Fibrin polymerization and its regulatory role in hemostasis

Proteolytic conversion of fibrinogen to fibrin results in self-assembly to form a three-dimensional clot matrix that subsequently becomes cross-linked by fXIIIa to form the central structural element of the in vivo thrombus. The process of fibrin formation and assembly leads to new properties that serve to regulate the rate and extent of clotting, cross-linking, and fibrinolysis. These are brought about by the ability of fibrin (1) to bind thrombin at a noncatalytic site, thus limiting its diffusability but at the same time preserving its catalytic potential; (2) to bind fXIII, regulate its activation to fXIIIa, and limit further activation of fXIII once fibrin cross-linking has occurred; and (3) to bind alpha 2-PI, tPA, and plasminogen and regulate the initiation and propagation of fibrinolysis. Additional interactions not covered in this review between fibrin(ogen), and other plasma proteins, cells or matrix components suggest other functions for fibrin that, along with those discussed above, define a critical role in modulating hemostasis, inflammation, and the wound healing process.     

Fibrinogen counteracts the antiadhesive effect of fibrin‐bound plasminogen by preventing its activation by adherent U937 monocytic cells

Summary. Background: Fibrinogen and plasminogen strongly reduce adhesion of leukocytes and platelets to fibrin clots, highlighting a possible role for these plasma proteins in surface‐mediated control of thrombus growth and stability. In particular, adsorption of fibrinogen on fibrin clots renders their surfaces non‐adhesive, while the conversion of surface‐bound plasminogen to plasmin by transiently adherent blood cells results in degradation of a superficial fibrin layer, leading to cell detachment. Although the mechanisms whereby these proteins exert their antiadhesive effects are different, the outcome is the same: the formation of a mechanically unstable surface that does not allow firm cell attachment. Objectives: Since fibrin clots in circulation are exposed to both fibrinogen and plasminogen, their combined effect on adhesion of monocytic cells was examined. Methods: Fibrin gels were coated with plasminogen and its activation by adherent U937 monocytic cells in the presence of increasing concentrations of fibrinogen was examined by either measuring ¹²⁵I‐labeled fibrin degradation products or plasmin amidolytic activity. Results: Unexpectedly, the antiadhesive effects of two fibrin binding proteins were not additive; in fact, in the presence of fibrinogen, the effect of plasminogen was strongly reduced. An investigation of the underlying mechanism revealed that fibrinogen prevented activation of fibrin‐bound plasminogen by cells. Confocal microscopy showed that fibrinogen accumulates in a thin superficial layer of a clot, where it exerts its blocking effect on activation of plasminogen. Conclusion: The results point to a complex interplay between the fibrinogen‐ and plasminogen‐dependent antiadhesive systems, which may contribute to the mechanisms that control the adhesiveness of a fibrin shell on the surface of hemostatic thrombi.     

A unique property of a plasma proteoglycan, the C1q inhibitor. An anticoagulant state resulting from its binding to fibrinogen.

The C1q inhibitor, C1qI, an approximately 30-kD circulating chondroitin-4 sulfate proteoglycan, displayed concentration-dependent prolongation of plasma and fibrinogen solution clotting times. Under factor XIIIa catalyzed cross-linking conditions and maximum C1qI concentrations, minor amounts of clot formed displaying complete gamma-gamma dimer formation but virtually no alpha-polymer formation. The anticoagulant effect was undiminished by its binding to C1q, by increased ionic strength, and by CaCl2, but was abolished by incubation of C1qI with chondroitinase ABC. 125I-labeled C1qI bound to immobilized fibrinogen, fibrin monomer, fibrinogen plasmic fragments D1 and E, and fibrin polymers. Occupancy on the E domain required uncleaved fibrinopeptides together with another structure(s), and it did not decrease binding of thrombin to fibrinogen. Occupancy on the D domain did not decrease the fibrinogen binding to fibrin monomer. We conclude that the E domain occupancy impaired fibrinopeptide cleavage, and occupancy on the D domain impaired polymerization, both steric hindrance effects. C1qI binding to fibrinogen explains at least in part the well-known fibrin(ogen) presence in immune complex-related lesions, and the fibrinogen presence in vascular basement membranes and atheromata. We postulate that fibrin binding by resident basement membrane proteoglycans provides dense anchoring of thrombus, substantially enhancing its hemostatic function.     

