Fibrin Alpha-chain Research Articles

Co-Administration of Factor XIII with Hemostatic Agents in Hemophilia Promotes Clot Stability and Composition

Hemophilia A results from the absence, deficiency, or inhibition of factor VIII. Bleeding is treated with use of two broad classes of hemostatic agents; replacement factors (recombinant or plasma-derived human FVIII [FVIII] or factor IX) or bypassing agents (recombinant activated factor VIIa [rFVIIa], plasma-derived anti-inhibitor coagulant complex [FEIBA], or recombinant porcine FVIII [rpFVIII]). Despite treatment with hemostatic agents, some hemophilia patients experience refractory bleeding. Factor XIII-A2B2 (FXIII) is a thrombin-activated protransglutaminase. Activated FXIII (FXIIIa) crosslinks fibrin to stabilize clots and increase red blood cell retention during clot contraction, and therefore, clot weight; these functions have been specifically associated with fibrin alpha-chain crosslinking and production of high molecular weight (HMW) crosslinked fibrin species. Although pilot studies have indicated that in hemophilia co-administration of FXIII with hemostatic agents improves hemostasis, the biochemical mechanisms evoked during co-administration have yet to be elucidated. We hypothesize that co-administration of FXIII with hemostatic agents enhances hemostasis in hemophilia by accelerating FXIII activation, increasing fibrin crosslinking, and improving the composition of contracted clots.We first analyzed in vitrotissue factor-activated coagulation in FVIII-deficient plasmas (n=5) with or without buffer (normal saline with 0.75% bovine serum albumin), plasma-derived FXIII (2 U/mL), and/or recombinant human FVIII (1 U/mL). Reactions were performed at 37°C and quenched with urea/EDTA at varying time points. Clot lysates were separated on 10% Tris-glycine gels, blotted, probed for FXIII(a) and fibrin(ogen), and analyzed by densitometry. Compared to buffer, FXIII, or FVIII-treated hemophilic plasma, FVIII+FXIII co-treatment increased the formation rate and level of FXIIIa. Furthermore, compared to buffer or FXIII-treated hemophilic plasma, both FVIII-treatment and FVIII+FXIII co-treatment increased the formation rate and level of crosslinked fibrin species (gamma chain and HMW species). Notably, compared to FVIII-treatment (alone), FVIII+FXIII co-treatment increased the formation rate and level of HMW crosslinked fibrin species.Next, we analyzed clot formation in whole blood obtained from untreated FVIII-deficient (n=2) or FVIII-inhibitor (n=6) patients. Citrated whole blood was pre-incubated at 37°C for 30 minutes with or without buffer, FXIII (2 U/mL), rFVIIa (25 nM), FEIBA (1 U/mL), or rpFVIII (1 U/mL), alone and in combination with FXIII, and clot formation was triggered with tissue factor and recalcification. In thrombelastography assays, compared to buffer or FXIII-treated hemophilic whole blood, rFVIIa, FEIBA, and rpFVIII treatments alone or with FXIII co-treatment shortened the time to clot formation (clot time [R]). Compared to buffer, FXIII, FEIBA or rpFVIII-treated hemophilic whole blood, rFVIIa-treatment (alone) and co-treatment with rFVIIa+FXIII or FEIBA+FXIIII increased clot stability (area under the elastic curve). In whole blood clot contraction assays, compared to buffer, FXIII, rFVIIa, FEIBA, or rpFVIII-treated hemophilic whole blood, co-treatment with rFVIIa+FXIII, FEIBA+FXIII, or rpFVIII+FXIII significantly increased clot weight.Our data show that in hemophilic plasmas, co-administration of FXIII with hemostatic agents accelerates FXIIIa activation, resulting in accelerated and increased HMW species formation. Furthermore, in hemophilic whole blood, co-administration of FXIII with hemostatic agents improved clot biophysical characteristics and increased clot weight. Collectively, these data suggest co-administration of FXIII with conventional hemostatic agents may promote clot stability and improve the composition of contracted clots by enhancing fibrin alpha-chain crosslinking. DisclosuresNo relevant conflicts of interest to declare.

