Polyphosphate nanoparticles enhance the fibrin stabilization by histones more efficiently than linear polyphosphates.

INTRODUCTION: Beyond the three-dimensional fibrin network, the mechanical and lytic stability of thrombi is supported by the matrix of neutrophil extracellular traps (NETs) composed of polyanionic DNA meshwork with attached proteins including polycationic histones. Polyphosphates represent another type of polyanions, which in their linear form are known to enhance the fibrin stabilizing effects of DNA and histones. However, in vivo polyphosphates are also present in the form of nanoparticles (PolyP-NP), the interference of which with the fibrin/NET matrix is poorly characterized. AIMS: To compare the effects of linear and nanoparticulate polyphosphates, and their combinations with relevant NET components (DNA, histone H3) on fibrin formation, structure, and lysis in in vitro assays focusing on histone-polyphosphate interactions.
METHODS: Transmission electron microscopy and dynamic light scattering for stability of the PolyP-NP preparations. Turbidimetry for kinetics of fibrinogen clotting by thrombin and fibrin dissolution by tissue-type plasminogen activator/plasminogen. Scanning electron microscopy for fibrin structure. Surface plasmon resonance for strength of histone-PolyP interactions.
RESULTS: Both linear PolyP and PolyP-NP accelerated the fibrin formation and slowed down its dissolution and these effects were strongly dependent on the number of individual PolyP particles and not on their size. Addition of DNA did not modify significantly the PolyP-NP effects on fibrin formation and lysis. Both linear and nanoparticulate PolyP counteracted the effect of histone in the acceleration of fibrinogen clotting by thrombin. PolyP-NP, but not linear PolyP enhanced the prolongation of lysis time in fibrin containing histone and caused more pronounced thickening of the fibrin fibers than the linear form. Finally, PolyP-NP bound weaker to histone than the linear form.
CONCLUSIONS: The interaction of PolyP with histone was a stronger modulator of fibrin formation and lysis than its interaction with DNA. In addition, the PolyP nanoparticles enhanced the thrombus stabilizing effects of histone more effectively than linear PolyP.

Introduction

Neutrophil extracellular traps (NETs) and polyphosphates (PolyPs) are both known as key players of (immuno)thrombosis. Their procoagulant and prothrombotic nature, as well as their interactions in circulation have been recently reviewed in detail [1]. Our previous studies on the effects of NET components on structural and lytic stability of fibrin and plasma clots revealed that DNA, histones and their combinations increase the mechanical stability of clots and hamper tissue-type plasminogen activator (tPA) mediated clot lysis [2,3]. Furthermore, we found, that linear PolyPs stabilize fibrin via size-dependent modulation of its structure and kringle-dependent inhibition of plasmin-mediated fibrinolysis [4], while in combination with NET components, linear PolyPs enhanced the clot stabilizing effects of DNA and histones [5]. In addition to the linear form, nanoparticulate polyphosphates (PolyP-NPs) have also been shown to localize at the surface of activated platelets and activate the intrinsic pathway of coagulation [6], but in contrast to the linear PolyP effects, little is known about their contribution to the stabilization of thrombi by NETs.

In this study, we aimed 1) to develop a new method for preparing stable, pure PolyP-NPs that are suitable for in vitro applications to study their role in hemostasis, and 2) to compare and characterize the effects of the linear and nanoparticulate forms of PolyPs and the combined effects of DNA, histones and PolyPs on the kinetics of clotting, structure and lytic susceptibility of fibrin clots in vitro.

Materials and methods

Plasminogen depleted human fibrinogen, alkaline phosphatase, short-chain polyphosphate (PolyP-Lin-45 with an average polymer length of 45 monomers), histones from calf thymus type VIIIS (arginine-rich histone H3) were obtained from Merck Kft. (Budapest, Hungary). Polyphosphate, Medium Chain (PolyP-Lin-100) and Polyphosphate, Long Chain (PolyP-Lin-700) were from Kerafast (Boston, MA, USA). Bovine thrombin was purchased from Serva (Heidelberg, Germany) and further purified by ion-exchange chromatography on sulfopropyl-Sephadex yielding a preparation with a specific activity of 2,100 IU/mg [7] and 1 IU/ml was considered equivalent to approximately 10.7 nM by active site titration [8]. Plasminogen and plasmin were prepared as previously described [9].

Preparation of PolyP-NPs

Pure PolyP nanoparticles are labile, so we developed a novel technique for preparing stable PolyP nanoparticles based on improvements of earlier work [10,11]. The detailed procedure is as follows. Ca2+-PolyP nanoparticles were generated by adding linear PolyP45 (1 mM) to CaCl2 (1.67 mM) in BSA-TBS buffer (8 mM Tris-HCl, pH 9, containing 1 mg/ml bovine serum albumin). Ca2+-precipitation was allowed to proceed at room temperature for 20 min under continuous vigorous stirring to achieve homogeneity. Thereafter large aggregates were dispersed by ultrasound sonication with a Branson Sonifier 250 (BRANSON Ultrasonics Corp., Danbury, CT, USA) for 10 min (20 kHz horn frequency, 65 W) with a 5-min pause at half-time. Remnant large aggregates were removed by filtrating them sequentially through a syringe filter of 1.2 μm and then through a filter of 0.22 μm pore size. Finally, the filtrate with the remaining PolyP-NPs was retained and stored at 4°C.

Determination of phosphate monomer and polyphosphate nanoparticle concentration

The phosphate monomer concentration of both linear and nanoparticulate polyphosphates was determined by a modified version of the Fiske-Subbarow method [12]. Alkaline phosphatase (20 U/l) was added to polyphosphate solutions and mixtures were incubated for 2 hours at 37°C to cleave phosphate polymers to monomers. Then a mixture of 4.4%(w/v) ascorbic acid and 4.4%(w/v) trichloroacetic acid (TCA) was added to the samples followed by centrifugation for 2 min at 5,000g. The liquid phase was withdrawn and after the addition of 0.2%(w/v) ammonium molybdate the samples were incubated for 1 hour at 50°C. Following the color reaction, the absorbance of the samples and a dilution series of NaH2PO4 standard were measured in a CLARIOstar spectrophotometer (BMG Labtech, Ortenberg, Germany) at 700 nm. Particle concentration of the PolyP-NP preparations was estimated considering the phosphate monomer concentration of the samples, the average density of Ca2+-PolyP-NPs (3.14 g/cm3) [13] and the radius of the particles calculated according to our transmission electron microscopy and dynamic light scattering measurements using sphere approximation [14]. The preparations of applied polyphosphates are summarized in Table 1.

Table 1

Designations of polyphosphates used in this study.

