Correlation between Fibrin Fibrillation Kinetics and the Resulting Fibrin Network Microstructure

Abstract

Fibrin, the prominent extracellular matrix in early wound tissue, is discussed to influence immune cells and healing. The nature of fibrinogen/fibrin to form fibrillary networks is frequently exploited to engineer microenvironments for cellular analysis. This study focuses on revealing the correlation of fibril formation kinetic and the resulting network microstructure of engineered 3D fibrin networks. Different concentrations of fibrinogen (1–3 mg mL⁻¹), thrombin (0.01–0.15 U mL⁻¹), sodium chloride (40–120 mm), and calcium chloride (1–10 mm) are applied to assess the impact on the fibril growth kinetics by turbidity analysis and on the resulting fibril and pore diameter by laser scanning microscopy. The results highlight a direct influence of the sodium chloride concentration on fibrillation kinetics and reveal a strong correlation between fibrillation kinetics and network microstructure. With the assumption of a first‐order growth kinetic, an increase of the growth constant k (0.015–0.04 min⁻¹) is found to correlate to a decrease in fibril diameter (1–0.65 µm) and pore diameter (11–5 µm). The new findings enable an easy prediction of 3D fibrin network microstructure by the fibril formation kinetic and contribute to an improved engineering of defined scaffolds for tissue engineering and cell culture applications.

Engineering of defined 3D fibrin networks is frequently used to mimic wound matrices and investigate immune cells in vitro. Here, a direct correlation between fibrin fibrillation kinetics and resulting fibrin network topology is revealed and sheds light into the mechanism of fibril formation in dependence on buffer NaCl concentration.

1. Introduction

The extracellular matrix (ECM) is an important regulator of cellular functions during many physiological and pathological processes. Biochemical and biophysical properties of the ECM are crucial parameters that vary time‐ and condition‐dependent and can control cell proliferation, differentiation, migration, and immune cell activation.^{[ 1} , ² , ^{3 ]} A prominent example of ECM proteins is fibrinogen. Fibrinogen circulates in the blood at a concentration of about 9 µm (or 1.5–4 mg mL^{−1[ 4 ]}) while the level increases during inflammation.^{[ 5 ]} At sites of tissue injury, the enzymatic transformation of fibrinogen into fibrin by activated thrombin is the trigger to enter the blood coagulation cascade and induces the formation of 3D fibrillary networks. This provisional wound matrix is mainly composed of fibrin, fibronectin, and proteoglycans and serves as a shield against bleeding, a scaffold for the invasion of local and recruited cells, and a reservoir of cytokines and growth factors.^{[ 5} , ⁶ , ⁷ , ^{8 ]} As the name implies, the provisional matrix is a temporary construct undergoing degradation and replacement during wound repair. Thus, a critical distinction between an “early provisional matrix” directly after vascular injury and a “late provisional matrix” (mainly cell‐derived) is suggested.^{[ 7 ]} Due to the obvious, important role of a fibrin matrix during wound healing, this acute‐phase ECM protein is used in tissue engineering applications and has potential to operate as a biomimetic wound matrix to investigate wound healing and cell behavior in vitro.^{[ 9} , ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ^{17 ]}

The in vitro preparation of fibrin networks benefits from extensive investigations over the last decades. Details about the molecular structure of fibrinogen, its transformation into fibrin and the subsequent polymerization or fibrillation process were previously summarized.^{[ 18} , ¹⁹ , ^{20 ]} In short, fibrinogen is a 340 kDa dimeric glycoprotein, with each dimer consisting of three polypeptide chains termed Aα, Bβ, and γ. Fibrinogen molecules contain two outer D domains and a central E domain where each set of α, β, and γ chains is linked by disulfide bonds at their N‐termini. While the α and γ chains are stabilized by one and two interchain disulfide bonds, β chains are linked to the α chain and to the γ chain by one disulfide‐bridge, respectively. Between the central E domain and the two distal D domains, the polypeptide chains form a rod‐like, α‐helical, coiled‐coil region. Fibrinogen polymerization is initiated by selectively cleaving the fibrinopeptides A (FibpA), first, and fibrinopeptides B (FibpB) more slowly or readily after protofibril and fibril assembly from the N‐termini of the Aα and Bβ chains by thrombin. By this enzymatic reaction, thrombin exerts an important initial impact on the following fibrillation kinetic. The exposure of the peptide sequences, termed knobs A and B, in the central E domain as well as the detachment of the αC domains from the central E domain is followed as a consequence. By the interaction of knobs A and B with the complementary holes a and b, that are present in the D domains of adjacent now termed fibrin monomers, half‐staggered dimers are formed resulting in double‐stranded protofibrils when linear fibril growth proceeds. While interaction of the A:a complementary binding sites is necessary for protofibril formation, interaction of B:b sites is not, but contributes to strengthen the protofibrils. Protofibrils are known to have a diameter of about 11 nm^{[ 21 ]} and a critical length prior the start of lateral assembly, ranging from 500 to 600 nm^{[ 4} , ¹⁹ , ^{21 ]} depending on fibrillation condition. As next, lateral assembly of protofibrils occurs to form thicker fibrils.^{[ 19} , ²² , ^{23 ]} Finally, the loose fibrin fibrils are crosslinked by the thrombin activated factor XIIIa (FXIIIa) to form a highly organized and stable structure.^{[ 24 ]} In conclusion, fibrin fibril assembly is a sequential and time‐dependent process characterized by three steps: Step I) Conversion of fibrinogen into fibrin by activated thrombin; Step II) linearly aggregation of fibrin monomers in a half‐staggered manner to form oligomers—so‐called two‐stranded protofibrils; Step III) laterally aggregation of protofibrils to form fibers which further aggregate and branch resulting in a fibrillary 3D network that is finally crosslinked by FXIIIa.^{[ 10} , ²² , ²³ , ^{24 ]} These basic processes of Step I to III are outlined in Figure  1 .

