Factor XIII Binding Research Articles

Factor XIII is a newly identified binding partner for platelet collagen receptor GPVI‐dimer—An interaction that may modulate fibrin crosslinking

BackgroundIn the fibrin‐forming process, thrombin cleaves fibrinogen to fibrin, which form fibrils and then fibers, producing a gel‐like clot. Thrombin also activates coagulation factor XIII (FXIII), which crosslinks fibrin γ‐chains and α‐chains, stabilizing the clot. Many proteins bind to fibrin, including FXIII, an established regulation of clot structure, and platelet glycoprotein VI (GPVI), whose contribution to clot function is largely unknown. FXIII is present in plasma, but the abundant FXIII in platelet cytosol becomes exposed to the surface of strongly activated platelets. ObjectivesWe determined if GPVI interacts with FXIII and how this might modulate clot formation. MethodsWe measured interactions between recombinant proteins of the GPVI extracellular domain: GPVI‐dimer (GPVI‐Fc2) or monomer (GPVIex) and FXIII proteins (nonactivated and thrombin‐activated FXIII, FXIII subunits A and B) by ELISA. Binding to fibrin clots and fibrin γ‐chain crosslinking were analyzed by immunoblotting. ResultsGPVI‐dimer, but not GPVI‐monomer, bound to FXIII. GPVI‐dimer selectively bound to the FXIII A‐subunit, but not to the B‐subunit, an interaction that was decreased or abrogated by the GPVI‐dimer–specific antibody mFab‐F. The GPVI‐dimer–FXIII interaction decreased the extent of γ‐chain crosslinking, indicating a role in the regulation of clot formation. ConclusionsThis is the first report of the specific interaction between GPVI‐dimer and the A‐subunit of FXIII, as determined in an in vitro system with defined components. GPVI‐dimer–FXIII binding was inhibitory toward FXIII‐catalyzed crosslinking of fibrin γ‐chains in fibrin clots. This raises the possibility that GPVI‐dimer may negatively modulate fibrin crosslinking induced by FXIII, lessening clot stability.

Factor XIII crosslinking in fibrinogen αc(233-425) is assisted by electrostatic and hydrophobic residues in αc(389-403)

The transglutaminase Factor XIII (FXIII) generates fibrinolytic-resistant blood clots by crosslinking reactive glutamines and lysines between fibrin and other proteins. The fibrin(ogen) αC region (233-425) encompasses three reactive glutamines (Q237, Q328, and Q366) and a FXIII binding site (αC 389 – 403). αC E396 was reported to be instrumental for FXIII binding, but its absence diminishes but does not prevent crosslinking. This project seeks to identify additional residues within Fbg αC 389 – 403 that accelerate transglutaminase activity toward αC based on interactions with complementary residues in FXIII.

Effect of Binding Site Mutations in Fibrinogen αC (233-425) On FXIII Substrate Specificity

In blood coagulation, the transglutaminase Factor XIII (FXIII) catalyzes the crosslinking of specific glutamine and lysine residues between fibrin and other coagulation proteins to generate fibrin clots resistant to fibrinolysis. The αC region (233-425) of the fibrinogen alpha chain contains three reactive glutamines (Q237, Q328, Q366) and a putative FXIII binding region (αC 389-403), where E396 is reported to be the key binding residue. FXIII can be activated proteolytically by thrombin with low calcium concentration (∼4 mM Ca2+, FXIIIA°) or non-proteolytically with high calcium concentration (>25 mM Ca2+, FXIII A°), both leading to FXIIIA conformational sets that vary in transglutaminase activity. These features were explored further by assaying glutamine reactivities (Fbg αC WT, E396A, and αC 389Stop) with the lysine mimic glycine ethyl ester (GEE) by MALDI-ToF mass spectrometry. Across all αC variants, the reactivity ranking from FXIII A° and FXIII A° was Q237 >> Q366 ∼ Q328. Neither the αC E396A (233-425) nor the αC 389Stop (233-388) mutant prevented transglutaminase activity from FXIII A° or FXIII A°. However, the Q237-GEE crosslinking rate for either mutant was hindered compared to WT for both FXIIIA forms. Removal of the putative FXIII binding region in αC 389Stop had less of an adverse effect on FXIII A°-catalyzed GEE crosslinking to Q237 than αC E396A. Additionally, the Q237-GEE crosslinking in αC 389Stop was slightly faster with FXIII A° than with A°, in contrast to WT and E396A where FXIII A' crosslinking was faster. Overall for the FXIII A° studies, the Q-reactivities were WT > E396A > 389Stop whereas with FXIII A°, they were relatively similar. Differences in transglutaminase function between FXIII A° and FXIII A° involve more than activity and include how they interface with substrates like Fbg αC.