Designation	Type and form of polyphosphate
PolyP	any type, linear or nanopaticulate
PolyP-Lin	any type, linear
PolyP-Lin-45	45 monomer average size, linear
PolyP-Lin-100	100 monomer average size, linear
PolyP-Lin-700	700 monomer average size, linear
PolyP-NP	45 monomer average size, nanoparticulate
PolyP-Linm	45 monomer average size, linear at 330 μM monomer equivalent
PolyP-Linp	45 monomer average size, linear at 1.96×1012 particle/l

Characterization of PolyP-NPs by transmission electron microscopy (TEM)

To visualize and to measure the size of the polyphosphate nanoparticles samples were mounted on Formwar-Carbon coated 300-mesh nickel grids (Nisshin EM Co. Ltd. Tokyo, Japan) and stained with 2%(w/v) uranyl acetate. Samples were examined using a JEOL JEM-1011 electron microscope (JEOL, Ltd., Tokyo, Japan) at an accelerating voltage of 80 kV. Images were taken by Morada 11 Megapixel camera (Olympus, Tokyo, Japan) using iTEM software (Olympus) [15].

Characterization of PolyP-NPs by dynamic light scattering (DLS)

The stability of polyphosphate nanoparticles was monitored by measuring the size distribution of PolyP-NP samples for 6 days with DLS. Furthermore, the average particle size was checked before each measurement to validate that the nanoparticle diameter is within the target range of 100–200 nm [6]. This was performed using an ALV goniometer with a Melles Griot diode-pumped solid-state laser at 457.5 nm wavelength (type: 58 BLD 301). The intensity of the scattered light was measured at 90° and the autocorrelation function was calculated using an IBM PC-based data acquisition system developed in the Department of Biophysics and Radiation Biology of Semmelweis University (Budapest, Hungary). Particle size distributions were determined by the maximum entropy method [16].

Turbidimetric measurements of fibrin formation and dissolution

In order to maximize the modulator effects we used the highest final PolyP concentration achievable in the reaction mixtures during the turbidimetric measurements which was 330 μM in terms of phosphate monomer that corresponds to 1.96×1012 PolyP particle/l in terms of particle concentration. PolyPlin concentrations were set to match these values: PolyP-Linp samples of 1.96×1012 PolyP particle/l and PolyP-Linm samples of 330 μM monomer concentration were prepared. The equivalence of the concentrations of the three different PolyP solutions is illustrated in S1 Fig.

Formation and dissolution of fibrin clots were followed in 96-well microtiter plates by measuring the light absorbance at 340 nm at 37°C with a CLARIOstar spectrophotometer (BMG Labtech, Ortenberg, Germany). Fibrinogen (7 μM) containing plasminogen (0.2 μM) was clotted by thrombin (2 nM) with or without the modulators (5 μg/ml DNA, 20 μM histone or the above-mentioned concentrations of PolyPs) and their combinations at 100 μl total volume. Regarding the different Ca2+ content of our PolyP-Lin (0 mM) and PolyP-NP preparations (1.67 mM), Ca2+-free and Ca2+-bearing control samples were used. After turbidity reached the plateau phase 90 μl 140 nM tPA was layered on the surface to trigger lysis, and the measurement was continued until complete dissolution of the clots. Kinetics of clotting and lysis were characterized by the time to reach half-maximal turbidity on the ascending and descending part of the turbidimetric curves (clotting time50 and lysis time50, respectively).

Characterization of the structure of fibrin clots by scanning electron microscopy (SEM)

Fibrinogen (7 μM) was clotted by thrombin (2 nM) in the presence of modulators and their combinations at the concentrations used in the turbidimetric measurements. After complete clotting, fibrin was washed 3 times with 100 mM Na-cacodylate pH 7.2 buffer and fixed in 1%(v/v) glutaraldehyde for 30 min, then dehydrated in a series of ethanol dilutions [20–96%(v/v)], 1:1 mixture of 96%(v/v) ethanol/acetone and pure acetone followed by critical point drying with CO2 in an E3000 Critical Point Drying Apparatus (Quorum Technologies, Newhaven, UK). The specimens were then mounted on adhesive carbon discs, sputter-coated with gold in an SC7620 Sputter Coater (Quorum Technologies) and images were taken with scanning electron microscope EVO40 (Carl Zeiss GmbH, Oberkochen, Germany). The images were analyzed to determine the diameter of the fibrin fibers as described previously [2,17].

Histone-fibrinogen gelation

Previous studies demonstrated the gelation of histone-fibrinogen mixtures and its consequences on maximal turbidity during clotting assays [18]. We investigated the impact of this phenomenon to avoid misleading interpretations of our results with histones.

Fibrinogen at 7 μM was mixed with histones at various concentrations (in the range of 0–26 μM) in the presence or absence of 50 nM plasmin in 96-well titration plates and the turbidity of the samples was monitored by a CLARIOstar spectrophotometer at 340 nm for 150 min at 37°C. Samples (10 μl) were withdrawn at 0, 60 and 150 minutes and treated with 90 μl 1%(w/v) sodium dodecyl sulfate (SDS) in 100 mM Tris-HCl, 100 mM NaCl, pH 8.2 and polyacrylamide gel electrophoresis (PAGE) was performed on a 4–15% gradient gel followed by silver staining.

Surface plasmon resonance (SPR) measurements of histone-PolyP interactions

The interaction of histone and PolyP was evaluated with surface plasmon resonance (SPR) technology on SR7500DC system (Reichert Technologies Life Sciences, NY, USA) with standard software SPRAutolink 1.1.14-T provided by the manufacturer for reporting and analyzing data. The running buffer was 10 mM HEPES-NaOH 150 mM NaCl pH 7.4 containing Tween-20 at 0.005 w/w% (HBS-T) in all experiments, except when PolyP-NP was measured, where 1.67 mM CaCl2 was also added. One of the channels on a dextran coated gold chip (Reichert SR7000 chip, 500 kDa carboxymethyl-dextran, part. no. 13206066) was coated non-covalently with 0.25 mg/ml Histone H3 in 10 mM Na-acetate pH 5.5 at a flow rate of 25 μl/min for 7 min [19]. After the coating, the chip was washed and equilibrated with the running buffer for ~10 min. For the interaction measurements 1 mM PolyP solutions were injected at 25 μl/min flow rate for 7 min. Results are reported in relative units (RU) as a difference in the signal between the histone and the reference channels. After each measurement, the chip surface was stripped and regenerated using 6 M guanidine hydrochloride in unbuffered ultrapure water, injected at 25 μl/min for 7 min. Guanidine was chosen to prevent precipitation of Ca2+ salts that is observed with more common stripping methods. The system was then washed with pure water and re-equilibrated with running buffer for a subsequent non-covalent coating step.

Statistical analysis

The distribution of the data on fiber diameter was analyzed using an algorithm described previously: theoretical distributions were fitted to the empirical data sets and compared using Kuiper’s test and Monte Carlo simulation procedures running under MatlabR2018b (The Mathworks, Natick, MA) [20]. Kolmogorov-Smirnov test was used for statistical evaluation of differences in distribution of other experimental datasets in this report (Statistics and Machine Learning Toolbox v.11.4 of MatlabR2018b) at p<0.05.

