Blood coagulation factor IX: structural insights impacting hemophilia B therapy.

Coagulation factor IX plays a central role in hemostasis through interaction with factor VIIIa to form the factor X-activating complex at the site of injury. The absence of factor IX activity results in the bleeding disorder hemophilia B. This absence of activity can arise either from a lack of circulating factor IX protein or from mutations that decrease the activity of factor IX. This review focuses on analyzing the structure of factor IX with respect to molecular mechanisms that are at the basis of factor IX function. Proteolytic activation of factor IX to activated factor IX(a) and subsequent structural rearrangements are insufficient to generate fully active factor IXa. Multiple specific interactions between factor IXa, the cofactor VIIIa, and physiological substrate factor X further alter the factor IXa structure to realize the full enzymatic activity of factor IXa. Factor IXa also interacts with inhibitors, extravascular proteins, and cellular receptors that clear factor IX(a) from circulation. Hemophilia B is treated by replacement of the missing factor IX by plasma-derived protein, a recombinant bioequivalent, or via gene therapy. An understanding of how the function of factor IX is tied to structure is leading to modified forms of factor IX that have increased residence time in circulation, higher functional activity, protection from inhibition, and even activity in the absence of factor VIIIa. These modified forms of factor IX have the potential to significantly improve therapy for patients with hemophilia B.

Hypertonicity and/or acidosis induce marked rheological changes under hypoxic conditions in sickle trait red blood cells.

Deformability and sickling of red blood cells (RBCs) from individuals with sickle cell trait (SCT) was evaluated under harsh biophysical conditions that mimic certain vascular beds in vivo. RBC deformability in osmotic-gradient ektacytometry was decreased in HbAS (SCT) compared to HbAA (wild-type) RBCs at supraphysiological osmolalities. RBC deformability was also measured by oxygen-gradient ektacytometry. Whereas RBC sickling was not observed under isotonic and neutral pH conditions, hypertonicity and acidosis alone or in combination induced reversible sickling of SCT RBC. These data suggest that hyperosmolality and/or acidosis enhance hypoxia-induced sickling of SCT RBC.

Red blood cell microvesicles activate the contact system, leading to factor IX activation via 2 independent pathways.

Storage lesion-induced, red cell-derived microvesicles (RBC-MVs) propagate coagulation by supporting the assembly of the prothrombinase complex. It has also been reported that RBC-MVs initiate coagulation via the intrinsic pathway. To elucidate the mechanism(s) of RBC-MV-induced coagulation activation, the ability of storage lesion-induced RBC-MVs to activate each zymogen of the intrinsic pathway was assessed in a buffer system. Simultaneously, the thrombin generation (TG) assay was used to assess their ability to initiate coagulation in plasma. RBC-MVs directly activated factor XII (FXII) or prekallikrein, but not FXI or FIX. RBC-MVs initiated TG in normal pooled plasma and in FXII- or FXI-deficient plasma, but not in FIX-deficient plasma, suggesting an alternate pathway that bypasses both FXII and FXI. Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner. Similarly, purified kallikrein activated FIX in buffer and initiated TG in normal pooled plasma, as well as FXII- or FXI-deficient plasma, but not FIX-deficient plasma. Dual inhibition of FXIIa by corn trypsin inhibitor and kallikrein by soybean trypsin inhibitor was necessary for abolishing RBC-MV-induced TG in normal pooled plasma, whereas kallikrein inhibition alone was sufficient to abolish TG in FXII- or FXI-deficient plasma. Heating RBC-MVs at 60°C for 15 minutes or pretreatment with trypsin abolished TG, suggesting the presence of MV-associated proteins that are essential for contact activation. In summary, RBC-MVs activate both FXII and prekallikrein, leading to FIX activation by 2 independent pathways: the classic FXIIa-FXI-FIX pathway and direct kallikrein activation of FIX. These data suggest novel mechanisms by which RBC transfusion mediates inflammatory and/or thrombotic outcomes.

Computationally Driven Discovery in Coagulation.

