Unusual Surface Coagulation Activation Patterns of Crystalline and Amorphous Silicate‐Based Biominerals

Activation of coagulation cascades, especially FX and prothrombin, prevents blood loss and reduces mortality from hemorrhagic shock. Inorganic salts are efficient but cannot stop bleeding completely in hemorrhagic events, and rebleeding carries a significant mortality risk. The coagulation mechanism of biominerals has been oversimplified in the past two decades, limiting the creation of novel hemostats. Herein, at the interface, the affinity of proteins, the protease activity, fibrinolysis, hydration shell, and dynamic microenvironment are monitored at the protein level. Proteomic analysis reveals that fibrinogen and antithrombin III's affinity for kaolin's interface causes a weak thrombus and rebleeding during hemostasis. Inspiringly, amorphous bioactive glass (BG) with a transient‐dynamic ion microenvironment breaches the hydration layer barrier and selectively and slightly captures procoagulant components of kiniogen‐1, plasma kallikrein, FXII, and FXI proteins on its interface, concurrently generating a continuous biocatalytic interface to rapidly activate both intrinsic and extrinsic coagulation pathways. Thus, prothrombin complexes are successfully hydrolyzed to thrombin without platelet membrane involvement, speeding production of high‐strength clots. This study investigates how the interface of inorganic salts assists in coagulation cascades from a more comprehensive micro‐perspective that may help elucidate the clinical application issues of kaolin‐gauze and pave the way to new materials for managing hemorrhage.


