John R. Shainoff

Stable Complex of Fibrinogen and Fibrin

Fibrin-stabilizing factor acts on monomeric fibrin combined with fibrinogen to form a stable complex that is soluble at physiologic pH and ionic strength. The complex has been isolated by chromatography on agarose gel and characterized by sedimentation rate near 24 Sw.20 at both pH 5.3 and 7.4, by coagulability when treated with thrombin, and by content of fibrinopeptides. Formation of the complex could provide a pathway for solubilization of monomeric fibrin produced in circulating blood.

FIBRINOPEPTIDE B IN FIBRIN ASSEMBLY AND METABOLISM: PHYSIOLOGIC SIGNIFICANCE IN DELAYED RELEASE OF THE PEPTIDE*

The delayed release of peptide B that accelerates towards the end of fibrin formation unmasks accessory (b-) epitopes for monomer interaction. Ultracentrifuge and chromatographic analysis of the composition and dissociation of soluble complexes formed by monomers in fibrinogen solution indicate that the b-epitope augments aggregation by acting cooperatively with the a-epitope to reinforce rather than cross-bridge oligomer assembly. Monomer/fibrinogen association by coordinated interactions through both epitopes is strengthened by an additional order of magnitude over associations (10(7) and 1.6 X 10(6) M-1) through the a- and b-epitopes individually, without affecting oligomer thickness. It is suggested that the delayed release of B has purpose in allowing early complexes to dissociate for (1) rapid equilibration across interstitial fluids, and for (2) rapid uptake by phagocytic cells which depend on access to the a-epitope for monomer absorption. In late stages of coagulation, stabilization of oligomer assembly imparted by the b-epitope blocks both equilibration of fibrin concentrations and phagocytic clearance of the fibrin to localize deposition.

Low factor XIIIA levels are associated with increased blood loss after coronary artery bypass grafting.

Current hematologic approaches to minimize postoperative bleeding have focused principally on antifibrinolytic agents. To explore whether a need might exist to promote clot stabilization independent of steps that might be taken to prevent lysis, we followed levels of the functional A-chain of factor XIII (fibrin stabilizing factor) immunologically in 19 patients undergoing coronary artery bypass grafting. The levels of factor XIIIA together with alterations in fibrinogen were followed at five stages of operation: (1) initial catheter placement (control), (2) heparinization, (3) initiation of cardiopulmonary bypass, (4) discontinuation of cardiopulmonary bypass, and (5) heparin neutralization with protamine sulfate. Significant (p < 0.05) inverse correlations were observed between postoperative chest-tube drainage volumes and levels of XIIIA at stages 1 through 3, and borderline associations (p < 0.1) were observed for stages 4 and 5. Pronounced losses of factor XIIIA accompanied initiation of cardiopulmonary bypass, when levels fell to 43% +/- 12% (standard deviation) of the control value, significantly below the 59% +/- 9% of the control value expected from hemodilution. By comparison, fibrinogen concentrations fell only to the extent attributable to hemodilution, unaccompanied by substantial degradation as indicated by electrophoretic, functional, and immunologic assays. There was a reversible heparin-induced precipitation of fibrin complexes and fibrinogen dimers from the blood on initiation of hypothermia, but these components returned to the circulation on restoration of normothermia. This precipitation was unrelated to losses of factor XIIIA. The findings warrant inference that XIIIA supplementation in deficient states should be considered as an adjunct to other therapies for postoperative bleeding.

Deposition of modified fibrinogen within the aortic intima

Abstract Substantial quantities of an insoluble form of fibrinogen differing from fibrin were found to occur within the intima of arteriosclerotic aortas. Identification was based on the fact that thrombin digests of reduced and alkylated protein from the tissue yielded fibrinopeptides in quantities that were stoichiometrically related to the composition of fibrinogen. The material could be extracted from the tissue with 5 M guanidine-HCI but not with common salt solutions or 40% urea. As judged from yields of fibrinopeptides, lesion bearing segments contained largest quantities averaging the equivalent of 3.6 mg of fibrinogen per g wet tissue with only 1 of 15 specimens giving none. The determinations for normal appearing segments from atherosclerotic vessels were occasionally as high; 10 of 15 fell below 0.5 mg/g. None was found in the aortic media. A tripeptide (glycylprolylarginine) located in a relatively thrombin-resistant segment of fibrinogen immediately behind fibrinopeptide A was measured and compared with the fibrinopeptides. There was not a large disparity in content of this peptide in relation to fibrinopeptides as occurs in fibrin clots, indicating that clot formation may not be the principle pathway to deposition of the protein related to fibrinogen in the aortic intima. It is suggested that the deposits are formed by a process analogous to the formation and stabilization of cryoprofibrin in blood.

Fibrinogen and fibrin: biochemistry and pathophysiology.

Fibrinogen is a thrombin-coagulable glycoprotein occurring in the blood of vertebrates. The primary structure of the alpha, beta, and gamma polypeptide chains of human fibrinogen is known from amino acid and nucleic acid sequencing. The intact molecule has a trinodular, dimeric structure and is functionally bivalent. Thrombin cleaves short peptides from the amino termini of the alpha and beta chains exposing polymerization sites that are responsible for the formation of fibrin fibers and appearance of a clot. The major physiological function of fibrinogen is the formation of fibrin that binds together platelets and some plasma proteins in a hemostatic plug. In pathological situations, the network entraps large numbers of erythrocytes and leukocytes forming a thrombus that may occlude a blood vessel. Fibrinogen and fibrin are multifunctional proteins. Fibrinogen is indispensable for platelet aggregation; it also binds to several plasma proteins, however, the biological function of this interaction is not completely understood. Fibrin is an essential matrix for regulation of fibrinolysis and for facilitation of cell attachment in wound healing.