Bleeding frequency and severity within clinical categories of hemophilia A are highly variable and the origin of this variation is unknown. Solving this mystery in coagulation requires the generation and analysis of large data sets comprised of experimental outputs or patient samples, both of which are subject to limited availability. In this review, we describe how a computationally driven approach bypasses such limitations by generating large synthetic patient data sets. These data sets were created with a mechanistic mathematical model, by varying the model inputs, clotting factor, and inhibitor concentrations, within normal physiological ranges. Specific mathematical metrics were chosen from the model output, used as a surrogate measure for bleeding severity, and statistically analyzed for further exploration and hypothesis generation. We highlight results from our recent study that employed this computationally driven approach to identify FV (factor V) as a key modifier of thrombin generation in mild to moderate hemophilia A, which was confirmed with complementary experimental assays. The mathematical model was used further to propose a potential mechanism for these observations whereby thrombin generation is rescued in FVIII-deficient plasma due to reduced substrate competition between FV and FVIII for FXa (activated factor X).

The Art and Science of Building a Computational Model to Understand Hemostasis.

Computational models of various facets of hemostasis and thrombosis have increased substantially in the last decade. These models have the potential to make predictions that can uncover new mechanisms within the complex dynamics of thrombus formation. However, these predictions are only as good as the data and assumptions they are built upon, and therefore model building requires intimate coupling with experiments. The objective of this article is to guide the reader through how a computational model is built and how it can inform and be refined by experiments. This is accomplished by answering six questions facing the model builder: (1) Why make a model? (2) What kind of model should be built? (3) How is the model built? (4) Is the model a "good" model? (5) Do we believe the model? (6) Is the model useful? These questions are answered in the context of a model of thrombus formation that has been successfully applied to understanding the interplay between blood flow, platelet deposition, and coagulation and in identifying potential modifiers of thrombin generation in hemophilia A.

Mouse models of hemostasis.

Hemostasis is the normal process that produces a blood clot at a site of vascular injury. Mice are widely used to study hemostasis and abnormalities of blood coagulation because their hemostatic system is similar in most respects to that of humans, and their genomes can be easily manipulated to create models of inherited human coagulation disorders. Two of the most widely used techniques for assessing hemostasis in mice are the tail bleeding time (TBT) and saphenous vein bleeding (SVB) models. Here we discuss the use of these methods in the evaluation of hemostasis, and the advantages and limits of using mice as surrogates for studying hemostasis in humans.

In vitro activation of coagulation by human neutrophil DNA and histone proteins but not neutrophil extracellular traps.

NETosis is a physiologic process in which neutrophils release their nuclear material in the form of neutrophil extracellular traps (NETs). NETs have been reported to directly promote thrombosis in animal models. Although the effects of purified NET components including DNA, histone proteins, and neutrophil enzymes on coagulation have been characterized, the mechanism by which intact NETs promote thrombosis is largely unknown. In this study, human neutrophils were stimulated to produce NETs in platelet-free plasma (PFP) or in buffer using phorbol myristate actetate or calcium ionophore. DNA and histone proteins were also separately purified from normal human neutrophils and used to reconstitute chromatin using a salt-gradient dialysis method. Neutrophil stimulation resulted in robust NET release. In recalcified PFP, purified DNA triggered contact-dependent thrombin generation (TG) and amplified TG initiated by low concentrations of tissue factor. Similarly, in a buffer milieu, DNA initiated the contact pathway and amplified thrombin-dependent factor XI activation. Recombinant human histones H3 and H4 triggered TG in recalcified human plasma in a platelet-dependent manner. In contrast, neither intact NETs, reconstituted chromatin, individual nucleosome particles, nor octameric core histones reproduced any of these procoagulant effects. We conclude that unlike DNA or individual histone proteins, human intact NETs do not directly initiate or amplify coagulation in vitro. This difference is likely explained by the complex histone-histone and histone-DNA interactions within the nucleosome unit and higher-order supercoiled chromatin leading to neutralization of the negative charges on polyanionic DNA and modification of the binding properties of individual histone proteins.

Abnormal joint and bone wound healing in hemophilia mice is improved by extending factor IX activity after hemarthrosis.

