Ability of Polyphosphate and Nucleic Acids to Trigger Blood Clotting: Some Observations and Caveats.

Polyphosphate plays several roles in coagulation and inflammation, while extracellular DNA and RNA are implicated in thrombosis and as disease biomarkers. We sought to compare the procoagulant activities of polyphosphate versus DNA or RNA isolated from mammalian cells. In a recent study, we found that much of the procoagulant activity of DNA isolated from mammalian cells using Qiagen kits resisted digestion with nuclease or polyphosphatase, and even resisted boiling in acid. These kits employ spin columns packed with silica, which is highly procoagulant. Indeed, much of the apparent procoagulant activity of cellular DNA isolated with such kits was attributable to silica particles shed by the spin columns. Therefore, silica-based methods for isolating nucleic acids or polyphosphate from mammalian cells are not suitable for studying their procoagulant activities. We now report that polyphosphate readily co-purified with DNA and RNA using several popular isolation methods, including phenol/chloroform extraction. Thus, cell-derived nucleic acids are also subject to contamination with traces of cellular polyphosphate, which can be eliminated by alkaline phosphatase digestion. We further report that long-chain polyphosphate was orders of magnitude more potent than cell-derived DNA (purified via phenol/chloroform extraction) or RNA at triggering clotting. Additional experiments using RNA homopolymers found that polyG and polyI have procoagulant activity similar to polyphosphate, while polyA and polyC are not procoagulant. Thus, the procoagulant activity of RNA is rather highly dependent on base composition.

Enzymatically oxidized phospholipids restore thrombin generation in coagulation factor deficiencies.

Hemostatic defects are treated using coagulation factors; however, clot formation also requires a procoagulant phospholipid (PL) surface. Here, we show that innate immune cell-derived enzymatically oxidized phospholipids (eoxPL) termed hydroxyeicosatetraenoic acid-phospholipids (HETE-PLs) restore hemostasis in human and murine conditions of pathological bleeding. HETE-PLs abolished blood loss in murine hemophilia A and enhanced coagulation in factor VIII- (FVIII-), FIX-, and FX-deficient human plasma . HETE-PLs were decreased in platelets from patients after cardiopulmonary bypass (CPB). To explore molecular mechanisms, the ability of eoxPL to stimulate individual isolated coagulation factor/cofactor complexes was tested in vitro. Extrinsic tenase (FVIIa/tissue factor [TF]), intrinsic tenase (FVIIIa/FIXa), and prothrombinase (FVa/FXa) all were enhanced by both HETE-PEs and HETE-PCs, suggesting a common mechanism involving the fatty acid moiety. In plasma, 9-, 15-, and 12-HETE-PLs were more effective than 5-, 11-, or 8-HETE-PLs, indicating positional isomer specificity. Coagulation was enhanced at lower lipid/factor ratios, consistent with a more concentrated area for protein binding. Surface plasmon resonance confirmed binding of FII and FX to HETE-PEs. HETE-PEs increased membrane curvature and thickness, but not surface charge or homogeneity, possibly suggesting increased accessibility to cations/factors. In summary, innate immune-derived eoxPL enhance calcium-dependent coagulation factor function, and their potential utility in bleeding disorders is proposed.

Multiplexed silicon photonic sensor arrays enable facile characterization of coagulation protein binding to nanodiscs with variable lipid content.

Interactions of soluble proteins with the cell membrane are critical within the blood coagulation cascade. Of particular interest are the interactions of γ-carboxyglutamic acid-rich domain-containing clotting proteins with lipids. Variability among conventional analytical methods presents challenges for comparing clotting protein-lipid interactions. Most previous studies have investigated only a single clotting protein and lipid composition and have yielded widely different binding constants. Herein, we demonstrate that a combination of lipid bilayer nanodiscs and a multiplexed silicon photonic analysis technology enables high-throughput probing of many protein-lipid interactions among blood-clotting proteins. This approach allowed direct comparison of the binding constants of prothrombin, factor X, activated factor VII, and activated protein C to seven different binary lipid compositions. In a single experiment, the binding constants of one protein interacting with all lipid compositions were simultaneously determined. A simple surface regeneration then facilitated similar binding measurements for three other coagulation proteins. As expected, our results indicated that all proteins exhibit tighter binding (lower Kd ) as the proportion of anionic lipid increases. Interestingly, at high proportions of phosphatidylserine, the Kd values of all four proteins began to converge. We also found that although koff values for all four proteins followed trends similar to those observed for the Kd values, the variation among the proteins was much lower, indicating that much of the variation came from the kinetic binding (kon) of the proteins. These findings indicate that the combination of silicon photonic microring resonator arrays and nanodiscs enables rapid interrogation of biomolecular binding interactions at model cell membrane interfaces.

Polyphosphate and RNA Differentially Modulate the Contact Pathway of Blood Clotting.