Fibrin(ogen) exacerbates inflammatory joint disease through a mechanism linked to the integrin alphaMbeta2 binding motif

Fibrin deposition within joints is a prominent feature of arthritis, but the precise contribution of fibrin(ogen) to inflammatory events that cause debilitating joint damage remains unknown. To determine the importance of fibrin(ogen) in arthritis, gene-targeted mice either deficient in fibrinogen (Fib-) or expressing mutant forms of fibrinogen, lacking the leukocyte receptor integrin alphaMbeta2 binding motif (Fibgamma390-396A) or the alphaIIbbeta3 platelet integrin-binding motif (FibgammaDelta5), were challenged with collagen-induced arthritis (CIA). Fib- mice exhibited fewer affected joints and reduced disease severity relative to controls. Similarly, diminished arthritis was observed in Fibgamma390-396A mice, which retain full clotting function. In contrast, arthritis in FibgammaDelta5 mice was indistinguishable from that of controls. Fibrin(ogen) was not essential for leukocyte trafficking to joints, but appeared to be involved in leukocyte activation events. Fib- and Fibgamma390-396A mice with CIA displayed reduced local expression of TNF-alpha, IL-1beta, and IL-6, which suggests that alphaMbeta2-mediated leukocyte engagement of fibrin is mechanistically upstream of the production of proinflammatory mediators. Supporting this hypothesis, arthritic disease driven by exuberant TNF-alpha expression was not impeded by fibrinogen deficiency. Thus, fibrin(ogen) is an important, but context-dependent, determinant of arthritis, and one mechanism linking fibrin(ogen) to joint disease is coupled to alphaMbeta2-mediated inflammatory processes.     

Fibrin structure and wound healing

Fibrinogen and fibrin play an important role in blood clotting, fibrinolysis, cellular and matrix interactions, inflammation, wound healing, angiogenesis, and neoplasia. The contribution of fibrin(ogen) to these processes largely depends not only on the characteristics of the fibrin(ogen) itself, but also on interactions between specific-binding sites on fibrin(ogen), pro-enzymes, clotting factors, enzyme inhibitors, and cell receptors. In this review, the molecular and cellular biology of fibrin(ogen) is reviewed in the context of cutaneous wound repair. The outcome of wound healing depends largely on the fibrin structure, such as the thickness of the fibers, the number of branch points, the porosity, and the permeability. The binding of fibrin(ogen) to hemostasis proteins and platelets as well as to several different cells such as endothelial cells, smooth muscle cells, fibroblasts, leukocytes, and keratinocytes is indispensable during the process of wound repair. High-molecular-weight and low-molecular-weight fibrinogen, two naturally occurring variants of fibrin, are important determinants of angiogenesis and differ in their cell growth stimulation, clotting rate, and fibrin polymerization characteristics. Fibrin sealants have been investigated as matrices to promote wound healing. These sealants may also be an ideal delivery vehicle to deliver extra cells for the treatment of chronic wounds.     

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

Adhesion and spreading of cultured human umbilical vein endothelial cells on fibrin surfaces of varying structure were characterized to understand better the interactions occurring between endothelium and fibrin at sites of vascular injury. Fibrin prepared with reptilase, which cleaves only fibrinopeptide A from fibrinogen, and fibrin prepared with thrombin, which cleaves both fibrinopeptide A and fibrinopeptide B, equally supported endothelial cell adhesion. In contrast, only fibrin made with thrombin mediated endothelial cell spreading, as assessed by fluorescence microscopy of cells stained with rhodamine phalloidin to identify actin stress fibers or by scanning electron microscopy. Fibrin prepared with reptilase failed to support cell spreading. To further investigate the role of the amino terminus of the fibrin beta chain after fibrinopeptide B cleavage in promoting cell spreading, protease III from Crotalus atrox venom was used to specifically cleave the amino-terminal 42 residues of the fibrinogen B beta chain. After clotting with thrombin, this fibrin derivative lacking B beta 1-42 failed to support significant cell spreading. Spreading on fibrin was unaffected by depletion of Weibel-Palade bodies from endothelial cells, indicating that the spreading was independent of stimulated von Willebrand factor release. We conclude that endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and involves residues 15-42 of the fibrin beta chain.     