Wound healing requires interactions between coagulation, inflammation, angiogenesis, cellular migration, and proliferation. Healing in dermal wounds of hemophilia B mice is delayed when compared with hemostatically normal wild-type (WT) mice, with abnormal persistence of iron deposition, inflammation, and neovascularity. We observed healing following induced joint hemorrhage in WT and factor IX (FIX) knockout (FIX-/-) mice, examining also parameters previously studied in an excisional skin wound model. Hemostatically normal mice tolerated this joint bleeding challenge, cleared blood from the joint, and healed with minimal pathology, even if additional autologous blood was injected intra-articularly at the time of wounding. Following hemarthrosis, joint wound healing in hemophilia B mice was impaired and demonstrated similar abnormal histologic features as previously described in hemophilic dermal wounds. Therefore, studies of pathophysiology and therapy of hemophilic joint bleeding performed in hemostatically normal animals are not likely to accurately reflect the healing defect of hemophilia. We additionally explored the hypothesis that the use of a FIX replacement protein with extended circulating FIX activity could improve synovial and osteochondral wound healing in hemophilic mice, when compared with treatment with unmodified recombinant FIX (rFIX) in the established joint bleeding model. Significantly improved synovial wound healing and preservation of normal osteochondral architecture are achieved by extending FIX activity after hemarthrosis using glycoPEGylated FIX when compared with an equivalent dose of rFIX. These results suggest that treating joint bleeding only until hemostasis is achieved may not result in optimal joint healing, which is improved by extending factor activity.

Assessing the impact of product inhibition in a chromogenic assay.

Chromogenic substrates (CS) are synthetic substrates used to monitor the activity of a target enzyme. It has been reported that some CSs display competitive product inhibition with their target enzyme. Thus, in assays where enzyme activity is continuously monitored over long periods of time, the product inhibition may significantly interfere with the reactions being monitored. Despite this knowledge, it is rare for CSs to be directly incorporated into mathematical models that simulate these assays. This devalues the predictive power of the models. In this study, we examined the interactions between a single enzyme, coagulation factor Xa, and its chromogenic substrate. We developed, and experimentally validated, a mathematical model of a chromogenic assay for factor Xa that explicitly included product inhibition from the CS. We employed Bayesian inference, in the form of Markov-Chain Monte Carlo, to estimate the strength of the product inhibition and other sources of uncertainty such as pipetting error and kinetic rate constants. Our model, together with carefully calibrated biochemistry experiments, allowed for full characterization of the strength and impact of product inhibition in the assay. The effect of CS product inhibition in more complex reaction mixtures was further explored using mathematical models.

Prophylactic efficacy of BeneFIX vs Alprolix in hemophilia B mice.

FIX binds tightly to collagen IV. Furthermore, a FIX mutant, FIXK5R, which binds better than wild-type FIX to collagen IV, provides better hemostasis than wild-type FIX, long after both are undetectable in the plasma. There is also credible evidence of extravascular FIX. Here, we use the saphenous vein bleeding model to compare the efficacy of recombinant FIXFc (Alprolix) and wild-type FIX (BeneFIX) in hemophilia B mice 7 days postinfusion. Although the terminal half-life of Alprolix is significantly longer than that of BeneFIX, at equal doses Alprolix is not better at controlling bleeding 7 days postinfusion, presumably because of the extravascular FIX. Both BeneFIX and Alprolix exhibit a linear response in clotting efficacy up to 150 IU/kg, where they appear to saturate an extravascular compartment, because there is no additional prophylactic benefit from higher doses. A robust pool of extravascular FIX is clearly observed surrounding blood vessels, localized to the same region as collagen IV, in 2 representative human tissues: liver and skeletal muscle. We see no increased risk for thrombosis at 250 IU/kg FIX at 6 hours postinfusion. In summary, 7 days postinfusion into hemophilia B mice, BeneFIX and Alprolix are hemostatically indistinguishable despite the latter's increased half-life. We predict that doses of FIX ∼3 times higher than the currently recommended 40 to 50 IU/kg will, because of FIX's large extravascular compartment, efficiently prolong prophylactic hemostasis without thrombotic risk.

