Fibrinolysis: A Primordial System Linked to the Immune Response

The fibrinolytic system provides an essential means to remove fibrin deposits and blood clots. The actual protease responsible for this is plasmin, formed from its precursor, plasminogen. Fibrin is heralded as it most renowned substrate but for many years plasmin has been known to cleave many other substrates, and to also activate other proteolytic systems. Recent clinical studies have shown that the promotion of plasmin can lead to an immunosuppressed phenotype, in part via its ability to modulate cytokine expression. Almost all immune cells harbor at least one of a dozen plasminogen receptors that allows plasmin formation on the cell surface that in turn modulates immune cell behavior. Similarly, a multitude of pathogens can also express their own plasminogen activators, or contain surface proteins that provide binding sites host plasminogen. Plasmin formed under these circumstances also empowers these pathogens to modulate host immune defense mechanisms. Phylogenetic studies have revealed that the plasminogen activating system predates the appearance of fibrin, indicating that plasmin did not evolve as a fibrinolytic protease but perhaps has its roots as an immune modifying protease. While its fibrin removing capacity became apparent in lower vertebrates these primitive under-appreciated immune modifying functions still remain and are now becoming more recognised.     

Plasminogen Binding and Activation at the Mesothelial Cell Surface Promotes Invasion through a Collagen Matrix

Plasminogen (Plg) activation to the serine protease plasmin (Pla) plays a key role in regulating wound healing and fibrotic responses, particularly when bound to cell surface receptors. Our previous work suggested that mesothelial cells bind Plg at the cell surface, though no Plg receptors were described for these cells. Since mesothelial cells contribute to injury responses, including cellular differentiation to a mesenchymal-like phenotype and extracellular matrix remodeling, we hypothesized that Plg binding would promote these responses. Here, we confirm that Plg binds to both pleural and peritoneal mesothelial cells via the lysine-binding domain present in Plg, and we demonstrate the presence of three Plg receptors on the mesothelial cell surface: α-Enolase, Annexin A2, and Plg-R_KT. We further show that bound-Plg is activated to Pla on the cell surface and that activation is blocked by an inhibitor of urokinase plasminogen activator or by the presence of animal-derived FBS. Lastly, we demonstrate that Plg promotes mesothelial cell invasion through a type I collagen matrix but does not promote cellular differentiation or proliferation. These data demonstrate for the first time that mesothelial cells bind and activate Plg at the cell surface and that active Pla is involved in mesothelial cell invasion without cell differentiation.     

The Multifaceted Role of Plasminogen in Cancer

Fibrinolytic factors like plasminogen, tissue-type plasminogen activator (tPA), and urokinase plasminogen activator (uPA) dissolve clots. Though mere extracellular-matrix-degrading enzymes, fibrinolytic factors interfere with many processes during primary cancer growth and metastasis. Their many receptors give them access to cellular functions that tumor cells have widely exploited to promote tumor cell survival, growth, and metastatic abilities. They give cancer cells tools to ensure their own survival by interfering with the signaling pathways involved in senescence, anoikis, and autophagy. They can also directly promote primary tumor growth and metastasis, and endow tumor cells with mechanisms to evade myelosuppression, thus acquiring drug resistance. In this review, recent studies on the role fibrinolytic factors play in metastasis and controlling cell-death-associated processes are presented, along with studies that describe how cancer cells have exploited plasminogen receptors to escape myelosuppression.     

Fibrin Network Formation and Lysis in Septic Shock Patients

Background: Septic shock patients are prone to altered fibrinolysis, which contributes to microthrombus formation, organ failure and mortality. However, characterisation of the individual patient’s fibrinolytic capacity remains a challenge due to a lack of global fibrinolysis biomarkers. We aimed to assess fibrinolysis in septic shock patients using a plasma-based fibrin clot formation and lysis (clot–lysis) assay and investigate the association between clot–lysis parameters and other haemostatic markers, organ dysfunction and mortality. Methods: This was a prospective cohort study including adult septic shock patients (n = 34). Clot–lysis was assessed using our plasma-based in-house assay. Platelet count, activated partial thromboplastin time (aPTT), international normalised ratio (INR), fibrinogen, fibrin D-dimer, antithrombin, thrombin generation, circulating fibrinolysis markers and organ dysfunction markers were analysed. Disseminated intravascular coagulation score, Sequential Organ Failure Assessment (SOFA) score and 30-day mortality were registered. Results: Three distinct clot–lysis profiles emerged in the patients: (1) severely decreased fibrin formation (flat clot–lysis curve), (2) normal fibrin formation and lysis and (3) pronounced lysis resistance. Patients with abnormal curves had lower platelet counts (p = 0.05), more prolonged aPTT (p = 0.04), higher lactate (p < 0.01) and a tendency towards higher SOFA scores (p = 0.09) than patients with normal clot–lysis curves. Fibrinogen and fibrin D-dimer were not associated with clot–lysis profile (p ≥ 0.37). Conclusion: Septic shock patients showed distinct and abnormal clot–lysis profiles that were associated with markers of coagulation and organ dysfunction. Our results provide important new insights into sepsis-related fibrinolysis disturbances and support the importance of assessing fibrinolytic capacity in septic shock.     