Introduction
Trauma is a global socioeconomic problem, claiming many lives up to nearly 6 million people annually. Uncontrollable bleeding is responsible for 80% of mortality in both military and civilian traumas. In addition, 56%-87% of mortality occurs before patients arrive at the hospital due to massive hemorrhages without having the chance to receive proper treatment. [1][2][3] It reported that the usage of hemostatic agents in the event of massive hemorrhages ensures the safety of many patients; however, the effectiveness of current hemostatic agents needs to be improved, necessitating extensive research in this field in the near future. [4] The enhancement of hemostatic performance needs to be guided by comprehensive mechanisms. The coagulation mechanism is a sequence of interrelated protein dissolution processes that transform zymogen into a trypsin-like enzyme and produce thrombin. [5,6] Thrombin can induce fibrinogen and platelets to form a hemostatic thrombus. The blood coagulation enzymatic process may occur at any pace but depends on the creation of multimolecular complexes on the cell surface by the zymogen, cofactors, and transition enzymes. [6] The coagulation cascade response consists mainly of two planned processes: FX activation, with a reaction rate of 10 3 levels, and prothrombin activation, with a reaction rate of 10 6 levels. This potent amplification effect makes modulation of coagulation crucial. [7] Clarifying the structure-activity link between materials and thrombin activation is crucial for developing innovative therapeutic strategies to promote fast hemostasis and increase the survival rate of patients with major bleeding. [8,9] Presently, mineral salt-impregnated gauze, [1,10,11] chitosanbased hemostatic powder, gauze, and sponge, [12,13] polyurethane foam, [2,14] expandable compression sponge, [2] sealant, [15] etc. are the most commonly used hemostatic materials for the control of massive hemorrhage in a prehospital emergency. Through chemical activation of coagulation factors, blood cell action, physical expansion and compression, adsorption and concentration, the coagulation mechanism of these hemostatic materials primarily accelerates the normal coagulation speed of the body and promotes the control of massive bleeding at the wound site. [9,16] QuikClot battle gauze (QCG), a nonwoven gauze loaded with inorganic kaolin salts, is the sole biomaterial associated with zero percent mortality in pig models with fatal inguinal injuries. However, QCG cannot entirely prevent major bleeding with a local application; after 30 min, there is still a 25% chance of limb movement hemorrhage. [17] Although kaolin has shown greater efficacy in stopping massive bleeding, its procoagulant process remains unexplained. Recently, Fan et al. revealed that zeolite with a microporous crystal structure activated thrombin at the molecular level. The prothrombin conjugates formed on the inorganic surface of calcium ion exchange zeolite (CA zeolite) demonstrated thrombin protein conversion to thrombin. [18] The mechanism of kaolin is generally known as the surface electronegativity of kaolin activating the protein coagulation factor (FXII), subsequently activating the endogenous coagulation system. [19] However, this mechanism oversimplifies the hemostatic process and fails to adequately explain the positive and negative effects of kaolin on proteases.
On the entire coagulation cascade, crystal structure, particle size, surface properties (such as topological structure, electronegativity, roughness, and hydration ability, etc.), ionic microenvironment, protein adsorption kinetics, and other parameters will either have a synergistic or competitive effect. [20][21][22] Also, the selective adsorption of proteins on the surface of substances regulates the activation of the blood coagulation system. [18,23] However, the difference in protein adsorption between inorganic salt crystals and amorphous structure interfaces, as well as the effectiveness of its activation of coagulation-related enzyme sources, is an interesting phenomenon that merits comparative study.
Bioactive glass (BG) is an amorphous inorganic synthetic substance with a shelf life of three years and little toxicity to the human body. Since its discovery by Hench et al. in 1971, the US Food and Drug Administration (FDA) has approved the use of silicate and borate bioactive glasses (BGs) (45S5, 13-93, 13-93B) for orthopedic surgery and bone defects (PERIOGLAS, NOVABONE), dental care (Prevest Micron Bioactive, SENSO-DYNE), and wound healing (DermFactor, and MIRRAGEN Advanced Wound Matrix. [24][25][26][27][28][29] It may regulate immunology (such as macrophage polarization), angiogenesis, stem cell destiny, and bacterial behavior through a dynamic functional ion microenvironment, surface mineralization, and topology, hence promoting tissue repair and regeneration. [24][25][26][27][28][29] BGs, like zeolites and kaolinbased hemostatic agents, can activate the coagulation cascade, have a favorable degradation profile, and repair and regenerate tissues. Therefore, the BGs show great potential as hemostatic materials. However, fifty years after the invention of bioactive glass, only a handful of clinical papers have shown its use in the emergency treatment of major bleeding. Although mesoporous silicate and its composites have opened up a new route for BG in the area of hemostasis, its biosafety, stability, and large-scale industrial manufacturing remain the most challenging barriers to utilization. [30][31][32][33] With the change in composition and structure, the expression and mechanism of this kind of material's coagulation function are still unknown.
Based on prior research on inorganic salts and spongy hemostatic biomaterials, [31,34,35] biological mechanisms of the interface dynamic ion microenvironment in chronic wound healing, and bone defect repair, [36][37][38] the progressive work has been carried out. Following analysis of the coagulation pattern on the interface of crystalline kaolin, amorphous bioactive glasses (BGs) with transient-dynamic ion microenvironment interface were created to break down hydration shell hinder, thereby obtaining a novel inorganic salt hemostat with a unique active interface and the coagulation mode. In this work, we produced a large number of silicate (bioglass 13-93, abbreviated as 0B), borosilicate (BSG, abbreviated as 1B, 1.5B, and 2B), and borate bioactive glasses (13-93B, abbreviated as 3B). The consistency of kaolin and BGs in inducing blood coagulation was elucidated by in vitro and in vivo evaluations of hemostatic performance. At the protein level, we compared the coagulation cascade reaction triggered by kaolin and bioactive glass, analyzed the activation efficiency of the material at the protease source in each step, and monitored the minute-by-minute rate at which kaolin and BGs stimulated thrombin production. Therefore, we have acquired the crystal form and the amorphous structure surface-specific coagulation mode. The high adherence of kaolin to fibrinogen may be one of the most important contributors to rebleeding after hemostasis. Compared to kaolin, amorphous bioactive glass is substantially more effective in activating protease, promoting enzymatic hydrolysis, and accelerating thrombin production. Moreover, we discovered that when boron was added to BGs, the surface electronegativity decreased, diminishing its coagulationpromoting effects. 0B and its composites are the next generation of hemostatic agents for preventing severe bleeding and rebleeding and are also excellent therapeutic materials for bone repair and wound healing.