Results

Generation of stable polyphosphate nanoparticles

First, we aimed to develop a novel technique for preparing stable PolyP nanoparticles (PolyP-NP). Our method was based on previous work by Morrissey et al. with further modifications [10]. The structure and stability of the generated nanoparticles were investigated by TEM and DLS. According to TEM micrographs small, electron-dense Ca2+-polyphosphate nanoparticles were formed with diameters in the range of 80–150 nm (Fig 1A). The size of the particles according to DLS was similar: peak volume probabilities were observed at 110–200 nm particle size (Fig 1B). The slightly larger size values with DLS were probably due to differences in the hydration state of the samples: for TEM the samples were dehydrated, while in DLS samples preserved their hydration shell. The nanoparticle stability was monitored by measurements of the size distribution in the PolyP-NP preparation with DLS. Till the 6th storage day no change in the median diameter of PolyP-NPs was observed suggesting that neither aggregation, nor disintegration of the PolyP-NPs occurred in the preparations for at least 5 days stored in CaCl2-BSA-TBS at 4°C (Fig 2).

Fig 1

Structure and size distribution of polyphosphate nanoparticles (PolyP-NPs).

PolyP-NPs were formed by precipitation of soluble PolyP-Lin-45 (1 mM) with CaCl2 (1.67 mM) in BSA-TBS, then sonicated and filtered through a syringe filter with a pore size of 220 nm. Panel A: Transmission electron micrographs of PolyP-NP (TEM). Panel B: Fractional volume distribution of PolyP-NPs according to their particle size in PolyP-NP preparation examined by dynamic light scattering (DLS). The distribution curves of two different PolyP-NP preparations are shown to illustrate the minimal and maximal median values of particle size in the PolyP-NP preparations used in this study.

Fig 2

Stability of polyphosphate nanoparticles (PolyP-NPs).

The particle size of the PolyP-NPs was monitored by dynamic light scattering (DLS) for 6 days as a measure of the preparation stability. Median particle diameters and interquartile range are shown (n = 100).

Fibrin formation and lysis in the presence of polyphosphates

All examined types of polyphosphate preparations (PolyP-Linm, PolyP-NP, PolyP-Linp) decreased the clotting time50 and increased the lysis time50, but the size of their effect differed (Table 2). Despite the difference in particle size (large aggregates in PolyP-NP and isolated molecules of 45 monomers in PolyP-Linp) the magnitude of the effect on the time course of clotting and lysis was similar for the preparations with identical number of particles (PolyP-NP and PolyP-Linp; 19 and 12% faster clotting, 1.76-, and 1.28-fold slower lysis, respectively, compared to the PolyP-free control). However, particles of the same structure exerted stronger effects at higher numbers (PolyP-Linm vs. PolyP-Linp; 45 and 12% faster clotting, 3.08-, and 1.28-fold slower lysis, respectively). These data show that the modulation of the kinetics of fibrin formation and dissolution depends primarily on the number of individual PolyP particles and not on their size.

Table 2

Effects of different types of polyphosphates on fibrin formation and clot lysis.

		No additive	Ca2+	PolyP-Linm	PolyP-Linp	PolyP-NP
clotting time50 (min)	median	52.5	38.56	28.62	46.17	31.11
	interquartile range	14.28	8.75	6.43	5.46	7.27
	P-value (vs. no additive)	-	3.30×10−8	1.08×10−14	7.60×10−3	-
	P-value (vs. Ca2+)	3.30×10−8	-	-	-	1.24×10−5
lysis time50 (min)	median	12.01	17.21	37.03	15.39	30.32
	interquartile range	6.28	11.79	5.52	9.27	8.31
	P-value (vs. no additive)	-	1.30×10−3	2.45×10−17	0.27	-
	P-value (vs. Ca2+)	1.30×10−3	-	-	-	5.64×10−17

Mixture of fibrinogen (7 μM) and plasminogen (0.2 μM) was clotted by thrombin (2 nM) in the presence or absence of linear PolyP (PolyP-Linm or PolyP-Linp, 330 μM and 1.96×1012 particle/l, respectively) or PolyP-NP (330 μM which corresponds to 1.96×1012 particle/l) and the turbidity was monitored at 340 nm. At the plateau phase tPA was added to the surface of the clots and measurement was continued until complete lysis. The time corresponding to half maximal turbidity was used as a measure of reaction kinetics during both clotting and lysis (clotting-, and lysis time50). P-values are shown according to Kolmogorov-Smirnov test in comparison with the relevant control (n = 20).

Combined effects of DNA and polyphosphates on fibrin formation and lysis

In vivo blood clots are known to contain polyanions (e.g. PolyPs and DNA) and their interplay as modulators of blood clotting and lysis is potentially relevant and partially revealed [4,21]. According to our turbidimetry data, the presence of DNA neither altered clotting time50 alone, nor modulated the enhancement of clotting by PolyPs (Fig 3A and 3C). In the fibrinolytic assay DNA behaved in a Ca2+-dependent manner. In a Ca2+-free milieu, DNA doubled the lysis time50 similarly to PolyP-Linm, but in combination the two polyanions did not show any additivity of their effects (Fig 3B and 3D). In the presence of Ca2+, the lysis was not affected by DNA, while PolyP-NP increased lysis time50 by 45% and this anti-fibrinolytic effect was only slightly moderated by DNA.

Fig 3

Combined effects of DNA and polyphosphates on fibrin formation and lysis.

Fibrinogen (7 μM) containing plasminogen (0.2 μM) was clotted by thrombin (2 nM) in the presence or absence of DNA (50 μg/ml), linear PolyP (330 μM) or PolyP-NP (330 μM) and the absorbance at 340 nm (Turbidity) was measured (Panel A). At the plateau phase tPA (140 nM) was layered on the surface of clots and measurement was continued until complete lysis (Panel B). Arrows indicate the time to reach half-maximal turbidity (clotting time50 and lysis time50) in the ascending and descending phase of the representative turbidimetric curves in panels A and B, respectively. Median values and interquartile range (error bars) of clotting time50 (Panel C) and lysis time50 (Panel D) are shown in black (no Ca2+) or grey (1.67 mM Ca2+). P-values for differences of the indicated pairs of datasets are shown according to Kolmogorov-Smirnov test (n = 25).

Combined effects of histones and polyphosphates on fibrin formation and lysis

Similarly to extracellular DNA, histones play critical roles in host defense, exerting procoagulant, proinflammatory and antifibrinolytic effects (reviewed in [22]). Their known impact on clotting and lysis kinetics was confirmed by our measurements showing a concentration-dependent decrease in clotting-, and prolongation of lysis times (Table 3). The plausible, electric charge-dependent interplay of histones and polyphosphates during in vivo thrombus formation justifies comparative evaluation of the effect of histones with the histone+PolyP combinations on the kinetics of clotting and lysis.

