Cross-linking of Plasminogen Activator Inhibitor 2 and α2-Antiplasmin to Fibrin(ogen)

Ritchie, Heather; Lawrie, Laura; Crombie, Patricia W.; Mosesson, Michael W.; Booth, Nuala A.

doi:10.1074/jbc.m002901200

Cited by 67 publications

(67 citation statements)

References 48 publications

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“…Importantly, our results extend their findings to identify that C3 becomes cross-linked to fibrin during clot formation in purified and plasma-based systems. These data support specific interactions between C3, FXIII and fibrin, and, with our previous results indicating the presence of two high affinity binding sites for C3 on fibrin (Howes et al, 2012), suggest that C3 may become cross-linked to fibrin a-chains, consistent with the cross-linking sites of other FXIIIa substrates (Ritchie et al, 2000). FXIIIa initially binds to glutamine containing-substrates prior to cross-linking and, given the structural similarity between the FXIII-B subunit and complement regulatory proteins (Rodriguez de Cordoba et al, 1988), the interactions between C3 and FXIII-B may be important for initial substrate recognition.…”

supporting

confidence: 65%

Complement C3 is a substrate for activated factor XIII that is cross‐linked to fibrin during clot formation

et al. 2012

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NKG2D-mediated immunity underlying paroxysmal nocturnal haemoglobinuria and related bone marrow failure syndromes. British Journal of Haematology, 146,[538][539][540][541][542][543][544][545] Hanaoka, N., Murakami, Y., Nagata, M., Nagakura, S., Yonemura, Y., Sonoki, T., Kinoshita, T. & Nakakuma, H. (2012) Complement C3 is a substrate for activated factor XIII that is cross-linked to fibrin during clot formation Complement C3 is the main effector protein of the complement system and plays a major role in innate immunity. A growing body of evidence indicates complex interactions between the complement and coagulation cascades (Oikonomopoulou et al, 2012), which are likely to be beneficial in the context of protection following injury. We previously identified C3 as a novel clot component and demonstrated that C3 binds to fibrin with high affinity and prolongs fibrinolysis in a purified system and plasma milieu (Howes et al, 2012), consistent with results of several clinical studies (Schroeder et al, 2010;Hess et al, 2012;Howes et al, 2012).In the present study we further explored the mechanisms by which C3 becomes incorporated into clots, by evaluating the interactions between C3 and factor XIII (FXIII). Using 5-(biotinamido)pentylamine (BPNH2) in microplate-based cross-linking assays (full details of all methods are provided Appendix S1), BPNH2 was incorporated into immobilized C3 and fibrinogen (positive control) in the presence of thrombin-activated FXIII (FXIIIa) but not zymogen FXIII (FXIIIA2B2) in a concentration-dependent manner ( Fig 1A). Time-dependent incorporation of BPNH2 to C3 in the fluid phase was also observed in the presence of FXIIIa but not (Fig 1B). C3 in a plasma milieu was cross-linked to immobilized fibrinogen in a time-dependent and FXIIIadependent manner (Fig 1C). BPNH2 was cross-linked to both the C3a and C3b chains, with incorporation increasing with incubation time (Fig 1D), indicating the presence of accessible glutamine residues in both C3 chains. These data indicate that C3 is a substrate for FXIIIa that is cross-linked to fibrinogen even in the presence of physiological concentrations of other plasma substrates for FXIIIa.Cross-linking of C3 to itself and to fibrin within a clot environment was also evaluated. No high molecular weight (HMW) species were observed upon incubation of C3 with FXIIIa in the absence of fibrinogen, indicating C3 is not cross-linked to itself (Fig 2A, B). Characteristic HMW fibrin multimer formation was observed in all samples containing fibrinogen and FXIIIa (Fig 2A, C). Fibrinogen and C3 incubated together in the absence of FXIIIa did not produce HMW products (Fig 2A, B), whereas in the presence of FXIIIa HMW multimers cross-reacting with anti-C3c antibody were identified (Fig 2D-F). Three HMW bands appeared after 5 min, two of which also cross-reacted with anti-fibrin(ogen) antibody and remained over 24 h (Fig 2E, F, arrows). The third band was absent after 5 min, suggesting incorporation into larger HMW cross-linked products. A fourth C3-containing H...

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supporting

confidence: 65%

Complement C3 is a substrate for activated factor XIII that is cross‐linked to fibrin during clot formation

et al. 2012

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“…78,79 Cross-linking of these proteins makes the clot less susceptible to lysis. FXIIIa cross-links fibronectin, which alters the mechanical properties of the clot by increasing fiber thickness and clot permeability.…”