Hemostasis In Vitro
The coagulation performance of BGs with varied ion compositions was primarily evaluated using the recalcification method. [31,35] In Figure 1a and Supplementary Figure S1, the clotting time of BGs rose as the boron concentration of the BSGs increased but was still significantly lower for the 1B, 1.5B, and 2B groups compared to the blank control group. However, there was no significant difference in the clotting time between the 3B and the control. On the other hand, the pro-coagulant activity of 0B without boron was comparable to that of kaolin and MMT. The clotting time of the 0B (100 mg) group was 78 ± 6 s, about 442 s faster than that of the blank (n = 3, ****p < 0.001). When the size of the 0B group increased from 10 mm to 400 mm ( Figure S2, Supporting Information), the clotting time was initially prolonged but then slightly decreased (Figure 1b). Furthermore, the ambient temperature diminished the material's activation of the coagulation so that the coagulation performance might drop. [39] In Figure 1c, the effect of temperature on clotting time has been deeply analyzed. Compared to higher temperatures (37°C), a lower temperature (25°C) prolonged the clotting period by roughly 5.4 min. In either low-or high-temperature settings, 0B, kaolin, and MMT exhibited a rapid pro-coagulation effect, while the low-temperature setting slightly extended the coagulation time. Also, the impact of material dose on coagulation was investigated in Figure 1d. Even at low dosages, 0B, kaolin, and MMT retained robust coagulation effects. In Figure 1e,f and Figure S3, Supporting Information, the influence of materials on plasma coagulation was further investigated. 0B was discovered to have the same capability as kaolin and MMT in triggering and A time sweep of rheology on blood coagulation was performed at 37°C: g) Representative rheological diagram of the phase transition process from sol (G") to thrombus gel (G') after blood and material interaction. h) The phase transition point of blood sol to thrombosis gel. i) The G' of thrombus at 600 s. (j) Photos of thrombus state after blood and material interaction tested by rheometer.
stabilizing plasma coagulation. Altogether, these results demonstrated that 0B promoted blood coagulation efficiently and that boron doping might impair BG's pro-coagulant characteristics.
The rheological assessment was also done to confirm the initial coagulation time of BG and the strength of the thrombus formation, since the recalcification procedure was not very precise.
When the blood began to solidify, the storage modulus (G') exceeded the loss modulus (G") until equilibrium was reached, indicating that the thrombus gel formation from fibrinogen to fibrin was complete. Based on Figure 1g,h and Figure S4, Supporting Information, 0B could form thrombus gel faster than kaolin. ( ### p < 0.005). MMT exhibited no significant difference from 0B www.advancedsciencenews.com www.advhealthmat.de in the gel transformation process of blood solution; however, its thrombus intensity was significantly lower ( #### p < 0.001) (Figure 1g-j). Additionally, the tendency of BGs to boost blood coagulation was consistent with the recalcification procedure described above. Conclusively, 0B may induce coagulation more quickly in vitro than kaolin and MMT and produce a more homogenous and stable network structure with blood components and fibrin.

Hemostasis In Vivo
Uncontrollable massive hemorrhage was one of the leading causes of high mortality. Although a pig model with a 6 mm incision in the femoral artery was proposed to test the clinical efficiency of materials, [40] the cost was too high for the initial research. Thus, it is essential to develop a small-or mediumsized animal model of severe bleeding. An SD rat tail amputation model, an SD rat femoral artery, vein, and nerve inguinal disconnection model, a SD rat liver resection model, a New Zealand white rabbit X-incision severe liver injury model, and a New Zealand rabbit femoral artery puncture with a 1.6 mm-diameter needle model were made to test how well materials stop bleeding.

The Hemostatic Effect of Materials on An SD rat Model of Tail Amputation
The rat's tail was cut in order to assess the hemostatic efficacy in vivo (Figure 2a-c). After ≈8 min, the blank group ceased to bleed. The hemostatic time and blood loss in the kaolin and 0B groups were significantly different from those in the blank group. It demonstrated that both kaolin and 0B have superior hemostatic characteristics in the context of vein amputation of tail. As depicted in Figure 2a, it was noteworthy that 0B could be rapidly invaded by blood throughout the experiment and create a thrombus community with blood more quickly.