The Pl(A2) polymorphism of integrin beta(3) enhances outside-in signaling and adhesive functions

Genetic factors are believed to influence the development of arterial thromboses. Because integrin alpha(IIb)beta(3) plays a crucial role in thrombus formation, we analyzed receptor adhesive properties using Chinese hamster ovary and human kidney embryonal 293 cells overexpressing the Pl(A1) or Pl(A2) polymorphic forms of alpha(IIb)beta(3). Soluble fibrinogen binding was no different between Pl(A1) and Pl(A2) cells, either in a resting state or when alpha(IIb)beta(3) was activated with anti-LIBS6. Pl(A1) and Pl(A2) cells bound equivalently to immobilized fibronectin. In contrast, significantly more Pl(A2) cells bound to immobilized fibrinogen in an alpha(IIb)beta(3)-dependent manner than did Pl(A1) cells. Disruption of the actin cytoskeleton by cytochalasin D abolished the increased binding of Pl(A2) cells. Compared with Pl(A1) cells, Pl(A2) cells exhibited a greater extent of polymerized actin and cell spreading, enhanced tyrosine phosphorylation of pp125(FAK), and greater fibrin clot retraction. These adhesion differences appear to depend on a signaling mechanism sensitive to receptor occupancy. Thus, the Pl(A2) polymorphism altered integrin-mediated functions of adhesion, spreading, actin cytoskeleton rearrangement, and clot retraction.     

Human fibrinogen

The structure and physical properties of human fibrinogen and fibrin are reviewed along with methods for the detection of products of their metabolism. Interactions of human fibrinogen with thrombin, factor XIII, plasminogen, glycoprotein IIb/IIIa, and other proteins are related to their relevance to thrombosis and hemostasis. To the extent information is available, the structural determinants of these interactions are delineated, and kinetic and thermodynamic parameters associated with the interactions are listed. Individual steps in the reaction pathway for the conversion of fibrinogen to cross-linked fibrin are characterized. The altered hemostatic properties of mutational variants of fibrinogen are related to their altered structure. The structures of the genes coding for the polypeptide chains of fibrinogen are discussed along with the current state of knowledge of the control and regulation of fibrinogen synthesis. Fibrinogen catabolism and fibrinolysis are also reviewed.     

4‐Thio‐deoxyuridylate‐modified thrombin aptamer and its inhibitory effect on fibrin clot formation,platelet aggregation and thrombus growth on subendothelial matrix

Summary. Background: The consensus thrombin aptamer C15‐mer is a single‐stranded DNA of 15 nucleotides [d(GGTTGGTGTGGTTGG)] that was identified by the selection of thrombin‐binding molecules from a large combinatorial library of oligonucleotides. It is capable of inhibiting thrombin at nanomolar concentrations through binding to a specific region within thrombin exosite 1. As has been shown in our earlier studies, the 4‐thio‐deoxyuridylate (s4dU)‐containing oligonucleotides have high affinity for a number of proteins, due to the reduced hydrophilic character of the modified oligonucleotide. Methods: Three different analogs of the original thrombin‐inhibiting sequence, in which some of the thymidylate residues were replaced by 4‐thio‐deoxyuridylates, were synthesized. The inhibitory effect of modified aptamers was tested on thrombin‐catalyzed fibrin clot formation and fibrinopeptide A release from fibrinogen, thrombin‐induced platelet aggregation/secretion, and the formation of thrombus on coverslips coated with human collagen type III, thrombin‐treated fibrinogen or subendothelial matrix of human microvascular endothelial cells. Results: As compared with the C15‐mer, the analog with the sequence GG(s4dU)TGG(s4dU)G(s4dU)GGT(s4dU)GG (UC15‐mer) showed a 2‐fold increased inhibition of thrombin‐catalyzed fibrin clot formation, fibrinopeptide A release, platelet aggregation and secretion in human plasma and thrombus formation on thrombin‐treated fibrinogen surfaces under flow conditions. Concerning the inhibition of thrombin‐induced fibrin formation from purified fibrinogen and activation of washed platelets, UC15‐mer was 3‐fold and twelve‐fold more effective than C15‐mer, respectively. Conclusion: The replacement of four thymidylate residues in C15‐mer by 4‐thio‐deoxyuridylate resulted in a new thrombin aptamer with increased anticoagulant and antithrombotic properties.     

