Jonathan H. Foley

Insights into thrombin activatable fibrinolysis inhibitor function and regulation.

Fibrinolysis is initiated when the zymogen plasminogen is converted to plasmin via the action of plasminogen activators. Proteolytic cleavage of fibrin by plasmin generates C‐terminal lysine residues capable of binding both plasminogen and the plasminogen activator, thereby stimulating plasminogen activator‐mediated plasminogen activation and propagating fibrinolysis. This positive feedback mechanism is regulated by activated thrombin activatable fibrinolysis inhibitor (TAFIa), which cleaves C‐terminal lysine residues from the fibrin surface, thereby decreasing its cofactor activity. TAFI can be activated by thrombin alone, but the rate of activation is accelerated when in complex with thrombomodulin. Plasmin is also known to activate TAFI. TAFIa has no known physiologic inhibitors and consequently, its primary regulatory mechanism involves its intrinsic thermal instability. The rate of TAFI activation and stability of the active form, TAFIa, function in maintaining its concentration above the threshold value required to down‐regulate fibrinolysis. Although all methods to quantify TAFI or TAFIa have their limitations, epidemiologic studies have indicated that elevated TAFI levels are correlated with an increased risk of venous thrombosis. Major efforts have been made to develop TAFI inhibitors that can either directly interfere with TAFIa activity or impair its activation. However, the anti‐inflammatory properties of TAFIa might complicate the development and application of a TAFIa inhibitor that aims to increase the efficiency of thrombolytic therapy.

Cross Talk Pathways Between Coagulation and Inflammation

Anatomic pathology studies performed over 150 years ago revealed that excessive activation of coagulation occurs in the setting of inflammation. However, it has taken over a century since these seminal observations were made to delineate the molecular mechanisms by which these systems interact and the extent to which they participate in the pathogenesis of multiple diseases. There is, in fact, extensive cross talk between coagulation and inflammation, whereby activation of one system may amplify activation of the other, a situation that, if unopposed, may result in tissue damage or even multiorgan failure. Characterizing the common triggers and pathways are key for the strategic design of effective therapeutic interventions. In this review, we highlight some of the key molecular interactions, some of which are already showing promise as therapeutic targets for inflammatory and thrombotic disorders.

Hi-Fi SELEX: A High-Fidelity Digital-PCR Based Therapeutic Aptamer Discovery Platform.

Current technologies for aptamer discovery typically leverage the systematic evolution of ligands by exponential enrichment (SELEX) concept by recursively panning semi‐combinatorial ssDNA or RNA libraries against a molecular target. The expectation is that this iterative selection process will be sufficiently stringent to identify a candidate pool of specific high‐affinity aptamers. However, failure of this process to yield promising aptamers is common, due in part to (i) limitations in library designs, (ii) retention of non‐specific aptamers during screening rounds, (iii) excessive accumulation of amplification artifacts, and (iv) the use of screening criteria (binding affinity) that does not reflect therapeutic activity. We report a new selection platform, High‐Fidelity (Hi‐Fi) SELEX, that introduces fixed‐region blocking elements to safeguard the functional diversity of the library. The chemistry of the target‐display surface and the composition of the equilibration solvent are engineered to strongly inhibit non‐specific retention of aptamers. Partition efficiencies approaching 106 are thereby realized. Retained members are amplified in Hi‐Fi SELEX by digital PCR in a manner that ensures both elimination of amplification artifacts and stoichiometric conversion of amplicons into the single‐stranded library required for the next selection round. Improvements to aptamer selections are first demonstrated using human α‐thrombin as the target. Three clinical targets (human factors IXa, X, and D) are then subjected to Hi‐Fi SELEX. For each, rapid enrichment of ssDNA aptamers offering an order‐nM mean equilibrium dissociation constant (Kd) is achieved within three selection rounds, as quantified by a new label‐free qPCR assay reported here. Therapeutic candidates against factor D are identified. Biotechnol. Bioeng. 2015;112: 1506–1522.

Polyphosphate suppresses complement via the terminal pathway

Polyphosphate, synthesized by all cells, is a linear polymer of inorganic phosphate. When released into the circulation, it exerts prothrombotic and proinflammatory activities by modulating steps in the coagulation cascade. We examined the role of polyphosphate in regulating the evolutionarily related proteolytic cascade complement. In erythrocyte lysis assays, polyphosphate comprising more than 1000 phosphate units suppressed total hemolytic activity with a concentration to reduce maximal lysis to 50% that was 10-fold lower than with monophosphate. In the ion- and enzyme-independent terminal pathway complement assay, polyphosphate suppressed complement in a concentration- and size-dependent manner. Phosphatase-treated polyphosphate lost its ability to suppress complement, confirming that polymer integrity is required. Sequential addition of polyphosphate to the terminal pathway assay showed that polyphosphate interferes with complement only when added before formation of the C5b-7 complex. Physicochemical analyses using native gels, gel filtration, and differential scanning fluorimetry revealed that polyphosphate binds to and destabilizes C5b,6, thereby reducing the capacity of the membrane attack complex to bind to and lyse the target cell. In summary, we have added another function to polyphosphate in blood, demonstrating that it dampens the innate immune response by suppressing complement. These findings further establish the complex relationship between coagulation and innate immunity.

