Fibrin monomer preparation. Fibrin monomer was prepared by clotting fibrinogen with thrombin as described 16 with modifications. Thrombin was diluted to 1.
The clot was washed in 5 mL of 0. The repolymerization procedure was repeated twice. Polymerization turbidity curves. Polymerization of fibrinogen or fibrin monomer was measured by turbidity changes with time at nm using a Shimadzu UV spectrophotometer equipped with thermostatic cuvette holder Shimadzu Corp, Tokyo, Japan.
Transmission and scanning electron microscopy. Final concentration of fibrinogen was 0. After 1 hour at room temperature, 0. This solution was replaced with a fresh one three times during 1 hour. After the clot was fixed, the Parafilm was carefully removed followed by rinsing, dehydration, and critical-point drying.
Characterization of recombinant human fibrinogen. To accurately compare recombinant fibrinogen with plasma fibrinogen, we further purified commercial plasma fibrinogen by immunoaffinity chromatography with the same protocol used for recombinant fibrinogen. Under nonreducing conditions Fig 1 A , both samples appear as two high molecular weight bands, comparable to the previously described high molecular weight HMW and low molecular weight LMW fibrinogens.
Thus, the chains in recombinant fibrinogen that were modified with carbohydrate have the same molecular weight as those known to be modified in plasma fibrinogen. We also examined fibrin monomers prepared from plasma and recombinant fibrinogens Fig 1 C and found that the fibrin samples show comparable changes indicative of fibrinopeptide loss.
A through C were stained with Coomassie blue; D was stained with Schiff reagent. Panels A, B, and D are fibrinogen; C is fibrin; lane 1 samples are plasma protein and lane 2 samples are recombinant protein. Molecular weight markers for the nonreduced gel is indicated at the left and for the reduced gels at the right.
We confirmed the identity of these bands by Western blot analysis, as shown in Fig 2. Using a polyclonal antiserum that reacts with all three chains lanes 1 and 2 , we confirmed the similarity of recombinant fibrinogen to plasma fibrinogen.
Recombinant fibrinogen had a dominant band at 67 kD, a secondary band at 55 kD, and a faint band at 64 kD. Plasma fibrinogen had a dominant band at 67 kD, a secondary band at 64 kD, and a faint band at 55 kD.
Western blot analysis of recombinant and plasma fibrinogens. Lanes 1, 3, 5, and 7 were recombinant fibrinogen and 2, 4, 6, and 8 were plasma fibrinogen. Thrombin-catalyzed release of fibrinopeptides. The reactions were performed at ambient temperature and the released fibrinopeptides were separated by reversed-phase HPLC.
The data were plotted as percent fibrinopeptide release and were fitted to progress curves assuming the reactions are first order, as previously described. These data showed that the rate of fibrinopeptide release from recombinant fibrinogen was essentially the same as from plasma fibrinogen. Progress curves of fibrinopeptide release. The data were fitted to first-order rate equations, assuming FpA is released before FpB, as described.
Thrombin-catalyzed fibrin polymerization. Polymerization was measured as the change in turbidity at nm, as described. Representative curves are shown in Fig 4. We characterized the curves by two quantitative measures—the lag period, which represents the time required for protofibril formation, and the maximum slope, which reflects the rate of assembly of protofibrils. This analysis showed that, on average, the lag period was about 1. Polymerization of plasma and recombinant fibrinogens. Polymerization was initiated by the addition of thrombin at time 0 0.
Polymerization of fibrin monomers. To circumvent the contribution of thrombin catalysis to polymerization, 5 we examined polymerization of fibrin monomers prepared from each of these fibrinogens. Polymerization was initiated by diluting fibrin monomers dissolved in 0. Representative curves are shown in Fig 5 , and the quantitative data are presented in Table 2.
On average, we found that the lag period with recombinant fibrin monomer was 1. Polymerization of plasma and recombinant fibrin monomers. FXIIIa catalyzed crosslinking of fibrin. We, therefore, concluded that the kinetics of crosslink formation and the nature of the crosslinked products were comparable for recombinant and plasma fibrinogens.
Electron microscopy of fibrinogen and fibrin clots. We examined individual fibrinogen molecules by electron microscopy of samples prepared by rotary shadowing. The appearance of more than molecules was analyzed with particular attention focused toward additional nodules, indicated by arrows, either near the central domain or near the lateral domains.
Representative molecules are shown in Fig 7 , and the quantitative data are presented in Table 3. Clearly, the overall shape of the molecules of the recombinant fibrinogen was essentially the same as of the plasma fibrinogen. The fraction of molecules containing a fourth nodule was reduced with recombinant fibrinogen.
Transmission electron microscopy of individual fibrinogen molecules. Recombinant fibrinogen is shown in A and plasma fibrinogen is shown in B. The smaller panels contain molecules selected to illustrate the various structures with four nodules. We also examined clot structure by scanning electron microscopy. The appearance of clots prepared from recombinant and plasma fibrinogens, and their respective fibrin monomers, were very similar Fig 8.
Clots prepared from plasma fibrinogen and recombinant fibrinogen Fig 8 A and C both showed extensively branched fiber networks. As has been previously seen, 5 the degree of lateral aggregation and the pore size were significantly greater for clots prepared from fibrin monomers Fig 8 C and D versus those prepared by thrombin activation of fibrinogen Fig 8 A and B. This was true for both recombinant and plasma fibrinogens. The diameters of several hundred fibers were measured from micrographs of clots formed from plasma fibrinogen and recombinant fibrinogen.