A Narrative Review on Plasminogen Activator Inhibitor-1 and Its (Patho)Physiological Role: To Target or Not to Target?

Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of plasminogen activators (PAs) and is therefore an important inhibitor of the plasminogen/plasmin system. Being the fast-acting inhibitor of tissue-type PA (tPA), PAI-1 primarily attenuates fibrinolysis. Through inhibition of urokinase-type PA (uPA) and interaction with biological ligands such as vitronectin and cell-surface receptors, the function of PAI-1 extends to pericellular proteolysis, tissue remodeling and other processes including cell migration. This review aims at providing a general overview of the properties of PAI-1 and the role it plays in many biological processes and touches upon the possible use of PAI-1 inhibitors as therapeutics.     

The position of the structurally autonomous kringle 2 domain influences the functional features of tissue-type plasminogen activator

Tissue-type plasminogen activator (t-PA) is composed of structurallyautonomous domains. From the N-terminus of t-PA, a finger-likedomain (F), an epidermal growth factor-like domain (G), twokringle domains (Kl and K2) and a serine protease domain (P)can be discerned. The K2 domain of t-PA is known to be involvedin lysine binding, fibrin binding and fibrin-dependent plasminogenactivation. To study the functional autonomy of the K2 domainin t-PA we constructed, with the aid of a cassette t-PA gene[Rehberg et al. (1989) Protein Engng, 2,371–377], mutantt-PA genes coding for four molecules (FGK1K2P, FGK2K1P, GK1K2Pand GK2K1P) in which the K2 domain was placed in two differentpositions in t-PA. The DNAs of wild-type t-PA and the t-PA variantswere expressed in Chinese hamster ovary cells and the recombinantproteins were purified by affinity chromatography.All moleculeswere expressed in their single-chain form and could be convertedto their two-chain form. With these molecules, lysine binding,fibrin binding and fibrin-dependent plasminogen activation werestudied. All variants showed affinity for lysyl-Sepharose andaminohexyl-Sepharose. Reversal of the K domains (FGK2K1P versusFGK2K1P and GK1K2P versus GK2K1P) resulted in a 23–47%weaker interaction to both lysyl-Sepharose and aminohexyl-Sepharose.Deleting the F domain (FGK1K2P versus GK1K2P and FGK2K1P versusGK2K1P) resulted in a 20–70% improvement of the interactionslysyl-Sepharose and aminohexyl-Sepharose. All variants boundto a forming fibrin clot. Reversal of the K domains (FGK1K2Pversus FGK2K1P) reduced fibrin binding. In the presence of thelysine analogue -amino caproic acid, only FGK1K2P bound to fibrin.All variants activated plasminogen. In the absence of fibrinogenCNBr fragments (mimic of fibrin), the reversal of the K domain(FGK2K1P) resulted in a 2-fold improved plasminogen activation.In the presence of a fibrin mimic, the plasminogen activationsof the F domain deletion analogues GK1K2P and GK2K1P were foundto be decreased 2- to 4-fold. From these results we concludedthat the function of t-PA in lysine binding, fibrin bindingand fibrin-dependent plasminogen activation is dependent onthe correct spatial orientation of the K2 domain within thet-PA molecule     

Role of Shear Stress and tPA Concentration in the Fibrinolytic Potential of Thrombi