The Hemostatic Effect of Materials On an SD rat Model of Fatal Femoral Artery, Vein, and Nerve Amputation
When the femoral artery, vein, and nerve at the groin root of the SD rat' right leg were surgically severed, the groin root suffered a considerable hemorrhage. In this situation, the 0B group unexpectedly improved the survival rate of SD rats over time. As shown in Figure 2d, the survival rate of the 0B group was ≈73%, whereas that of the kaolin and gauze groups was only ≈33% and 25%, respectively. Furthermore, when a substantial volume of blood was generated during the procedure, the kaolin group could not infiltrate into the blood as well as the 0B group, demonstrating that kaolin has a lower wettability. In Figure 2e, a more intuitive representation of the hemostasis process corroborates this conclusion.

The Hemostatic Impact of Materials on the Liver Resection Model in SD Rats
Injury of parenchymal organs like the liver or spleen was also a common source of significant bleeding and substantial blood artery damage. In Figure S5, Supporting Information (n = 6), the clotting time at the location of liver trauma was about 1 min for the 0B and kaolin groups, nearly 70% faster than the blank group. Besides, when 0B or kaolin was used, the blood loss at the location of liver trauma was only 1.68 ± 0.94 g and 1.65 ± 0.94 g, respectively, a 46% and 47% decrease over the blank group (*p < 0.05) (Figure 2f). The visual images taken during hemostasis revealed that the 0B and kaolin groups showed outstanding hemostatic efficacy without any organ damage (Figure 2g).
In a model of liver injury with bleeding, both kaolin and 0B were better at getting the blood to clot and stopping the bleeding quickly. Without a doubt, the liver injury model couldn't tell the difference between kaolin and 0B's ability to stop bleeding. While 0B demonstrated a considerable hemostatic advantage over kaolin in the disconnection model of the femoral artery, nerve, and vein of the SD rat. It illustrated that the feasibility of the hemorrhage model should be determined by the capacity to distinguish between materials' performance differences.

Hemostasis in An Extensive Liver Incision of A New Zealand White Rabbit
In Figure 3a, an incompressible abdominal hemorrhage was modeled by making an X-shaped incision (2 cm ×2 cm ×1 cm) in the liver tissue of New Zealand rabbits using a scalpel. [35] When utilizing 0B, complete hemostasis at the injury site was achieved within 10 min (n = 6), but not in the blank group (n = 6) in Figure 3a,b. Moreover, the blood loss in the 0B, kaolin, and MMT groups was only 3.64 ± 1.86 g (***p < 0.005), 5.15 ± 0.99 g (**p < 0.01), and 3.76 ± 2.08 g (***p < 0.005), respectively, compared to 10.78 ± 0.94 g in the blank group ( Figure 3c). In addition, Figure 3d suggested that the material groups have the capability to gather RBCs at the location of liver injury hemostasis, although this must be confirmed by more investigations in vitro.

Hemostasis in A Severed Femoral Artery Injury of a New Zealand White Rabbit
The femoral artery was peeled off, and then a 1.6 mm diameter syringe needle was used to make a bilateral penetration, causing to bleed (Figure 4a). Next, 0B, kaolin, MMT, or gauze were applied in Figure 4b. They all took less than 10 min to stop bleeding (n = 4), except gauze (n = 4). As expected in Figure 4c, 0B, like kaolin or MMT, provided statistically significant hemostatic superiority, reducing blood loss from 27.27 ± 3.54 g (gauze group) to 5.93 ± 2.01 g (0B group). Additionally, after hemostasis, the materials' influence on the distal organs of New Zealand rabbits was investigated ( Figure S6, Supporting Information). The H&E staining of the 0B, kaolin, and gauze groups indicated almost no significant abnormalities in all organs. However, small emboli developed in the distal lung in the MMT group, suggesting that MMT might induce distal embolism. It is also one of the main reasons the FDA banned WoundStat's (MMT) use in the hemostatic domain in 2013. [34,41]  In vivo hemostasis of tail amputation of SD rats. a) Real-time hemostatic effect, b) blood loss, and c) clotting time. d) The survival rate of SD rats in the hemorrhage of lethal femoral artery, vein, and nerve amputation after being treated with either 0B, kaolin, or gauze. e) Hemostatic photographs of the groin site taken for the first 10 min of trauma for the 0B and kaolin groups. f) Blood loss at the location of the liver trauma (the mean ± SD (n = 6), *p < 0.05 versus gauze group). g) Hemostatic images depicting the trauma location on the liver, as well as a schematic of the liver following surgical resection.