Table 3

Effects of histone on fibrin formation and lysis.

		No additive	Histone 3.5 μM	Histone 7 μM	Histone 13 μM	Histone 20 μM
clotting time50 (min)	median	53.63	35.65	30.68	22.50	11.08
	interquartile range	15.26	8.20	4.28	9.68	7.79
	P-value	-	8.71×10−5	2.86×10−7	2.86×10−7	2.86×10−7
lysis time50 (min)	median	14.62	31.18	40.00	44.82	59.95
	interquartile range	6.10	16.13	24.72	16.78	6.78
	P-value	-	1.10×10−3	1.70×10−4	3.99×10−6	3.28×10−6

Fibrinogen (7 μM) containing plasminogen (0.2 μM) was clotted by thrombin (2 nM) in the presence of histone at various concentrations. Reaction kinetics was monitored and lysis induced as in Fig 3. P-values are shown according to Kolmogorov-Smirnov test in comparison with no additive (n = 15).

The effect of histone on clotting time (clotting time50 decreased by 79% in a Ca2+-free and by 73% in a Ca2+-bearing milieu) was opposed by all of the polyphosphates: histone+PolyP-Linm, histone+PolyP-Linp and histone+PolyP-NP combinations increased clotting time50 3.3-, 2.5- and 1.6-fold, respectively, compared to histone’s effect alone (Fig 4A and 4C). Histone’s effect on lysis time50 (a 4.1-fold increase in the absence of Ca2+ and a 2.5-fold increase in the presence of Ca2+) was not influenced by histone+PolyP-Linm or histone+PolyP-Linp combinations, but it was enhanced by histone+PolyP-NP (Fig 4B and 4D). The PolyP-NP’s effect on lysis time50 (increase by 76%) was remarkably strengthened by histone (increased further by 118%). According to these findings we can conclude, that 1) in fibrin polymerization all types of polyphosphates oppose the accelerating effect of histone, but the histone effect is least hampered by PolyP-NP; 2) in fibrinolysis only the polyphosphate in nanoparticulate form (and not the linear one) acts in synergism with histones to inhibit the process.

Fig 4

Combined effects of histone and polyphosphates on fibrin formation and lysis.

Fibrinogen (7 μM) mixed with plasminogen (0.2 μM) was clotted by thrombin (2 nM) in the presence or absence of histone (20 μM), linear PolyP-Linm (330 μM), linear PolyP-Linp (1.96×1012 particle/l equivalent to 330 μM) or nanoparticulate (1.96×1012 particle/l) polyphosphate and the absorbance at 340 nm (Turbidity) was measured (Panel A). At plateau phase tPA (140 nM) was layered on the surface of clots and measurement was continued until complete lysis (Panel B). Arrows indicate the time to reach half-maximal turbidity (clotting time50 and lysis time50) in the ascending and descending phase of the representative turbidimetric curves in panels A and B, respectively. Median values and interquartile range (error bars) of clotting time50 (Panel C) and lysis time50 (Panel D) are shown in black (no Ca2+) or grey (1.67 mM Ca2+). P-values for differences of the indicated pairs of datasets are shown according to Kolmogorov-Smirnov test (n = 15).

Histone-fibrinogen aggregation and its effect on fibrinogen digestion by plasmin

During the investigation of the effects of histone alone or in combination with PolyPs on fibrinogen clotting and lysis, we observed increased turbidity at the onset of clot formation (Fig 4A) and prolonged fibrin lysis time (Table 3, Fig 4D). Our results confirmed the earlier findings of histone-fibrinogen gelation: incubating fibrinogen with histone resulted in increasing histone-fibrinogen aggregation (Fig 5A), and fibrinogen became resistant to lysis by plasmin (Fig 5B) [18].

Fig 5

Histone-fibrinogen aggregation and its effect on fibrinogen digestion by plasmin.

Fibrinogen solution (7 μM) was added to histone (0, 6 or 20 μM) in the absence (black bars in panel A, a-b lanes in panel B) or presence (grey bars in panel A, c-h lanes of panel B) of plasmin (50 nM). Panel A: Aggregation was monitored by turbidimetry for 150 min. Mean maximal turbidity ± SD and P-values according to Kolmogorov-Smirnov test for differences between the indicated pairs of datasets are shown (n = 15). Panel B: Digestion of fibrinogen-histone mixtures by plasmin. Non-reducing samples were taken for SDS-PAGE at 0, 60 or 150 min and electrophoresis was performed on a 4–15% polyacrylamide gel followed by silver staining. Panel B: a, c, d: Fibrinogen; b: Histone (20 μM); e, f: Fibrinogen + histone (20 μM); g, h: Fibrinogen + histone (6 μM).

Combined effects of histones and polyphosphates on fibrin structure

The kinetics of fibrin formation are known to affect the ultimate clot architecture (fiber size, branching density, porosity), whereas the fibrin structure is an essential determinant of fibrinolysis (reviewed in [23]). According to the results reported in the preceding sections, histones (and not DNA) modulated significantly the polyphosphate effects on fibrin formation and lysis. Thus, modification of the fibrin structure in the presence of polyphosphates in combination with histones could be hypothesized as a mechanism underlying our current findings. We characterized one parameter of the fibrin structure—the fiber thickness. In line with our earlier results [2], histone itself increased the median fibrin fiber diameter by 19% (Fig 6C). PolyP-Linm and its combination with histone increased the fiber diameter by 13% and 23%, respectively (not shown). Ca2+ itself reduced fiber diameter by 11%, while PolyP-NP and histone+PolyP-NP combination increased it by 23% and 28%, respectively. In summary, the examined modulators and their combinations all increased the fibrin fiber diameter, however to a different extent and the combination of a polyanionic modulator (polyphosphates in different form) and a polycationic modulator (histone) did not neutralize, but rather reinforced their standalone effects.

Fig 6

Effects of histone and PolyP-NP on clot structure.

Representative scanning electron micrographs of fibrin prepared in the absence (panel A) or presence of modulators (histone+PolyP-NP (panel B), histone or PolyP-NP) as described in Materials and methods. Panel C: The diameters of 300 fibrin fibers were measured from each of 3 independent fibrin sample images and the probability density function (PDF) of the diameter values is shown. The empirical data are shown as a histogram with bins of equal area (each bin has equal product of density and span of data), whereas the grey line indicates the lognormal theoretical distribution that fitted best these data. All differences in the median fiber diameters were significant at p<0.05, according to Kuipers’s test of the distributions [20].