The resolution of arterial thrombi is critically dependent on the endogenous fibrinolytic system. Using well-established and complementary whole blood models, we investigated the endogenous fibrinolytic potential of the tissue-type plasminogen activator (tPA) and the intra-thrombus distribution of fibrinolytic proteins, formed ex vivo under shear. tPA was present at physiologically relevant concentrations and fibrinolysis was monitored using an FITC-labelled fibrinogen tracer. Thrombi were formed from anticoagulated blood using a Chandler Loop and from non-anticoagulated blood perfused over specially-prepared porcine aorta strips under low (212 s⁻¹) and high shear (1690 s⁻¹) conditions in a Badimon Chamber. Plasminogen, tPA and plasminogen activator inhibitor-1 (PAI-1) concentrations were measured by ELISA. The tPA–PAI-1 complex was abundant in Chandler model thrombi serum. In contrast, free tPA was evident in the head of thrombi and correlated with fibrinolytic activity. Badimon thrombi formed under high shear conditions were more resistant to fibrinolysis than those formed at low shear. Plasminogen and tPA concentrations were elevated in thrombi formed at low shear, while PAI-1 concentrations were augmented at high shear rates. In conclusion, tPA primarily localises to the thrombus head in a free and active form. Thrombi formed at high shear incorporate less tPA and plasminogen and increased PAI-1, thereby enhancing resistance to degradation.     

From Plasminogen to Plasmin: Role of Plasminogen Receptors in Human Cancer

Cell surface-associated proteolysis mediated by plasmin (PLA) is an essential feature of wound healing, angiogenesis and cell invasion, processes that are dysregulated in cancer development, progression and systemic spread. The generation of PLA, initiated by the binding of its precursor plasminogen (PLG) to the cell surface, is regulated by an array of activators, inhibitors and receptors. In this review, we will highlight the importance of the best-characterized components of the PLG/PLA cascade in the pathogenesis of cancer focusing on the role of the cell surface-PLG receptors (PLG-R). PLG-R overexpression has been associated with poor prognosis of cancer patients and resistance to chemotherapy. We will also discuss recent findings on the molecular mechanisms regulating cell surface expression and distribution of PLG-R.     

Involvement of residues 296-299 in the enzymatic activity of tissue-type plasminogen activator

The tetra-alanine substitution variant KHRR 296–299 AAAAof tissue-type plasminogen activator (t-PA) was previously shownto have enhanced fibrin specificity and enhanced activity inthe presence of fibrin compared with the wild-type form of themolecule. The structural requirements for these alterationsin enzymatic activity were investigated by constructing severalamino acid substitution variants at each of the positions from296 to 299 and evaluating their activities under a variety ofconditions. Effects on plasminogen activator activity were commonamong the point mutants at positions 296–299; nearly allhad a phenotype similar to the KHRR 296–299 AAAA variant.The greatest effects on enzymatic function were found with multiplesubstitution variants, but some single charge reversals andproline substitutions had substantial effects. The enhancedfibrin specificity of KHRR 296–299 AAAA t-PA results inless fibrinogenolysis than seen with wild-type t-PA. Approximatelyfour times greater concentration of KHRR 296–299 AAAAcompared with wild-type t-PA was required to consume 50% ofthe fibrinogen in human plasma.     

The Role of Fibrinolytic System in Health and Disease

The fibrinolytic system is composed of the protease plasmin, its precursor plasminogen and their respective activators, tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), counteracted by their inhibitors, plasminogen activator inhibitor type 1 (PAI-1), plasminogen activator inhibitor type 2 (PAI-2), protein C inhibitor (PCI), thrombin activable fibrinolysis inhibitor (TAFI), protease nexin 1 (PN-1) and neuroserpin. The action of plasmin is counteracted by α2-antiplasmin, α2-macroglobulin, TAFI, and other serine protease inhibitors (antithrombin and α2-antitrypsin) and PN-1 (protease nexin 1). These components are essential regulators of many physiologic processes. They are also involved in the pathogenesis of many disorders. Recent advancements in our understanding of these processes enable the opportunity of drug development in treating many of these disorders.     