Figure 1.

Schematic illustration of a typical three‐phase turbidity–time curve of fibrin fibrillation and network formation with underlying steps of the fibrillation process. Fibrin fibrillation can be followed by light scattering revealing three typical, time‐dependent phases caused by molecular transitions during the fibrin network formation which on the other hand can be divided into three steps of fibrillation. Phase 1 covers Step I (fibrinogen‐to‐fibrin transition due to cleaving of fibrinopeptide (Fibp) A and B by thrombin) and Step II (linear protofibril growth up to a critical length) of fibrin fibrillation and runs without change in optical density because of the small size of protofibrils. Phase 2 is caused by Step III where nucleation of fibrils from protofibrils, lateral fibril growth, and network formation occur leading to a significant increase in optical density. After completion of lateral fibril growth and during final crosslinking of loose fibrils via the action of thrombin and factor XIIIa, the turbidity–time curve ends in a plateau of Phase 3.

Different physicochemical parameters and their effect on the fibrillation kinetic and/or on the resulting network properties were investigated. It has been reported that fibrinogen and thrombin concentration,^{[ 10} , ²² , ²⁵ , ^{26 ]} sodium chloride (NaCl),^{[ 10} , ²² , ^{23 ]} calcium chloride (CaCl₂),^{[ 27} , ²⁸ , ^{29 ]} zinc chloride,^{[ 30 ]} and chloride ions,^{[ 31 ]} ionic strength,^{[ 10 ]} pH,^{[ 26 ]} and other buffer components^{[ 32 ]} have significant impact. The resulting complexity of fibrin self‐assembling, the variance of preparation protocols and, additionally, the partial usage of commercially available unpurified—fibronectin contaminated—fibrinogen compounds, often hinders direct comparisons between different studies. However, the general observation that the microstructure of fibrin networks is kinetically determined is very interesting with respect to the need to engineer structurally defined fibrin networks for in vitro and in vivo applications. The multistep fibrillation process can be easily observed by light‐scattering experiments revealing typical three‐phase turbidity curves that correspond to the three steps of fibrin network formation, as described above. Phase 1 is a so‐called lag‐phase equivalent to the processes in steps I and II where no increase of the turbidity is observed due to the small size of protofibrils. Phase 2 corresponds to processes in step III where turbidity significantly increases due to lateral assembly of protofibrils and initial network formation. Phase 3 ends up with a plateau of constant turbidity which is dependent on the final network microstructure. A typical three‐phase turbidity‐time curve including the sequential Steps I‐III of fibrinogen‐to‐fibrin‐to network transition, as described above, is summarized in Figure 1.

As the above‐mentioned buffer parameters are known to affect the self‐assembly kinetic of fibrin fibrils as well as fibrin network microstructure; however, a quantitative understanding and description with options for bioengineering implementations are missing. As a motivation for our presented work, we hypothesized a direct correlation between fibrillation kinetics and the resulting network microstructure similar to our investigation on such correlations with respect to collagen type I fibrillation.^{[ 33 ]} Therein the growth constant of the lateral growth phase of collagen fibrils in terms of a first‐order rate reaction was shown to be dependent on the applied fibrillation conditions (e.g., buffer) and correlates with the final diameter of collagen fibrils. Thus, a quantitative analysis of the fibrillation kinetics can be assumed to have potential to be used as a parameter predicting network microstructures based on plain light‐scattering analysis of fibrillation kinetics in case of fibrin as well. Such a new approach of predicting fibrin network microstructures by simple fibrillation kinetic investigations would facilitate the process of adaptation of suitable fibrillation conditions for the reconstitution of defined 3D network microstructures in bioengineering of cell culture scaffolds.

