Quantification of citrullinated histones: Development of an improved assay to reliably quantify nucleosomal H3Cit in human plasma.

BACKGROUND: Recent data propose a diagnostic and prognostic capacity for citrullinated histone H3 (H3Cit), a marker of neutrophil extracellular traps (NETs), in pathologic conditions such as cancer and thrombosis. However, current research is hampered by lack of standardized assays. OBJECTIVES: We aimed to develop an assay to reliably quantify nucleosomal H3Cit in human plasma. METHODS: We assessed the common practice of in vitro enzymatically modified histone H3 as calibration standards and the specificity of available intrapeptidyl citrulline antibodies. Based on our findings, we developed and validated a novel assay to quantify nucleosomal H3Cit in human plasma. RESULTS: We show that enzymatically citrullinated H3 proteins are compromised by high enzyme-dependent lot variability as well as instability in plasma. We furthermore demonstrate that the majority of commercially available antibodies against intrapeptidyl citrulline display poor specificity for their reported target when tested against a panel of semi-synthetic nucleosomes containing distinct histone H3 citrullinations. Finally, we present a novel assay utilizing highly specific monoclonal antibodies and semi-synthetic nucleosomes containing citrulline in place of arginine at histone H3, arginine residues 2, 8, and 17 (H3R2,8,17Cit) as calibration standards. Rigorous validation of this assay shows its capacity to accurately and reliably quantify nucleosomal H3Cit levels in human plasma with clear elevations in cancer patients compared to healthy individuals. CONCLUSIONS: Our novel approach using defined nucleosome controls enables reliable quantification of H3Cit in human plasma. This assay will be broadly applicable to study the role of histone citrullination in disease and its utility as a biomarker.

Chromatin structure and its chemical modifications regulate the ubiquitin ligase substrate selectivity of UHRF1.

Mitotic inheritance of DNA methylation patterns is facilitated by UHRF1, a DNA- and histone-binding E3 ubiquitin ligase that helps recruit the maintenance DNA methyltransferase DNMT1 to replicating chromatin. The DNA methylation maintenance function of UHRF1 is dependent on its ability to bind chromatin, where it facilitates monoubiquitination of histone H3 at lysines 18 and 23, a docking site for DNMT1. Because of technical limitations, this model of UHRF1-dependent DNA methylation inheritance has been constructed largely based on genetics and biochemical observations querying methylated DNA oligonucleotides, synthetic histone peptides, and heterogeneous chromatin extracted from cells. Here, we construct semisynthetic mononucleosomes harboring defined histone and DNA modifications and perform rigorous analysis of UHRF1 binding and enzymatic activity with these reagents. We show that multivalent engagement of nucleosomal linker DNA and dimethylated lysine 9 on histone H3 directs UHRF1 ubiquitin ligase activity toward histone substrates. Notably, we reveal a molecular switch, stimulated by recognition of hemimethylated DNA, which redirects UHRF1 ubiquitin ligase activity away from histones in favor of robust autoubiquitination. Our studies support a noncompetitive model for UHRF1 and DNMT1 chromatin recruitment to replicating chromatin and define a role for hemimethylated linker DNA as a regulator of UHRF1 ubiquitin ligase substrate selectivity.

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.

In vitro and in vivo characterization of a reversible synthetic heparin analog.

BACKGROUND: The global supply of unfractionated heparin (UFH) and all commercially available low molecular weight heparins (LMWH) remain dependent on animal sources, such as porcine intestine or bovine lung. Recent experience has shown that contamination of the supply chain (with over-sulfated chondroitin sulfates) can result in lethal toxicity. Fondaparinux is currently the only commercially available synthetic analog of heparin. We recently described a new class of chemoenzymatically synthesized heparin analogs. One of these compounds (S12-mer) is a dodecasaccharide consisting of an antithrombin-binding moiety with repeating units of IdoA2S-GlcNS6S and two 3-O-sulfate groups that confer the ability to bind protamine. OBJECTIVE/METHODS: We sought to further characterize this new compound in vitro using biochemical and global coagulation assays and in vivo using thrombosis and hemostasis assays. RESULTS: The anticoagulant activities of the Super 12-mer (S12-mer) and Enoxaparin in anti-factor Xa and plasma-based thrombin generation assays were roughly equivalent with a 50% reduction in peak thrombin generation occurring at approximately 325nM. When protamine was titrated against a fixed concentration of S12-mer in plasma or blood, the S12-mer displayed a significant restitution of thrombin generation and clot formation. In vivo, S12-mer inhibited venous thrombosis to a similar extent as Enoxaparin, with similar bleeding profiles. CONCLUSIONS: These data show that the S12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the ability to ensure purity and homogeneity from batch to batch, the S12-mer is a promising new synthetic heparin analog with a potentially enhanced safety profile.

Red blood cells and thrombin generation in sickle cell disease.