A Fibrin-Specific Monoclonal Antibody from a Designed Phage Display Library Inhibits Clot Formation and Localizes to Tumors In Vivo

Fibrin formation from fibrinogen is a rare process in the healthy organism but is a pathological feature of thrombotic events, cancer and a wide range of inflammatory conditions. We have designed and constructed an antibody phage display library (containing 13 billion clones) for the selective recognition of the N-terminal peptide of fibrin alpha chain. The key structural feature for selective fibrin binding was a K94E mutation in the VH domain. From this library, an antibody was isolated (termed AP2), which recognizes the five N-terminal amino acids of fibrin with high affinity (Kd=44nM), but does not bind to fibrinogen. The AP2 antibody could be expressed in various formats (scFv, small immune protein and IgG) and inhibited fibrin clot formation in a concentration-dependent manner. Moreover, the AP2 antibody stained the fibrin-rich provisional stroma in solid tumors but did not exhibit any detectable staining toward normal tissues. Using a radioiodinated antibody preparation and quantitative biodistribution studies in tumor-bearing mice, AP2 was shown to selectively localize to fibrin-rich F9 murine teratocarcinomas, but not to SKRC-52 human kidney cancer xenografts. Collectively, the experiments indicate that the AP2 antibody recognizes fibrin in vitro and in vivo. The antibody may facilitate the development of fibrin-specific therapeutic agents.

Abstract 5483: The Inflammatory Protein Complement C3 is a Novel Substrate for Factor XIII and Impairs Fibrin Clot Lysis

Introduction: Inflammation is fundamentally important in the pathogenesis of cardiovascular disease (CVD) and increasing evidence supports a functional role for complement C3. Using a functional proteomics approach we have identified zymogen and activated C3 in perfused solubilized clots, suggesting C3 influences thrombus formation. Hypothesis: In this study we assessed the hypothesis that complement C3 interacts with coagulation and fibrinolytic factors to influence thrombosis. Methods and Results: Analysis of 123 paired plasma and serum samples revealed that 24.8% (SD 7.6) of plasma C3 was incorporated into the clot. To explore the mechanism of incorporation we investigated binding interactions between C3, fibrin and FXIII using ELISA based binding assays and surface plasmon resonance and found that C3 bound to immobilized fibrinogen and FXIII in a concentration dependent manner. Further analysis of the interactions between C3 and FXIII demonstrated that activated FXIII (FXIIIa) incorporated a known FXIIIa substrate, 5-(biotinamido)pentylamine, into immobilized C3 in a concentration dependent manner and FXIIIa also induced the formation of high-molecular weight C3 polymers, indicating that C3 is a FXIII substrate. Furthermore, FXIIIa incorporated plasma C3 into immobilized fibrin and Western blot analysis of clots formed from purified fibrinogen and C3 indicated high molecular weight bands that cross-reacted with specific antibodies against C3 and fibrinogen, suggesting that C3 is cross-linked to fibrin alpha chains. In turbidimetric assays using purified proteins C3 prolonged fibrin clot lysis times in a concentration dependent manner and Western blot analysis of lysed clots indicated that C3 was cleaved by plasmin, suggesting impaired fibrinolysis is due to substrate competition for plasmin. Increased plasma C3 concentration was significantly associated with increased ex vivo plasma clot density and prolonged fibrinolysis times, after adjusting for fibrinogen concentrations, to support our in vitro results. In conclusion, taken together these data indicate that complement C3 influences key processes involved in fibrin formation and fibrinolysis supporting a role for C3 in cardiovascular thrombosis.

