Neutralizing the pathological effects of extracellular histones with small polyanions

Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9–1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.

Histones (400 µg ml -1) were mixed with 400 µg ml -1 of the polyanions mCBS, MTS and heparin, the resultant precipitates pelleted by centrifugation and the supernatants collected. a, Histone concentration in supernatants measured by QUBITÒ protein assay and percent histones precipitated calculated. b, Polyanion concentration in supernatants measured by the DMMB assay and percent polyanions precipitated calculated. Data presented as mean ± s.e.m. (n=3 biological replicates) and analyzed by two-way ANOVA with Tukey's correction for multiple comparisons. Source data are provided as a Source Data File.

Supplementary Fig. 5 | Detection of free histones in plasma and effect of mCBS and MTS on
histone plasma levels. a, Mice injected i.v. with histones (50 mg kg -1 ) in normal saline (NS) had their blood collected 1 min post injection. Free histones present in the resultant plasma were absorbed to heparin coupled beads, the eluted histones run on SDS-PAGE and visualized with the Sypro Ruby stain. The 0 min sample represents plasma collected from mice injected with NS alone. A 25 kDa unidentified, heparin-binding, protein present in mouse plasma was used as a loading control (data representative of 3 gels). b, Effect of injection of mCBS or MTS (100 mg kg -1 ) 10 min prior to histones on circulating histone levels 1-2 min after histone injection (data representative of 2 gels). c, The effect quantified by densitometry. Data presented as mean ± s.e.m. (n=5-6 mice/group) and analyzed by one-way ANOVA with Tukey's correction for multiple comparisons. Source data are provided as a Source Data File.

Supplementary Fig. 7 | DNase1 enzymatic activity is not inhibited by heparin or the SPAs mCBS and MTS.
Calf thymus DNA (CTDNA) (500 ng ml -1 ) was incubated for 10 min at 37 o C alone or with DNase1 (1 µg ml -1 ) and in the presence or absence of heparin or the SPAs mCBS and MTS (100 µg ml -1 ). DNA content of samples was quantified using PicoGreen, a highly sensitive fluorescent DNA detection reagent. Under the experimental conditions described above ~85% of the CTDNA was degraded and mCBS, MTS or heparin had no significant effect on the amount of undegraded DNA present at the end of the assay, although in the presence of each of the three polyanions there was slight inhibition of uptake of the PicoGreen dye by the undegraded CTDNA control (~5-13%, heparin>MTS>mCBS). Data presented as mean ± s.e.m. (n=3 biological replicates) and analyzed by two-way ANOVA with Tukey's correction for multiple comparisons. Source data are provided as a Source Data File. Supplementary Fig. 9 | Effect of mCBS on clotting time. Blood from a healthy unmedicated volunteer was collected into a sodium citrate vacutainer and analysed within 2 h of collection using rotational thromboelastometry (ROTEM). Whole blood (300 µl) was supplemented with mCBS 200 µg ml -1 in water or an equivalent volume of water for 1 min then loaded onto the ROTEM machine, according to the manufacturer's instructions, with the NATEM (non-activated), EXTEM (tissue factor activated), INTEM (contact pathway activated) and FIBTEM (tissue factor activated + cytochalasin-D neutralization of platelets) assays performed. Results of each assay (3 replicates) are presented as the fold-change above the value obtained in the water control sample run at the same time. Data presented as mean ± s.e.m. and analyzed by two-way ANOVA with Sidak's correction for multiple comparisons. Source data are provided as a Source Data File. Fig. 10 | Comparison of anticoagulant effect of mCBS with low-molecular weight-(LMWH) and unfractionated heparin (UFH). a, Blood from a healthy unmedicated volunteer was collected into a sodium citrate vacutainer and analysed within 2 h of collection using rotational thromboelastometry (ROTEM). Whole blood (300 µl) was supplemented with a titration of compounds in water or an equivalent volume of water for 1 min then loaded onto the ROTEM machine according to the manufacturer's instructions with the NATEM (non-activated) assay used due to its greater sensitivity to heparins ( Supplementary Fig 9). b, as in a however a direct comparison was made between the compounds at the concentrations indicated at the same time rather than across assays. Results are expressed as a percentage of the water control sample run at the same time. Source data are provided as a Source Data File. Fig. 11 | Initial gating strategies, based on forward (FSC) and side (SSC) scatter, for FACS data. a, Gating strategy to remove debris from HMEC-1 cytotoxicity studies (with and without histone treatment) as described in Supplementary Fig. 1, Fig. 1a,c,d and Fig. 3a. b, Gating strategy to assess RBC aggregation (with and without histone treatment) as described in Fig. 2a,c,d. c, Gating strategy to assess platelet aggregation (with and without histone treatment) as described in Fig. 2e,f. d, Gating strategy to assess histone-mediated HMEC-1 Ca 2+ flux in viable cells (with and without CBS/MTS treatment) as described in Fig. 3d. After FSC/SSC gating for viable HMEC-1, singlets gated and Ca 2+ flux measured for 1 min after addition of CBS or MTS (0 min), and 1 min, 3 min and 9 min after histone addition.