In sum, comprehensive investigations on fibrin self‐assembly and network characteristics have been realized. However, missing quantitative studies with respect to a correlation of fibrin fibrillation kinetics and the resulting network microstructure for the various relevant reconstitution parameters including physiologically relevant fibrinogen concentrations, thrombin concentrations allowing the preparation of reproducible networks at reasonably slow self‐assembly speed, and various buffer ions ask for a more detailed investigation. Therefore, the aim of our new study was to explore influences of reconstitution conditions (buffer components and concentration of fibrinogen, thrombin, CaCl₂, and NaCl) on the kinetics of fibrin fibrillation and dissect correlations between kinetics and the resulting 3D network microstructure. The revealed kinetic‐structure‐relationship contributes to the understanding of fibrin network formation and an improved engineering of well‐defined 3D fibrin matrices.

2. Results and Discussion

2.1. Microstructure of Fibrillary Fibrin Networks in Dependence on Fibrillation Conditions

To enable engineering of topologically defined fibrin networks, we investigated the mean pore and fibril diameter of fibrin networks in dependence on different fibrillation conditions, namely: concentration of fibrinogen (1, 2, and 3 mg mL⁻¹), thrombin (0.01, 0.05, 0.1, and 0.15 U mL⁻¹), NaCl (40, 60, 80, 100, and 120 mm), and CaCl₂ (0, 1, 1.5, 2, 2.5, 5, and 10 mm). Mean pore and fibril diameter were determined from confocal image stacks of fluorescently stained fibrin networks by an automated analysis tool.

Representative network structures induced by varying fibrinogen, thrombin, NaCl, and CaCl₂ concentration are shown by confocal images in Figure  2A–M. This overview clearly demonstrates the possibility to control the resulting network structure by the investigated fibrillation conditions. While increasing fibrinogen and NaCl concentrations resulted in a continuous decrease of the mean pore diameter (Figure 2A–C,G,H), a decrease in pore diameter with increasing thrombin content was only observed between 0.01 and 0.05 U mL⁻¹ (Figure 2D,E). Higher thrombin concentrations (0.1 and 0.15 U mL⁻¹) induced no further pore diameter decrease (Figure 2E,F). In contrast, an increase in CaCl₂ concentration from 1 to 10 mm induced an increase of the mean pore diameter (Figure 2K–M), reflecting an opposing trend compared to fibrinogen, thrombin, and NaCl. However, clear differences were only seen between a low, 1 mm, and high, 10 mm, CaCl₂ concentration.

Figure 2.

Representative cLSM images on the impact of fibrinogen, thrombin, NaCl, and CaCl₂ concentrations on the resulting fibrin network structure. A–C) Fibrinogen at 1, 2, and 3 mg mL⁻¹; D–F) thrombin at 0.01, 0.05, and 0.15 U mL⁻¹; G–I) NaCl at 60, 80, and 120 mm; K–M) CaCl₂ at 1, 5, and 10 mm. The fibrillation buffer was 20 mm HEPES containing 1 mg mL⁻¹ fibrinogen, 0.05 U mL⁻¹ thrombin (THR), 2.5 mm CaCl₂, and 100 mm NaCl if not otherwise stated. Images show one confocal plane of z‐stacks with 5 µm image distance. Image size: 160 × 160 µm².

The quantitative analysis of pore and fibril diameter is summarized in Figure  3 . An increase in fibrinogen concentration from 1 to 3 mg mL⁻¹ resulted in a strong decrease in pore diameter from 6.2 to 4.3 µm (Figure 3A). An increasing NaCl concentration (60–120 mm) strongly decreased the pore diameter from 10.7 to 4.8 µm (Figure 3E). For thrombin, we found only a very weak dependence of pore diameter on thrombin concentration. A fivefold increase in thrombin concentration (from 0.01 to 0.05 U mL⁻¹) induced a small decrease in pore diameter from 7.3 to 6.2 µm (Figure 3C), while an even higher amount of thrombin up to 0.15 U mL⁻¹ only slightly reduced the mean pore diameter to 5.1 µm, also with larger scatter. Additional tests at a thrombin concentration above 0.15 U mL⁻¹ (namely 0.3 U mL⁻¹) led to fibrillation times in a range of seconds such that a preparation of defined and homogeneous networks and the analysis of full‐range turbidity‐time curves were not possible. In contrast to fibrinogen, thrombin and NaCl, an increasing CaCl₂ concentration increased the mean pore diameter (Figure 3G). Increasing the CaCl₂ level from 1 to 10 mm led to an increase in pore diameter from 4.6 to 6.3 µm. However, this effect on pore diameter is comparatively low and accompanied by a high data variance at low CaCl₂ content (1.5 and 2.5 mm). When comparing the investigated mean fibril diameter in dependence on the different reconstitution conditions it is obvious that only NaCl (60–120 mm) and CaCl₂ (2–10 mm) have a strong influence (Figure 3F,H). While NaCl induced a decrease from 1.01 to 0.69 µm, CaCl₂ induced an increase from about 0.67 to 0.8 µm. In contrast, varying concentrations of fibrinogen and thrombin left the mean fibril diameter almost unaffected at about 0.72 µm. However, a very low thrombin concentration of 0.01 U mL⁻¹ results in a fibril diameter of 0.83 µm.