Anti-thrombosis Repertoire of Blood-feeding Horsefly Salivary Glands

Blood-feeding arthropods rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands although their saliva has been thought to contain wide range of physiologically active molecules. In traditional Eastern medicine, horseflies are used as anti-thrombosis material for hundreds of years. By proteomics coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which exert mainly on anti-thrombosis functions, were identified and characterized from 60,000 pairs of salivary glands of the horsefly Tabanus yao Macquart (Diptera, Tabanidae). They are: (I) ten fibrin(ogen)olytic enzymes, which hydrolyze specially alpha chain of fibrin(ogen) and are the first family of fibrin(ogen)olytic enzymes purified and characterized from arthropods; (II) another fibrin(ogen)olytic enzyme, which hydrolyzes both alpha and beta chain of fibrin(ogen); (III) ten Arg-Gly-Asp-motif containing proteins acting as platelet aggregation inhibitors; (IV) five thrombin inhibitor peptides; (V) three vasodilator peptides; (VI) one apyrase acting as platelet aggregation inhibitor; (VII) one peroxidase with both platelet aggregation inhibitory and vasodilator activities. The first three families are belonging to antigen five proteins, which show obvious similarity with insect allergens. They are the first members of the antigen 5 family found in salivary glands of blood sucking arthropods to have anti-thromobosis function. The current results imply a possible evolution from allergens of blood-sucking insects to anti-thrombosis agents. The extreme diversity of horsefly anti-thrombosis components also reveals the anti-thrombosis molecular mechanisms of the traditional Eastern medicine insect material.

Purification and characterization of a novel fibrinolytic protease from Fusarium sp. CPCC 480097

A novel fibrinolytic enzyme from Fusarium sp. CPCC 480097, named Fu-P, was purified to electrophoretic homogeneity using ammonium sulfate precipitation and ion exchange and gel filtration chromatography. Fu-P, a single protein had a molecular weight of 28 kDa, which was determined by SDS-PAGE and gel filtration chromatography. The isoelectric point of Fu-P determined by isoelectric focusing electrophoresis (IEF) was 8.1, and the optimum temperature and pH value were 45 degrees C and 8.5, respectively. Fu-P cleaved the alpha-chain of fibrin (ogen) with high efficiency, and the beta-chain and gamma-gamma (gamma-)-chain with lower efficiency. Fu-P activity was inhibited by EDTA and PMSF, and the enzyme exhibited a high specificity for the chymotrypsin substrate S-2586. Fu-P was therefore identified as a chymotrypsin-like serine metalloprotease. The first 15 amino acids of the N-terminal sequence of Fu-P were Q-A-S-S-G-T-P-A-T-I-R-V-L-V-V and showed no homology with that of other known fibrinolytic enzymes. This protease may have potential applications in thrombolytic therapy and in thrombosis prevention.

Evidence that alpha2-antiplasmin becomes covalently ligated to plasma fibrinogen in the circulation: a new role for plasma factor XIII in fibrinolysis regulation.

Plasma alpha2-antiplasmin (alpha2AP) is a rapid and effective inhibitor of the fibrinolytic enzyme plasmin. Congenital alpha2AP deficiency results in a severe hemorrhagic disorder due to accelerated fibrinolysis. It is well established that in the presence of thrombin-activated factor XIII (FXIIIa), alpha2AP becomes covalently ligated to the distal alpha chains of fibrin or fibrinogen at lysine 303 (two potential sites per molecule). Some time ago we showed that alpha2AP is covalently linked to plasma fibrinogen . That singular observation led to our hypothesis that native plasma factor XIII (FXIII), which is known to catalyze covalent cross-linking of fibrinogen in the presence of calcium ions, can also incorporate alpha2AP into fibrinogen in the circulation. We now provide evidence that FXIII incorporates I 125-labelled alpha2AP into the Aalpha-chain sites on fibrinogen or fibrin. We also measured the content of alpha2AP in isolated plasma fibrinogen fractions by ELISA and found that substantial amounts were present (1.2-1.8 moles per mole fibrinogen). We propose that alpha2AP becomes ligated to fibrinogen while in the circulation through the action of FXIII, and that its immediate presence in plasma fibrinogen contributes to regulation of in vivo fibrinolysis.