Impact of Non-Vitamin K Antagonist Oral Anticoagulants From a Basic Science Perspective.

The biochemical properties of the non-vitamin K antagonist oral anticoagulants (NOACs) and their differences from the mechanism of action of vitamin K antagonists contribute to their properties as anticoagulants. These properties include as follows: (1) Inhibiting a single protease is much less effective at inhibiting coagulation than is inhibiting at multiple steps. Thus, the dose-response relationship between NOAC level and intensity of anticoagulation is shallower and more linear than that of vitamin K antagonists. This partially accounts for the greater safety of NOACs than vitamin K antagonists reported in some studies. (2) Because they are small molecules, NOACs can reach their target proteases in locations that plasma protease inhibitors, such as antithrombin, cannot. (3) NOACs compete with substrates for binding at the active site of the target protease and that binding is reversible. When the drug level falls, the drug dissociates from its target, and protease activity is restored. Thus, there is the possibility of a rebound in procoagulant activity if the drug is abruptly terminated. (4) The effects of a NOAC can be overcome by increasing the amount of substrate available for the target protease or the amount of protease produced. This property may contribute to the safety of NOACs and their potential reversibility by coagulation factor concentrates. The biochemical properties of NOACs contribute to their suitability for use in conditions that require a predictable moderate degree of anticoagulation when administered orally at a consistent dose. Their effects can be overcome by a sufficiently strong procoagulant stimulus. This characteristic likely contributes to their generally reduced risk of serious bleeding. However, they are not well suited for use in settings that require a profound degree of anticoagulation.

Single synonymous mutation in factor IX alters protein properties and underlies haemophilia B.

BACKGROUND: Haemophilia B is caused by genetic aberrations in the F9 gene. The majority of these are non-synonymous mutations that alter the primary structure of blood coagulation factor IX (FIX). However, a synonymous mutation c.459G>A (Val107Val) was clinically reported to result in mild haemophilia B (FIX coagulant activity 15%-20% of normal). The F9 mRNA of these patients showed no skipping or retention of introns and/or change in mRNA levels, suggesting that mRNA integrity does not contribute to the origin of the disease in affected individuals. The aim of this study is to elucidate the molecular mechanisms that can explain disease manifestations in patients with this synonymous mutation. METHODS: We analyse the molecular mechanisms underlying the FIX deficiency through in silico analysis and reproducing the c.459G>A (Val107Val) mutation in stable cell lines. Conformation and non-conformation sensitive antibodies, limited trypsin digestion, activity assays for FIX, interaction with other proteins and post-translation modifications were used to evaluate the biophysical and biochemical consequences of the synonymous mutation. RESULTS: The Val107Val synonymous mutation in F9 was found to significantly diminish FIX expression. Our results suggest that this mutation slows FIX translation and affects its conformation resulting in decreased extracellular protein level. The altered conformation did not change the specific activity of the mutated protein. CONCLUSIONS: The pathogenic basis for one synonymous mutation (Val107Val) in the F9 gene associated with haemophilia B was determined. A mechanistic understanding of this synonymous variant yields potential for guiding and developing future therapeutic treatments.

Polyphosphates rock! A role in thrombosis?

In this issue of Blood, Zhu et al have established, in human blood, that factor XIa and polyphosphate make significant contributions to thrombus formation. This makes these molecules good targets for therapeutic intervention.

FVIIa as used pharmacologically is not TF dependent in hemophilia B mice.

Activated factor VII is approved for treating hemophilia patients with autoantibodies to their factor IX or FVIII; however, its mechanism of action remains controversial. Some studies suggest that FVIIa requires tissue factor (TF) for function and that the reason for the high dose requirement is that it must compete with endogenous FVII for tissue factor. Others suggest that FVIIa binds platelets where it activates FX directly; the high concentration required would result from FVIIa's weak affinity for phospholipids. We address this question by infusing a chimera of mouse FIX (Gla and EGF1) with FVIIa (EGF2 and catalytic domain) into hemophilia B mice. This mutant has no TF-dependent activity because it cannot functionally bind TF at physiologically relevant concentrations. In vivo, this mutant is as effective as mouse FVIIa in controlling bleeding in hemophilia B mice. Our results suggest that the hemostatic effect of pharmacologic doses of FVIIa is TF independent.

