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Specific molecular interactions mediated by the N-terminus of fibrinogen's Bbeta chain were revealed using laser tweezers-based force spectroscopy. We examined interactions between fibrinogen fragments representing the center of the molecule, NDSK, desA-NDSK, and desAB-NDSK, and two recombinant fibrinogens, gammaD364H and gammaD364A, which have nonfunctional gamma-chain polymerization sites to prevent the dominant knob-hole binding. Interactions between desA-NDSK, where the N-terminus of the Bbeta chain is present, and the fibrinogen variants showed a complex spectrum of rupture forces which disappeared with desAB-NDSK, lacking both FpA and FpB. The interactions between desA-NDSK and gammaD364H or gammaD364A were inhibited by addition of soluble FpB, but not FpA or the polymerization inhibitor peptides GPRP and GHRP. When gammaD364H fibrinogen was replaced with its X-fragment lacking alphaC- domains or with fragment D, the strongest component of the rupture force spectrum disappeared, suggesting interactions between the uncleaved FpB and the alphaC-domain. Electron microscopy confirmed the binding of desA-NDSK to either D or E regions of fibrinogen as well as to alphaC-domains. The data demonstrate the existence of weak transient interactions within and between fibrin molecules mediated by the N-terminus of the fibrinogen Bbeta chain.

The functions of the alphaC domains of fibrinogen in clotting and fibrinolysis, which have long been enigmatic, were determined using recombinant fibrinogen truncated at Aalpha chain residue 251. Scanning electron microscopy and confocal microscopy revealed that the fibers of alpha251 clots were thinner and denser, with more branch points than fibers of control clots. Consistent with these results, the permeability of alpha251 clots was nearly half that of control clots. Together, these results suggest that in normal clot formation, the alphaC domains enhance lateral aggregation to produce thicker fibers. The viscoelastic properties of alpha251 fibrin clots differed markedly from control clots; alpha251 clots were much less stiff and showed more plastic deformation, indicating that interactions between the alphaC domains in normal clots play a major role in determining the clot's mechanical properties. Comparing factor XIIIa cross-linked alpha251 and control clots showed that gamma chain cross-linking had a significant effect on clot stiffness. Plasmin-catalyzed lysis of alpha251 clots, monitored with both macroscopic and microscopic methods, was faster than lysis of control clots. In conclusion, these studies provide the first definitive evidence that the alphaC domains play an important role in determining the structure and biophysical properties of clots and their susceptibility to fibrinolysis.

The peptide antibiotic ramoplanin is highly effective against several drug-resistant gram-positive bacteria, including vancomycin-resistant Enterococcus faecium (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), two important opportunistic human pathogens. Ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by binding to Lipid intermediates I and II at a location different than the N-acyl-D-Ala-D-Ala dipeptide site targeted by vancomycin. Lipid I/II capture physically occludes these substrates from proper utilization by the late-stage PG biosynthesis enzymes MurG and the transglycosylases. Key structural features of ramoplanin responsible for antibiotic activity and PG molecular recognition have been discovered by antibiotic semisynthetic modification in conjunction with NMR analyses. These results help define a minimalist ramoplanin pharmacophore and introduce the possibility of generating ramoplanin-derived peptide or peptidomimetic antibiotics for use against VRE, MRSA, and related pathogens.

The peptide antibiotic ramoplanin inhibits bacterial peptidoglycan (PG) biosynthesis by interrupting late-stage membrane-associated glycosyltransferase reactions catalyzed by the transglycosylase and MurG enzymes. The mechanism of ramoplanin involves sequestration of lipid-anchored PG biosynthesis intermediates, physically occluding these substrates from proper utilization by these enzymes. In this report, we describe the first molecular-level details of the interaction of ramoplanin with PG biosynthesis intermediates. NMR analysis in conjunction with chemical dissection of the PG monomer revealed that the ramoplanin octapeptide D-Hpg-D-Orn-D-alloThr-Hpg-D-Hpg-alloThr-Phe-D-Orn recognizes MurNAc-Ala-gamma-D-Glu pyrophosphate, the minimum component of PG capable of high-affinity complexation and fibril formation. Ramoplanin therefore recognizes a PG binding locus different from the N-acyl-D-Ala-D-Ala moiety targeted by vancomycin. Because ramoplanin is structurally less complex than glycopeptide antibiotics such as vancomycin, peptidomimetic chemotherapeutics derived from this recognition sequence may find future use as antibiotics against vancomycin-resistant Enterococcus faecium, methicillin-resistant Staphylococcus aureus, and related pathogens.