Leukocyte engagement of fibrin(ogen) via the integrin receptor alphaMbeta2/Mac-1 is critical for host inflammatory response in vivo

The leukocyte integrin alpha(M)beta(2)/Mac-1 appears to support the inflammatory response through multiple ligands, but local engagement of fibrin(ogen) may be particularly important for leukocyte function. To define the biological significance of fibrin(ogen)-alpha(M)beta(2) interaction in vivo, gene-targeted mice were generated in which the alpha(M)beta(2)-binding motif within the fibrinogen gamma chain (N(390)RLSIGE(396)) was converted to a series of alanine residues. Mice carrying the Fibgamma(390-396A) allele maintained normal levels of fibrinogen, retained normal clotting function, supported platelet aggregation, and never developed spontaneous hemorrhagic events. However, the mutant fibrinogen failed to support alpha(M)beta(2)-mediated adhesion of primary neutrophils, macrophages, and alpha(M)beta(2)-expressing cell lines. The elimination of the alpha(M)beta(2)-binding motif on fibrin(ogen) severely compromised the inflammatory response in vivo as evidenced by a dramatic impediment in leukocyte clearance of Staphylococcus aureus inoculated into the peritoneal cavity. This defect in bacterial clearance was due not to diminished leukocyte trafficking but rather to a failure to fully implement antimicrobial functions. These studies definitively demonstrate that fibrin(ogen) is a physiologically relevant ligand for alpha(M)beta(2), integrin engagement of fibrin(ogen) is critical to leukocyte function and innate immunity in vivo, and the biological importance of fibrinogen in regulating the inflammatory response can be appreciated outside of any alteration in clotting function.     

Mechanism of ancrod anticoagulation: A direct proteolytic effect on fibrin

Fibrin formed in response to ancrod, reptilase, or thrombin was reduced by beta-mercaptoethanol and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It was found that ancrod progressively and totally digested the alpha-chains of fibrin monomers at sites different than plasmin; however, further digestion of fibrin monomers by either reptilase or thrombin was not observed. Highly purified ancrod did not activate fibrin-stabilizing factor (FSF); however, the reptilase preparation used in these experiments, like thrombin, activated FSF and thereby promoted cross-link formation. Fibrin, formed by clotting purified human fibrinogen with ancrod, reptilase, or thrombin for increasing periods of time in the presence of plasminogen, was incubated with urokinase and observed for complete lysis. Fibrin formed by ancrod was strikingly more vulnerable to plasmin digestion than was fibrin formed by reptilase or thrombin. The lysis times for fibrin formed for 2 hr by ancrod, reptilase, or thrombin were 18, 89, and 120 min, respectively. Evidence was also obtained that neither ancrod nor reptilase activated human plasminogen. These results indicate that fibrin formed by ancrod is not cross-linked and has significantly degraded alpha-chains: as expected, ancrod-formed fibrin is markedly susceptible to digestion by plasmin.     