A novel extracellular protease with fibrinolytic activity from the culture supernatant of Cordyceps sinensis: purification and characterization

A novel serine protease with fibrinolytic activity named CSP was purified from the culture supernatant of the fungus Cordyceps sinensis, a kind of Chinese herbal medicine. Analysis of the purified enzyme by SDS-PAGE indicated that CSP was a single polypeptide chain with an apparent molecular weight of 31 kDa, and N-terminal sequencing revealed that the first ten amino acid residues of the enzyme were Ala-Leu-Ala-Thr-Gln-His-Gly-Ala-Pro-Trp-. When casein was used as a substrate, the proteolytic activity of CSP reached its maximum at pH 7.0 and 40 degrees C. The effect of chemical agents on the enzyme activity indicated that CSP is a serine protease with a free cysteine residue near the active site. It hydrolysed fibrinogen, fibrin and casein with a high efficiency, while hydrolysing bovine serum albumin (BSA) and human serum albumin (HSA) to a lesser extent. CSP was found to be a plasmin-like protease, but not a plasminogen activator, and it preferentially cleaved the A alpha chain of fibrinogen and the alpha-chain of fibrin. Therefore, the extracellular protein CSP may represent a potential new therapeutic agent for the treatment of thrombosis.

Association of citrullinated proteins with synovial exosomes

In addition to releasing proteins and mediators, cells also release membrane vesicles (exosomes and apoptotic blebs) into the extracellular environment. Apoptotic blebs contain multiple autoantigens, but few data are available concerning the protein content of exosomes. Exosomes are formed during an immune response and can directly stimulate T cells or bind to dendritic cells. The aim of this study was to identify the nature of synovial exosomes from patients with different rheumatic diseases and to examine their potential autoantigenic content, which may be involved in the induction of an autoimmune response. Synovial exosomes from patients with rheumatoid arthritis (RA), patients with reactive arthritis, and patients with osteoarthritis were purified, analyzed by electron microscopy, and labeled with immunogold to detect IgG and IgM molecules. Autoantigen content was identified by 2-dimensional electrophoresis-immunoblotting and subsequent mass spectrometry. In order to investigate the presence of citrullinated proteins, immunoblotting with anticitrulline antibodies was performed. Citrullinated proteins were observed in all exosome preparations, in contrast to other autoantigenic proteins (e.g., BiP and heterogeneous nuclear RNP A2) that were previously observed in RA and other autoimmune diseases. These citrullinated proteins included the fibrin alpha-chain fragment, fibrin beta-chain, fibrinogen beta-chain precursor, fibrinogen D fragment, and the Sp alpha (CD5 antigen-like protein) receptor. Purification of synovial exosomes led to the detection of citrullinated fibrinogen and citrullinated Sp alpha associated with IgM and IgG. Synovial exosomes contain citrullinated proteins, which are known to be autoantigens in RA. Although immune mechanisms in which exosomes carry citrullinated peptides could play an important role in the induction and distribution of citrullinated proteins, there must be a specific recognition of these proteins that is unique to the RA immune system.

Factor XIIIa Mediated Attachment of S. aureus Fibronectin-Binding Protein A (FnbA) to Fibrin: Identification of Gln103 as a Major Cross-Linking Site

In the present study we investigated the role of factor XIIIa reactive Gln and Lys sites of staphylococcal FnbA receptor in cross-linking reaction with alpha chains of fibrin. For this purpose we produced two recombinant FnbA mutants in which either a single Gln103 site (1Q FnbA) or all identified reactive Gln103, 105, 783, 830 and Lys157, 503, 620, 762 sites (4Q4K FnbA) were substituted with Ala residues. The results of FXIIIa-catalyzed incorporation of dansylcadaverine and dansylated peptide patterned on the NH2-terminal segment of fibronectin revealed that the reactivity of Gln substrate sites was drastically reduced in 1Q FnbA and 4Q4K FnbA mutants, while the reactivity of Lys substrate sites was only moderately decreased in 4Q4K FnbA. When it was tested in the FXIIIa-mediated fibrin cross-linking reaction, the 1Q FnbA mutant exhibited about 70-85% reduction in reactivity compared to that of the wild-type FnbA. These results demonstrate that FnbA participates in cross-linking to alpha chains of fibrin predominantly via its Gln103 reactive site. Several minor sites, including residues replaced in 4Q4K FnbA mutant, contributed to an additional 15-30% of the total fibrin cross-linking reactivity of FnbA. Comparison of amino acid sequences that follow the major reactive Gln site in FnbA and several known substrate proteins revealed that FXIIIa displays a preference for the glutamine residue in an xQAxBxPx sequence, where Q represents reactive glutamine, x is any amino acid residue, A is a polar residue, B is either valine or leucine, and P is proline.