Fate of fibrinopeptides in the reaction between human plasmin and fibrinogen

Abstract The trichloroacetic acid-soluble fragments that are released during initial stages of attack of plasmin upon fibrinogen consist in large part of N-terminal segments from the βB chain. The β B fragments could be distinguished from others because they release fibrinopeptide B when treated with thrombin. Release of the β B fragments was partially preceded by faster release of only one other trichloroacetic acid-soluble fragment and followed by slower release of at least three and subsequently many others. No loss of coagulability of the fibrinogen occurred until the overall reaction with plasmin had progressed well beyond the stage of β B fragment release. Fibrinopeptide B itself was not cleaved from the fragments by plasmin until a late stage in the overall reaction. As could be predicted from studies of Iwanaga, S., Wallen, P., Grondahl, N., Henschen, A., and Blombach, B. [(1967) Biochim. Biophys. Acta 147, 606–609] fibrinopeptide A was fully retained as part of the trichloroacetic acid-precipitable hulk of the degraded fibrinogen up to the terminal stage. Gel electrophoresis showed that the large C-terminal fragment of the αA chain which others had described as initial product was released over the same period taken for release of the β B fragments. The rapid loss of B due to release of the β B fragments, and the retention of A contrasted sharply with the converse order in which fibrinopeptides are released from fibrinogen by thrombin. Full content of fibrinopeptide B in some preparations of cryoprofibrin indicated formation of this fibrinogen-fibrin complex is not critically dependent upon degradation by plasmin.

Zonal immobilization of proteins

Abstract A gel matrix that can be used in sequence to separate proteins and then immobilize them was obtained by incorporating into agarose an aldehydic ligand with readily controllable reactivity. The gel was prepared by etherifying agarose with glycidol and subsequently oxidizing with periodate. It provided an inert matrix equivalent to ordinary agarose for separating proteins at neutral or acidic pH, but rapidly absorbed them through formation of stable alkyl amine linkages on exposure to either alkaline or concentrated NaCNBH 3 . Thus, the protein could be fixed without use of denaturants. The ability to array proteins electrophoretically on an immobilizing substrate opens new possibilities for analysis of complex mixtures by providing means for carrying out affinity binding assays in relation to physical properties of the protein, and for performing multiple tests of composition without loss or spread.

Role of fibrinogen in fibrin transport: Chromatographic studies

Several physical criteria indicate that the soluble complexes formed by molecular fibrin in fibrinogen solution consist of large aggregates, but gel-filtration chromatography frequently portrays them as having permeation characteristics equivalent to fibrinogen itself. An equilibrium exists between dissociation and formation of the complexes within both the gel and transporting medium, so that equilibration of fibrin across the gel should proceed in parallel with fibrinogen under conditions favorable to rapid attainment of equilibrium. This concept is illustrated by the following phenomena: 1) In absence of fibrinogen, fibrin monomers aggregated into immobile strands. 2) In dilute fibrinogen at 37°C, soluble complexes were formed and traversed the column at rate commensurate with fibrinogen as though fully dissociable into monomers. 3) Physiologic concentrations of fibrinogen at 37°C accelerated elution of the complexes to a degree indicative of substantially suppressed dissociability into species smaller than dimers in the more concentrated fibrinogen. 4) On lowering temperature to 25°C, elution advanced to rates approaching the maximum for poorly permeating complexes. 5) The poor permeation at 25°C was attributable to reduced rates of dissociation of the complexes rather than a change in their size, because permeation became fully equivalent to fibrinogen when adequate time on the order of six hours was allowed for equilibration. It is proposed that analogous transport through dissociation and reassembly of the complexes across permeability barriers occurs in vivo, and may function in transendothelial deposition and clearance of fibrin.

Microangiopathic hemolytic anemia in renal allotransplantation: Report of a successfully treated case and review of the literature

The development of microangiopathic hemolytic anemia after renal transplantation in a 17 year old white boy is reported, and the literature is reviewed. In this patient microangiopathic hemolytic anemia developed 6 weeks after renal transplantation during a second episode of rejection. Light, fluorescence and electron microscopy demonstrated the renal vascular lesion associated with this syndrome. In contrast to the other four previously reported cases of microangiopathic hemolytic anemia associated with renal allotransplantation, this patient had complete resolution of the microangiopathic hemolytic anemia with heparin therapy and improved allograft function, presumably with diminution of the vascular lesion. He survived a complicated early period after renal transplantation and has shown no recurrence of microangiopathic hemolytic anemia in the 18 months since transplantation. Special red blood cell and fibrinogen studies are discussed.

Further evidence for the existence of angiotensinogen stimulating activity (ASA) after nephrectomy.

Summary After nephrectomy, plasma or serum acquires the property of stimulating angiotensinogen (A) synthesis in liver slices from normal rats. The possibility that this angiotensinogen stimulating activity (ASA) may be an artifact arising from exposure of the tissue to the high levels of A in the test material has been examined. It has now been found that ASA and A can be separated by electrophoresis on starch powder and gradient elution from DEAE cellulose. The identification of ASA as a distinct component of the renal-pressor system raises new prospects for investigation of control mechanisms within the system.