Fibg390-396A Limits Metastatic Potential Via a Mechanism Independent of Fibrin-Myeloid Cell Interactions, but Prospectively through the Disruption of FXIII-Mediated Fibrin Crosslinking

Previous studies have shown that fibrin(ogen) supports metastasis. In order to better define the mechanisms coupling fibrin(ogen) to metastasis, we compared metastatic potential in immunocompetent mice carrying specific structure/function alterations in fibrinogen and controls. FibrinogenAEK mice, which have a germ-line mutation in the thrombin cleavage site that essentially "locks" fibrinogen in its soluble state, exhibited diminished metastatic potential relative to FibWT mice. Notably, previous studies have established that factor XIII is also a significant determinant of metastasis. Taken together, these studies suggest that stable fibrin polymer formation is important in the metastatic process. However, FibAEK retained significant metastatic potential relative to mice with complete fibrinogen deficiency, indicating that fibrinogen possesses prometastatic properties in the absence of polymer formation. Fibrin(ogen) has also been shown to mediate inflammatory cell functions through direct engagement of leukocyte integrins independently of its role in platelet aggregation. Given previous studies demonstrating that myeloid cells are promote metastatic potential, we hypothesized that fibrin(ogen)-mediated leukocyte engagement represents one mechanism by which fibrinogen drives metastasis. Consistent with this view, mice expressing a mutant fibrinogen lacking the y chain binding motif for the leukocyte integrin αMβ2 (Fibγ390-396A) had diminished metastatic potential in both spontaneous and experimental metastasis assays. Furthermore, fate analyses revealed that Fibγ390-396A results in diminished survival of newly-formed metastatic foci. The early reduction in metastatic potential observed in Fibγ390-396A mice suggests that fibrin(ogen) promotes metastasis by recruiting myeloid cells to the early metastatic niche. In order to explore this hypothesis, we performed experimental metastasis assays in immunocompetent mice in which macrophages or neutrophils were specifically depleted using either clodronate or an anti-Ly6G antibody. In contrast to mice carrying the Fibγ390-396A mutation, depletion of macrophages or neutrophils had no significant impact on the early survival of metastatic foci. These studies suggest that expression Fibγ390-396A limits metastatic potential via a mechanism independent of interactions with myeloid cells. In addition to disrupting fibrin-αMβ2 interactions, elimination of the γ chain 390-396a binding motif has also been shown to limit factor XIII binding to fibrinogen. Given the established importance of FXIII in metastasis, it is conceivable that even subtle alterations in the kinetics of fibrin cross-linking resulting from expression of Fibγ390-396A limits metastatic potential. Disclosures Palumbo: Ionis Pharmaceuticals: Research Funding.

Abstract 147: Plasma Factor XIII Binding to Cold-stored Platelets Results in Increased Fibrin Crosslinking and Clot Strength

Currently, platelets (PLTs) stored at room temperature (RT) for 5-7 days with gentle agitation are exclusively used for transfusion although FDA recently clarified that apheresis PLTs stored at 4°C for up to 72 hours may be used for treating active hemorrhage. We have demonstrated that cold (4C) storage of PLT is an attractive alternative to RT storage since it better preserves the PLT metabolic reserves, in vitro responses to agonists of activation, aggregation and physiologic inhibitors, as well as adhesion to thrombogenic surfaces. In this study, we tested the hypothesis that 4C-stored PLT will form clots with mechanical strength superior to those from RT-stored PLT due to higher hemostatic potential. From rheological measurements, we observed that the clots formed from 5 day 4C-stored PLTs are significantly stiffer (elastic modulus) and stronger (critical stress) than those formed from RT-stored PLT but comparable to fresh PLT (Fig. A). We also observed from ultrastructural microscopy that the fibrin fibers in clots from cold-stored PLT were thinner with more branch points than those from RT-stored PLTs, indicating the presence of increased crosslinks (Fig. B, C). Finally, molecular analysis revealed an increase FXIII transglutaminase activity due to the binding of plasma FXIII/fibrinogen to the surface of 4C-stored PLTs (Fig D).In conclusion, we have shown that cold-induced plasma FXIII binding to PLT surface result in increased fibrin crosslinking and enhanced clot strength. Our data, together with the benefit of reduced risk of late thrombosis due to their rapid clearance in vivo , underscores the consideration of 4C-stored PLT for acute response to hemorrhage. Figure 1 (A) RT-stored platelets form clots with less stiffness (n=5); (B) Representative SEM images of clots (n=3) (C) Clots from 4C-stored platelets have higher cross-linking density (n=5) (D) In FXIII-deficient plasma, 4C-stored platelets form clots with higher stiffness than fresh platelets (n=4)