Influence of HLA-DR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrin

Rheumatoid arthritis (RA), a chronic inflammatory joint disease, develops in patients expressing particular HLA-DR alleles. RA patients' sera contain antibodies to a post-translationally modified form of fibrin on which arginyl residues are transformed in citrullins. We tested whether HLA-DR alleles influence the production of anti-citrullinated fibrin antibodies in RA patient sera and whether the replacement of arginyl residues by citrullyl residues on fibrin peptides could modify their binding to RA-associated HLA-DR molecules and their recognition by T cells in RA patients and controls. We found that RA-associated HLA-DR alleles are also associated with presence of anti-citrullinated fibrin antibodies in RA patient sera. Multiple peptides from the alpha and beta chain of fibrin are capable to bind many HLA-DR alleles. RA-associated HLA-DR alleles are good fibrin peptide binders. However, citrullination does not influence fibrin peptide binding to HLA-DR or fibrin peptide recognition by T cells. Finally, peripheral blood T cells that recognize native or citrullinated fibrin peptides are common in RA patients and very uncommon in normal controls. These results suggest that citrullination of fibrin has nothing to do with peptide/HLA-DR/T cell interaction and is merely involved in the definition of B-cell epitopes.

Safety and Efficacy of Alfimeprase for Restoring Function in Occluded Central Venous Catheters: Interim Results of a Phase 2, Multicenter, Randomized, Double-Blind Study (NuCath).

Alfimeprase, a recombinantly produced 22.6 kilodalton metalloproteinase, is a genetically modified variant of fibrolase. Alfimeprase, like fibrolase, proteolytically cleaves both the alpha and beta chains of fibrin(ogen) independent of plasminogen activation to plasmin and directly dissolves thrombi. Readily abundant alpha-2 macroglobulin acts as an endogenous circulating alfimeprase inactivator, forming a covalent, irreversible bond with alfimeprase which results in an inactive complex. Based on the direct thrombolytic effect of alfimeprase, rapid activity was hypothesized. We performed a Phase 2, randomized, double-blind, controlled, dose-ranging study to compare the safety and efficacy of one or two instillations of three intraluminal doses of alfimeprase (0.3 mg; 1.0 mg; and 3.0 mg) and Cathflo® Activase® (Genentech) 2.0 mg in reestablishing patency to occluded central venous access devices (CVADs). This report describes an interim analysis of 48 patients with CVAD withdrawal occlusion who were enrolled and randomized to treatment. Catheter patency was assessed at 5, 15, 30, and 120 minutes after each dose of study drug. Adverse events including bleeding events were assessed for a 30-day period after exposure to study drug. Cumulative patency rates are shown in the table. All study arms had similar patency rates at 120 minutes after the first dose. The alfimeprase 3.0 mg dose produced the highest patency rate at 120 minutes after the second dose. All three alfimeprase doses were more effective than Cathflo® Activase® during the first 30 minutes of treatment. The alfimeprase 1.0 mg and 3.0 mg doses resulted in ≥ 50% patency restoration rates at 15 minutes compared to 0% for Cathflo® Activase®. No intracranial hemorrhage, major hemorrhagic, or embolic events were reported in any treated patients at 5 days. Efficacy and safety results of this study support further evaluation of alfimeprase doses ranging from 1.0 mg to 3.0 mg for treatment of occluded CVADs.Cumulative Patency Rate (%)Alfimeprase 0.3Alfimeprase 1Alfimeprase 3Cathflo ActivaseN=13N=14N=9N=12Baseline0000D 1; 5 min1514440D 1; 15 min1550570D 1; 30 min30505725D 1; 120 min46505750D 2; 5 min46505758D 2; 15 min46505767D 2; 30 min46505767D 2; 120 min46507867D=dose; min=minutes after dose

Biophysical characterization of fibrinogen Caracas I with an Aalpha-chain truncation at Aalpha-466 Ser: identification of the mutation and biophysical characterization of properties of clots from plasma and purified fibrinogen.