Plasma fibrinogen is a mixture of multiple molecular forms arising mainly through alternative mRNA processing and subsequent posttranslational modification. Recombinant fibrinogen is synthesized without alternative mRNA processing in a cultured cell system that may generate novel posttranslational modifications. Thus, to show that recombinant fibrinogen can serve as a functional model for plasma fibrinogen, we have examined the conversion of fibrinogen to fibrin, comparing the recombinant with the plasma protein. We examined the kinetics of (1) thrombin-catalyzed fibrinopeptide release, (2) thrombin-catalyzed polymerization of fibrinogen, (3) the polymerization of fibrin monomers, and (4) FXIIIa-catalyzed cross-link formation. We saw small differences in polymerization, suggesting that the ordered assembly of protofibrils and fibers was not identical. In all other analyses, we found that plasma fibrinogen and recombinant fibrinogen were remarkably similar. Using electron microscopy, we examined the structures of individual fibrinogen molecules and fibrin clots. Individual fibrinogen molecules were predominantly three nodule structures for both recombinant and plasma proteins. Both samples also displayed four nodule structures, but fewer four nodule structures were found with recombinant fibrinogen. Fibrin clot structures were essentially indistinguishable. We concluded that recombinant fibrinogen can serve as a accurate model for plasma fibrinogen.

The role of the carboxyl-terminal portion of the alpha chains of fibrin (alpha C domains) in clot formation was investigated by transmission and scanning electron microscopy and turbidity studies of clots made from preparations of molecules missing one or both of these domains. Highly purified and entirely clottable preparations of bovine fragment X monomer, one containing primarily molecules missing a single alpha C domain (fragment X1) and the other consisting of molecules missing both alpha C domains (fragment X2), were used for these experiments. These preparations were characterized by various methods, including the complete determination of the amino- and carboxyl-termini of all peptides and fragments. These preparations formed clots on dilution to neutral pH. In all cases, clots observed by either scanning or transmission electron microscopy were made up of a branched network of fibers, similar to those formed by thrombin treatment of intact fibrinogen, suggesting that the alpha C domains are not necessary for protofibril and fiber formation or branching. However, both the fiber and clot structure varied with the different fractions, indicating that the alpha C domains do participate in polymerization. The rate of assembly, as indicated by the lag period and maximum rate of turbidity increase, as well as the final turbidity, was decreased with removal of the alpha C domains, suggesting that they accelerate polymerization. preparations of isolated alpha C fragment added to fibrin monomer have striking effects on the turbidity curves, showing a decrease in the rate of polymerization in a dose-dependent manner but not complete inhibition. Electron microscopy of fibrin monomer desA molecules at neutral pH showed that most of the alpha C domains, like those in fibrinogen, remain associated with the central region. Thus, it appears that normally with thrombin cleavage of fibrinogen the effects of the interactions of alpha C domains observed here will be most significant for lateral aggregation.

The locations of the carboxyl-terminal two thirds of the A alpha chains, or the alpha C domains, were determined for fibrinogen and some of its derivatives by electron microscopy of rotary-shadowed preparations. A monoclonal antibody, G8, to the carboxyl-terminal 150 amino acids of the A alpha chain, binds near the central region of fibrinogen, indicating that the alpha C domains of most molecules are not normally visible because they are on or near the amino-terminal disulfide knot. At pH 3.5, fibrinogen and fibrin monomers appear to be similar, with a projection terminating in a small globular domain from each end of most molecules. In contrast, fragment X monomers, produced by cleavage of the alpha C domains from fibrinogen with plasmin, show no such projections. When fibrin monomer is brought to neutral pH under conditions where polymerization is delayed, individual molecules are still visible showing the alpha C domains as a single additional nodule near the central region. Moreover, analysis of clusters of molecules reveals some intermolecular associations via the alpha C domains. A 40-kDa fragment comprising the alpha C domain has been isolated from a plasmin digest of fibrinogen and characterized by SDS-polyacrylamide gel electrophoresis and determination of amino-terminal amino acid sequences. Electron microscopy of alpha C fragments reveals individual globular structures, as well as oligomeric aggregates. The addition of alpha C fragments to fibrin monomer followed by dilution to neutral pH to initiate polymerization results in lower turbidity, longer lag period, and slower maximum rate of turbidity increase. Also, electron microscopy reveals complexes of alpha C fragments with fibrin monomer at neutral pH. It appears that the free alpha C fragments can bind to the alpha C domains of fibrin, competing with the normal alpha C domain interactions involved in polymerization.