Figure 3.

Mean pore and fibril diameter of fibrin networks in dependence on A,B) fibrinogen, C,D) thrombin, E,F) NaCl, and G,H) CaCl₂ concentration. If not otherwise stated nonvaried components are present at following concentrations: fibrinogen at 1 mg mL⁻¹ in 20 mm HEPES, 0.05 U mL⁻¹ thrombin, 2.5 mm CaCl₂, and 100 mm NaCl. Box‐and‐whiskers plots indicate median at line, mean at square, independent replicates at dots (n = 5), boxes from 25th to 75th percentiles, and whiskers from minimum to maximum values. Statistical analyses revealed significant differences (*) at the significance level of p < 0.05 either to all other samples or between individual samples as indicated by horizontal lines (nonparametric Mann–Whitney test).

In our study we were interested in analyzing and engineering fibrin networks that can be prepared for in vitro cell culture experiments to mimic in vivo situations. On the basis of our initial screening experiments (Figure 3) we focused on experimental conditions with 1) a fixed thrombin concentration of 0.05 U mL⁻¹ to ensure a moderate fibrillation time and 2) a variation of concentrations of fibrinogen in the physiological range and 3) NaCl as a strongly influencing parameter. CaCl₂, as possible buffer component to vary fibrin network properties, too, did not fulfill these expectations as a suitable parameter due to the small range of variation of networks topology and large scatter in data. Therefore, it was not further investigated.

2.2. Quantitative Assessment of Fibrin Fibrillation Kinetics

As the fibrillation kinetics was hypothesized to correspond to the network microstructure, we investigated the fibrin fibrillation kinetics in dependence on the two most potent buffer parameters influencing the resulting network microstructure, namely the concentration of fibrinogen (at 1, 2, and 3 mg mL⁻¹) and NaCl (at 40–120 mm). Figure  4A,B shows typical turbidity–time curves of the fibrillation process. At used conditions almost no lag phase is observed with a very fast increase in turbidity shortly after initiation of fibrillation. From earlier studies^{[ 10 ]} and own preliminary experiments this behavior can be accounted to the used concentrations of fibrinogen and thrombin. At much lower concentrations the so‐called lag phase in turbidity measurements can be observed due to the prolonged time needed for enzymatic cleavage of fibrinogen and protofibril formation.

Figure 4.

Investigation of fibrillation kinetics in dependence on NaCl and fibrinogen concentration and derivation and NaCl‐dependent presentation of the lateral fibril growth rate k _lat. A,B) Turbidity–time curves at varying fibrinogen (at 100 mm NaCl) or NaCl (at 2 mg mL⁻¹ fibrinogen) concentration. Turbidity was analyzed at 1 min intervals, every 16th value is visualized for clarity. C) The final turbidity increase after fibrillation Δτ _p determined at different fibrinogen and NaCl concentration. D) Linear fit for dτ/dt _max over fibrinogen concentration proving a first‐order rate process of the lateral fibril growth. E) k _lat as derived from the fits in (D) in dependence on NaCl concentration. General buffer composition was 20 mm HEPES, 0.05 U mL⁻¹ thrombin, and 2.5 mm CaCl₂.

However, such conditions usually lead to very sparsely distributed fibrils and nonstable 3D fibrin networks, not applicable in tissue engineering and cell culture approaches. Therefore, such conditions were not investigated herein in detail.

A quantitative analysis of the turbidity‐time curves revealed details on fibrin fibrillation kinetics. As expected, an increase in fibrinogen concentration is reflected by an increased final plateau turbidity Δτ _p with a linear dependence on fibrinogen concentration (Figure 4C). Accordingly, no dependence of Δτ _p is observed for an increased NaCl concentration at constant fibrinogen concentration. Only for very high NaCl concentrations of 120 mm a small decrease of Δτ _p was found, which is accounted for by a very fast fibril formation process leading to very thin fibrin fibrils, too, and the nonlinear dependence of turbidity on fibril diameter,^{[ 34} , ^{35 ]} see also the discussion further below.

Next, we proofed the assumption of a first‐order growth kinetics of the fibrillation process in dependence on fibrinogen concentration c _fng with the initial growth rate dτ/dt _max ≈ c _fng, similar to approaches used for fibrillation of collagen I networks,^{[ 33} , ³⁶ , ^{37 ]} see also Methods section. The plot of maximum slope of the turbidity‐time curves dτ/dt _max over fibrinogen concentration c _fng clearly shows the validity of the assumption with a linear increase of the slope with c _fng for all investigated conditions (Figure 4D). This finding indicates that the later growth phase of fibrin fibril formation can be described by a first‐order rate process together with a linear correlation of protofibril formation on fibrinogen concentration.