Transglutaminase Factor XIII Cross-Links Reactive Glutamines in Disordered Regions of Fibrinogen αC

Fibrinogen is the most abundant protein involved in blood coagulation and has been implicated in cardiovascular disease. During the formation of a blood clot, thrombin cleaves fibrinopeptides A and B from fibrinogen (AαBβγ)2 to form fibrin monomers (αβγ)2. Fibrin polymerization proceeds with the formation of protofibrils and a hard clot is only generated when the transglutaminase factor XIII (FXIII) introduces covalent γ-glutamyl-e-lysyl bonds between specific reactive glutamines and lysines in the fibrin clot network. The αC region (Aα221-610) of fibrinogen contains numerous reactive glutamines in the flexible αC-connector (221-392) and a FXIII binding site within αC (371-425). MALDI-TOF mass spectrometry assays and 2D 15N-1H HSQC NMR studies have been carried out on fibrinogen αC (233-425), a segment containing three known reactive glutamines (Q237, Q328, and Q366). FXIIIa crosslinks these three glutamines to the lysine mimic glycine ethyl ester (GEE) in the order Q237 >> Q366 > Q328. To better characterize this system, single site mutants have been generated in which the reactive glutamines (Q) are individually replaced with inactive asparagines (N). Work with these mutants suggests that FXIIIa does not require crosslinking to occur in a sequential manner. Each glutamine can be cross-linked independently of the other. Due to its highly flexible nature, the fibrinogen αC region has not been observed by X-ray crystallography. 2D HSQC NMR studies on 15N-labeled αC (233-425) suggest that this protein is intrinsically disordered. FXIIIa substrate specificity for specific reactive glutamines is predicted to be controlled by a combination of neighboring residues and the presence of regions of disorder. The knowledge gained from these studies can be used for subsequent assay and drug designs.

The blood clotting Factor XIIIa forms unique complexes with amyloid-beta (Aβ) and colocalizes with deposited Aβ in cerebral amyloid angiopathy.

Cerebral amyloid angiopathy (CAA) is a key pathological hallmark of Alzheimer's disease (AD) characterized by accumulation of amyloid-beta (Aβ) protein in blood vessel walls. CAA impairs vessel functioning, affects blood brain barrier integrity and accelerates cognitive decline of AD patients. Unfortunately, mechanisms underlying Aβ deposition in the vessel wall remain largely unknown. Factor XIIIa (FXIIIa) is a blood-derived transglutaminase crucial in blood coagulation by cross-linking fibrin molecules. Evidence is mounting that blood-derived factors are present in CAA and may play a role in protein deposition in the vessel wall. We therefore investigated whether FXIIIa is present in CAA and if FXIIIa cross-link activity affects Aβ aggregation. Using immunohistochemistry, we investigated the distribution of FXIIIa, its activator thrombin and in situ FXIIIa activity in CAA in post-mortem AD tissue. We used surface plasmon resonance and Western blot analysis to study binding of FXIIIa to Aβ and the formation of FXIIIa-Aβ complexes, respectively. In addition, we studied cytotoxicity of FXIIIa-Aβ complexes to cerebrovascular cells. FXIIIa, thrombin and in situ FXIIIa activity colocalize with the Aβ deposition in CAA. Furthermore, FXIIIa binds to Aβ with a higher binding affinity for Aβ1-42 compared with Aβ1-40 . Moreover, highly stable FXIIIa-Aβ complexes are formed independently of FXIIIa cross-linking activity that protected cerebrovascular cells from Aβ-induced toxicity in vitro. Our data showed that FXIIIa colocalizes with Aβ in CAA and that FXIIIa forms unique protein complexes with Aβ that might play an important role in Aβ deposition and persistence in the vessel wall.

Specific effects of fibrinogen and the γA and γ'-chain fibrinogen variants on angiogenesis and wound healing.