The prothrombotic nature of sickle cell disease (SCD) is evidenced by the chronically elevated levels of almost all coagulation activation biomarkers, and an increased incidence of certain thrombotic events, including venous thromboembolism. Numerous studies have attempted to define the extent and elucidate the mechanism of the observed increase in thrombin generation in SCD patients in vivo. In general, these studies were performed using thrombin generation assays in platelet poor or platelet rich plasma and showed little difference in endogenous thrombin potential between the SCD cohort and healthy matched controls. In SCD, erythrocytes and monocytes have been demonstrated to exhibit procoagulant characteristics. Thus, the absence of these cellular components in standard thrombin generation assays may fail to reflect global hypercoagulability in the whole blood of patients with SCD. We were therefore surprised to see no difference in net thrombin generation in tissue factor-initiated initiated clotting of whole blood from patients with SCD. However, we are continuing to reconcile these seemingly disparate observations by slight modifications of the whole blood model that include alternative coagulation triggers and a re-examination of the net thrombin generation when the protein/protein S system is simultaneously interrogated.

Thrombin generation and fibrin clot formation under hypothermic conditions: an in vitro evaluation of tissue factor initiated whole blood coagulation.

BACKGROUND: Despite trauma-induced hypothermic coagulopathy being familiar in the clinical setting, empirical experimentation concerning this phenomenon is lacking. In this study, we investigated the effects of hypothermia on thrombin generation, clot formation, and global hemostatic functions in an in vitro environment using a whole blood model and thromboelastography, which can recapitulate hypothermia. METHODS: Blood was collected from healthy individuals through venipuncture and treated with corn trypsin inhibitor, to block the contact pathway. Coagulation was initiated with 5pM tissue factor at temperatures 37°C, 32°C, and 27°C. Reactions were quenched over time, with soluble and insoluble components analyzed for thrombin generation, fibrinogen consumption, factor (f)XIII activation, and fibrin deposition. Global coagulation potential was evaluated through thromboelastography. RESULTS: Data showed that thrombin generation in samples at 37°C and 32°C had comparable rates, whereas 27°C had a much lower rate (39.2 ± 1.1 and 43 ± 2.4 nM/min vs 28.6 ± 4.4 nM/min, respectively). Fibrinogen consumption and fXIII activation were highest at 37°C, followed by 32°C and 27°C. Fibrin formation as seen through clot weights also followed this trend. Thromboelastography data showed that clot formation was fastest in samples at 37°C and lowest at 27°C. Maximum clot strength was similar for each temperature. Also, percent lysis of clots was highest at 37°C followed by 32°C and then 27°C. CONCLUSIONS: Induced hypothermic conditions directly affect the rate of thrombin generation and clot formation, whereas global clot stability remains intact.

Platelets do not express the oxidized or reduced forms of tissue factor.

BACKGROUND: Expression of tissue factor (TF) antigen and activity in platelets is controversial and dependent upon the laboratory and reagents used. Two forms of TF were described: an oxidized functional form and a reduced nonfunctional form that is converted to the active form through the formation of an allosteric disulfide. This study tests the hypothesis that the discrepancies regarding platelet TF expression are due to differential expression of the two forms. METHODS: Specific reagents that recognize both oxidized and reduced TF were used in flow cytometry of unactivated and activated platelets and western blotting of whole platelet lysates. TF-dependent activity measurements were used to confirm the results. RESULTS: Western blotting analyses of placental TF demonstrated that, in contrast to anti-TF#5, which is directed against the oxidized form of TF, a sheep anti-human TF polyclonal antibody recognizes both the reduced and oxidized forms. Flow cytometric analyses demonstrated that the sheep antibody did not react with the surface of unactivated platelets or platelets activated with thrombin receptor agonist peptide, PAR-1. This observation was confirmed using biotinylated active site-blocked factor (F)VIIa: no binding was observed. Likewise, neither form of TF was detected by western blotting of whole platelet lysates with sheep anti-hTF. Consistent with these observations, no FXa or FIXa generation by FVIIa was detected at the surface of these platelets. Similarly, no TF-related activity was observed in whole blood using thromboelastography. CONCLUSION AND SIGNIFICANCE: Platelets from healthy donors do not express either oxidized (functional) or reduced (nonfunctional) forms of TF.

The role of the red cell membrane in thrombin generation.

Red blood cells have historically been viewed as innocent bystanders in the process of blood coagulation and thrombin generation; however a century of clinical evidence linking red blood cells to thrombosis suggests the contrary. In this brief review, the biochemical evidence for red blood cell involvement in thrombin generation is evaluated. It is concluded that in addition to platelets, red blood cells actively participate in thrombin generation. A sub-fraction of red blood cells express phosphatidylserine on their surface and unlike platelets, red blood cells produce thrombin through the meizothrombin pathway, which has interesting consequences in the context of clot formation and stabilization.