Based on that, we are able to determine a lateral growth constant k _lat for the lateral growth of fibrin fibrils from protofibrils for different NaCl concentrations. We find k _lat to increase from 0.015 ± 0.004 min⁻¹ at 40 mm NaCl up to 0.039 ± 0.003 min⁻¹ at 120 mm NaCl indicating an increase in k _lat of about 0.0003 min⁻¹ per 1 mm increase in NaCl. The plot in Figure 4E even indicates a linear dependence of the lateral growth constant on NaCl concentration. This finding suggests NaCl as a straightforward parameter to adjust fibrillation kinetics.

2.3. Correlation between Fibrillation Kinetics and Resulting Network Microstructure

The observed decrease in pore and fibril diameter with increasing NaCl concentration in combination with the linear increase of k _lat underlines a correlation between buffer condition (NaCl), fibrillation kinetic, and resulting network microstructure. Due to the linear dependence of the lateral growth constant on NaCl concentration, NaCl was used to modulate fibrillation kinetics. Figure  5A,B shows fibril and pore diameter in dependence on k _lat at a constant fibrinogen concentration. Clear trends of decreasing fibril and pore diameter with increasing k _lat are observed. One can note that both plots imply a similar weak inverse power law behavior, too.

Figure 5.

Correlation between lateral fibril growth rate k _lat and A) fibril and B) pore diameter at varying NaCl concentration. C) Observed dependence between pore and fibril diameter at different fibrinogen and NaCl concentration. D) Pore diameter depending on an inverse square root of ionic strength while extrapolated linear fit functions cross at about 200 mm NaCl and 2 µm pore diameter. General buffer composition was 20 mm HEPES, 0.05 U mL⁻¹ thrombin, and 2.5 mm CaCl₂.

This dependence can be used to check appropriate fibrillation conditions (e.g., molar level of NaCl) to generate a required network topology. Furthermore, at constant fibrinogen concentration the different fibrillary network topologies should comprise the same amount and volume of fibrin fibrils. Hence, from first‐principles geometric considerations a linear correlation of pore diameter to fibril diameter can be expected, which is proofed by the plots in Figure 5C for three different fibrinogen concentrations. To note, in a recent small‐angle X‐ray scattering (SAXS) study slight variations of fibrin packing density were found for fibrin networks at very high fibrinogen concentrations,^{[ 34 ]} which might lead to small deviations from our assumptions. However, there changes in fibrin packing density were also discussed to be based on the specific fibrinogen preparation conditions in the cited study.

Furthermore, conclusions on the underlying fibrillary growth process have to be discussed, which are sketched in Figure  6 . As noted above, the lateral growth of fibrin fibrils was revealed as a first‐order rate process in dependence on fibrinogen concentration. In there a linear correlation between k _lat and NaCl was found, too. This points on a direct influence not only of the amount of fibrin and preformed protofibrils but also on an influence of NaCl on fibrin monomer and/or protofibril interactions during the formation of thick fibrin fibrils in step III of fibrillation. Under the used conditions, linear protofibril growth, including fibrinogen to fibrin conversion, and subsequent formation of fibrillation nuclei (protofibrils of sufficient length) were not analytically accessible by turbidity (no lag phase was observed). When protofibrils reach a critical length they immediately start to nucleate, a process which depends on protofibril interaction, concentration, and transport.

Figure 6.

Summary of the observed NaCl dependent fibrin network microstructure and the found correlation to the first‐order rate process of lateral aggregation.

As protofibril‐to‐fibril association is preferred over fibrin monomer‐to‐monomer or protofibril‐to‐protofibril association to yield more or longer fibrils, one can assume that the number and length of final fibrils and thus pore structure is defined very early during network formation during a fibril nucleation process. Such a nucleation process with an early definition of the final network microstructure was previously reported for similar fibril networks of collagen I.^{[ 33 ]} With the above assumption, the number of fibrillary nuclei defines the kinetic of lateral growth as protofibrils will primarily assemble to the growing fibril nuclei. Such a fibrin network formation mechanism fits the found first‐order lateral growth kinetics as protofibril concentration and nucleation conditions at constant NaCl concentration will depend on fibrinogen concentration.

Now it is important to note, that lateral growth is affected by NaCl concentration in terms of influencing the lateral growth constant and the resulting fibril diameter, without changing the first order of the growth process. Hence, it is reasonable to assume that NaCl concentration primarily influences the fibril nucleation process leading to different numbers of nuclei and, hence, a different resulting network topology. As a consequence, an increase in NaCl concentration (at constant fibrinogen concentration) triggers the formation of more nuclei leading to a faster growth of fibrils (increase in k _lat) by the availability of more nuclei and results in thinner and more network fibrils, comparable to a smaller fibril diameter and pore diameter. How NaCl might influence this early (nucleation) phase of fibrin network formation, whether by control of protofibril formation (e.g., control of thrombin activity) or protofibril interaction is further discussed below.