In a newly formed wound, the natural fibrin network provides the first temporary matrix for tissue repair. Topical application of fibrin to a new wound may improve wound healing. A matrix of the common natural γ' fibrin variant may further improve wound healing because it is expected to have a different architecture and this will influence angiogenesis, because it possesses increased thrombin and factor XIII binding and decreased platelet binding, when compared with the common γA fibrin matrix. Our objective was to determine the effect of fibrinogen and its γA and γ' variants on angiogenesis and wound healing. We used in vitro angiogenesis models and an in vivo rat full-thickness excisional wound healing model. When comparing γA and γ' fibrin in vitro, more tube-like structures were formed on day 7 in γA fibrin than in γ' fibrin (13.83±6.12 AU vs. 6.1±1.46 AU). Wounds treated with fibrin demonstrated improved healing in vivo with more perfusion (47%±3% vs. 26%±4%, p<0.01 in placebo) and higher CD34 density score (2.0±0.4 vs. 2.8±0.1, p<0.01) on day 21 with fibrin matrices when compared with placebo-treated wounds. Increased perfusion was observed in γA fibrin-treated wounds on day 21 (53%±10% vs. 41%±7% for γ' fibrin). The other parameters showed slightly improved (not significant) wound healing with γA fibrin compared with γ' fibrin matrices. In conclusion, the use of fibrin and fibrin variant matrices offers an interesting methodology to stimulate the wound healing process.

Factor XIII activity mediates red blood cell retention in venous thrombi.

Venous thrombi, fibrin- and rbc-rich clots triggered by inflammation and blood stasis, underlie devastating, and sometimes fatal, occlusive events. During intravascular fibrin deposition, rbc are thought to become passively trapped in thrombi and therefore have not been considered a modifiable thrombus component. In the present study, we determined that activity of the transglutaminase factor XIII (FXIII) is critical for rbc retention within clots and directly affects thrombus size. Compared with WT mice, mice carrying a homozygous mutation in the fibrinogen γ chain (Fibγ390-396A) had a striking 50% reduction in thrombus weight due to reduced rbc content. Fibrinogen from mice harboring the Fibγ390-396A mutation exhibited reduced binding to FXIII, and plasma from these mice exhibited delayed FXIII activation and fibrin crosslinking, indicating these residues mediate FXIII binding and activation. FXIII-deficient mice phenocopied mice carrying Fibγ390-396A and produced smaller thrombi with fewer rbc than WT mice. Importantly, FXIII-deficient human clots also exhibited reduced rbc retention. The addition of FXIII to FXIII-deficient clots increased rbc retention, while inhibition of FXIII activity in normal blood reduced rbc retention and produced smaller clots. These findings establish the FXIII-fibrinogen axis as a central determinant in venous thrombogenesis and identify FXIII as a potential therapeutic target for limiting venous thrombosis.

Binding of plasma factor XIII to thrombin-receptor activated human platelets

Platelet-bound coagulation factor XIII (FXIII) is targeted and concentrated at the site where platelet-rich thrombi are formed. Previous studies were in disagreement about the nature of FXIII binding to platelets. In this study, thrombin-receptor activating peptide (TRAP)-stimulated human whole blood and washed platelets were analysed by flow cytometry for the binding of FXIII using a monoclonal antibody against the A subunit of FXIII (FXIII-A). Here, we demonstrate that FXIII-A positivity significantly increased on activated platelets in whole blood compared to unstimulated sample, but not in washed platelets. GPIIb/IIIa receptor plays an essential role in FXIII binding, as fibrinogen receptor antagonist eptifibatide and fibrinogen binding inhibitor RGDS tetrapeptide significantly prevented the binding of FXIII. Furthermore, stimulated platelets from a patient with severe type I Glanzmann thrombasthenia showed insignificant FXIII-A positivity versus healthy controls. In addition, basal negligible amount of FXIII on washed platelets was only slightly increased when highly purified plasma FXIII (FXIII-A(2)B(2)) was added upon platelet activation by TRAP. Similarly, no remarkable FXIII-A positivity was observed when we used FXIII-A(2)B(2) with gammaA/gammaA fibrinogen. However, gammaA/gamma' fibrinogen significantly augmented FXIII binding on TRAP-stimulated platelets in the presence of non-activated FXIII-A(2)B(2). We conclude that FXIII-A(2)B(2) of plasma origin binds to thrombin-receptor activated platelets via GPIIb/IIIa receptor-bound fibrinogen with gamma'-chain and is not capable of direct platelet binding.

The effect of fibrinogen concentrate on thrombocytopenia.