Histone-PolyP binding

We recorded the SPR sensorgrams of four different polyphosphate forms (nanoparticulate form and linear variants of three differently lengths) flowing over histone non-covalently immobilized on carboxymethyl-dextran surface in the SPR cell (Fig 7). We employed a non-covalent immobilization strategy to prevent chemical modifications of the histone that could change the positive charge of its lysine and arginine residues conceivable in the targeted interactions (as described elsewhere, [19]). This strategy also circumvented the potential problem of histone denaturation, as each run started with freshly immobilized histone material. Polyphosphates were applied at concentrations spanning the range 0.1–1 mM (concentration in monomeric phosphate equivalents) (S1 File). We found that the nanoparticle form interacted weakly with histone with estimated dissociation equilibrium constant in the millimolar range (S1 File), whereas the linear polyphosphate forms resulted in a decline in the signal (Fig 7). This latter finding is consistent with a strong linear polyphosphate-histone binding that partially stripped the surface of non-covalently attached histone. There was no significant difference between the linear polyphosphate forms of different size (45, 100 or 700 monomeric units) at equivalent monomeric phosphate concentrations. To sum up, all forms of polyphosphate were able to bind histone, but with a marked qualitative difference in the strength of binding of nanoparticulate and linear forms. The stripping effect could be of interest, if polyphosphates and histones interact under flow conditions in vivo.

Fig 7

Representative SPR sensorgrams of the interaction of different polyphosphate forms and histone.

Histone, non-covalently attached to a carboxymethyl-dextran surface, was exposed to a flow of polyphosphate of the indicated type at 1 mM concentration as described in Materials and methods. The nanoparticulate form of polyphosphate shows a small, but positive SPR signal, indicative of an ordinary weak interaction. In contrast, the linear polyphosphate forms strip the non-covalently bound histone from the chip, irrespective of their length.

Discussion

Neutrophil extracellular traps were first described as novel players of the innate immune system against bacterial infection [24]. In addition to NETosis, neutrophils contribute to the localization of microbial infection by promoting blood coagulation [21]. Furthermore, there is growing evidence that NETs are key players in the formation of pathological thrombi [25]. In our previous works we focused on the effects of NET components (histones and DNA) on thrombus formation, structure and stability [2,3,26]. Besides DNA, another biorelevant, negatively charged polyanion, polyphosphate emerged as a potential modulator of thrombus formation and clot lysis during the last decade [1]. Polyphosphates are stored within the cells in the presence of divalent metal ions and upon platelet activation Ca2+-precipitated PolyPs are released in addition to the soluble, linear form. PolyP-NPs are proven to associate to platelet membrane and to be more potent activators of the contact pathway of blood coagulation than the linear form [6]. This finding raised the need to compare the effects of these two forms of polyphosphates on thrombus formation. Multifaceted interactions of linear polyphosphates with thrombogenic factors have been observed. Linear polyphosphates accelerate blood clotting by activating the contact pathway and activation of factor V, which counteracts the action of a major anticoagulant protein, tissue factor pathway inhibitor [27]. They also delay clot lysis through enhancing the action of thrombin-activatable fibrinolysis inhibitor [27]. Linear polyphosphates induce directly platelet aggregation and the long-chain variants are more potent in this action [28]. Linear polyphosphates modulate platelet activation indirectly, too. They induce von Willebrand factor release from endothelial cells through a high mobility group box 1 dependent mechanism resulting in platelet string formation [29]. Furthermore, the coexistence of neutrophils and platelets at the site of (immune)thrombosis allows for interplay between NET components and polyphosphates [1]. However, linear polyphosphates are unstable in circulation because of the presence of phosphatases. Thus, it is important to assess, if the particulate form of the polyphosphates that is more resistant to degradation can exert the same prothrombotic effects as their linear counterpart. To this end we produced a stable suspension of polyphosphate nanoparticles and in the present study we focused on one aspect of the multifaceted role of polyphosphates in thrombus formation, structure and lysis. We compared the effects of linear and nanoparticulate polyphosphates alone and in combination with NET components (DNA, histone) utilizing a simplified thrombin-mediated fibrin clot model.

Changes in the kinetics of clotting and lysis: The role of the nanoparticulate form emerges

When polyphosphates acted alone or in combination with DNA the linear form exerted the most pronounced effect on both clotting and lysis time50 values. According to our present observations the main determinant of these PolyP effects was the number of particles and DNA did not significantly modify the PolyP effects on the clotting and lysis kinetics. However, when acting together with histone, a new perspective emerged. Firstly, histone and PolyPs modified each other’s effects during both clotting and lysis. Secondly, histone exerted its effects on PolyP-Linm and PolyP-NP in a different way: the former was hindered (during clotting) or slightly enhanced (during lysis), while the latter was remarkably reinforced during both clot formation and lysis. Based on these findings we conclude that: 1) differences in the intensity of interactions between DNA-PolyPs and histone-PolyPs can be attributed to the overall charges of the molecules: opposite charges in the case of histones and PolyPs are accompanied by stronger interactions; 2) the disparity of the histone-PolyP-Linm and histone-PolyP-NP interplay in fibrin turnover suggests differences in their molecular interactions. The latter interpretation was confirmed by SPR measurements. Both linear (PolyP-Linm) and nanoparticulate PolyP bound to histone, but their SPR signals diverged suggesting that through stronger binding to the histone, the linear variants competed successfully with the non-covalent immobilization forces of the carboxymethyl-dextran matrix, whereas the weaker interactions of the PolyP-NP were not able to detach the complexes from the solid support. An important in vivo implication of this finding could be a more pronounced procoagulant and antifibrinolytic effects of polyphosphates in nanoparticulate form in extracellular compartments, where they are retained by the immobilized histone (e.g. in the meshwork of NETs) and enhance the prothrombotic effect of the latter in contrast to the linear alternates, which would rather contribute to the removal of the histone under flow.

Modification of fibrin structure: A plausible mechanism for the combined effects of histone and polyphosphates on the lytic stability of composite clots

According to earlier studies with pure fibrin, accelerated clot formation (e.g. by higher thrombin concentration) results in thinner fibers [30-32]. In our current study the fibrin fiber diameter was increased by histone, polyphosphates and their combinations despite accelerated clotting phase. Our earlier studies with composite clots have clarified the ultrastructural background of these effects using a small-angle X-ray scattering (SAXS) technique [2]. We have demonstrated that both histone and DNA cause fibrin fiber thickening based on distinct molecular mechanisms: histone interferes with the lateral organization of protofibrils, resulting in lower protofibril density and thicker fibers, while DNA preserves the protofibril-to-protofibril distance, but disrupts the regular longitudinal alignment of the fibrin monomers [2]. As for PolyP, it was shown earlier by others, that the linear form incorporates into polymerizing fibrin clots [33], which may be the reason for its fiber thickening effect. We have recently shown that linear polyphosphates enhance the fiber-thickening effect of histones [5], and our current results provide evidence that in the form of nanoparticles they are even stronger structural modifiers.