Locating the unpaired cysteine of tissue-type plasminogen activator

Variants of tissue-type plasminogen activator (t-PA) were constructedwith selected cysteines replaced by alanine to evaluate therole of an unpaired cysteine, which has been presumed to bein the growth factor module. C75A, C83A, C84A and CC83–84AAvariants of t-PA were expressed transiently in human embryonickidney cells. The biochemical properties of these variants providedexperimental evidence to identify the unpaired cysteine in t-PA.Assays of amidolytic activity, plasminogen activation (in thepresence or absence of fibrinogen or fibrin), plasma clot lysis,fibrin binding, clearance in mice, and interaction with a panelof monoclonal antibodies were performed as the basis for comparingthese variants with wild-type t-PA. In all assays, C83A t-PAwas biochemically equivalent to wild-type t-PA. C75A t-PA, C84At-PA and CC83-84AA t-PA variants exhibited reduced activitiesin a variety of functional assays. These variants displayedtwo- to threefold lower activity in fibrinogen or fibrin stimulatedplasminogen activation, and fivefold reduced plasma clot lysisactivity compared with that of wild-type t-PA. The affinityof C75A t-PA and C84A t-PA for fibrin was decreased more thantwo orders of magnitude compared with C83A t-PA or wild-typet-PA. Plasma clearance of C75A t-PA and C84A t-PA was reduced2-fold in mice. The C75A, C84A and CC83–84AA variantsdisplayed significantly decreased reactivity with anti-tPA monoclonalantibodies specific for finger/growth factor domain epitopes.These data are consistent with a disulfide linkage of Cys75with Cys84 and that Cys83 exists as an unpaired sulfhydryl.The significance of the unpaired cysteine is as yet undeterminedsince C83A t-PA and wild-type t-PA are functionally equivalent.     

Characterising the Subsite Specificity of Urokinase-Type Plasminogen Activator and Tissue-Type Plasminogen Activator using a Sequence-Defined Peptide Aldehyde Library

Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are two serine proteases that contribute to initiating fibrinolysis by activating plasminogen. uPA is also an important tumour-associated protease due to its role in extracellular matrix remodelling. Overexpression of uPA has been identified in several different cancers and uPA inhibition has been reported as a promising therapeutic strategy. Although several peptide-based uPA inhibitors have been developed, the extent to which uPA tolerates different tetrapeptide sequences that span the P1–P4 positions remains to be thoroughly explored. In this study, we screened a sequence-defined peptide aldehyde library against uPA and tPA. Preferred sequences from the library screen yielded potent inhibitors for uPA, led by Ac-GTAR-H (K_i=18 nm ), but not for tPA. Additionally, synthetic peptide substrates corresponding to preferred inhibitor sequences were cleaved with high catalytic efficiency by uPA but not by tPA. These findings provide new insights into the binding specificity of uPA and tPA and the relative activity of tetrapeptide inhibitors and substrates against these enzymes.     

A Novel Volume-Stable Collagen Matrix Induces Changes in the Behavior of Primary Human Oral Fibroblasts,Periodontal Ligament,and Endothelial Cells

The aim of the present study was to investigate the influence of a novel volume-stable collagen matrix (vCM) on early wound healing events including cellular migration and adhesion, protein adsorption and release, and the dynamics of the hemostatic system. For this purpose, we utilized transwell migration and crystal violet adhesion assays, ELISAs for quantification of adsorbed and released from the matrix growth factors, and qRT-PCR for quantification of gene expression in cells grown on the matrix. Our results demonstrated that primary human oral fibroblasts, periodontal ligament, and endothelial cells exhibited increased migration toward vCM compared to control cells that migrated in the absence of the matrix. Cellular adhesive properties on vCM were significantly increased compared to controls. Growth factors TGF-β1, PDGF-BB, FGF-2, and GDF-5 were adsorbed on vCM with great efficiency and continuously delivered in the medium after an initial burst release within hours. We observed statistically significant upregulation of genes encoding the antifibrinolytic thrombomodulin, plasminogen activator inhibitor type 1, thrombospondin 1, and thromboplastin, as well as strong downregulation of genes encoding the profibrinolytic tissue plasminogen activator, urokinase-type plasminogen activator, its receptor, and the matrix metalloproteinase 14 in cells grown on vCM. As a general trend, the stimulatory effect of the vCM on the expression of antifibrinolytic genes was synergistically enhanced by TGF-β1, PDGF-BB, or FGF-2, whereas the strong inhibitory effect of the vCM on the expression of profibrinolytic genes was reversed by PDGF-BB, FGF-2, or GDF-5. Taken together, our data strongly support the effect of the novel vCM on fibrin clot stabilization and coagulation/fibrinolysis equilibrium, thus facilitating progression to the next stages of the soft tissue healing process.     