In summary, our results demonstrate a quantitative correlation between the fibrillation kinetic described by a first‐order rate process and the resulting fibrin network microstructure in terms of fibril and pore diameter. The determination of k _lat from turbidity measurements is a straightforward and easy method to predict fibrin network microstructure. Furthermore, NaCl concentration of the buffer during fibrin fibrillation is shown to serve as a tool to control pore and fibril diameter. By applying a corresponding correlation function, NaCl or essentially k _lat can be used to adjust specific pore and fibril diameters in fibrin networks.

2.4. Impact of Buffer Chloride Ions on Fibrin Fibrillation Kinetics

As shown above, NaCl has a strong impact on fibrillation kinetics and the resulting network structure by decreasing pore and fibril diameter at increasing NaCl concentration also in agreement with previous studies.^{[ 10} , ²² , ³¹ , ^{38 ]} Na⁺ ions are well known to have a direct impact on thrombin via the allosteric enhancement of thrombin activity and, thus, drive the fibrinopeptide cleavage and subsequent fibrin monomer formation.^{[ 39} , ⁴⁰ , ^{41 ]} Cl⁻ ions were specifically shown to control fibril growth, not only by affecting electrostatic interactions between fibrin protofibrils during lateral aggregation.^{[ 26} , ^{31 ]}

As the Na⁺‐dependent thrombin activity is important during Step I and II of the fibrillation (see also Figures 1 and 6), an impact of NaCl concentration on the overall fibrillation kinetics and also the subsequent Step III, lateral fibril growth, can be speculated. However, in pre‐experiments we observed no or no stable network formation at low NaCl concentration (40 mm, at 0.05 U mL⁻¹ thrombin). Furthermore, at a variation of thrombin between 0.05 and 0.15 U mL⁻¹ (at constant NaCl, 100 mm) no changes in turbidity–time curves were observed (data not shown). These results imply that thrombin activity, either controlled by Na⁺ concentration or amount of thrombin, did not control network formation kinetics in Step III and network topology in our experimental conditions.

In the context of the influence of Cl⁻ ions the effect of kosmotrope and chaotrope monovalent anions was discussed, pointing out the dominant importance of chaotropes such as Cl⁻ to interact with proteins. It was shown that Cl⁻ ions impair lateral aggregation of protofibrils leading to a smaller size of fibrin fibrils. This effect was argued by an impairment of deprotonation of amino acid end groups on fibrin, important for lateral fibril aggregation. A convoluting influence of Cl⁻ on the aggregation promoting effect of fibrinopeptide cleavage was also observed. By this explanation Cl⁻ ions as chaotrope ions bind water molecules weakly, tend to interact with basic groups of proteins, and impair the lateral aggregation of protofibrils by influences on ionization and electrostatic interactions, finally leading to smaller fibril diameters.^{[ 31 ]} However, the complex process of kinetic formation of lateral aggregation requires cumulative effects of many weak interactions along protofibrils of sufficient length.^{[ 10 ]} In the light of our new results we want to discuss additional options which are apparent from our analysis. As shown in Figure 4B, phase 1 of fibrin network formation—linear protofibril growth—is not strongly affected by NaCl concentration at our experimental conditions. Our topology analysis of resulting network microstructure implied the dominance of the nucleation in protofibril assembly to laterally growing fibrils, determining early in lateral growth the final number of these fibrils.

Within this interpretation we also want to discuss electrostatic interactions between protofibrils as well as steric and fluctuation interactions between protofibrils to be other important parameters determining fibrin network microstructure, considering possible influences of Cl⁻ therein. αC regions, which are separated from the central E region in the fibrin monomer, have been reported to influence lateral aggregation of fibrils and to have a positive net charge, while fibrin itself has a net negative charge.^{[ 42} , ^{43 ]} It is therefore feasible that electrostatic interactions between these charged regions are also affected by Cl⁻ ion presence. An interesting hint toward this option is given by our data in Figure 5D showing pore diameter in dependence on an inverse square root of ionic strength I _c (or NaCl concentration). This relation resembles a direct dependence on the Debye length (λ _D ≈ I _c ^−0.5) as an important parameter for surface charge screening in electrolyte solutions. Interestingly, these plots give an almost linear dependence and an extrapolation toward higher ionic strength—higher NaCl concentration—results in a crossing of all linear fits around a NaCl concentration of 200 mm. This NaCl concentration was previously found to almost inhibit lateral aggregation and fibrin network formation at otherwise similar buffer conditions.^{[ 31 ]} In light of this report the found crossing of the linear fits at 200 mm would be related to a vanishing dependence of pore diameter on fibrinogen concentration, which should agree to vanishing fibrin network formation similar to the reported study. Hence, our finding suggests that electrostatic screening of protofibril surface charges explains the dependence of fibrin network formation in dependence on NaCl.