Materials and Methods
Unless stated otherwise, reactions were performed in oven-dried glassware with reagents and dry solvents purchased from commercial sources. Thin-layer chromatography (TLC) was performed using aluminium plates coated with silica gel 60 F254 (Merck). Reactions monitored by TLC were visualized by UV light and heated upon reaction with ethanolic H2SO4 (5% v/v). Flash chromatography was performed using Silica Gel 60 (0.040-0.063 mm).
NMR spectra were recorded on a Bruker Avance 400 or 600 MHz (Ultra-shield) spectrometer. NMR data acquisition and processing were performed with Mestrenova software. 1  IR spectra were recorded on a Bruker ALPHA-P FT-IR spectrometer. Low resolution mass spectra (LRMS) were acquired in either positive or negative ion mode as indicated on a Bruker Daltonics Esquire 3000 ESI MS; high-resolution mass spectra (HRMS) data were acquired at either Griffith University's FTMS Facility on a Bruker QTOF mass spectrometer or at the Smart Water Research Centre on an Agilent 6530 Q-TOF mass spectrometer using Agilent Jetstream ESI.
All final compounds were purified by HPLC using a gradient of H2O/CH3CN in 10 mM ammonium acetate at 1 mL/min through a Phenomenex 5 μm C18 guard column.

General procedure A for per-O-sulfation (co-solvent approach)
Standard Protocol: To the polyol sugar (1 mmol) was added sulfur trioxide-trimethylamine complex (SO3•TMA, 1.5 to 5 equiv per OH), followed by the addition of anhydrous DMF (1.2 mL/per 1 mmol of SO3•TMA) and anhydrous DCE. The preferred reaction concentration is between 10 to 50 mM.
The resulting suspension was heated under Argon at 80 -90 o C for 10 min to 1 h. Upon cooling, the mixture was co-evaporated with chlorobenzene to remove DMF under reduced pressure. The crude residue was then dissolved in water and filtered through a pad of cotton. The transparent and brownish liquid was further purified through size exclusion chromatography (SEC) on Sephadex G-25 using water as the eluent. Combined fractions were concentrated to obtain the final sulfated compound which was dissolved in de-ionized water and directly subjected to ion-exchange chromatography (DOWEX 50Wx8, Na + form). The combined fractions were lyophilized to yield the final sulfated product as a sodium salt.

General procedure B for per-O-sulfation (conventional approach)
Standard Protocol: To a solution of the starting material in anhydrous DMF (60 mL/per mmol of starting material) was added sulfur trioxide-trimethylamine complex (SO3•TMA, 1.5 to 5 equiv per OH) under Argon. The mixture was stirred at 50 -60 °C until reaction completion was indicated by 1 H-NMR spectroscopy. Upon cooling, the reaction mixture was quenched with Et3N and coevaporated with chlorobenzene to remove DMF (bath temperature is not over 30 °C) under reduced pressure. The crude residue was dissolved in water and filtered through a pad of cotton. The transparent and brownish liquid was further purified through size exclusion chromatography (SEC) on Sephadex G-25 using water as eluent. Combined fractions were concentrated to obtain the final sulfated compound as a triethylammonium salt which was dissolved in de-ionized water and directly subjected to ion-exchange column (DOWEX 50Wx8, Na + form). The combined fractions were lyophilized to yield the final sulfated product as a sodium salt.