Antifibrinolytic effect of single apo(a) kringle domains: relationship to fibrinogen binding

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] areassociated with an increased risk for the development of atheroscleroticdisease which may be attributable to the ability of Lp(a) toattenuate fibrinolysis. A generally accepted mechanism for thiseffect involves direct competition of Lp(a) with plasminogenfor fibrin(ogen) binding sites thus reducing the efficiencyof plasminogen activation. Efforts to determine the domainsof apolipoprotein(a) [apo(a)] which mediate fibrin(ogen) interactionshave yielded conflicting results. Thus, the purpose of the presentstudy was to determine the ability of single KIV domains ofapo(a) to bind plasmin-treated fibrinogen surfaces as well todetermine their effect on fibrinolysis using an in vitro clotlysis assay. A bacterial expression system was utilized to expressand purify apo(a) KIV ₂ , KIV ₇ , KIV ₉ Cys (which lacks theseventh unpaired cysteine) and KIV ₁₀ which contains a stronglysine binding site. We also expressed and examined three mutantderivatives of KIV ₁₀ to determine the effect of changing criticalresidues in the lysine binding site of this kringle on bothfibrin(ogen) binding and fibrin clot lysis. Our results demonstratethat the strong lysine binding site in apo(a) KIV ₁₀ is capableof mediating interactions with plasmin-modified fibrinogen ina lysine-dependent manner, and that this kringle can increase in vitro fibrin clot lysis time by ~43% at a concentrationof 10 µM KIV ₁₀ . The ability of the KIV ₁₀ mutant derivativesto bind plasmin-modified fibrinogen correlated with their lysinebinding capacity. Mutation of Trp ⁷⁰ to Arg abolished bindingto both lysine–Sepharose and plasmin-modified fibrinogen,while the Trp ⁷⁰ Phe and Arg ³⁵ Lys substitutions each resultedin decreased binding to these substrates. None of the KIV ₁₀ mutant derivatives appeared to affect fibrinolysis. Apo(a) KIV ₇ contains a lysine- and proline-sensitive site capable of mediatingbinding to plasmin-modified fibrinogen, albeit with a lowerapparent affinity than apo(a) KIV ₁₀ . However, apo(a) KIV ₇ had no effect on fibrinolysis in vitro . Apo(a) KIV ₂ andKIV ₉ Cys did not bind measurably to plasmin-modified fibrinogensurfaces and did not affect fibrinolysis in vitro .     

Domain-domain interactions in hybrids of tissue-type plasminogen activator and urokinase-type plasminogen activator

Fibrin-dependent plasminogen activation by tissue-type plasminogenactivator (t-PA) is in part associated with the presence ofthe kringle 2 domain in t-PA. Within this kringle 2 domain alysyl-binding site has been described. The plasminogen to plasminconversion by urokinase-type plasminogen activator (u-PA), incontrast to that of t-PA, is not enhanced in the presence offibrin. Within the u-PA kringle domain no lysyl-binding siteis found. To study whether introduction of a lysyl-binding sitein the u-PA kringle domain will make u-PA a fibrin-dependentplasminogen activator, three stretches of amino acid residuesof the u-PA kringle domain (A²⁸-Q³³, D⁵⁵-N⁵⁷ and G⁶⁷-V⁷²) weresubstituted by three stretches of amino acids from the correspondingpositions of the kringle 2 domain of t-PA (M²⁸-K³³, D⁵⁵-D⁵⁷and N⁶⁷-W⁷²). These changes resulted in the creation of thelysyl-binding site consensus of the kringle 2 domain (K³³, D⁵⁵,D⁵⁷, W⁶² and W⁷²) in the u-PA kringle. However, the resultingu-PA mutant did not interact with lysyl-Sepharose, nor did itdisplay fibrin-enhanced plasminogen activation in the presenceof soluble fibrin mimic. When the kringle domain of u-PA wasreplaced by the kringle 2 domain of t-PA, similar results wereobtained. The hybrid protein hardly interacted with lysyl-Sepharoseand the plasminogen activation was not enhanced in the presenceof fibrin mimic However, the N-terminal fragment isolated fromthis hybrid molecule (consisting of growth factor domain andkringle 2 domain) did interact with lysyl-Sepharose, suggestingthat in the hybrid molecule a functional lysyl-binding siteis present but not operational. Indeed, lysine analogue (e-amino-caproicacid) sensitive binding of isolated t-PA kringle 2 domain tou-PA could be observed. The modified u-PA kringle, the wildtype u-PA kringle and the kringle 2 of the u-PA hybrid werealso placed N-terminal of the protease domain of t-PA. As expected,the t-PA mutant consisting of the kringle 2 domain and the proteasedomain bound to lysyl-Sepharose and showed fibrin-dependentplasminogen activation. Further, the hybrid molecule consistingof the u-PA kringle placed N-terminal of the t-PA protease domaindid not display these features. Introduction of the modifiedu-PA kringle N-terminal of the t-PA protease domain resultedin a very weak interaction with lysyl-Sepharose. Despite thehigh overall similarity in primary structure of the modifiedu-PA kringle and t-PA kringle 2 (68%), no fibrin-dependent plasminogenactivation of this hybrid molecule was observed. The above-mentionedresults question the concept that the structural auto-nomousdomains within hybrid plasminogen activators t-PA and u-PA functionas autonomous domains and suggest that interactions betweenthe kringle and the protease domain in hybrid molecules stronglyinfluences their functional features     