Fibrinogen Caracas I is a dysfibrinogenemia with a mild bleeding tendency; a novel nonsense mutation, in the gene coding the Aalpha-chain, identified in this study as G4731T, giving rise to a new stop codon at Aalpha-Glu 467. Fibrinogen from two family members, the mother and sister of the propositus, both heterozygous for the mutation were studied, analyzing clots made from both plasma and purified fibrinogen. Clot structure and properties were characterized by turbidity, permeation, scanning electron microscopy and rheological studies. Permeation through Caracas I plasma clots was decreased, consistent with the decreased final turbidity. As shown by scanning electron microscopy, plasma clots from the patients were composed of very thin fibers, with increased fibrin density and reduced pore size. Viscoelastic measurements revealed that fibrinogen Caracas I plasma clots were much stiffer and less subject to compaction. These results demonstrate a key role of the carboxyl-terminal alpha chains of fibrin in lateral aggregation during polymerization and reinforce the utility of studying plasma clots. It is important to point out that the biophysical studies with fibrinogen purified by two different methods yielded contradictory results, which can be accounted for by selective purification of certain molecular species as seen by two-dimensional electrophoresis.

Structural alterations in hereditary dysfibrinogens.

Dysfibrinogens can be grossly divided in two groups: (1) defective thrombin-catalyzed conversion of fibrinogen molecules to fibrin monomers, and (2) defective fibrin polymerization due to structural alterations in polymerization sites, that include "A" and "a" sites, end-to-end D:D abutment surfaces, and lateral association sites involving the carboxyl terminal region of the fibrin alpha-chain. Recently, a number of mutations in the fibrinogen genes have been identified, and many of these encode changes that occur in regions of fibrinogen that have been elucidated by high-resolution structural studies. Here we focus on the structure-function relationships of fibrinogen that can be inferred from studies involving these abnormal molecules.

Congenital alpha(2)-plasmin inhibitor deficiencies: a review.

Congenital alpha(2)-plasmin inhibitor deficiencies: a review.

From perinuclear factor to citrulline, a target structure for autoantibodies in rheumatoid arthritis

Antiperinuclear factor (APF) and the so-called antikeratin (AKA), have been described 37 and 22 years ago, respectively. Now, their diagnostic value in Rheumatoid Arthritis (RA) was confirmed on thousands of patients. In the past few years the molecular targets of these RA-specific serum IgG antibodies were identified : first those of AKA were shown to be acidic variants of filaggrin in human epidermis and (pro)filaggrin-related proteins in rat oesophagus epithelium ; secondarily the target of APF in human buccal mucosa cells was demonstrated to correspond to tissue-specific forms of (pro)filaggrin. APF and AKA were proved to constitute two overlapping subgroups of antibodies belonging to a same family of antifilaggrin autoantibodies (AFA). All these AFA-targetted proteins were demonstrated to be deiminated i.e. having their arginyl residues (arginines involved in peptidic bonds) transformed into citrullyl residues (citrullines). This post-translational enzymatic modification is due to a peptidyl-arginine deiminase. AFA are unreactive with native recombinant human filaggrin but become highly reactive after enzymatic deimination of the protein. Moreover synthetic peptides derived from the human filaggrin sequence are reactive with AFA only when their arginyl residues have been substituted by citrullyl residues (citrullinated peptides). Therefore AFA are directed to peptidic epitopes in which citrullyl residues play a pivotal role, nevertheless the neighbouring residues are important since certain are permissive and participate to generation of AFA epitopes whereas others are non-permissive and make the reactivity with AFA impossible. Identification of the molecular targets of AFA allowed the development of new tests for their detection, using either (pro)filaggrin extracted from epithelia or deiminated recombinant filaggrins and/or filaggrin-derived citrullinated peptides. Several of these new tests prove to be highly efficient in the diagnosis of RA and largely more performant than the reference APF and AKA tests. They show that at least 2 out of 3 RA patients develop the very specific antifilaggrin B autoimmunity. In rheumatoid synovial tissue, (pro)filaggrin was confirmed to be absent, however several deiminated proteins were detected. Among them, only two proteins were highly reactive with AFA. They were identified as the alpha and beta chains of fibrin. Deiminated fibrin therefore appears as the major synovial target of AFA and probably correspond to their genuine target. In RA patients, the proportion of AFA among IgG was recently found to be largely higher in the synovial interstitium than in synovial fluid and serum, moreover AFA were shown to be secreted by plasma cells of the rheumatoid pannus. These results strongly suggest that the chronic conflict between the locally secreted AFA / antifibrin autoantibodies and the fibrin deposits particularly prominent in the RA synovium, play a central role in the pathophysiology of RA.