General procedure C for one-pot per-O-acetylation and bromination
To the mixture of free sugar (20 mmol) and glacial acetic acid (100 mL) was added acetyl bromide (1.5 equiv per OH) at room temperature. The resultant creamy mixture was subsequently heated at 60 o C for 45 -55 min until the reaction mixture turned clear, which indicated that the reaction was complete. The hot reaction mixture was carefully poured into a beaker pre-charged with cracked ice (300 g). The mixture was stirred until a white solid precipitated (~10 min), upon which another portion of cold water (60 mL) was added and the mixture stirred for a further 10 min. The suspension was then filtered through a sintered funnel and washed with cold water (30 mL x 3) to obtain the precipitated product that was then further dissolved in DCM (100 mL). The DCM layer was washed with brine, dried over Na2SO4, filtered and concentrated under reduced pressure at below 35 o C to yield the target glycosyl bromide that was then directly used in the following glycosylation reaction.
General procedure D for b-glycosylation using Ag2CO3 as promoter To a mixture of per-O-acetylated glycosyl bromide (25 mmol), anhydrous DCM (80 mL), anhydrous MeOH (80 mL), and activated 3 Å molecular sieves (7 g) was added silver carbonate (Ag2CO3, 7.5 g, 27.5 mmol, 1.1 equiv). The resultant mixture was stirred in the absence of light for 16 h. The reaction mixture was then purified through a plug of silica and eluted with EtOAc. The collected fractions were concentrated to give the crude product as a brownish solid that was directly used in the next step.

General procedure E for Zemplén de-O-acetylation
To the suspension mixture of per-O-acetylated sugar in anhydrous MeOH (100 mL) was added a small piece of Na (172 mg, 7.5 mmol, 0.3 equiv) at room temperature. The mixture was then stirred overnight in order to ensure completion of de-O-acetylation. The final solution was concentrated and dried under vacuum overnight to obtain the crude methyl glycoside that was then directly used for sulfation.   NaHCO3 aqueous solution and brine, dried over Na2SO4, filtered and concentrated to obtain the crude product. Further purification was performed by either flash column chromatography or trituration.

General procedure G for benzylation
To a solution of substrate and benzyl bromide (1.1 equiv/per OH) in anhydrous N,Ndimethylformamide (DMF, 1 mL for 1 mmol of substrate) in an ice bath was portion-wise added 60% NaH (1.2 equiv/per OH). The reaction was allowed to warm up to room temperature until H2 evolution ceased. The resultant mixture was stirred for a further two hours and the reaction carefully quenched with water at 0 °C. Upon removal of DMF by rotary evaporation, the crude mixture was diluted with EtOAc, washed with 1% aqueous HCl solution, water, saturated NaHCO3 aqueous solution, brine, dried over Na2SO4, filtered and concentrated to obtain the crude benzylated product.
Further purification was performed by flash column chromatography.

General procedure H for removal of benzylidene acetal
To a solution of benzylidenated substrate (0.3 mmol) in DCM (5 mL) was added trifluoroacetic acid (TFA, 5 equiv) at 0 o C. The solution was gradually warmed to room temperature and stirred for 1-2 h. Upon completion of reaction, TFA was removed by co-evaporation with toluene to furnish the crude product that was then washed with saturated NaHCO3 aqueous solution, brine, dried over Na2SO4, filtered and concentrated to obtain the crude 4',6'-diol. Further purification was performed by flash column chromatography to yield the debenzylidenated product.

