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 functions of the ␣C domains of fibrinogen in clotting and fibrinolysis, which have long been enigmatic, were determined using recombinant fibrinogen truncated at A␣ chain residue 251. Scanning electron microscopy and confocal microscopy revealed that the fibers of ␣251 clots were thinner and denser, with more branch points than fibers of control clots. Consistent with these results, the permeability of ␣251 clots was nearly half that of control clots. Together, these results suggest that in normal clot formation, the ␣C domains enhance lateral aggregation to produce thicker fibers. The viscoelastic properties of ␣251 fibrin clots differed markedly from control clots; ␣251 clots were much less stiff and showed more plastic deformation, indicating that interactions between the ␣C domains in normal clots play a major role in determining the clot's mechanical properties. Comparing factor XIIIa cross-linked ␣251 and control clots showed that ␥ chain crosslinking had a significant effect on clot stiffness. Plasmin-catalyzed lysis of ␣251 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 ␣C domains play an important role in determining the structure and biophysical properties of clots and their susceptibility to fibrinolysis. IntroductionFibrinogen, which is essential for both platelet aggregation and the fibrin clot formation, is made up of 3 pairs of polypeptide chains, (A␣B␤␥) 2 . It is a fibrous protein 45 nm in length with globular regions at each end and in the middle. Each fibrinogen molecule contains 2 ␣C domains, which are the carboxyl terminal two-thirds of the A␣ chains. The ␣C domains, each made up of a globular and an extended portion, are juxtaposed to one another near the central globular region of fibrinogen where they interact intramolecularly. [1][2][3][4] During the conversion of fibrinogen to fibrin, there is a large-scale conformational change such that the ␣C domains dissociate from the central region, allowing them to interact intermolecularly, adding protofibrils to a fiber. 2,4,5 Indeed, isolated ␣C fragments, which were removed proteolytically from fibrinogen, can bind to normal fibrin, competing with the normal ␣C domain interactions and thereby impairing polymerization. 5 Thus, the ␣C domain interactions appear critical for the enhancement of lateral aggregation during fibrin polymerization. 4,6,7 Several dysfibrinogenemias with amino acid substitutions in or truncations of the ␣C domains showed striking effects on polymerization, clot structure and fibrinolysis. [8][9][10] While the results from these and other dysfibrinogenemias are intriguing and give us many clues to the function of the ␣C domains, interpretation of the results is still somewhat confused by other accompanying changes to the molecules (such as disruption of disulfides or attachment of albumin) and the fact that most of these cases are heterozygous. [11][12][13][14][15][16] Studies using pr...

No abstract

No abstract