The hypothesis that the administration of fibrinogen concentrate enables restoration of impaired clot formation and increased bleeding in severe thrombocytopenia was tested. Thirty pigs were anesthetized, instrumented for blood sampling (routine coagulation tests, modified thrombelastography ROTEM, hemodynamic monitoring and platelet apheresis to a target below 30 x 10(9) L(-1) after splenectomy. Thereafter 10 each of the animals randomly received two apheresis platelet concentrates, 250 mg kg(-1) fibrinogen concentrate or normal saline solution. A standardized liver injury was subsequently inflicted to induce uncontrolled hemorrhage. Median (Q1, Q3) clot firmness increased significantly more in thrombocytopenic pigs after fibrinogen administration (42 mm (41, 43) to 60 mm (57, 63)) than following platelet transfusion (40 mm (37, 45) to 52 mm (48, 55), P = 0.0004) or placebo (45 mm (41, 48) to 45 mm (43, 46), P = 0.0002). Median blood loss velocity after liver injury was significantly less with fibrinogen (33 mL min(-1), P = 0.005) than with platelets (62 mL min(-1), P = 0.037) or saline (84 mL min(-1), P = 0.005), and median survival time after liver injury was 55 min in the fibrinogen, 26 min in the platelet (P = 0.035) and 19 min in the saline group (P = < 0.0001). These data show for the first time that impaired clot formation during thrombocytopenia improves with administration of fibrinogen concentrate, which results in a slowdown of blood loss and prolonged survival.

An Investigation of Factor XIII Binding to Recombinant γ′/γ′ and γ/γ′ Fibrinogen.

Increased levels of the alternatively spliced γ′ chain of fibrinogen have been measured in patients suffering from coronary artery disease (Lovely et al. Thromb. Haemost. 2002; 88:26–31). Others have suggested that the γ′ chain serves as a binding site for factor XIII (Siebenlist et al. Biochemistry 1996; 35:10448–10453), thus increasing the amount of cross-linking and therefore the stability of the γ′-containing clots (Falls and Farrell JBC 1997; 272:14251–14256). We have expressed recombinant γ′/γ′ homodimer and γ/γ′ heterodimer fibrinogen variants in CHO cells and characterized their polymerization by turbidity. Compared to normal recombinant γ/γ fibrinogen, γ′/γ′ fibrinogen displayed a slower rate of polymerization and a lower final absorbance. γ/γ′ Heterodimer fibrinogen had both a polymerization rate and final absorbance that was intermediate between γ/γ and γ′/γ′. These observations suggest that the presence of the γ′ chain slows lateral aggregation and leads to the formation of thinner fibers. These variants were then used to investigate the binding of factor XIII to fibrinogen. We designed an ELISA assay to measure this binding by immobilizing fibrinogen on a 96-well plate, incubating with factor XIII, and measuring bound factor XIII with an anti-factor XIII A-subunit antibody. Normal recombinant γ/γ fibrinogen bound factor XIII zymogen (A2B2) with a KD of 4.1 x 10−8 M +/− 1.3 x 10−8 M, similar to previous data for plasma fibrinogen. Surprisingly, both γ′/γ′ and γ/γ′ fibrinogen variants bound factor XIII zymogen with the same KD as did γ/γ fibrinogen. As shown by ELISAs with antibodies specific for either γ or γ′, the C-termini of both the γ and the γ′ chains are accessible on this immobilized fibrinogen. These findings suggest that the C-terminus of the γ′ chain is not a critical element in the binding of factor XIII zymogen. Our findings differ from previously reported findings. Further studies are in progress to reconcile these differences.

The structure and biological features of fibrinogen and fibrin.

Fibrinogen and fibrin play important, overlapping roles in blood clotting, fibrinolysis, cellular and matrix interactions, inflammation, wound healing, and neoplasia. These events are regulated to a large extent by fibrin formation itself and by complementary interactions between specific binding sites on fibrin(ogen) and extrinsic molecules including proenzymes, clotting factors, enzyme inhibitors, and cell receptors. Fibrinogen is comprised of two sets of three polypeptide chains termed A alpha, B beta, and gamma, that are joined by disulfide bridging within the N-terminal E domain. The molecules are elongated 45-nm structures consisting of two outer D domains, each connected to a central E domain by a coiled-coil segment. These domains contain constitutive binding sites that participate in fibrinogen conversion to fibrin, fibrin assembly, crosslinking, and platelet interactions (e.g., thrombin substrate, Da, Db, gamma XL, D:D, alpha C, gamma A chain platelet receptor) as well as sites that are available after fibrinopeptide cleavage (e.g., E domain low affinity non-substrate thrombin binding site); or that become exposed as a consequence of the polymerization process (e.g., tPA-dependent plasminogen activation). A constitutive plasma factor XIII binding site and a high affinity non-substrate thrombin binding site are located on variant gamma' chains that comprise a minor proportion of the gamma chain population. Initiation of fibrin assembly by thrombin-mediated cleavage of fibrinopeptide A from A alpha chains exposes two EA polymerization sites, and subsequent fibrinopeptide B cleavage exposes two EB polymerization sites that can also interact with platelets, fibroblasts, and endothelial cells. Fibrin generation leads to end-to-middle intermolecular Da to EA associations, resulting in linear double-stranded fibrils and equilaterally branched trimolecular fibril junctions. Side-to-side fibril convergence results in bilateral network branches and multistranded thick fiber cables. Concomitantly, factor XIII or thrombin-activated factor XIIIa introduce intermolecular covalent epsilon-(gamma glutamyl)lysine bonds into these polymers, first creating gamma dimers between properly aligned C-terminal gamma XL sites, which are positioned transversely between the two strands of each fibrin fibril. Later, crosslinks form mainly between complementary sites on alpha chains (forming alpha-polymers), and even more slowly among gamma dimers to create higher order crosslinked gamma trimers and tetramers, to complete the mature network structure.