General procedure I for hydrogenolysis
A solution of protected saccharide and palladium hydroxide on carbon (20 wt. % loading, 10 times the weight of starting material) in phosphate buffer (20 mM, pH = 7.0, 200 µL/per mg of starting material) and MeOH (200 µL/per mg of starting material) was charged with a hydrogen balloon. The resultant mixture was stirred at room temperature for 2 days. The reaction mixture was filtered through celite, and the filtrate was concentrated and purified using a Sephadex G-25 column eluted with water. After concentration, the residue was dissolved in water and passed through an ion-exchange column of DOWEX 50W-X8 (Na + form). The collected fractions were lyophilized to give the target product as a sodium salt.

General procedure J for TBDPS protection
To a continuously stirred solution of benzylidenated starting material (0.5 mmol) in DMF (30 mL) at room temperature was added Et3N (1.5 equiv), imidazole (0.5 equiv) and finally TBDPSCl (1.2 equiv). The mixture was stirred for a further 3 h. Another portion of TBDPSCl (0.6 equiv) was added and the reaction was stirred for another 24 h. The resultant mixture was quenched with MeOH, diluted with EtOAc (200 mL), washed with 1% HCl aqueous solution, water, saturated NaHCO3 aqueous solution, brine, dried over Na2SO4, filtered and concentrated to furnish the silylated crude mixture that was further purified using silica column chromatography to yield the pure protected product.

General procedure K for removal of TBDPS group
To a solution of protected saccharide (0.31 mmol) in THF (10 mL
mCBS is presented in 10 ml glass vials fitted with a rubber stopper and seal, as a sterile, concentrate solution (70 mg ml -1 ) formulated in phosphate buffer. At this concentration the solution is isotonic (~300 mOsml kg -1 ) with a pH of ~7.5. The drug product solution (labelled as STC3141) was diluted and prepared for dosing according to the clinical protocol, but is anticipated to be diluted to concentrations in the range of 1.5-28 mg ml -1 (mCBS heptasodium salt) using saline for injection, ready for intravenous (i.v.) infusion.

Nonclinical Pharmacology
The in vitro primary pharmacodynamic data indicates that mCBS exhibits the following properties: • Protective against histone induced cell cytotoxicity in a concentration dependent manner and is able to reverse the damaging effect of histones • Has a protective effect against histone-mediated RBC aggregation and able to inhibit histoneinduced RBC aggregation dose dependently; also has a protective effect against histoneinduced RBC lysis, able to attenuate histone-induced RBC fragility and reverse histonemediated RBC toxicity • Prevents histone-mediated platelet aggregation and degranulation in a dose-response manner • Has minimal anticoagulant activity compared to low molecular weight heparin (LMWH) and unfractionated heparin (UFH) Based on in vitro data, an effective concentration range for mCBS against histone-mediated toxic effects with histones at 400 μg ml -1 , which is close to the maximum concentration of circulating histones detected clinically, appears to be 50-200 μg ml -1 .
Two in vivo pharmacodynamic (PD) studies, one in the mouse and the other in the rabbit, respectively, showed that mCBS can mediate a reduction in histone-induced systemic toxicity. Single intraperitoneal (i.p.) doses of 6.25, 25 or 100 mg kg -1 (as sodium salt of mCBS) in the mouse showed a dose related reduction in cell injury induced by histones (50 mg kg -1 ), as shown by a reduction in the biomarkers alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and creatinine (Crea).
The higher dose of mCBS, 100 mg kg -1 , showed near complete abolishment of the histone-induced increase in these biomarkers. An in vivo study in the rabbit showed i.v. administration of 50 or 100 mg kg -1 mCBS dose-proportionally decreased the levels of histone coated Tc-nanoparticles lodging in rabbit lung, thus indicating mCBS has the potential to reduce the accumulation of histones in the lung during disease, such as sepsis. Based on these in vivo PD data, the human equivalent dose (HED) of the pharmacologically active doses of mCBS, range from approximately 0.5 mg kg -1 in the mouse (i.p. route) up to 32 mg kg -1 in the rabbit (i.v. route) (expressed as sodium salt of mCBS).
The in vitro human Ether-a-go-go Related Gene (hERG) study indicated no potential liability for cardiac toxicity mediated via this mechanism at concentrations up to 2 mM. Lack of an effect on the cardiovascular system was also confirmed in vivo in the dog where i.v. infusion of doses up to 2785 mg kg -1 day -1 mCBS (free base) for 14 days had no effect on the morphology of the P-QRS-T wave complex or heart rate, PR, QRS duration, QT and QTc intervals. No effect on respiratory parameters (respiratory rate, tidal volume and minute volume) was seen following a single i.v. injection of mCBS in male rats at doses up to 848 mg kg -1 (free base). No effect on the central nervous system was indicated by detailed observation of rat behavior (general behavior, sensorimotor or autonomic) following 14 days continuous dosing (3000 mg kg -1 day -1 ) .