Tissue-type plasminogen activator variants with domain duplications and rearrangements

The interactions between tPA domains that are important forcatalysis are poorly understood. We have probed the functionof interdomain interactions by generating tPA variants in whichdomains are duplicated or rearranged. The proteins were expressedin a transient mammalian expression system and tested in vitrofor their ability to activate plasminogen, induce fibrinolysisand bind to a forming fibrin clot. Duplication of the heavychain domains of tPA produced enzymatically active tPA variants,many of which demonstrated similar in vitro amidolytic and fibrinolyticactivity and similar fibrin affinity to the parent molecule.Zymographic analysis of the domain duplication tPA variantsshowed one major active species for each variant. Selectionof the residues duplicated and the interdomain spacing werefound to be critical considerations in the design of tPA variantswith duplicated domains. We also rearranged the domains of tPAsuch that kringle 1 replaced the second kringle domain and viceversa. An analysis of these variants indicates that the firstkringle domain can confer fibrin affinity to a tPA variant andfunction in place of kringle 2. Therefore, in wild-type tPA,the functions of kringle 1 and kringle 2 must be dependent partiallyon their orientation within the heavy chain of the protein.The functional autonomy of the heavy and light chains of tPAis demonstrated by the activity of a tPA variant in which theorder of the heavy and light chains was reversed.     

Natural and engineered plasmin inhibitors: applications and design strategies

The serine protease plasmin is ubiquitously expressed throughout the human body in the form of the zymogen plasminogen. Conversion to active plasmin occurs through enzymatic cleavage by plasminogen activators. The plasminogen activator/plasmin system has a well-established function in the removal of intravascular fibrin deposition through fibrinolysis and the inhibition of plasmin activity; this has found widespread clinical use in reducing perioperative bleeding. Increasing evidence also suggests diverse, although currently less defined, roles for plasmin in a number of physiological and pathological processes relating to extracellular matrix degradation, cell migration and tissue remodelling. In particular, dysregulation of plasmin has been linked to cancer invasion/metastasis and various chronic inflammatory conditions; this has prompted efforts to develop inhibitors of this protease. Although a number of plasmin inhibitors exist, they commonly suffer from poor potency and/or specificity of inhibition that either results in reduced efficacy or prevents clinical use. Consequently, there is a need for further development of high-affinity plasmin inhibitors that maintain selectivity over other serine proteases. This review summarises clearly defined and potential applications for plasmin inhibition. The properties of naturally occurring and engineered plasmin inhibitors are discussed in the context of current knowledge regarding plasmin structure, specificity and function. This includes design strategies to obtain the potency and specificity of inhibition in addition to controlled temporal and spatial distribution tailored for the intended use.     

Fibrinogen and Antifibrinolytic Proteins: Interactions and Future Therapeutics

Thrombus formation remains a major cause of morbidity and mortality worldwide. Current antiplatelet and anticoagulant therapies have been effective at reducing vascular events, but at the expense of increased bleeding risk. Targeting proteins that interact with fibrinogen and which are involved in hypofibrinolysis represents a more specific approach for the development of effective and safe therapeutic agents. The antifibrinolytic proteins alpha-2 antiplasmin (α2AP), thrombin activatable fibrinolysis inhibitor (TAFI), complement C3 and plasminogen activator inhibitor-2 (PAI-2), can be incorporated into the fibrin clot by FXIIIa and affect fibrinolysis by different mechanisms. Therefore, these antifibrinolytic proteins are attractive targets for the development of novel therapeutics, both for the modulation of thrombosis risk, but also for potentially improving clot instability in bleeding disorders. This review summarises the main properties of fibrinogen-bound antifibrinolytic proteins, their effect on clot lysis and association with thrombotic or bleeding conditions. The role of these proteins in therapeutic strategies targeting the fibrinolytic system for thrombotic diseases or bleeding disorders is also discussed.