The consequences of the changes of fiber size in terms of lytic susceptibility of fibrin depend on the cause of this structural modification. If higher thrombin activity generates fibrin monomers at higher rate, their fast polymerization results in densely branched fibrin meshwork composed of thin fibers that is resistant to lysis [34, reviewed in 35]. However, if the cause of changes in fibrin architecture is incorporation of modifier molecules, the thicker fibers are more resistant to lysis. Because the structure of the major fibrin-degrading protease, plasmin is optimal for the inter-protofibril distances within modifier-free fibrin fibers (reviewed in [23]), looser packaging of the protofibrils in the fibers would decrease its catalytic efficiency, as we have previously described for the breakdown of fibrin containing histones [2] or combination of histone and linear polyphosphate [5]. This interpretation is supported by the current result that the nanoparticulate PolyP with the strongest steric effect on the fiber geometry enhances stronger the anti-fibrinolytic action of histones than the linear variants. The known anti-fibrinolytic effect of histone-induced aggregation of fibrinogen [36] was observed in our experimental setting too, and could contribute as an independent factor to the lytic resistance of the composite clots.

Introducing a novel method to prepare stable PolyP-NPs

In recent years, different techniques were developed for generating stable nanoparticulate PolyPs. Colloidal confinement of PolyP on gold or attaching PolyP to silica nanoparticles resulted in enhanced procoagulant function with stability for even 24 weeks, thus potential candidates were developed for the treatment of internal injuries or a range of other haemorrhagic scenarios [37,38]. However, there is limited experience with generating pure PolyP-NPs, moreover, developed particles are stable only for a few hours [10]. Our paper presents a further improved version of previously described method for preparing pure PolyP-NPs [10,11]. The generated particles were of a size similar to those secreted by platelets in vivo (100–200 nm [6]), retained the former introduced polyphosphate functions on clot forming and lysis and were stable for at least 5 days stored in BSA-TBS at 4°C.

Conclusions

In the past decade, the role of a secondary scaffold formed by activated neutrophil granulocytes has been conceived as an essential factor in thrombus evolution beyond the formation of a 3-dimensional fibrin network. The main effector of NETs, histone possesses several cytotoxic and prothrombotic effects and its overall impact is modified by polyanions (DNA, heparin derivatives and polyphosphates) in the microenvironment of clots. Heparinoids counteract, while DNA and polyphosphates enhance the clot stabilizing effects of histone. The focus of the present work was the comparison of the effects of linear and nanoparticulate polyphosphates alone and in combination with relevant NET components on thrombus formation, structure, and lysis. We found that the PolyP-histone interaction is more significant than the PolyP-DNA interplay probably due to differences in molecular charges: opposite charges are accompanied by stronger interactions. In addition, PolyP nanoparticles enhance the thrombus stabilizing effects of histone more effectively than the linear form. The underlying mechanism of this difference could be related to the weaker PolyP-NP/histone binding observed by us. The weak binding of PolyP-NP to histones allows for independent action of these fibrin-stabilizing factors with consequent additivity of their effects on fibrin fiber thickening and lytic resistance. In contrast, the stronger linear PolyP-histone interaction counteracts more efficiently the accelerating effect of histones in clotting, but their complex formation abrogates the independence of their individual effects on fibrin structure and lysis and thus their combined action is not additive in the stabilization of fibrin. Therefore, nanoparticulate PolyP may be a relevant target of antithrombotic or thrombolytic agents and the novel method presented here for preparing stable PolyP-NPs may serve this line of investigation.

Relation of the monomeric and particular concentrations of the PolyP preparations.

(TIF)

Click here for additional data file.

Original SPR signal data for the interaction of histone and polyphosphates in nanoparticulate and linear form.

(ZIP)

Click here for additional data file.

28 Jan 2022

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Reviewer #1: In this manuscript “ Polyphosphate nanoparticles enhance the fibrin stabilization by histones more efficiently than linear polyphosphates” Lovas et al demonstrated Polyphosphate nanoparticle with histone is the stronger modulator of fibrin formation than the linear poly-p. The work is interesting.

However, I have some minor questions:

1. Authors did not used in-vivo system to clarify their effect of nano-particle

2. Aim was not elucidated clearly

3. “PolyP-NP bound 44 weaker to histone and caused more pronounced thickening of the fibrin fibers than linear polyp” ….please clarify it.

4. Authors used turbidimetric method in their experiment. In there any effect on platelet aggregation?

5. 0.005% Tween?

6. Authors may discuss the brief mechanism (2-3 sentences) in context of their nano-particle comparison-NET-histone-DNA, which was missing in conclusion section.

Reviewer #2: In this article, Lovas et al analyzed the fibrin polymerization from fibrinogen and clot lysis (plasmin) in presence of polyphosphate, polyphosphate nanoparticle NP, DNA and histone.

In the context of NETs and thrombosis, interplay between histones, DNA, fibrinogen are very crucial.

Author found that polyphosphate nanoparticle binding to histones are less than its linear conformers. Most probably, this is the reason why polyphosphate nanoparticle show less inhibition of polyphosphate nanoparticle-mediated inhibition of histone-dependent clot formation in comparison to the linear one. However, it would be difficult to conclude as DNA may have similar effects.

Overall, experiments were conducted logically, but still lack in details.

Major:

1. polyphosphate nanoparticle synthesis should be described elaborately.

2. Fig 1B should be labeled clearly to understand the 2 graph (112 & 188) are these 2 different molecules/preparation ??

3. What is the significance of stability (fig2 ). If it is not physiologically significant, need to be removed or transferred to suppl.

4. Figure 3A, 3B, 4A, 4B curve should be labeled. otherwise it is difficult to understand.

Presence of Ca in polyphosphate nanoparticle preparation may interfere the experiment. Linear polyphosphate experiments should contain similar Ca.

5. Why polyphosphate nanoparticle induce better clot formation and less clot lysis ? explanation should be added in discussion.

6.Table 3. histone concentration is too high. 0.5 to 5 uM concentration is clinically relevant and should be studied accordingly. Which type (CTH/H3/H4) of histone used ? Is all the histones have same effects ? need to be verified.

7. What is the effect of polyphosphate nanoparticle on thrombin activity, as polyphosphate can accelerate this. Moreover, TAFI can be activated in presence of polyphosphate. Is polyphosphate nanoparticle interfere TAFI activity (experiment or explanation required).

8. Polyphosphate and Histones interact and induce VWF from endothelial cells and platelets. VWF is one of the important factor for coagulation, inflammation and thrombosis. In the context of NETs, VWF plays a key role by interacting with fibrin, histones and DNA.

Author should mentioned these research.

**********

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Reviewer #2: Yes: Indranil Biswas

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5 Mar 2022

Reviewer #1: In this manuscript “ Polyphosphate nanoparticles enhance the fibrin stabilization by histones more efficiently than linear polyphosphates” Lovas et al demonstrated Polyphosphate nanoparticle with histone is the stronger modulator of fibrin formation than the linear poly-p. The work is interesting.