Pharmacokinetics and metabolism in animals.
Overall, single dose pharmacokinetic (PK) parameters in rats indicated linearity between doses of 16.3 and 81.5 mg kg -1 (free base) given as a single i.v. bolus, with peak exposure at administration, a wide distribution through the body tissues (approximately 48 l kg -1 ) and slow clearance ( In the rat 14-day i.v. infusion study, over the dose range 300 to 3000 mg kg -1 day -1 , the mCBS plasma steady-state concentrations (Css) were reached within 5 h post start of infusion in all animals. The mean Css ranged from 20.9 to 242 μg ml -1 , while mean AUC0-336 (AUC0-360) ranged from 6970 (7010) to 81000 (81400) μg h -1 ml -1 . After the end of infusion, the mean mCBS plasma concentrations declined rapidly at a mean estimated t. value ranging from 0.653 to 0.742 h and a mean Cl ranging from 0.47 to 0.6 l h -1 kg -1 . The mean volume of distribution (Vz) ranged from 481 to 590 ml kg -1 , suggesting that mCBS is largely distributed among tissues. The conclusion from this study was that In vitro, protein binding of mCBS in the human, rat and dog plasma was low (19-23%). Minimal in vitro metabolism was seen for mCBS in human, rat and dog liver microsomes under phase 1 or phase 2 conditions.