Fibrinogen Bethesda: a congenital dysfibrinogenemia with delayed fibrinopeptide release

A dysfibrinogenemia (fibrinogen Bethesda) was detected in a 9 yr old male of Mexican-English extraction who had a lifelong history of mild bleeding diathesis. The prothrombin and partial thromboplastin times were moderately prolonged; the thrombin and Reptilase times were markedly prolonged. The plasma fibrinogen level was normal by conventional methods but was markedly reduced by the Clauss method. Results of all other tests for clotting factors, fibrinolysis, antithrombin levels, clot stabilization, and fibrin(ogen) degradation products were normal. The patient's plasma and fibrinogen inhibited the clotting of normal plasma or fibrinogen by thrombin. Family studies revealed that the propositus' mother and two siblings exhibited these abnormalities to a lesser degree and indicated an autosomal dominant inheritance. Fibrinogen Bethesda was similar to normal fibrinogen in the following respects: metabolic turnover time (measured in the propositus' mother); immunodiffusion, ultracentrifugal, electrophoretic (on cellulose acetate or polyacrylamide gel), and chromatographic (on DEAE-cellulose) characteristics; sialic acid content; and aggregation of fibrin monomers. By contrast, fibrinogen Bethesda gave an abnormal immunoelectrophoretic pattern especially when whole plasma (as opposed to purified fibrinogen) was examined, and it showed a pronounced decrease in the rate of fibrinopeptide release by thrombin. This decrease, which was shown to involve both fibrinopeptides A and B, distinguishes fibrinogen Bethesda from previously reported dysfibrinogenemias.     

Methods for the determination of factor XIII/XIIIa

Activated factor XIII (FXIIIa) plays an important role in the final stage of the coagulation cascade by covalent crosslinking of fibrin strands. As a transglutaminase FXIIIa catalyses the generation of intermolecular amide bonds between lysine and glutamine residues resulting in a complex three-dimensional clot structure. Enhanced clot stability is supported by covalent binding of cytosceleton factors like actin and myosin. Moreover, the clot is protected against premature lysis by the incorporation of fibrinolysis inhibitors like alpha(2)-antiplasmin and thrombin activatable fibrinolysis inhibitor (TAFI). A covalent crosslinking of the fibrin strands with extra-cellular matrix proteins like fibronectin, vitronectin, collagen and von Willebrand factor (vWF) immobilizes the clot at the site of injury. Moreover FXIII supports the healing process of damaged tissue. In this review assays for determination of FXIIIa activity, FXIII concentration and identification of the FXIIIVal34Leu polymorphism are shown.     

Soluble fibrin degradation products potentiate tissue plasminogen activator-induced fibrinogen proteolysis.

Despite its affinity for fibrin, tissue plasminogen activator (t-PA) administration causes systemic fibrinogenolysis. To investigate the mechanism, t-PA was incubated with plasma in the presence or absence of a fibrin clot, and the extent of fibrinogenolysis was determined by measuring B beta 1-42. In the presence of fibrin, there is a 21-fold increase in B beta 1-42 levels. The potentiation of fibrinogenolysis in the presence of fibrin is mediated by soluble fibrin degradation products because (a) the extent of t-PA induced fibrinogenolysis and clot lysis are directly related, (b) once clot lysis has been initiated, fibrinogenolysis continues even after the clot is removed, and (c) lysates of cross-linked fibrin clots potentiate t-PA-mediated fibrinogenolysis. Fibrin degradation products stimulate fibrinogenolysis by binding t-PA and plasminogen because approximately 70% of the labeled material in the clot lysates binds to both t-PA- and plasminogen-Sepharose, and only the bound fractions have potentiating activity. The binding site for t-PA and plasminogen is on the E domain because characterization of the potentiating fragments using gel filtration followed by PAGE and immunoblotting indicates that the major species is (DD)E complex, whereas minor components include high-molecular weight derivatives containing the (DD)E complex and fragment E. In contrast, D-dimer is the predominant species found in the fractions that do not bind to the adsorbants, and it has no potentiating activity. Thus, soluble products of t-PA-induced lysis of cross-linked fibrin potentiate t-PA-mediated fibrinogenolysis by providing a surface for t-PA and plasminogen binding thereby promoting plasmin generation. The occurrence of this phenomenon after therapeutic thrombolysis may explain the limited clot selectivity of t-PA.     