Interactions of human fibrinogens with factor XIII: roles of calcium and the gamma' peptide.

Plasma factor XIII is the zymogen of the transglutaminase factor XIIIa. This enzyme catalyzes the formation of isopeptide cross-links between fibrin molecules in nascent blood clots that greatly increase the mechanical stability of clots and their resistance to thrombolytic enzymes. We have characterized the solution interactions of factor XIII with two variants of fibrinogen, the soluble precursor of fibrin. Both the predominant fibrinogen gamma(A)/gamma(A) and the major variant gamma(A)/gamma' form complexes with a 2 fibrinogen:1 factor XIII ratio. The absence of detectable concentrations of 1:1 complexes in equilibrium mixtures containing free factor XIII and 2:1 complexes suggests that this interaction is cooperative. Factor XIII binds fibrinogen gamma(A)/gamma' approximately 20-fold more tightly than fibrinogen gamma(A)/gamma(A), and the interaction with fibrinogen gamma(A)/gamma' (but not fibrinogen gamma(A)/gamma(A)) is accompanied by a significant release of Ca(2+). Taken together, these results suggest that the strikingly anionic gamma' C-terminal sequence contains features that are important for factor XIII binding. Consistent with this notion, a synthetic 20-residue polypeptide containing the gamma' sequence was found to associate with factor XIII in a 2:1 molar ratio and act as an efficient competitor for fibrinogen gamma(A)/gamma' binding.

Characterization of the reciprocal binding sites on human alpha-thrombin and factor XIII A-chain.

Solution- and solid-phase techniques were used to probe Factor XIII A-chain-alpha-thrombin interactions. Alpha-thrombin activated Factor XIII more efficiently (Km = 0.83 +/- 0.08 x 10(-7) M; V/K = 14.90 +/- 3.20 x 10(-3) min(-1)) than beta-thrombin (Km = 6.14 +/- 1.26 x 10(-7) M; V/K = 3.30 +/- 1.00 x 10(-3) min(-1)) or gamma-thrombin (Km = 6.25 +/- 1.15 x 10(-7) M; V/K = 3.00 +/- 0.80 x 10(-3) min(-1)). Immobilized FPR-alpha-thrombin bound plasma Factor XIII (Kd = 0.17 +/- 0.04 x 10(-7) M) > Factor XIIIa (Kd = 0.69 +/- 0.18 x 10(-7) M) > liver transglutaminase (Kd = 4.73 +/- 1.01 x 10(-7) M) > Factor XIII A-chain (Kd = 49.00 +/- 9.40 x 10(-7) M). FPR-alpha-thrombin and alpha-thrombin also bound immobilized Factor XIII A-chain with affinities inversely related to protease activity: maximal binding at 1.36 x 10(-7) M and 13.6 x 10(-7) M, respectively. Plasma Factor XIII, transglutaminase, and dithiothreitol competitively inhibited Factor XIII A-chain binding to FPR-alpha-thrombin: IC50 = 1.0 x 10(-7) M, 3.0 x 10(-6) M and 1.52 x 10(-4) M, respectively. Transglutaminase also inhibited Factor XIII binding to alpha-thrombin (IC50 = 2.0 x 10(-6) M). Thrombin-binding site was localized to G38-M731 fragment of Factor XIII A-chain, probably within homologous regions (N72-A493) of transglutaminase. R320-E579 of alpha-thrombin was Factor XIII A-chain binding site. Intra-B-chain disulfides in alpha-thrombin were essential for binding but not catalytic H363 or residues R382-N394 and R443-G475. These studies propose a structural basis for Factor XIII activation, provide a regulatory mechanism for Factor XIIIa generation, and could eventually help in the development of new structure-based inhibitors of thrombin and Factor XIIIa.