Hemostatic Mechanism of BG and Kaolin
Based on in vitro and in vivo research, initial blood coagulation and in vivo hemostatic performance varied dramatically between kaolin and 0B. (Figures 1-4). So, what happened when blood interacted with materials? Various methodologies were employed to analyze the aforementioned occurrences, namely 1) interaction factors between the material and blood interface, 2) interaction of blood cells with materials, 3) zymogen activity, 4) protein adsorption, 5) thrombinogenesis, 6) plasmin activity, and 7) dynamic parameters of whole blood coagulation.

Instantaneous Contact Between the Surface of the Material and Blood
During the hemostasis phase of a massive hemorrhage, every minute counts; consequently, every detail is crucial. The coagulation process was disrupted when a chemical came into contact Figure 3. a) Injury to the liver in the exposed left medial lobe. b) Changes in the hemostatic state of the injured site with time. c) Blood loss at the area of liver injury (the mean ± SD (n = 6), *p < 0.05 versus Gauze group). d) Masson staining of injured liver tissue when it has already stopped bleeding by gauze, kaolin, MMT, and 0B.
with blood. The overlooked wettability properties might be one of the variables affecting the activation rate of the material in the coagulation system. Figure 5a depicted a macroscopic image of the bioactive glass 0B. While Figures S7-S9, Supporting Information, exhibited the preparation equipment, scanning electron micrograph (SEM), EDS-Mapping element analysis, and AFM test parameters of BGs. As depicted in Figure 5b,c, water might be instantly dispersed over 0B and MMT. However, it took 7 seconds to wet the surface of kaolin with water, which corresponds to the observed phenomena found in SD rats following deadly bleeding hemostasis. In addition to faster activation of the coagulation system under static conditions in vitro, 0B also activated the coagulation system faster in vivo because 0B infiltrated the turbulent blood flow more rapidly than kaolin, which exhibited time-dependent contact angles, namely contact angle hysteresis. [42] To assess the wettability of each component, the surface roughness of 0B, kaolin, and MMT was measured using AFM ( Figure 5d). As demonstrated in Figure 5e,f, the roughness of amorphous 0B was significantly greater than that of crystalline MMT and kaolin, indicating that the surface of 0B displayed more appropriate and quick wetting properties. Moreover, there were almost no hydration films on the 0B and 3B surfaces, but hydration films with thicknesses of 76.2 nm and 38.9 nm, respectively, formed on the kaolin and MMT surfaces, containing both strong-and weak-binding hydration layers (Figure 5g-n). [43] The regulated degradation of 0B or 3B could create a dynamic ion microenvironment, so preventing the formation of a hydration film on the surface of BG (Figure 5n). [43][44][45] Blood coagulation might be affected continuously and synergistically by the interfacial characteristics of 0B and the ionic environment. In light of the findings, one of the key reasons why the survival rate of SD rats in the kaolin group was significantly lower than that of group 0B in Figure 2d could be a difference in contact wettability between materials and blood and the surface hydration film. Evidently, the 0B's initial response was to immediately penetrate the wound, spread and cover the maximum surface area, and initiate and maintain coagulation.