The relative kinetics of clotting and lysis provide a biochemical rationale for the correlation between elevated fibrinogen and cardiovascular disease

BACKGROUND: Elevated plasma fibrinogen is a well known risk factor for cardiovascular disease. The mechanistic rationale for this is not known. OBJECTIVES: These studies were carried out to determine the fibrinogen concentration dependencies of clotting and lysis times and thereby determine whether these times rationalize the correlation between an increased risk of cardiovascular disease and elevated plasma fibrinogen. METHODS: The time courses of clot formation and lysis were measured by turbidity in systems comprising a) fibrinogen, thrombin and plasmin, or b) fibrinogen, thrombin, plasminogen and t-PA, or c) plasma, thrombin and t-PA. From the lysis times, k(cat) and K(m) values for plasmin action on fibrin were determined. RESULTS: The time to clot increased linearly from 2.9 to 5.6 minutes as the fibrinogen concentration increased from 1 to 9 microM and did not increase further as the fibrinogen concentration was raised to 20 microM. In contrast, the clot lysis time increased linearly over the input fibrinogen concentration range of 2 to 20 microM. A similar linear trend was found in the two systems with t-PA and plasminogen. Apparent K(m) and k(cat) values for plasmin were 1.1 +/- 0.6 microM and 28 +/- 2 min(-1), respectively. K(m) values for plasmin in experiments initiated with t-PA and plasminogen were 1.6 +/- 0.2 microM in the purified system and 2.1 +/- 0.9 microM in plasma. CONCLUSION: As the concentration of fibrinogen increases, especially above physiologic level, the balance between fibrinolysis and clotting shifts toward the latter, providing a rationale for the increased risk of cardiovascular disease associated with elevated fibrinogen.     

Inhibition of complement-mediated opsonization and phagocytosis of Streptococcus pyogenes by D fragments of fibrinogen and fibrin bound to cell surface M protein

The biological effects of the binding of fibrin(ogen) degradation products to M protein-bearing group A streptococci were investigated. Type 24 group A streptococci bind fibrinogen degradation products of the D family, but not fragment E. Binding appears to be mediated by M protein, since a large peptide of this molecule (pep M24) bound to fragments containing the terminal domains of the fibrinogen molecule (D, X, and Y), but not fragment E, and pep M24 inhibited the binding of digested fibrinogen to streptococcal cells. An M protein-binding site occurs on fragment D3 and, therefore, differs from several functional sites present on D1 but not D3, including the fibrin polymerization site, the two gamma chain crosslink sites, and the bindings sites for platelet fibrinogen receptor, staphylococcal clumping factor, and ionized calcium. Bound fibrinogen degradation products prevented deposition of C3 on the streptococcal cell surface, and, in consequence, prevented phagocytosis by neutrophils in nonimmune blood. The average affinity of D fragments for the streptococcal cell surface was approximately 30 times lower than that of native fibrinogen, and a terminal plasmic digest was approximately 50 times less potent in inhibiting opsonization by C3. However, physiologic concentrations of digested fibrinogen sufficed to inhibit opsonization and phagocytosis completely. Digests of crosslinked fibrin clot also inhibited opsonization, although slightly less effectively than did fibrinogen digests. The antiopsonic effect of fibrin(ogen) degradation products may be relevant to circumstances in which fibrin(ogen)olysis is occurring, e.g., exudation and suppuration.     