The Role of γA/γ′ Fibrinogen in Plasma Factor XIII Activation

Factor XIII zymogen activation is a complex series of events that involve fibrinogen acting in several different roles. This report focuses on the role of fibrinogen as a cofactor in factor XIII activation by thrombin. We demonstrate that fibrinogen has two distinct activities that lead to an increased rate of factor XIII activation. First, the thrombin proteolytic activity is increased by fibrin. The cleavage rates of both a small chromogenic substrate and the factor XIII activation peptide are increased in the presence of either the major fibrin isoform, gammaA/gammaA fibrin, or a minor variant form, gammaA/gamma' fibrin. This enhancement of thrombin activity by fibrin is independent of fibrin polymerization and requires only cleavage of the fibrinopeptides. Subsequently, gammaA/gamma' fibrinogen accelerates plasma factor XIII activation by a non-proteolytic mechanism. This increased rate of activation results in a slightly more rapid cross-linking of fibrin gammaA and gamma' chains and a significantly more rapid cross-linking of fibrin alpha chain multimers. Together, these results show that although both forms of fibrin increase the rate of activation peptide cleavage by thrombin, gammaA/gamma' fibrinogen also increases the rate of factor XIII activation in a non-proteolytic manner. A revised model of factor XIII activation is presented below.

Regulatory mechanism of fibrinolysis system by thrombin activatable fibrinolysis inhibitor (TAFI)

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a 60-kDa plasma protein that has been shown to be identical to plasma carboxypeptidase B (CPB) and carboxypeptidase U (CPU). TAFI is activated by thrombomodulin (TM)-bound thrombin and specifically removes the C-terminal Lys and Arg by its CPB activity. One of its target substrates is the C-terminal Lys residue in the alpha-chain of plasmin-digested fibrin, which is critical for plasminogen binding to fibrin. Thus, its removal seems to be the main mechanism through which TAFI inhibits fibrinolysis. In this article, relevance of C-terminal Lys of plasmin-digested fibrin in fibrinolysis is described, and then possible roles of TAFI and TM-bound thrombin in a cross-talk between coagulation and fibrinolysis are discussed.

Characterization of Wild-Type and Mutant α2-Antiplasmins: Fibrinolysis Enhancement by Reactive Site Mutant