Section: Cross-linked Proteins and Circulating Saltsmentioning

confidence: 99%

Genetic and Environmental Determinants of Fibrin Structure and Function

Scott

Ariëns

Grant

2004

ATVB

137

129

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Abstract-The formation of a fibrin clot is one of the key events in atherothrombotic vascular disease. The structure of the fibrin clot and the genetic and environmental factors that modify it have effects on its biological function. Alterations in fibrin structure and function have implications for the clinical presentation of vascular disease. This review briefly describes the key features involved in the formation of a fibrin clot, its typical structure, and function. This is followed by a review of the current literature on genetic and environmental influences on fibrin structure/function and the relationship to clinical disease. Key Words: fibrin Ⅲ hemostatis Ⅲ genetic Ⅲ environment Ⅲ atherothrombosis D iseases associated with the development of thrombosis are a major cause of morbidity and mortality in the developed world. Atherothrombotic vascular disorders develop over many decades and involve the interaction of classic atherogenic risk factors such as diabetes, hyperlipidemia, and hypertension with abnormalities of the inflammatory and hemostatic systems. Arterial disease develops in a high-pressure, high-flow system, with lipid deposition and smooth muscle hyperplasia occurring to form an atherosclerotic plaque. 1 Subsequently, the plaque becomes unstable and ruptures exposing a prothrombotic lipid core activating the clotting cascade and leading to the development of a platelet-rich fibrin blood clot and arterial thrombotic occlusion. The extent of this process determines clinical outcome, ranging from no clinically noticeable event to acute coronary artery syndromes including myocardial infarction (MI), cerebrovascular and peripheral vascular disease, or sudden death. The Formation of a Fibrin ClotFibrin is the major protein constituent of the blood clot and is formed from fibrinogen, a glycoprotein that circulates largely inactively, in the blood stream. Fibrinogen consists of 6 polypeptide chains (A␣, B␤, ␥) 2 held together by disulphide bonds in a molecule with bilateral symmetry (Figure 1). The molecule consists of 3 main structural regions, 2,3 including a central region (E), which contains fibrinopeptides A and B and the amino acid termini of all 6 polypeptide chains, 2 distal regions (D) connected to the E region by 2 ␣-helical coiled segments and the D regions containing the carboxyl termini of the B␤ and ␥ chains, and those of the A␣ chain, which extend to form relatively flexible ␣C-domains, each ending in a globular domain. 2,3 In situations of tissue injury and inflammation, thrombin is generated after cleavage of prothrombin by the Xase complex. Thrombin subsequently binds to fibrinogen and cleaves the amino termini of the fibrinogen A␣ and B␤ chains at region E. This results in the release of fibrinopeptides A and B from fibrinogen, producing fibrin and initiating fibrin clot formation. Release of fibrinopeptide A by thrombin is fast and exposes a polymerization site on the E region of fibrin. 4,5 This combines with a complementary binding site on the ␥ chain in the D region of an adjac...

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“…23 The principal inhibitor of plasmin, a2-antiplasmin, 24,25 is also cross-linked to fibrin AaC residue Lys303 by FXIII-A 2 * making the fibrin clot resistant to degradation. 20,26 Recently, Fraser et al 27 demonstrated that resistance of a fibrin clot to lysis was primarily dependent on FXIII-A 2 * cross-linking of a2-antiplasmin to fibrin AaC Lys303, rather than fibrin-fibrin cross-linking. We have recently identified a key residue on the aC of fibrin (AaGlu396) involved in the binding of the FXIII-A 2 * subunit.…”

Section: Introductionmentioning

confidence: 99%

“…19 In addition to cross-linking g and Aa chains for clot stabilization, FXIII-A 2 * has a key role in cross-linking proteins involved in clot formation and fibrinolysis to the fibrin(ogen) aC regions. These include plasminogen activator inhibitor 2, 20 thrombospondin, 21 von Willebrand factor, 22 and fibronectin. 23 The principal inhibitor of plasmin, a2-antiplasmin, 24,25 is also cross-linked to fibrin AaC residue Lys303 by FXIII-A 2 * making the fibrin clot resistant to degradation.…”

Section: Introductionmentioning

confidence: 99%

The activation peptide cleft exposed by thrombin cleavage of FXIII-A2 contains a recognition site for the fibrinogen α chain

Smith

Pease

Avery

et al. 2013

Blood

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Key Points• Fibrin aC389-402 binds a cleft in the b-sandwich domain of FXIII-A 2 * exposed only after cleavage of the activation peptide.• Binding of fibrin aC389-402 to FXIII-A 2 * regulates fibrin cross-linking and thus clot stabilization and fibrinolysis.Formation of a stable fibrin clot is dependent on interactions between factor XIII and fibrin. We have previously identified a key residue on the aC of fibrin(ogen) (Glu396) involved in binding activated factor XIII-A 2 (FXIII-A 2 *); however, the functional role of this interaction and binding site(s) on FXIII-A 2 * remains unknown. Here we (1) characterized the functional implications of this interaction; (2) identified by liquidchromatography-tandem mass spectrometry the interacting residues on FXIII-A 2 * following chemical cross-linking of fibrin(ogen) aC389-402 peptides to FXIII-A 2 *; and (3) carried out molecular modeling of the FXIII-A 2 */peptide complex to identify contact site (s) involved. Results demonstrated that inhibition of the FXIII-A 2 */aC interaction using aC389-402 peptide (Pep1) significantly decreased incorporation of biotinamidopentylamine and a2-antiplasmin to fibrin, and fibrin cross-linking, in contrast to Pep1-E396A and scrambled peptide controls. Pep1 did not inhibit transglutaminase-2 activity, and incorporation of biotinyl-TVQQEL to fibrin was only weakly inhibited. Molecular modeling predicted that Pep1 binds the activation peptide cleft (AP-cleft) within the b-sandwich domain of FXIII-A 2 * localizing aC cross-linking Q366 to the FXIII-A 2 * active site. Our findings demonstrate that binding of fibrin aC389-402 to the APcleft is fundamental to clot stabilization and presents this region of FXIII-A 2 * as a potential site involved in glutamine-donor substrate recognition. (Blood. 2013;121(11):2117-2126

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Cross-linking of Plasminogen Activator Inhibitor 2 and α2-Antiplasmin to Fibrin(ogen)

Cited by 67 publications

References 48 publications

Complement C3 is a substrate for activated factor XIII that is cross‐linked to fibrin during clot formation

Complement C3 is a substrate for activated factor XIII that is cross‐linked to fibrin during clot formation

Genetic and Environmental Determinants of Fibrin Structure and Function

The activation peptide cleft exposed by thrombin cleavage of FXIII-A2 contains a recognition site for the fibrinogen α chain

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