” binding to hole “a”; rather, this residue’s negative charge has a critical role in the electrostatic interactions that facilitate the important first step in fibrin polymerization.

The importance of “B:b” knob-hole interactions remain unclear from conflicting

biochemical data (3-8). In Chapter 3, we used recombinant variant BβD432A to shed light into this controversy. Fibrinogen residue Bβ432Asp is part of hole “b” that interacts with knob “B” whose sequence starts with GHRP. Because previous studies showed BβD432A has normal polymerization, we hypothesized that Bβ432Asp is not critical for knob “B” binding and that new knob-hole interactions would compensate for the loss of this Asp residue. To test this hypothesis, we solved the crystal structure of fragment D from BβD432A. Surprisingly, the structure (rfD-BβD432A+GH) showed the peptide GHRP was not bound to hole “b”. We then re-evaluated the polymerization of this variant by examining clot turbidity, clot structure and the rate of FXIIIa-crosslinking. The turbidity and the rate of γ-γ dimer formation for BβD432A were indistinguishable compared to normal fibrinogen. Scanning electron microscopy showed no significant differences between the clots of BβD432A and normal fibrinogen, but the thrombin-derived clots had thicker fibers than clots obtained from batroxobin, suggesting that cleavage of FpB is more important than “B:b” interactions. The normal polymerization of BβD432A, despite, the absence of “B:b” interactions suggests that occupancy of hole “b” may have little impact on polymerization; it is the loss of the negatively-charged FpB which relieves charge repulsion, rather than the gain of “B:b” interactions that is critical. Hence, electrostatic factors are more critical than actual “B:b” knob-hole binding in fibrin polymerization.

In addition to the knob-hole binding, other interactions like the end-to-end associations mediated by the D:D interface stabilize fibrin polymerization. Unlike knob-hole interactions, D:D interfacial interactions are not well-studied. In Chapter 4, we examined the role of D:D interactions in fibrin polymerization using a variant recombinant fibrinogen, γN308K, patterned after a naturally occurring dysfibrinogen. Previous studies of this variant showed impaired polymerization that was not improved by increasing calcium ion concentrations (9). We combined biochemical and structural data to provide the molecular basis for the impairment seen in γN308K. Plasmin protection assays showed γN308K require higher concentration of the knob “A” peptide mimic (GPRP) to achieve partial protection from digestion suggesting that γN308K has slightly impaired “A:a” interactions. Calcium ions imparted protection to γN308K similar to normal fibrinogen suggesting calcium ion binding was not altered in the variant. However, modeling the D:D interactions seen in normal crosslinked fibrin for γN308K showed potential steric and charge repulsion between γ308Lys and γ321Lys that may destabilize the γ1 calcium binding loop. Analysis of the electrostatic potential in the area where D:D interactions normally occur showed increased positive charge in γN308K suggesting possible repulsion between abutting fibrin molecules. Indeed, molecular packing in rfD-γN308K+GP crystals showed symmetric D:D contacts involving residues different from those observed in all previously reported fragment D and naturally crosslinked double D structures. In conclusion, analysis of γN308K fibrinogen suggests that γ308Asn is critical not only for charge complementarity of the D:D interface during end-to- end association of fibrin molecules but also important for “A:a” knob-hole interactions and calcium binding.