Another aspect needed to be discussed for the lateral protofibril interaction during fibril aggregation are the inherent mechanical properties of fibrils. While different absolute numbers for the persistence length and mechanical flexibility of fibrin monomers and protofibrils are reported,^{[ 19} , ⁴⁴ , ^{45 ]} it is clear also from studies with other self‐assembling biopolymers such as collagen, that buffer conditions (e.g., ionic strength and salt ions) influence the mechanical properties and curvature fluctuations of protofibrils and impact the self‐assembly process.^{[ 46 ]} From this perspective one should also discuss the found dependence of pore diameter and relate it to be indirectly triggered via the influence of NaCl concentration on the mechanical properties of protofibrils and a resulting influence on lateral aggregation.

3. Conclusion

With increasing demand to mimic physiological and pathological in vivo situations in lab experiments, there is also a need to tightly control the ECM microstructure of various wound healing stages including very early stages of freshly formed fibrin clots. The precise adjustment of fibrin network microstructure and mechanics will allow to better investigate the role of different cell types in wound healing, e.g., immune cells like macrophages. In our work we demonstrated that an adjustment of buffer conditions (including fibrinogen, thrombin, NaCl, and CaCl₂ concentration) and the straightforward determination of fibrillation kinetics by turbidity is a tool to produce such defined fibrin networks. Our study did not only show NaCl concentration as an easy‐to‐use parameter to adjust fibril diameter of fibrin networks, but we also quantitatively revealed a direct correlation between fibrillation kinetics and resulting network topology and shed light into the mechanism of fibril formation, which was not known up to now. In this context, we find nucleation of fibrils from protofibrils, as the most important impact of NaCl and, thus, an early determination of the final number of fibrils in the reconstituted fibrin network. Thus, our study provides new quantitative tools for characterization and adjustment of defined 3D fibrin networks for matrix engineering approaches in biomedicine and cell culture analysis and enables a better understanding of the complex process of fibrin network formation.

4. Experimental Section

Chemicals and Proteins

Human fibrinogen (FIB3; plasminogen, von Willebrand Factor, and fibronectin depleted) obtained from Enzyme Research Laboratories (Swansea, UK) was dissolved in sterile, double‐deionized water, aliquoted and stored at −80 °C. Human thrombin Sigma‐Aldrich (Taufkirchen, Germany) was dissolved in sterile, double‐deionized water, aliquoted and stored at −20 °C. HEPES (4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid), CaCl₂ was purchased from Roth (Karlsruhe, Germany), NaCl was obtained from AppliChem (Corston Bath, UK). Buffer component HEPES was dissolved in double‐deionized water, adjusted to pH 7.4 by titration with 1 m NaOH or 1 m HCl, and stored at concentrations of 20 mm after sterile filtration. Phosphate buffered saline (PBS) was purchased from Biochrom (Berlin, Germany). 5‐(and‐6)‐Carboxytetramethylrhodamine succinimidyl ester (TAMRA‐SE) was bought from Invitrogen (Carlsbad, USA).

Preparation of Fibrin Solutions and Networks

Covalently attached layers of 3D fibrin networks were prepared on poly(styrene‐alt‐maleic anhydride) copolymer coated glass coverslips similar to a procedure reported for layers of 3D collagen I networks.^{[ 47 ]} The methodology of polymer coating was published by Pompe et al.^{[ 48 ]} Fibrin networks were prepared by mixing fibrinogen, at final concentrations of 0.5, 1, 1.5, 2, and 3 mg mL⁻¹, with thrombin, at final concentrations of 0.01, 0.05, 0.1, 0.15, 0.2, and 0.25 National Institute of Health (NIH) U mL⁻¹, in buffer solution (20 mm HEPES) on ice. To prevent variations of buffer content by mixing different volumes of fibrinogen and thrombin to achieve the required range of concentration, a certain amount of a 10x buffer (HEPES) was added to the given volumes of fibrinogen and thrombin solutions. CaCl₂ and NaCl content in the buffer were adjusted to final concentrations of 1, 1.5, 2, 2.5, 5, or 10 mm and 0, 40, 50, 60, 80, 100, 120, or 150 mm, respectively. Details on the buffer composition applied are summarized in Table  1 . 50 µL of fibrillation solution was transferred on polymer coated glass coverslips to reconstitute fibrin networks for topological analysis. 90 µL of fibrillation solution was transferred in a 96‐well‐plate cavity for turbidity analysis of fibrillation kinetics. Fibrillation was always accomplished at 37 °C and 95% humidity for 70 min. Final 3D networks were rinsed and stored in PBS after fibrillation to prevent dehydration.

Table 1.

