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. (Circ Res.

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.

NCT00920478.

Summary. 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.

The complement system plays an integral part of a host's innate immunity, and its activation is highly dependent on the chemistry and structure of a "foreign" target surface. We determined that the conformational state of glycopolymer chains, defined by the grafting density (chains/nm(2)), on the nanoparticle (NP) surface acts as a "molecular switch" for complement activation and amplification, and the protein corona on the NP surface dictates this process. A grafting density threshold was determined, below which minimal complement activation was observed and above which substantial complement activation was detected. The glycopolymer-grafted NPs activated complement via the alternative pathway. The chemical structure of pendent sugar units on the grafted polymer was also an important determinant for complement activation. NPs grafted with glucose-containing polymer activated complement at a lower grafting density compared to NPs grafted with galactose-containing polymer. Analysis of complement activation products C3a and SC5b-9 followed a similar pattern. Complement activation on the NP surface was independent of particle size or concentration for a given conformational state of grafted polymer. To gain insight into a putative surface-dependent mechanism of complement activation, we determined the nature of adsorbed protein corona on various NPs through quantitative mass spectrometry. Elevated levels of two pro-complement proteins, factors B and C3, present on the NP surface grafted with glycopolymer chains at high grafting density compared to low grafting density surface, may be responsible for its complement activity. Galactose polymer modified NPs adsorbed more of the negative regulator of complement, factor H, than the glucose surface, providing an explanation for its lower level of complement activation.

Key Points Polyphosphate suppresses complement via the terminal pathway by destabilizing C5b,6, thereby reducing the lytic capacity of the membrane attack complex. Polyphosphate, a novel negative regulator of complement, bridges coagulation and complement and is a potential therapeutic target.

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 10(6) 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.

Coagulation factor I (fibrinogen) plays an essential role in the hemostatic system by bridging activated platelets and being the key substrate for thrombin in establishing a consolidating fibrin network. Fibrinogen is synthesized in the liver and the plasma concentration is 1 to 5-4.0 g/L. During recent 10 years, fibrinogen has been recognized to play an important role in controlling hemorrhage. Dilutional coagulopathy induced by colloid plasma expanders is characterized by fibrinogen deficiency and dysfunctional fibrin polymerization. Trauma and use of extracorporeal circulation is also known to reduce levels of fibrinogen. A series of laboratory experiments and experimental animal studies have suggested fibrinogen as a potent hemostatic agent. These data are supported by retrospective surveys as well as a series of prospective proof of principal clinical trials. This article provides a description of the biochemistry and mechanisms of fibrinogen as well as the etiology for developing fibrinogen deficiency. Furthermore, it summarizes laboratory and experimental data on the role of fibrinogen in dilutional coagulopathy and addresses laboratory monitoring issues. Finally, it lists retrospective and prospective studies, which have been designed to assess the clinical efficacy and safety of hemostatic intervention with fibrinogen concentrate.