During human blood clotting, alpha2-antiplasmin (alpha2AP) becomes covalently linked to fibrin when activated blood clotting factor XIII (FXIIIa) catalyzes the formation of an isopeptide bond between glutamine at position two in alpha2AP and a specific epsilon-lysyl group in each of the alpha-chains of fibrin. This causes fibrin to become resistant to plasmin-mediated lysis. We found that chemically Arg-modified alpha2AP, which lacked plasmin-inhibitory activity, competed effectively with native alpha2AP for becoming cross-linked to fibrin and as a consequence, enhanced fibrinolysis. Recombinant alpha2AP reported to date by other groups either lacked or possessed a low level of FXIIIa substrate activity. As a first step in the development of an engineered protein that might have potential as a localized fibrin-specific fibrinolytic enhancer, we expressed recombinant alpha2AP in Pichia pastoris yeast. Two forms of nonglycosylated recombinant alpha2AP were expressed, isolated and characterized: (1) wild-type, which was analogous to native alpha2AP, and (2) a mutant form, which had Ala substituted for the reactive-site Arg364. Both the wild-type and mutant forms of alpha2AP functioned as FXIIIa substrates with affinities and kinetic efficiencies comparable to those of native alpha2AP, despite each having an additional acetylated Met blocking group at their respective amino-termini. Wild-type recombinant alpha2AP displayed full plasmin inhibitory activity, while mutant alpha2AP had none. Neither the absence of glycosylation nor blockage of the amino-terminus affected plasmin-inhibitory or FXIIIa substrate activities of wild-type alpha2AP. When our mutant alpha2AP, which lacked plasmin-inhibitory function, was added to human plasma or whole blood clots, urokinase (UK)-induced clot lysis was enhanced in a dose-dependent manner, indicating that mutant alpha2AP augmented lysis by competing with native alpha2AP for FXIIIa-catalyzed incorporation into fibrin.

Interaction of Recombinant Procollagen and Properdin Modules of Thrombospondin-1 with Heparin and Fibrinogen/Fibrin

Many properties have been assigned to the procollagen and properdin (Type I) modules of thrombospondin-1 (TSP1) based on activities of large proteolytic fragments of TSP1 or peptides containing TSP1-derived sequences. To examine the activities of the modules more exactly, we expressed the first properdin module (P1); the third properdin module (P3); the first and second properdin modules (P12); the first, second, and third properdin modules (P123); and the procollagen module with the first, second, and third properdin modules (CP123) in the GELEX expression vector (GE1) using the baculovirus system. GE1 encodes the pre-pro sequence, the transglutaminase cross-linking site(s), the protease-sensitive site, and the gelatin binding domain from the amino terminus of rat fibronectin. All five recombinant proteins were expressed by insect cells, secreted into the culture medium, and purified by gelatin-agarose affinity chromatography. P123 shared with TSP1 a resistance to trypsin unless reduced and alkylated. P12/GE1, P123/GE1, and CP123/GE1 bound poorly to heparin-agarose except in the absence of sodium chloride, whereas peptides based on P2 are known to bind to heparin in up to 150 mM sodium chloride. In cross-linking experiments employing activated recombinant factor XIII and the transglutaminase cross-linking site in the fibronectin-derived sequence, P12/GE1, P123/GE1, CP123/GE1, and P3/GE1 but not P1/GE1 became incorporated into a fibrin clot more than GE1 alone. Analysis of the complex indicated that cross-linking was to the portion of the fibrin alpha-chain remaining in the D-dimer of plasmin digests. P123 also cross-linked to the Aalpha-chain of unclotted fibrinogen. P123 competed for 125I-TSP1 incorporation into the fibrin clot. P123 did not cross-link to plasminogen, histidine-rich glycoprotein, fibronectin, or plasma globulins other than fibrinogen/fibrin. These results indicate that the properdin modules of TSP1 specifically interact with fibrinogen/fibrin but not with heparin under physiologic conditions.

Coagulation Factor XIIIa Undergoes a Conformational Change Evoked by Glutamine Substrate: STUDIES ON KINETICS OF INHIBITION AND BINDING OF XIIIa BY A CROSS-REACTING ANTIFIBRINOGEN ANTIBODY

Coagulation factor XIIIa, plasma transglutaminase (endo-gamma-glutamine:epsilon-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen Aalpha-chain segment 529-539 was previously observed from analysis of end-stage plasma clots to block fibrin alpha-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit gamma-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFTNPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope Aalpha-(529-540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.