Prothrombin activation in blood coagulation: the erythrocyte contribution to thrombin generation.

Prothrombin activation can proceed through the intermediates meizothrombin or prethrombin-2. To assess the contributions that these 2 intermediates make to prothrombin activation in tissue factor (Tf)-activated blood, immunoassays were developed that measure the meizothrombin antithrombin (mTAT) and α-thrombin antithrombin (αTAT) complexes. We determined that Tf-activated blood produced both αTAT and mTAT. The presence of mTAT suggested that nonplatelet surfaces were contributing to approximately 35% of prothrombin activation. Corn trypsin inhibitor-treated blood was fractionated to yield red blood cells (RBCs), platelet-rich plasma (PRP), platelet-poor plasma (PPP), and buffy coat. Compared with blood, PRP reconstituted with PPP to a physiologic platelet concentration showed a 2-fold prolongation in the initiation phase and a marked decrease in the rate and extent of αTAT formation. Only the addition of RBCs to PRP was capable of normalizing αTAT generation. FACS on glycophorin A-positive cells showed that approximately 0.6% of the RBC population expresses phosphatidylserine and binds prothrombinase (FITC Xa·factor Va). These data indicate that RBCs participate in thrombin generation in Tf-activated blood, producing a membrane that supports prothrombin activation through the meizothrombin pathway.

The resuscitative fluid you choose may potentiate bleeding.

BACKGROUND: Trauma is the leading cause of death in the younger population in the United States, frequently from the development of hemorrhagic shock. Controversy exists over the type of volume resuscitation for restoring hemodynamic stability that should be used in hemorrhagic shock. Little is known about how various resuscitative paradigms affect the coagulation cascade, which is essential to controlling hemorrhagic shock. METHODS: We studied the effect of various resuscitative formulas on blood coagulation using a new model of whole blood in a controlled setting with corn trypsin inhibitor and a 5-pM stimulus of tissue factor. We investigated thrombin generation, fibrin formation, and platelet activation with four diluents: 0.9% NaCl (NS), lactated Ringer's solution (LR), 6% hydroxyethyl starch (HES), and 3% NaCl (HS), each from 0% to 75% blood dilution. Thrombin generation was measured periodically during a time course of 20 minutes in its complex with antithrombin III. Platelet activation and fibrinopeptide A (FPA) release were monitored in serum at a 20-minute time point. Fibrin clots were collected and weighed. RESULTS: The coagulation markers (thrombin generation, platelet activation, and FPA release) were significantly different by dilution (p < 0.001 in all) and diluent by dilution (p < 0.001 in all). Thrombin generation, platelet activation, and FPA release decreased the least with the diluents NS and LR. LR caused the least amount of variation in thrombin generation over the dilution course. HS produced the most dramatic change in all of the markers; no coagulation was seen between 30% to 75% dilution (p < 0.05). HES produced greater decreases in thrombin generation and FPA release than NS and LR. Fibrin clot mass decreased with a 10% to 20% dilution for NS and LR, whereas stable fibrin mass did not decrease with the diluents HES and HS at 10% to 20% dilutions. At >30% dilutions, HS produced no stable clots and HES dramatically decreased clot formation by 61% and maintained this level. CONCLUSIONS: LR and NS had the least effect on thrombin generation, clot formation, and platelet activation at various concentrations compared with HES and HS. This observational data suggests that volume expanders such as HES and HS may be detrimental in treatment of hemorrhagic shock.

Analysis of cell-cycle specific localization of the Rdi1p RhoGDI and the structural determinants required for Cdc42p membrane localization and clustering at sites of polarized growth.

The Cdc42p GTPase regulates multiple signal transduction pathways through its interactions with downstream effectors. Specific functional domains within Cdc42p are required for guanine-nucleotide binding, interactions with downstream effectors, and membrane localization. However, little is known about how Cdc42p is clustered at polarized growth sites or is extracted from membranes by Rho guanine-nucleotide dissociation inhibitors (RhoGDIs) at specific times in the cell cycle. To address these points, localization studies were performed in Saccharomyces cerevisiae using green fluorescent protein (GFP)-tagged Cdc42p and the RhoGDI Rdi1p. GFP-Rdi1p localized to polarized growth sites at specific times of the cell cycle but not to other sites of Cdc42p localization. Overexpression of Rdi1p led to loss of GFP-Cdc42p from internal and plasma membranes. This effect was mediated through the Cdc42p Rho-insert domain, which was also implicated in interactions with the Bni1p scaffold protein. These data suggested that Rdi1p functions in cell cycle-specific Cdc42p membrane detachment. Additional genetic and time-lapse microscopy analyses implicated nucleotide binding in the clustering of Cdc42p. Taken together, these results provide insight into the complicated nature of the relationships between Cdc42p localization, nucleotide binding, and protein-protein interactions.