Details on buffer composition applied in the experiments. Buffer is based on 20 mm HEPES

NaCl [mM]	CaCl2 [mM]	pH	Ionic strength [mol L−1]
40	2.5	7.4	0.048
60			0.068
80			0.088
100			0.108
120			0.128
100	1		0.103
	1.5		0.105
	2		0.106
	2.5		0.108
	5		0.115
	10		0.130

Characterization of Fibrin Network Topology (Microstructure)

Network topology was analyzed by the previously establish methods for network topology analysis to determine fibril and pore diameter as well as pore diameter distribution.^{[ 47 ]} Briefly, four fibrin networks per condition were prepared and subsequently fluorescently labeled using 50 µm TAMRA‐SE (prediluted to 0.17 m in dimethyl formamide) in PBS buffer for 1 h. Networks were subsequently rinsed in PBS and image stacks at three positions per network were taken using a cLSM with a 40 × /1.2 water immersion objective (LSM700, Carl Zeiss, Germany) at an image resolution of 1024 × 1024 pixels with 0.16 × 0.16 µm or 0.08 × 0.08 µm per pixel. A constant z‐stack size of 80 µm and image distance of 10 µm were applied. Topological analyses of image stacks were performed automatically via a home‐built Matlab script (Matlab R2016b; MathWorks, USA) and provided mean fibril and pore diameters as well as pore distribution and fibril orientation isotropy of the investigated networks. Briefly, pore diameter is analyzed by transferring original confocal gray‐scale images into binary thresholded images and segmentation into pore (black) and fibril (white) image area for each image of an image stack. Within an erosion algorithm using circular objects, a threshold of 50% eroded pore area is defined as pore diameter. Mean pore diameters are calculated as average per image stack. Using 2D autocorrelation of original images in combination with a Gaussian fit of a cross‐sectional autocorrelation curve the fibril diameter is analyzed per image. Mean fibril diameters are calculated as average per image stack. The Matlab script is freely available at https://git.sc.uni‐leipzig.de/pe695hoje/topology‐analysis.

Characterization of Fibrin Network Thickness

Network thickness was analyzed by acquiring three image z‐stacks per sample with a 5 µm image distance using bright‐field microscopy (AxioObserver.Z1, Zeiss) and a 10 × dry objective (Zeiss) to yield a z‐stack size of about 600 µm. Image resolution was 692 × 520 pixels at binning 2. Network thickness determination was automatically performed by tracking the appearance of scatter from the fibrillary network via edge‐filtering and gray‐scale leveling using a home‐built Matlab routine (Matlab R2016b; MathWorks).

Assessing Kinetic Parameters of Fibrin Fibrillation

For kinetic analysis, the fibrin fibrillation process was observed by turbidity at 405 nm for 2 h at 1 min intervals in a prewarmed (37 °C) plate reader (Tecan Infinite F200Pro, Tecan, Männedorf, Switzerland). Samples were measured as triplicates in five independent experiments (n = 5). A typical turbidity‐time curve τ(t) of fibrin network formation is described by a lag phase of linear fibril growth (constant turbidity at a low level during formation of protofibrils from fibrin monomers too small to increase turbidity), a lateral fibril growth phase (rapid rise in turbidity due to lateral assembly of protofibrils into thicker fibrils and network formation), and a final constant plateau of turbidity (finished fibril and network formation).^{[ 22} , ^{23 ]} Similar to quantitative assessments of fibrillary growth kinetics of collagen I networks,^{[ 33} , ³⁶ , ^{37 ]} the growth constant of the lateral growth phase of fibrin fibrils was determined herein. An apparent lateral growth constant k _lat can be determined with the assumption of a first‐order rate process. This approach also assumes a linear dependence of protofibril formation from fibrin monomer concentrations during the lag phase. Thus, the first‐order rate of disappearance of protofibrils during the lateral fibril growth phase c _proto(t) can be related to the change in turbidity τ(t) and the same time dependence and rate constants can be expected. The validity of these assumptions can be checked by the linearity of lateral growth rate (initial phase of τ(t) after lag phase with the maximum slope of turbidity–time curves) in dependence on fibrinogen concentration c _fng. The maximum slope dτ/dt _max was revealed from the first derivative of the smoothed (Savitzky‐Golay method with five points per array) averaged data (triplicate measurements). k _lat was determined from a linear fit of the plots of dτ/dt _max on c _fng. The plateau value of final maximum turbidity relative to background turbidity Δτ _p was also determined to check the assumed linearity of fibril formation on c _fng. All calculations were performed in OriginPro 2019 (OriginLab Corp.).

Statistical Analysis

Experiments were performed at least with four independent replicates (n = 5) and data are presented as mean and standard deviation or box plots, if not otherwise stated. Statistical significance and data visualization was performed using OriginPro 2019 (OriginLab Corp.) by a nonparametric Mann–Whitney test (*p < 0.05).

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

The author contribution is as follows: conceptualization, investigation, formal analysis, writing—original draft, editing (K.P.); investigation, formal analysis (L.S.‐J.); investigation, formal analysis (A.S.‐T.); conceptualization, formal analysis, writing—original draft, editing (T.P.).

Pietsch K., Storm‐Johannsen L., Schmidt‐Thomée A., Pompe T., Correlation between Fibrin Fibrillation Kinetics and the Resulting Fibrin Network Microstructure. Adv. Healthcare Mater. 2023, 12, 2202231. 10.1002/adhm.202202231

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.