Thus, there were no obvious differences when comparing clots formed from recombinant with plasma fibrinogen. Scanning electron micrographs of fibrin clots. Fibrin clots were prepared from thrombin and recombinant A or plasma C fibrinogen, and from recombinant B or plasma D fibrin monomer.
We initiated these studies to determine whether recombinant fibrinogen synthesized in mammalian cells is functionally similar to fibrinogen isolated from human plasma. This comparison is necessary to interpret other studies that examine the biochemical properties of variant recombinant fibrinogens.
We examined the biochemical properties that are critical to the conversion of fibrinogen to fibrin. The data presented here showed that recombinant fibrinogen is remarkably similar to plasma fibrinogen. We also examined these fibrinogen molecules by electron microscopy and found no significant differences in overall molecular shape. We found no difference in thrombin-catalyzed fibrinopeptide release, but did find differences in the lag period and maximal slope for the thrombin-catalyzed conversion of fibrinogen into fibrin polymers.
The data in Fig 4 and Table 2 suggest that protofibril assembly was slower with recombinant fibrinogen, while lateral aggregation was faster. The functional significance of these differences would appear to be minimal because the structures of clots Fig 8 A and C formed from these fibrinogens and thrombin are remarkably similar.
That is, the observed differences in turbidity measurements were not reflected as differences in the clot structures. This contrasts with other cases. For example, in comparison to normal plasma fibrinogen, fibrinogen Dusart has both a markedly different clot structure and profoundly different turbidity measurements—a threefold increased lag time and a fivefold decreased slope.
This conclusion is supported by the turbidity data obtained with fibrin monomers, as the maximum rate of polymerization with recombinant fibrin monomers was the same as that with plasma fibrin monomers. Moreover, the micrographs of clots formed from recombinant and plasma fibrin monomers were remarkably similar. These data support the conclusion that the minor species in plasma fibrinogen that arise from alternative RNA processing do not contribute significantly to the characteristics we have measured.
The electron microscopy EM data Table 3 indicated a small quantitative difference when comparing micrographs of plasma fibrinogen with micrographs of recombinant fibrinogen. Furthermore, as described below, other interpretations are reasonable. Finally, the data indicate that posttranslational modification of recombinant fibrinogen is similar to plasma fibrinogen. The relative amounts of these species differs between the recombinant protein and the plasma protein.
We conclude that recombinant fibrinogen is a good model for plasma fibrinogen for the examination of the conversion of fibrinogen to crosslinked fibrin. We can therefore continue to examine these structural and functional characteristics using recombinant variant fibrinogens.
We are grateful to Li Fang Ping who performed all of the tissue culture work and provided excellent technical assistance. HL to S. HL to J.
Address reprint requests to Susan T. Sign In or Create an Account. Sign In. Skip Nav Destination Content Menu. Close Abstract. Article Navigation. Gorkun , Oleg V. This Site. Google Scholar. Yuri I. Veklich , Yuri I. Fibrinogen is a large, complex glycoprotein composed of three pairs of polypeptides: two A a , two B b , and two g. These polypeptides are linked together by 29 disulphide bonds, some of which are depicted in Figure 2 below. The polypeptides are oriented so all six N-terminal ends meet to form the central E domain.
Two regions of coiled coil alpha helices stretch out on either side of the E domain, each consisting of one A a , one B b and one g polypeptide. Each coiled coil region ends in a globular D domain consisting of the C-terminal ends of B b and g , as well as part of A a.
The C-terminal end of A a then protrudes from each D domain as a long strand; these A a protuberances can interact with each other and with the E domain during fibrin clot cross-linking. Both the E and D domains contain important binding sites for the conversion of fibrinogen to fibrin, for fibrin assembly and cross-linking, and for platelet aggregation.
Bound calcium ions are important to help maintain the structure of fibrinogen. The N-terminal ends of both the A a and B b polypeptides are cleaved by thrombin in order to turn soluble fibrinogen into gel-forming fibrin. Once cleaved from fibrinogen, the N-terminal ends are known as fibrinopeptide A from A a polypeptide and fibrinopeptide B from B b polypeptide. Figure 2. TOP — polypeptide organisation of fibrinogen.
In order to form a blood clot, fibrinogen must first be cleaved by thrombin to remove the fibrinopeptides. Fibrin molecules can link together through the interaction of the E domain on one fibrin molecule to the D domains on four other fibrin molecules, thereby polymerising to form staggered oligomers that build up into protofibrils. As the fibrin oligomers aggregate, these protofibrils continue to lengthen to make long fibres that can wind around one another to make multi-stranded, thick bundles, and which can branch into a 3-dimentional network of entangled fibres, the fibrin clot.
Factor XIIIa cross-links glutamine residues on one fibrin molecule to the lysine residues on another fibrin molecule by forming strong isopeptide bonds.
This cross-linking occurs between the C-terminal ends A a protuberances of the A a polypeptides, as well as more slowly at other sites, such as between the C-terminal ends of g chains. These cross-links help strengthen the fibrin clot, making it more resistant to physical and chemical damage.
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