Differential contribution of FXa and thrombin to vascular inflammation in a mouse model of sickle cell disease.

Activation of coagulation and vascular inflammation are prominent features of sickle cell disease (SCD). Previously, we have shown that inhibition of tissue factor (TF) attenuates activation of coagulation and vascular inflammation in mouse models of SCD. In this study, we examined the mechanism by which coagulation proteases enhance vascular inflammation in sickle BERK mice. To specifically investigate the contribution of FXa and thrombin, mice were fed chow containing either rivaroxaban or dabigatran, respectively. In addition, we used bone marrow transplantation to generate sickle mice deficient in either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) on nonhematopoietic cells. FXa inhibition and PAR-2 deficiency in nonhematopoietic cells attenuated systemic inflammation, measured by plasma levels of interleukin-6 (IL-6). In contrast, neither thrombin inhibition nor PAR-1 deficiency in nonhematopoietic cells affected plasma levels of IL-6 in sickle mice. However, thrombin did contribute to neutrophil infiltration in the lung, independently of PAR-1 expressed by nonhematopoietic cells. Furthermore, the TF-dependent increase in plasma levels of soluble vascular cell adhesion molecule-1 in sickle mice was not mediated by FXa or thrombin. Our data indicate that TF, FXa, and thrombin differentially contribute to vascular inflammation in a mouse model of SCD.

Reversal of dabigatran effects in models of thrombin generation and hemostasis by factor VIIa and prothrombin complex concentrate.

BACKGROUND: The oral thrombin inhibitor dabigatran has the drawbacks that it does not have a validated antidote. Data from animal studies and plasma coagulation assays suggest that prothrombin complex concentrate (PCC) or recombinant factor VIIa (FVIIa) might reverse dabigatran anticoagulation. METHODS: Cellular elements make a significant contribution to hemostasis. Our goals were to (1) test the hypothesis that both FVIIa and a 4-factor PCC improve parameters of thrombin generation in the presence of dabigatran in a cell-based model; and (2) determine whether results in a cell-based model correlate with hemostasis in vivo. RESULTS: PCC reversed dabigatran effects on the rate, peak, and total amount of thrombin but did not shorten the lag (n = 6 experiments in triplicate). By contrast, FVIIa shortened the lag, increased the rate and peak, but did not improve total thrombin (n = 6). Effects of PCC were seen at both therapeutic and markedly supratherapeutic dabigatran levels, whereas beneficial effects of FVIIa decreased as the dabigatran level increased. The PCC effect was reproduced by adding prothrombin, factor X, and factor IX. At therapeutic dabigatran levels, both PCC and FVIIa normalized hemostasis time in a mouse saphenous vein bleeding model. CONCLUSIONS: A cell-based model reflects the effects on thrombin generation of clinically relevant levels of FVIIa and PCC in the presence of dabigatran. Enhancing the rate of thrombin generation and peak thrombin level appear to correlate best with hemostasis in vivo. The ineffectiveness of FVIIa at supratherapeutic dabigatran levels may explain conflicting reports of its efficacy in dabigatran reversal.

Superactivated platelets: thrombus regulators, thrombin generators, and potential clinical targets.

Platelets contribute to hemostasis by forming the platelet plug and then contributing to coagulation by providing a catalytic surface where thrombin generation occurs efficiently. This catalytic activity, known as the platelet procoagulant response, is being recognized as a nuanced response. This review examines platelets' response to strong stimuli, which results in the formation of a platelet subpopulation (superactivated platelets) with several unique properties, including enhanced procoagulant activity. These platelets contribute uniquely to thrombus architecture and seem to have thrombus regulatory activity. Superactivated platelets' role in diseases of thrombosis and hemostasis, as either potentiating or mitigating factors, is not currently known, but may be an important pharmacological target.