However, I have some minor questions:

Response: We would like to thank the reviewer for the careful evaluation of our manuscript. We are very pleased that the reviewer has found our “work is interesting”. At the same time, the reviewer pointed out some possible gaps in the presentation and discussion of the results in the manuscript, and suggested some improvements in this respect. We have addressed all the comments of the reviewer in detail below and revised our manuscript accordingly. We believe that this has helped us to improve and strengthen the manuscript. The changes to the manuscript are highlighted in red. Itemized responses to each of the comments are summarized below.

1. Authors did not used in-vivo system to clarify their effect of nano-particle

Response: We thank the reviewer for this comment, because it pointed out that we haven’t emphasized that the aim of the study was to develop a formulation of polyphosphates that adequately represents their in vivo particulate form (as documented by our DLS and TEM data), and is sufficiently stable to be used in repeated series of in vitro measurements (as applied by us in the study of fibrin structure and lysis as an example of an in vitro application). Accordingly we modified the formulation of the study aims (l. 64-68): “In this study, we aimed 1) to develop a new method for preparing stable, pure PolyP-NPs that are suitable for in vitro applications to study their role in hemostasis, and 2) to compare and characterize the effects of the linear and nanoparticulate forms of PolyPs and the combined effects of DNA, histones and PolyPs on the kinetics of clotting, structure and lytic susceptibility of fibrin clots in vitro.”

2. Aim was not elucidated clearly

Response: We agree that the aim was not sufficiently detailed in the abstract. Accordingly we expanded it focusing on the in vitro aspect of the study (l. 30-32). “Aims-To compare the effects of linear and nanoparticulate polyphosphates, and their combinations with relevant NET components (DNA, histone H3) on fibrin formation, structure, and lysis in in vitro assays focusing on histone-polyphosphate interactions.”

3. “PolyP-NP bound 44 weaker to histone and caused more pronounced thickening of the fibrin fibers than linear polyp” ….please clarify it.

Response: We agree that the referred last sentence in the Results section of the Abstract was confusing. Accordingly we have paraphrased this part of the Abstract for clarity (l. 42-45). “PolyP-NP, but not linear PolyP enhanced the prolongation of lysis time in fibrin containing histone and caused more pronounced thickening of the fibrin fibers than the linear form. Finally, PolyP-NP bound weaker to histone, than the linear form.”

4. Authors used turbidimetric method in their experiment. In there any effect on platelet aggregation?

Response: In this study we used a simplified thrombin-mediated fibrin clot model, in which we tested the polyphosphate effects in the absence of platelets. However we agree with the reviewer that the known direct and indirect effects of linear polyphosphates on platelet activation represent a relevant line for future studies, in which the nanoparticulate formulation developed by us can be applied. Accordingly we added the following text in the Discussion including 2 new references (l. 400-409, 411-414): ”Linear polyphosphates induce directly platelet aggregation and the long-chain variants are more potent in this action [28]. Linear polyphosphates modulate platelet activation indirectly, too. They induce von Willebrand factor release from endothelial cells through a high mobility group box 1 dependent mechanism resulting in platelet string formation [29]…. However, linear polyphosphates are unstable in circulation because of the presence of phosphatases. Thus, it is important to assess, if the particulate form of the polyphosphates that is more resistant to degradation can exert the same prothrombotic effects as their linear counterpart.”

5. 0.005% Tween?

Response: The text was corrected (l. 175): “Tween-20 at 0.005 w/w%.

6. Authors may discuss the brief mechanism (2-3 sentences) in context of their nano-particle comparison-NET-histone-DNA, which was missing in conclusion section.

Response: We agree that a comment on the possible mechanism of the observed effects was missing in the conclusions section. Accordingly we have added the following text (l. 491-501): “We found that the PolyP-histone interaction is more significant than the PolyP-DNA interplay probably due to differences in molecular charges: opposite charges are accompanied by stronger interactions. In addition, PolyP nanoparticles enhance the thrombus stabilizing effects of histone more effectively than the linear form. The underlying mechanism of this difference could be related to the weaker PolyP-NP/histone binding observed by us. The weak binding of PolyP-NP to histones allows for independent action of these fibrin-stabilizing factors with consequent additivity of their effects on fibrin fiber thickening and lytic resistance. In contrast, the stronger linear PolyP-histone interaction counteracts more efficiently the accelerating effect of histones in clotting, but their complex formation abrogates the independence of their individual effects on fibrin structure and lysis and thus their combined action is not additive in the stabilization of fibrin.”

Reviewer #2: In this article, Lovas et al analyzed the fibrin polymerization from fibrinogen and clot lysis (plasmin) in presence of polyphosphate, polyphosphate nanoparticle NP, DNA and histone. In the context of NETs and thrombosis, interplay between histones, DNA, fibrinogen are very crucial. Author found that polyphosphate nanoparticle binding to histones are less than its linear conformers. Most probably, this is the reason why polyphosphate nanoparticle show less inhibition of polyphosphate nanoparticle-mediated inhibition of histone-dependent clot formation in comparison to the linear one. However, it would be difficult to conclude as DNA may have similar effects.

Overall, experiments were conducted logically, but still lack in details.

Response: We appreciate the positive comments about our work, including that it addresses “very crucial” research questions and that our “experiments were conducted logically”. We are grateful for the constructive comments and suggestions that we have addressed with revisions in the manuscript as itemized below.

Major:

1. polyphosphate nanoparticle synthesis should be described elaborately.

Response: In line with this suggestion, we added two sentences in the methods section so that it is apparent that the detailed recipe is there. The initial part of the section (l. 81-83) now reads: “Pure PolyP nanoparticles are labile, so we developed a novel technique for preparing stable PolyP nanoparticles based on improvements of earlier work [10,11]. The detailed procedure is as follows.” The final filtration step was also more detailed (l. 89-91) “Remnant large aggregates were removed by filtrating them sequentially through a syringe filter of 1.2 μm and then through a filter of 0.22 μm pore size. Finally, the filtrate with the remaining PolyP-NPs was retained and stored at 4 °C.”

2. Fig 1B should be labeled clearly to understand the 2 graph (112 & 188) are these 2 different molecules/preparation ??

Response: The figure legend has been changed in order to clarify the origin of the two curves in Fig. 1B (l. 215-217) “The distribution curves of two different PolyP-NP preparations are shown to illustrate the minimal and maximal median values of particle size in the PolyP-NP preparations used in this study.”

3. What is the significance of stability (fig2 ). If it is not physiologically significant, need to be removed or transferred to suppl.