Interaction of Blood Cells with Materials
Next, a substantial volume of blood infiltrated into the hemostats. Stronger water absorption encouraged the formation of the blood-material mixture, forming a thrombus. As expected, BG's water storage capacity was much lower than that of gauze, kaolin, and MMT (Figure S10a, Supporting Information), and its specific surface area was significantly less than that of clays (Figure 5h). The probable reason was that the water in BG could not be forcedly stored between particles due to the amorphous nature of BG ( Figure S10b, Supporting Information), which has weaker particle connections than crystalline layered clays. [46] Another factor for BG's limited water absorption was its inability to generate a hydration layer in Figure 5g-n. It could be conjectured that the activation of the coagulation process using BGs might be achieved by a continual collision between its surface and fresh blood, as opposed to kaolin and MMT's blood adsorption and later diffusion activation. There might be substantial differences in the mechanism of activation of the coagulation system between BG and clay surfaces. Also, it seemed that loading BG onto highly absorbent materials would be one of the most effective approaches to enhancing BG's hemostatic function in the future.
The interactions between materials, RBCs, and PLTs were investigated since they are closely associated with hemostasis. [31,35] The RBCs near the materials were ruptured with ionic water to release hemoglobin. And the absorbance of hemoglobin was positively correlated with the aggregation of RBCs. As shown in Figure S11a, Supporting Information, the relative absorbance of hemoglobin among the 0B, kaolin, and MMT groups was practically no different from the positive blank group (100% RBCs aggregation), stating that all of them had a proper capacity to aggregate RBCs. However, when the size of 0B increased from 10-40 μm (0B-1) to 40-50 μm (0B-2), the RBCs aggregation force on the surface of 0B-2 was diminished (Supplementary Figure  S12a). The size of materials interacting with blood components must be taken into account. Furthermore, SEM photographs confirmed that the interaction of 0B and kaolin with RBCs was somewhat due to physically adsorbed aggregation (Figure S12bg, Supporting Information). On the other hand, the RBCs aggregated by MMT suffered from apparent deformation, suggesting that MMT's aggregation impact on RBCs might be a combination of chemical and physical functions. [34] Masson staining of liver injury sites in New Zealand rabbits also proclaimed the aggregation of 0B on RBCs (Figure 3d). The sP-selectin Elisa kit was adopted to investigate the relationships between materials and PLTs, [35,47] and the release of sP-selectin was found to accompany the activation of PLTs. There was no significant change in the content of sP-selectin between the 0B and MMT groups compared to the blank group, as indicated in Figure S11b, Supporting Information. Yet, the kaolin group only partially activated PLTs, which is in line with previous studies. The formation of PF3a and the release of ADP that accompany platelet adherence to kaolin were possible symptoms of a change in the condition of the platelet membrane. [48,49] In general, despite 0B's limited capacity to activate PLTs, one thing was clear: 0B caused blood cell aggregation.

Process of Zymogen Activation upon the Materials
When the hemostat came into contact with blood, the coagulation system, classified into exogenous, endogenous, and common coagulation pathways, was triggered. [14,31,35] The unique property of BGs that is different from kaolin and MMT is the ionic microenvironment formed by its degradation, releasing coagulation-promoting ions such as SiO4 4− , Ca 2+ , and Mg 2+ . [38,40,50] What action did the ionic microenvironment contribute to BG's electronegativity in activating the coagulation cascade? To answer this question, the influence of materials, including the type of powder, suspension, and extract, on the plasma coagulation factors (PT, APTT, TT, and FIB), was determined (Figure 6a). As demonstrated in Figure S13, Supporting Information, powders and suspensions of 0B, kaolin, and MMT short-ened the APTT, but extracts showed no impact. According to previous research, [9,51] material electronegativity was relevant to the activation of the endogenous coagulation system. In Figure S14a, Supporting Information, further Zeta potential tests revealed that 0B and kaolin had the lowest Zeta potentials of roughly -30 mV. In contrast, the zeta potential of BGs grew steadily during the transition from silicate to borate until it reached approximately zero. In line with this, the stimulating impact of BGs on the coagulation process (Figure 1a,h), as well as the APTT, progressively tended to have no discernible effect. It revealed that the zeta potential rather than the ionic microenvironment markedly affected The TT appeared to lengthen as the amount of boron in BSGs (powder or suspension groups) increased (Figure 6d). Additionally, the FIB, which indicated the amount of fibrinogen, increased with the addition of BGs (powder, extract, or suspension), but decreased with the addition of kaolin and MMT (powder, extract, or suspension). The principle of TT and FIB tests was measuring the time fibrinogen was converted into fibrin, so the question is: What effect does the material have on thrombin and fibrinogen? Structurally, it was speculated that the unique borate ions released from the degradation of 1B, 1.5B, 2B, and 3B, different from 0B, might inhibit the TT reaction ( Figure S14c,d, Supporting Information). Surprisingly, we discovered that when diluted platelet-poor plasma (platelet-poor plasma was abbreviated as PPP.) was mixed with BGs during the FIB technique, it might spontaneously form a clot without being initiated by thrombin ( (Figure 6e and Figure S15, Supporting Information). As seen in Figure S14b, Supporting Information, the concentration of Ca 2+ rose rapidly as the BGs came into contact with the solution. Because of this, the Ca 2+ released by BGs could catalyze and promote fibrin formation. The greater BET and thicker hydration film of kaolin and MMT in Figure 5e,g-n might indicate that they prefer to adsorb large quantities of fibrinogen on their surface, [52,53] leading to a considerable decrease in fibrinogen in the FIB testing sample. These tests demonstrated that BGs, particularly 0B, might stimulate the endogenous coagulation system due to their surface electronegativity and facilitate fibrinogen-tofibrin conversion related to their ionic microenvironment. Besides, the strong adherence of kaolin and MMT to fibrinogen was probably one of the principal factors for the thrombus strength being lower than 0B.
To better understand the interaction between BGs and the coagulation system, the influence of BGs on several coagulation factors was investigated using a semi-automatic coagulation instrument. [31,35] The coagulation factors that were studied include: 1) exogenous system-related factors, such as FXII (HAGE-MAN), FXI (anti hemophilia globulin C), FIX (hemophilic globulin B), and FVIII (hemophilic globulin A), and 2) endogenous and common system-related factors, such as FVII (pro-coagulant), FII (prothrombin), FV (pro-coagulant), and FX (Stuart Prower factor). 0B effectively improved the activity of FXII and FXI in contrast to the blank group, as demonstrated in Figure 6f-i, without changing the activity of FIX and FVIII. Surprisingly, kaolin and MMT positively affected FXI [9,52] but restricted FIX and FVIII. Furthermore, kaolin activated FXII, [9,52] but MMT showed no effect. In the absence of FVII, 0B and MMT launched the exogenous coagulation system, while 1B and 2B did the reverse (Figure 6j-m). 0B significantly increased the activity of FX but did not significantly impact other common coagulation systemrelated factors. Even though 1B and 2B exhibited inhibitory effects on FVII and FV at low concentrations, there was no significant difference in their activities on PT (Figure 6c) at normal concentrations. In PT testing, it was probable that TF (FIII, tissue factor) had a greater competitive binding on FVII than 1B and 2B. On the one hand, it might be conjectured that materials like 0B and kaolin with enough lower Zeta potentials initiated the endogenous coagulation system (Figure 5j, 7b,f). On the other hand, boron-doped bioactive glass, kaolin, and MMT had a certain inhibitory function on some coagulation factors (Figure 6h,i,j,l,m). The pro-coagulant function of BGs may be regulated by the specific ratio of silicon and boron. Overall, 0B presented a better-boosting impact on the coagulation system than kaolin and MMT, consistent with the hemostatic effect in vivo and in vitro.