Platelet-associated factor XIII in platelet activation, adhesion, and clot stabilization.

Platelet-associated factor XIII provides a means by which to promote clot stabilization and platelet interaction with proteins of the coagulation and fibrinolytic pathways. In addition to its intracellular role within the platelet cytoplasm, activated factor XIII will bind to the surface of activated platelets. These platelets then participate in cell-cell or cell-clot interactions, thereby increasing the local concentration of factor XIIIa. The platelet-associated factor XIIIa may increase the amount of crosslinking in a fibrin clot, thereby contributing to the aging of the clot and the reduction in the degree of platelet binding. Clot resistance to fibrinolysis is enhanced by platelet factor XIIIa-mediated crosslinking of alpha 2-antiplasmin to fibrin. The binding of factor XIIIa to the platelet surface requires the activation of the platelet fibrinogen receptor, glycoprotein IIb-IIIa. Thus, platelet-associated factor XIIIa may be used as a marker of in vivo platelet activation. Since half of the factor XIII present in blood is provided by the platelets, it is not surprising that this form of factor XIII plays an important role in hemostasis.

Alpha-Chain cross-linking in fibrin(ogen) Marburg

The fibrinogen structural variant, Marburg (A alpha 1-460B beta gamma)2, is comprised of normal B beta and gamma chains but contains severely truncated A alpha chains that are missing approximately one half of their factor XIIIa cross-linking domain. Immunochemical studies of fibrin(ogen) Marburg were conducted to characterize the degree to which deletion of a defined A alpha-chain segment, A alpha 461-610, can affect the process of fibrin stabilization, ie, the factor XIIIa-mediated covalent interaction that occurs between alpha chains of neighboring fibrin molecules and between alpha chains and alpha 2 antiplasmin (alpha 2PI). The ability of Marburg (and control) alpha chains to serve as a substrate for factor XIIIa and undergo cross-linking was examined in an in vitro plasma clotting system. The capacity for alpha-chain cross-linking was evaluated both as the covalent incorporation of the small synthetic peptide, NQEQVSPLTLLK (which represents the first 12 amino acids of alpha 2PI and includes the factor XIIIa-sensitive glutamine residue responsible for the cross-linking of alpha 2PI to fibrin), and as the appearance of native (ie, natural), high-molecular-weight, cross-linked alpha-chain species. Antibodies specific for the (A)alpha and gamma/gamma-gamma chains of fibrin(ogen) and for the peptide and its parent protein, alpha 2PI (68 kD), were used as immunoblotting probes to visualize the various cross-linked products formed during in vitro clotting. Recalcification of Marburg plasma in the presence of increasing concentrations of peptide resulted in the formation of peptide-decorated Marburg alpha-chain monomers. Their size at the highest peptide concentration examined indicated the incorporation of a maximum of 3 to 4 mol of peptide per mole of alpha-chain. In the absence of alpha 2PI 1-12 peptide, the alpha chains of Marburg fibrin cross-linked to form oligomers and polymers, as well as heterodimers that included alpha 2PI. Both the peptide-decorated monomers and the native cross-linked alpha-chain species of Marburg fibrin were smaller than their control plasma counterparts, consistent with the truncated structure of the parent Marburg A alpha chain. Collectively, the findings indicate that, although deletion of the A alpha chain region no. 461-610 in fibrinogen Marburg prevents formation of an extensive alpha polymer network (presumably due to the absence of critical COOH-terminal lysine residues), it does not interfere with initial events in the fibrin stabilization process, namely, factor XIII binding and the ability of alpha chains to undergo limited cross-linking to one another and to alpha 2PI.