Toxicology.
Two Good Laboratory Practice (GLP) 14-day toxicity studies have been performed with mCBS administered by continuous i.v. infusion (24 h day -1) to rats and dogs.
The data from the 14-day rat toxicity study indicated a no-observed-adverse-effect level (NOAEL), following 14 days continuous i.v. infusion, of 300 mg kg -1 day -1 free base. At this level, mean (sexes combined) Css of 23.8 μg ml -1 , with an AUC0-360 of 7990 μg h -1 ml -1 was observed. Activated partial thromboplastin time (aPTT) was increased. The only notable macroscopic changes observed at 300 mg -1 kg -1 day -1 were enlarged bronchial and mediastinal lymph nodes, which was not observed in any of the animals after the 14-day recovery period. This dose was also associated with mild proximal tubular vacuolation/rarefaction in both kidneys which correlated with increased serum urea levels.
There was also mild accumulation of foamy Kupffer cells in the liver and minimal to mild accumulation of foamy macrophages in the ovaries and uterus of female animals and in the adrenals and in various lymph nodes of all treated animals.
Mid and high doses of 1000 and 3000 mg kg -1 day -1 administered to the rat for 14 days (continuous infusion) showed no hematology changes at the end of infusion on day 15 but decreased red blood cell count (RBC), hemoglobin and hematocrit and an increase in reticulocytes (absolute and relative) at the end of the recovery phase in animals dosed with 3000 mg kg -1 day -1 . aPTT was increased in a dose-related manner and showed reversibility after a 14-day recovery period. Adverse findings in the kidneys were accompanied by increases in serum creatinine and urea. At these two doses, pale discoloration in the kidneys and enlargement in a number of lymph nodes as well as microscopic changes in the kidney, liver, ovaries, uterus, adrenals, spleen and lungs were observed, largely increasing in frequency and severity in a dose-dependent manner. Changes in the thymus and mammary glands were noted in a number of the high dose animals after 14 days infusion. After the 14-day recovery period in the high-dose treated rats, most of the microscopic changes recorded were still observed, however, the incidence and/or severity was decreased suggesting an ongoing recovery process. Test item-related changes were no longer observed in the lung, thymus and mammary glands of the high-dose recovery cohort.
The data from the 14-day GLP dog toxicity study indicated a NOAEL (278.5 mg kg -1 day -1 ). At this level, mean (sexes combined) Css was observed at 49.85 μg ml -1 , with an AUC0-360 of 16750 μg h -1 ml -1 . In the dog, 278.5 mg kg -1 day -1 produced transient increases in aPTT as well as mild microscopic changes consisting of minimal to mild vacuolation of the proximal tubules of the kidney, minimal diffuse cell infiltrate in the liver and minimal to mild foamy cell accumulation of the iliac lymph node. The microscopic changes in kidneys, liver and lymph nodes were mild in severity, and therefore considered to be non-adverse.
The continuous i.v. infusion (24 h day -1 for 14 days) of 849 and 2785 mg kg -1 day -1 mCBS (the mid and high study doses respectively) in the dog produced transient decreases in platelet counts and increase in aPTT, as well as minimal to mild microscopic changes consisting mainly of cortical vacuolation (adrenals), vacuolation of tunica muscularis in the digestive tract (stomach, duodenum, ileum, cecum and colon), vacuolation of the joint capsule cells (femur), vacuolation of proximal tubules (kidneys) correlating with an increase in kidney weight, vacuolation of the submucosa (urinary bladder), diffuse cell infiltrate of the sinusoidal/portobiliary space (liver) correlating with an increase in liver weight and foamy cell accumulation of the lymph nodes (iliac, mandibular, mediastinal, mesenteric and pancreatic) and Peyer's patches (jejunum).
Additional changes seen at 2785 mg kg -1 day -1 mCBS included an increase in cholesterol, triglyceride, urea and creatinine serum levels. Microscopically, changes were similar to those observed at 849 mg kg -1 day -1 mCBS, but were observed at a slightly higher incidence and severity, and included also vacuolation of the tunica muscularis in the jejunum and rectum, vacuolation of the atrial myocardium (heart), as well as Kupffer cell hypertrophy in the liver.
At the end of the 14-day recovery period in dogs previously treated with 2785 mg kg -1 day -1 , all hematological, coagulation and clinical chemistry parameters had returned to baseline levels. All of the microscopic findings recorded in the study were still observed at a similar, or slightly lower incidence and severity after the recovery period, suggesting partial recovery and that a longer recovery period may be necessary before complete recovery is achieved. Kidney and liver weights were still increased after the 14-day recovery phase.
Based on the data above the pharmacology of the compound indicates prolongation of aPTT may be increased and platelet number reduced. Based on both the rat and dog data, the compound widely distributes into tissues. The vacuolation noted in tissues was widespread but generally minimal to mild in nature except for changes in the lymph nodes and femur which reached moderate and the kidney which reached severe. The kidney and liver also increased in weight and kidney was pale at necropsy. Thus, the main target organs appear to be the kidney and liver. Clinical protocols should include appropriate monitoring of hematology, coagulation, as well as include appropriate kidney and liver biomarkers.
The findings of accumulation of foamy macrophages (in the spleen and lymph nodes), foamy Kupffer cells (in the liver) and proximal tubular vacuolisation/rarefaction (in the kidneys) suggest an adaptive change of the mononuclear phagocytic system and kidneys in response to STC314, due to the phagocytosis and clearance of the test item and/or its degradation product(s). Other findings in the spleen were also considered to be an adaptive response to the activated phagocyte system. mCBS did not cause mutations in the GLP in vitro Ames test or produce chromosome aberrations in human peripheral lymphocytes. mCBS did not induce any genotoxic activity in the in vivo micronucleus assay performed in the continuous 14 day i.v. toxicology study in rats.
No studies investigating the effects of mCBS on fertility, fetal development or carcinogenicity have been performed in animals.