Response: We agree with the reviewer that Fig. 2 has no physiological relevance, but it is essential to illustrate the major methodological improvement achieved with our study, because the pure PolyP-NP preparations used in previous studies were stable for several hours only, whereas ours preserved their structure for several days. That’s why we would like to retain the figure in the main body of the paper. We also agree that the original text did not explain clearly how we interpret the stability of the preparation. Now we’ve corrected this gap expanding the main text on l. 206-207 “no change in the median diameter of PolyP-NPs was observed suggesting that neither aggregation, nor disintegration of the PolyP-NPs occurred in the preparations” and the figure legend l. 219-221“The particle size of the PolyP-NPs was monitored by dynamic light scattering (DLS) for 6 days as a measure of the preparation stability”.

4. Figure 3A, 3B, 4A, 4B curve should be labeled. otherwise it is difficult to understand.

Presence of Ca in polyphosphate nanoparticle preparation may interfere the experiment. Linear polyphosphate experiments should contain similar Ca.

Response: The representative original curves in Figs. 3A&B, 4A&B have been labelled in the graphs and the legend was adapted accordingly.

We agree with the reviewer concerning the effect of Ca in polyphosphate nanoparticle preparations. That’s why in all cases we compare the nanoparticle effects to controls containing the same amount of Ca. However, the suggestion to have similar Ca in the linear polyphosphate experiments is not feasible technically, because if Ca is added to the linear PolyP sample, it would precipitate in an uncontrolled manner, therefore it would not be a linear PolyP anymore. That’s why all control measurements for the effects of linear PolyP are performed in Ca-free medium.

5. Why polyphosphate nanoparticle induce better clot formation and less clot lysis ? explanation should be added in discussion.

Response: We agree with the reviewer that in the original Discussion we referred to our previous work on the fibrin structure/lysibility interrelations in the presence of histones alone or in combination with linear PolyP (Refs. 2, 5), but we didn’t provide any explanation for the difference in the effects of the two forms of PolyP. To address this point we have added the following text in the Discussion (l. 493-501): “In addition, PolyP nanoparticles enhance the thrombus stabilizing effects of histone more effectively than the linear form. The underlying mechanism of this difference could be related to the weaker PolyP-NP/histone binding observed by us. The weak binding of PolyP-NP to histones allows for independent action of these fibrin-stabilizing factors with consequent additivity of their effects on fibrin fiber thickening and lytic resistance. In contrast, the stronger linear PolyP-histone interaction counteracts more efficiently the accelerating effect of histones in clotting, but their complex formation abrogates the independence of their individual effects on fibrin structure and lysis and thus their combined action is not additive in the stabilization of fibrin.”

6.Table 3. histone concentration is too high. 0.5 to 5 uM concentration is clinically relevant and should be studied accordingly. Which type (CTH/H3/H4) of histone used ? Is all the histones have same effects ? need to be verified.

Response: We agree with the reviewer that histone concentrations up to 5 µM have been reported in circulating blood (for example in baboon sepsis model, Xu et al, Nat. Med. 2009; 15, 1318). However, when NETs are formed in thrombi the local concentration of histones is expected to be at least an order of magnitude higher. That’s why in studies of the role of NETs in thrombi histone concentrations up to 1,000 µg/mL (about 70 µM) are considered relevant (for example, Fuchs et al, Blood, 2011;118:3708). Thus, the concentration range chosen by us in Table 3 starts with a value relevant for the circulation and goes up to values that are typical for the local environment of the forming NETs. Concerning the type of histone used in our study we indicate in the Materials section (l. 73) that we used “histones from calf thymus type VIIIS (arginine-rich histone H3) “. We chose this type, because in our previous work (Refs. 2 and 5) we didn’t find any significant difference between the effects of the linker H1 and the core H3 histones on fibrin structure and lysis (as we indicated in Ref. 5). So we performed the extensive series of measurements reported now only with the H3 histone.

7. What is the effect of polyphosphate nanoparticle on thrombin activity, as polyphosphate can accelerate this. Moreover, TAFI can be activated in presence of polyphosphate. Is polyphosphate nanoparticle interfere TAFI activity (experiment or explanation required).

Response: In our study we used purified fibrinogen and monitored the kinetics of its conversion to fibrin by thrombin, as illustrated in Figs. 3A and 4A. Because fibrinogen is the natural substrate of thrombin, we think that the clotting time data reported in Figs. 3C and 4C are adequate indicators of the changes in thrombin activity. We agree that the known effects of linear polyphosphates on other hemostatic/fibrinolytic factors present in plasma prompt the evaluation of the nanoparticulate variants on the same factors, which could be a future application of the stable nanoparticles reported now. Accordingly we have expanded the Discussion section placing the utility of our polyphosphate formulation in the context of studies targeting other plasma factors, but emphasizing that the current work focused on a simplified pure fibrin experimental setup (l. 396-413): “Multifaceted interactions of linear polyphosphates with thrombogenic factors have been observed. Linear polyphosphates accelerate blood clotting by activating the contact pathway and activation of factor V, which counteracts the action of a major anticoagulant protein, tissue factor pathway inhibitor [27]. They also delay clot lysis through enhancing the action of thrombin-activatable fibrinolysis inhibitor [27]….. However, linear polyphosphates are unstable in circulation because of the presence of phosphatases. Thus, it is important to assess, if the particulate form of the polyphosphates that is more resistant to degradation can exert the same prothrombotic effects as their linear counterpart. To this end we produced a stable suspension of polyphosphate nanoparticles and in the present study we focused on one aspect of the multifaceted role of polyphosphates in thrombus formation, structure and lysis. We compared the effects of linear and nanoparticulate polyphosphates alone and in combination with NET components (DNA, histone) utilizing a simplified thrombin-mediated fibrin clot model.”

8. Polyphosphate and Histones interact and induce VWF from endothelial cells and platelets. VWF is one of the important factor for coagulation, inflammation and thrombosis. In the context of NETs, VWF plays a key role by interacting with fibrin, histones and DNA.

Author should mentioned these research.

Response: We agree with the reviewer that modulation of platelet function via VWF is an essential aspect of the polyphosphate effects in haemostasis. Accordingly we added the following text in the Discussion including 2 new references (l. 400-404): ”Linear polyphosphates induce directly platelet aggregation and the long-chain variants are more potent in this action [28]. Linear polyphosphates modulate platelet activation indirectly, too. They induce von Willebrand factor release from endothelial cells through a high mobility group box 1 dependent mechanism resulting in platelet string formation [29]”.

Submitted filename: Response_Reviewer comments_R1.docx

Click here for additional data file.

28 Mar 2022

Polyphosphate nanoparticles enhance the fibrin stabilization by histones more efficiently than linear polyphosphates

PONE-D-21-40112R1

Dear Dr.  Kolev

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Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

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Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

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Reviewer #1: (No Response)

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: (No Response)

Reviewer #2: Authors have addressed the reviewers suggestion and concern. Revised manuscript is improved in explanation of the experimental evidence.

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Reviewer #1: No

Reviewer #2: Yes: Indranil Biswas

6 Apr 2022

PONE-D-21-40112R1

Polyphosphate nanoparticles enhance the fibrin stabilization by histones more efficiently than linear polyphosphates

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