Fibrinogen altered by thrombin-catalyzed liberation of fibrinopeptide A was found to combine with native fibrinogen to form a cold-precipitable complex we have called "cryoprofibrin." The altered fibrinogen lacking fibrino-peptide A polymerized into fibrin, but not until conditions for equilibrium between its incorporation into both cryoprofibrin and fibrin were satisfied. At equilibrium, the concentration of cryoprofibrin was maintained at a threshold proportional to the concentration of fibrinogen. When the concentration of cryoprofibrin was below threshold, fibrin could be depolymerized and solubilized by fibrinogen with resultant formation of cryoprofibrin. Since threshold concentrations of cryoprofibrin appear necessary for precipitation of fibrin, the concentration of cryoprofibrin in plasma provides a basis for determining intravascular deposition of fibrin. Intravascular deposition of fibrin does not appear to occur normally in rabbits, because the concentration of cryoprofibrin in plasma from normal rabbits is far below the threshold for precipitation of fibrin. The applicability of cryoprofibrin as an indicator of fibrin deposition is demonstrated by the occurrence of levels of cryoprofibrin approaching the threshold for precipitation of fibrin in plasma from endotoxin-treated rabbits.
The current concept that the fibrinogen molecule can dissociate into subunits can be used to explain the conversion of fibrinogen to cryoprofibrin. As one possibility, the two residues of fibrinopeptide A contained in fibrinogen may be located on two separate subunits of the molecule; cryoprofibrin is produced when one of these subunits is replaced by a subunit altered by loss of fibrinopeptide A. Recombination of native subunits with subunits altered by loss of A would counter dissociation of cryoprofibrin and inhibit polymerization of subunits lacking fibrinopeptide A. As an alternate mechanism, two residues of A may be liberated concurrently from a single subunit. Cryoprofibrin would then correspond to a fibrinogen molecule, containing a subunit with two residues of A, in combination with an altered molecule containing a subunit lacking two residues of A.
Liberation of fibrinopeptide B did not contribute measurably to production of fibrin resulting from limited action of thrombin on rabbit fibrinogen. Both fibrin containing B but not A, and fibrin containing neither B nor A, as is produced by extensive action of thrombin, could be solubilized by fibrinogen.
Thrombin, or another enzyme utilizing tosyl-L-arginine methyl ester as substrate, appeared reversibly to inhibit polymerization of fibrin containing fibrinopeptide B. This enzyme and fibrinogen were the only proteins appearing to inhibit polymerization in plasma from normal rabbits.