The contact pathway of the plasma clotting cascade is dispensable for normal hemostasis, but contributes to thrombosis and serves as a bridge between inflammation and coagulation. This pathway is triggered upon exposure of plasma to certain anionic polymers and artificial surfaces. Recently, extracellular nucleic acids and inorganic polyphosphate (polyP) have been implicated as being important (patho)physiologically relevant activators of this pathway. However, mechanistic details regarding how nucleic acids or polyP modulate the individual reactions of the contact pathway have been lacking. In this study, we investigate the ability of RNA homopolymers and polyP to bind the primary constituents of the contact pathway: factor XIa, factor XIIa, and plasma kallikrein, in the presence and absence of high molecular weight kininogen (HK), an important cofactor in this pathway. We examine seven proteolytic activation reactions within the contact pathway and report that polyP greatly enhances the rate of all seven, while RNA is effective in supporting only a subset of these reactions. HK both enhances and suppresses these proteolytic activation reactions, depending on the specific reaction evaluated. Overall, we find that polyP is a potent mediator of contact pathway activation reactions in general, that RNA secondary structure may be important to its procoagulant activity, and that nucleic acids versus polyP may differentially modulate specific enzyme activation events within the contact pathway.

High-Resolution NMR Studies of Human Tissue Factor.

In normal hemostasis, the blood clotting cascade is initiated when factor VIIa (fVIIa, other clotting factors are named similarly) binds to the integral membrane protein, human tissue factor (TF). The TF/fVIIa complex in turn activates fX and fIX, eventually concluding with clot formation. Several X-ray crystal structures of the soluble extracellular domain of TF (sTF) exist; however, these structures are missing electron density in functionally relevant regions of the protein. In this context, NMR can provide complementary structural information as well as dynamic insights into enzyme activity. The resolution and sensitivity for NMR studies are greatly enhanced by the ability to prepare multiple milligrams of protein with various isotopic labeling patterns. Here, we demonstrate high-yield production of several isotopically labeled forms of recombinant sTF, allowing for high-resolution NMR studies both in the solid and solution state. We also report solution NMR spectra at sub-mM concentrations of sTF, ensuring the presence of dispersed monomer, as well as the first solid-state NMR spectra of sTF. Our improved sample preparation and precipitation conditions have enabled the acquisition of multidimensional NMR data sets for TF chemical shift assignment and provide a benchmark for TF structure elucidation.

Structure-Function Relationship of the Interaction between Tissue Factor and Factor VIIa.

Interactions between tissue factor and factor VIIa are the primary initiators of coagulation in hemostasis and certain thrombotic diseases. Tissue factor, an integral membrane protein expressed extensively outside of the vasculature, is the regulatory protein cofactor for coagulation factor VIIa. Factor VIIa, a trypsin-like serine protease homologous with other blood coagulation proteases, is weakly active when free in solution and must bind its membrane-bound cofactor for physiologically relevant activity. Tissue factor allosterically activates factor VIIa by several mechanisms such as active site positioning, spatial stabilization, and direct interactions with the substrate. Protein-membrane interactions between tissue factor, factor VIIa, and substrates all play critical roles in modulating the activity of this enzyme complex. Additionally, divalent cations such as Ca(2+) and Mg(2+) are critical for correct protein folding, as well as protein-membrane and protein-protein interactions. The contributions of these factors toward tissue factor-factor VIIa activity are discussed in this review.

Tissue Factor Residues That Modulate Magnesium-Dependent Rate Enhancements of the Tissue Factor/Factor VIIa Complex.

The blood coagulation cascade is initiated when the cell-surface complex of factor VIIa (FVIIa, a trypsin-like serine protease) and tissue factor (TF, an integral membrane protein) proteolytically activates factor X (FX). Both FVIIa and FX bind to membranes via their γ-carboxyglutamate-rich domains (GLA domains). GLA domains contain seven to nine bound Ca(2+) ions that are critical for their folding and function, and most biochemical studies of blood clotting have employed supraphysiologic Ca(2+) concentrations to ensure saturation of these domains with bound Ca(2+). Recently, it has become clear that, at plasma concentrations of metal ions, Mg(2+) actually occupies two or three of the divalent metal ion-binding sites in GLA domains, and that these bound Mg(2+) ions are required for full function of these clotting proteins. In this study, we investigated how Mg(2+) influences FVIIa enzymatic activity. We found that the presence of TF was required for Mg(2+) to enhance the rate of FX activation by FVIIa, and we used alanine-scanning mutagenesis to identify TF residues important for mediating this response to Mg(2+). Several TF mutations, including those at residues G164, K166, and Y185, blunted the ability of Mg(2+) to enhance the activity of the TF/FVIIa complex. Our results suggest that these TF residues interact with the GLA domain of FX in a Mg(2+)-dependent manner (although effects of Mg(2+) on the FVIIa GLA domain cannot be ruled out). Notably, these TF residues are located within or immediately adjacent to the putative substrate-binding exosite of TF.