Complement Activation in Arterial and Venous Thrombosis is Mediated by Plasmin

Thrombus formation leading to vaso-occlusive events is a major cause of death, and involves complex interactions between coagulation, fibrinolytic and innate immune systems. Leukocyte recruitment is a key step, mediated partly by chemotactic complement activation factors C3a and C5a. However, mechanisms mediating C3a/C5a generation during thrombosis have not been studied. In a murine venous thrombosis model, levels of thrombin–antithrombin complexes poorly correlated with C3a and C5a, excluding a central role for thrombin in C3a/C5a production. However, clot weight strongly correlated with C5a, suggesting processes triggered during thrombosis promote C5a generation. Since thrombosis elicits fibrinolysis, we hypothesized that plasmin activates C5 during thrombosis. In vitro, the catalytic efficiency of plasmin-mediated C5a generation greatly exceeded that of thrombin or factor Xa, but was similar to the recognized complement C5 convertases. Plasmin-activated C5 yielded a functional membrane attack complex (MAC). In an arterial thrombosis model, plasminogen activator administration increased C5a levels. Overall, these findings suggest plasmin bridges thrombosis and the immune response by liberating C5a and inducing MAC assembly. These new insights may lead to the development of strategies to limit thrombus formation and/or enhance resolution.

Interplay between fibrinolysis and complement: plasmin cleavage of iC3b modulates immune responses.

The plasmin(ogen) and complement systems are simultaneously activated at sites of tissue injury, participating in hemostasis, wound healing, inflammation and immune surveillance. In particular, the C3 proteolytic fragment, iC3b, and its degradation product C3dg, which is generated by cleavage by factor I (FI) and the cofactor complement receptor CR1, are important in bridging innate and adaptive immunity. Via a thioester (TE) bond, iC3b and C3dg covalently tag pathogens, modulating phagocytosis and adaptive immune responses.

Gas6 gains entry into the coagulation cascade.

In this issue of Blood, Robins and colleagues provide new insights into how Gas6 promotes thrombosis through contributions from platelets and from the vascular wall. By showing that Gas6 up-regulates the initiator of coagulation, tissue factor, in the endothelium, these studies may yield new and safer treatments for thrombotic disease.1

FXIII: mechanisms of action in the treatment of hemophilia A

Hemophilia is characterized by abnormal thrombin generation and impaired clot stability. FXIII promotes clot stability and may be a useful adjunct treatment for hemophilia.

Evaluation of and recommendation for the nomenclature of the CPB2 gene product (also known as TAFI and proCPU): communication from the SSC of the ISTH

J . H . FOLEY ,* P . Y . K IM,† D. HENDRIKS ,‡ J . MORSER ,§ A. G ILS ¶ and N . J . MUTCH,** FOR THE SUBCOMMITTEE ON F IBR INOLYS I S *Research Department of Haematology, University College London and Katherine Dormandy Haemophilia Centre and Thrombosis Unit, London, UK; †Thrombosis and Atherosclerosis Research Institute, McMaster University, Hamilton, ON, Canada; ‡Laboratory for Medical Biochemistry, University of Antwerp, Antwerp, Belgium; §Division of Hematology, School of Medicine, Stanford University, Stanford, CA, USA; ¶Laboratory for Therapeutic and Diagnostic Antibodies, KU Leuven, Leuven, Belgium; and **Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK

An in-vitro assessment of tranexamic acid as an adjunct to rFVIII or rFVIIa treatment in haemophilia A.

Haemophilia is characterised by defective thrombin generation, reduced clot stability and spontaneous bleeding. Treatment with factor VIII (FVIII) concentrate or bypassing agents (e.g. recombinant factor VIIa (rFVIIa)) is generally effective. Occasionally, haemostasis is not achieved, which may reflect a failure of factor concentrate to normalise clot stability. Tranexamic acid (TXA) is often used to aid haemostasis in surgery (e.g. joint replacements and dental procedures). Used routinely as an adjunct, it may enhance clot stability and allow effective, reliable, and cost-effective treatment at lower doses of factor concentrate. This study hypothesised that clot stabilising adjunct TXA is required in addition to factor substitution to normalise clot stability in whole blood from patients with severe haemophilia A. The in vitro effect of varying concentrations of recombinant FVIII or recombinant FVIIa and adjunct TXA on whole blood clot stability was measured by thromboelastometry. Coagulation was triggered by tissue factor and clots were challenged with tissue plasminogen activator. The area under the elasticity curve was the primary endpoint. High concentrations of FVIII and rFVIIa increased clot stability to levels that were not significantly different from controls (Mean ± SD: control 112,694 ± 84,115; FVIII 78,662 ± 74,126; rFVIIa 95,918 ± 88,492). However, the response was highly variable between individuals and demonstrates why some patients show clinical resistance to treatment. Addition of TXA resulted in normalised clot stability in all individuals, even when combined with the lowest doses of factor concentrate. The results support the concept that a more efficient, reliable and cost effective treatment may be obtained if TXA is combined with factor concentrates to treat individuals with haemophilia.