Anticoagulant Properties of mCBS
The anticoagulant activity of mCBS was investigated using rotational thromboelastometry (ROTEM) assays, these assays being performed by the Parish Laboratory, JCSMR. ROTEM analysis gives a holistic view of whole blood clotting as it provides information on the plasma coagulation cascade, the platelet contribution to clot formation as well as the rate of clot lysis. Clotting Time (CT) represents the time it takes from initiation of the clotting assay to the point at which a clot of 2 mm in size has formed and essentially represents the period from the activation of the plasma coagulation cascade to the point at which fibrin is formed. The subsequent contribution of platelets and fibrin can be determined by the amplitude of the clot, or size in mm, at various time points including 5, 10 and 20 min following the end of the CT.
The ROTEM assay can be manipulated to provide more specific information on the contribution of the extrinsic and intrinsic pathways of coagulation as well as the contribution of fibrin formation through the use of specific reagents added to the blood to initiate the assay. The type of reagent added determines the name of the assay hence the 'NATEM' assay is the non-activated assay; the 'EXTEM' is specific for the extrinsic pathway of coagulation; the 'INTEM' is specific for the intrinsic pathway of coagulation and the 'FIBTEM' is specific for the contribution of fibrin to clot formation.
The effect of mCBS on clotting parameters using the different ROTEM assays was investigated, with whole blood being supplemented with mCBS (200 µg ml -1 ) immediately prior to initiation of coagulation and the results being shown in Supplementary Fig. 9. It was found that mCBS, at a concentration (200 µg ml -1 ) that would be rarely if ever reached in patients, did not activate the extrinsic pathway of coagulation (EXTEM assay). This was an interesting result as it implies that the extrinsic pathway of coagulation that is triggered by tissue factor was not altered by the addition of mCBS. Furthermore, mCBS did not aid the contribution of fibrin to clot formation (FIBTEM assay) and only very weakly activated the intrinsic pathway (INTEM assay). In contrast, mCBS did exhibit some anticoagulant activity in the NATEM assay that measures coagulation in the absence of exogenous activators (Supplementary Fig. 9).
Stability studies were carried out at 5±3 °C, 25±2 °C and 40±2 °C, with levels of both mCBS and cellobiose sulfate (CBS) being tracked in formulated clinical material (i.e., mCBS in phosphate buffer, pH 7.5) via HPLC. Graphing the percent (%) change of mCBS and CBS relative to their starting amount (at T=0) revealed that mCBS levels remained unchanged after incubation at 5 °C and 25 °C for 25 months and 40 °C for 13 months, indicating that mCBS is a very stable compound, even when incubated in aqueous solution at 40 °C for 13 months (Supplementary Fig. 3a-c). In contrast, CBS levels declined rapidly and in a temperature dependent manner. Thus, at 5 °C approximately 50% of CBS had decomposed after 1 month and over 90% at 6-13 months ( Supplementary Fig. 3a), whereas at 25 °C and 40 °C essentially 100% of CBS had decomposed following storage in aqueous solution for only 1 month ( Supplementary Fig. 3b,c). These data indicate that CBS is highly unstable in aqueous solutions but the addition of a methyl group to the reducing terminus of CBS results in a molecule (mCBS) that is very stable when stored under aqueous conditions at a range of temperatures.
On the other hand, when stored as a powder at -20 °C for 25 months, both mCBS and CBS were found to be highly stable.
In additional studies, mCBS stability was assessed when formulated in different buffered aqueous solutions other than PBS, namely citrate and acetate buffered saline (pH 7.5). The data from these experiments revealed that the high stability of mCBS in PBS at 5 °C, 25 °C and 40 °C was recapitulated when mCBS was stored in citrate and acetate buffered solutions at pH 7.5.