The regulation by fibrinogen and fibrin of tissue plasminogen activator kinetics and inhibition by plasminogen activator inhibitor 1

BACKGROUND: Tissue plasminogen activator (tPA) is unusual in the coagulation and fibrinolysis cascades in that it is produced as an active single-chain enzyme (sctPA) rather than a zymogen. Two chain tPA (tctPA) is produced by plasmin but there are conflicting reports in the literature on the behaviour of sc- and tctPA and little work on inhibition by the specific inhibitor plasminogen activator inhibitor-1 (PAI-1) under physiological conditions. OBJECTIVES: To perform a systematic study on the kinetics of sctPA and tctPA as plasminogen activators and targets for PAI-1. METHODS: Detailed kinetic studies were performed in solution and in the presence of template stimulators, fibrinogen and fibrin, including native fibrin and partially digested fibrin. Numerical simulation techniques were utilized to cope with the challenges of investigating kinetics of activation and inhibition in the presence of fibrin(ogen). RESULTS: Enzyme efficiency (k(cat)/K(m)) was higher for tctPA than sctPA in solution with chromogenic substrate (3-fold) and plasminogen (7-fold) but in the presence of templates, such as fibrinogen and native or cleaved fibrin, the difference disappeared. sctPA was more susceptible to PAI-1 in buffer solution and in the presence of fibrinogen; however, in the presence of fibrin, PAI-1 inhibited more slowly and there was no difference between sc and tctPA. CONCLUSIONS: Fibrinogen and fibrin modulate the activity of tPA differently in regard to their activation of plasminogen and inhibition by PAI-1. Fibrinogen and fibrin stimulate tPA activity against plasminogen but fibrin protects tPA from PAI-1 to promote fibrinolysis.     

Fibrinogen deposition and macrophage-associated fibrin formation in malignant and nonmalignant lymphoid tissue.

Nonmalignant lymphoid tissue and tissue from patients with nodular sclerosis, Hodgkin's disease, and large cell lymphocytic lymphoma was examined by immunohistochemical techniques for the occurrence in situ of components of coagulation and fibrinolysis reaction pathways. Staining for material interpreted as fibrinogen was observed in abundance in both malignant and reactive lymphoid tissue. Fibrin also occurred to a variable extent but focally in all tissues. Components of coagulation pathways, including tissue factor, factor VII, factor X, and factor XIII ("a" subunit), were restricted to tissue macrophages. Double-labeling techniques revealed fibrin in direct apposition to tissue macrophages. We conclude that fibrinogen and fibrin occur in both benign and malignant lymphoid tissue and that the transformation of fibrinogen to fibrin is attributable to macrophage-initiated thrombin formation. We postulate that both systemic and local hypercoagulability associated with these disorders may be attributable to macrophage activation resulting in expression of procoagulant activity.     

Characterizing fibrin glue performance as modulated by heparin,aprotinin, and factor XIII

We describe the performance of fibrin glue (FG) as modulated by heparin, aprotinin, or factor XIII levels. In vitro tests and a rat kidney excision model demonstrated that the hemostatic efficacy of fibrin was not modulated by aprotinin. Overlapping rat skin sections demonstrated that adhesion strength (AS) was proportional to the area of overlap as well as to fibrinogen levels. AS was not modulated by exogenous heparin or aprotinin and was independent of the endogenous factor XIII in fibrinogen. SDS-PAGE developed by Coomassie or Western blots with anti-gamma chain antibody confirmed that normal skin sections contain adequate trans-glutaminase to maximally cross-link normal, as well as XIII-depleted, fibrin. Fibrin glue (FG) sprayed onto rat skin incision wounds with a dual channel spray applicator acted in 2 phases: initially (day 1), compared to wounds stapled without or treated with only thrombin, FG significantly increased breaking strength. In the second phase of wound healing (after day 3), all groups achieved increased but equivalent breaking strength. FG containing aprotinin (to 3000 U/m; Immuno, Behringwerke, Germany) exhibited initial tissue bonding strength equivalent to fibrin without aprotinin, but histological examination showed delayed fibrinolysis and a concomitant slower regeneration of granulation tissue. Thus, our data indicated that aprotinin was not particularly beneficial to wound healing and that the endogenous factor XIII level in the fibrinogen did not contribute significantly to skin bonding. Rather, the tissue supplied adequate trans-glutaminase activity required to crosslink fibrin to itself and to the tissue.