α-Chain Cross-Linking in Fibrin(ogen) Marburg

Abstract The fibrinogen structural variant, Marburg (A alpha 1–460B beta gamma)2, is comprised of normal B beta and gamma chains but contains severely truncated A alpha chains that are missing approximately one half of their factor XIIIa cross-linking domain. Immunochemical studies of fibrin(ogen) Marburg were conducted to characterize the degree to which deletion of a defined A alpha-chain segment, A alpha 461–610, can affect the process of fibrin stabilization, ie, the factor XIIIa- mediated covalent interaction that occurs between alpha chains of neighboring fibrin molecules and between alpha chains and alpha 2 antiplasmin (alpha 2PI). The ability of Marburg (and control) alpha chains to serve as a substrate for factor XIIIa and undergo cross- linking was examined in an in vitro plasma clotting system. The capacity for alpha-chain cross-linking was evaluated both as the covalent incorporation of the small synthetic peptide, NQEQVSPLTLLK (which represents the first 12 amino acids of alpha 2PI and includes the factor XIIIa-sensitive glutamine residue responsible for the cross- linking of alpha 2PI to fibrin), and as the appearance of native (ie, natural), high-molecular-weight, cross-linked alpha-chain species. Antibodies specific for the (A)alpha and gamma/gamma-gamma chains of fibrin(ogen) and for the peptide and its parent protein, alpha 2PI (68 kD), were used as immunoblotting probes to visualize the various cross- linked products formed during in vitro clotting. Recalcification of Marburg plasma in the presence of increasing concentrations of peptide resulted in the formation of peptide-decorated Marburg alpha-chain monomers. Their size at the highest peptide concentration examined indicated the incorporation of a maximum of 3 to 4 mol of peptide per mole of alpha-chain. In the absence of alpha 2PI 1–12 peptide, the alpha chains of Marburg fibrin cross-linked to form oligomers and polymers, as well as heterodimers that included alpha 2PI. Both the peptide-decorated monomers and the native cross-linked alpha-chain species of Marburg fibrin were smaller than their control plasma counterparts, consistent with the truncated structure of the parent Marburg A alpha chain. Collectively, the findings indicate that, although deletion of the A alpha chain region no. 461–610 in fibrinogen Marburg prevents formation of an extensive alpha polymer network (presumably due to the absence of critical COOH-terminal lysine residues), it does not interfere with initial events in the fibrin stabilization process, namely, factor XIII binding and the ability of alpha chains to undergo limited cross-linking to one another and to alpha 2PI.

A Microtiter Plate Assay for Factor XIII A-Chain-Fibrin Interactions

Factor XIII A-chain-fibrin interactions regulate factor XIIIa formation and fibrin cross-linking. A microtiter plate assay was developed for studying these interactions. Microtiter plate wells were coated with fibrinogen and converted to fibrin by thrombin. After blocking the wells with bovine serum albumin, factor XIII A-chain was added and binding was monitored by incubating first with anti-factor XIII followed by anti-rabbit IgG-alkaline phosphatase. Enzymatic hydrolysis of p-nitrophenyl phosphate was quantitated by the absorbance at 405 nm. Binding was specific, sensitive, rapid, saturable, and reversible, requiring only nanograms of either factor XIII or fibrin. Binding was time- and concentration-dependent and independent of divalent cations. The bound material was identified as factor XIII A-chain by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. Factor XIII binding was inhibited >75% by 250 mM sodium chloride or 250 nM anti-factor XIII IgG. The method was also suitable for demonstrating binding using 0.8% plasma or with r-factor XIII expressed in Saccharomyces cerevisiae or Escherichia coli. This method is suitable for identifying the binding sites that are important for plasma factor XIII activation and factor XIIIa activity.

Factor XIIIa Binding to Activated Platelets Is Mediated Through Activation of Glycoprotein IIb-IIIa

AbstractStabilization of a clot is dependent on fibrin cross-linking mediated by the transglutaminase, factor XIIIa (FXIIIa). In addition to fibrin stabilization, FXIIIa acts on a number of platelet-reactive proteins, including fibronectin and vitronectin, as well as the platelet proteins, glycoprotein (GP) IIb-IIIa, myosin, and actin. However, conditions inducing the platelet-activation dependent binding of FXIIIa have not been characterized nor have the sites mediating FXIIIa binding been identified. The generation of FXIIIa and consequent detection of FXIIIa on the platelet surface were compared with other thrombin-induced activation events; the rate at which FXIIIa bound to activated platelets was much slower than platelet degranulation or fibrin(ogen) binding. Whereas platelets could be rapidly induced to express a functional receptor for FXIIIa, the rate of FXIIIa binding to platelets is limited by the rate of conversion of FXIII to FXIIIa. Immunoprecipitation of radiolabeled platelets using polyclonal anti-FXIII A-chain antibody identified two proteins corresponding to GPIIb and GPIIIa. Preincubation of intact platelets with 7E3, a monoclonal antibody that blocks the fibrinogen binding site, or GRGDSP peptide inhibited FXIIIa binding by about 95% when measured by flow cytometry; FXIIIa binding to purified GPIIb-IIIa was also inhibited by 7E3. The binding of FXIIIa to purified GPIIb-IIIa was enhanced by the addition of fibrinogen, but not by that of fibronectin or thrombospondin, suggesting that FXIIIa also binds to fibrinogen associated with the complex. These observations suggest that activated platelets bearing FXIIIa may enhance stabilization of platelet-rich thrombi through surface-localized cross-linking events.