Specific Protein Adsorption at the Interface of the Materials by Proteomic Analysis
In addition to protease activation, adsorption may occur upon blood interaction with inorganic salts. As shown in Figure 7a, in stark contrast to crystalline kaolin and MMT, BG has a lower affinity for plasma proteins. This is directly connected to the specific surface area, electronegativity, and the capacity to produce a hydrated layer. When the protease colloid with a double electric layer and stable hydration layer in plasma comes into contact with the electronegative surface of kaolin, electrostatic interaction occurs, resulting in the formation of a stable, robust hydration layer. Further proteomic analysis of 0B and kaolin surfaces and their residual plasma (Figure 7b) revealed that, despite the difference in protein adsorption, kaolin, and 0B surfaces exhibited significant fibrinogen-selective adsorption. Specifically, the fibrinogen level in plasma following kaolin adsorption is almost null, demonstrating that kaolin has a very great affinity for fibrinogen. Also, the assessment of fibrinogen concentration (FIB, Figure 6e) demonstrates inferentially that kaolin and MMT have substantial adsorption on fibrinogen and that the plasma solution does not form fibrin clots even in the presence of thrombin. The fibrinogen that kaolin and MMT have adsorbed may be challenging to hydrolyze or to be released from its surface following hydrolysis. In addition, we discovered that kaolin exhibited a clear affinity for apolipoprotein.
In-depth investigation of plasma coagulation-related protein composition revealed that 0B and kaolin exhibited particular preferential adsorption on kinigen-1 (Figure 7f,g) (high molecular weight kininogen HMWK is a single chain protein that participates in the activation of the endogenous coagulation pathway and promotes the activation of FXIIa to FXI). Additionally, 0B has considerable and specific adsorption on plasma kallikrein (which participates in the activation of the endogenous coagulation pathway and supports the activation of FXIIa to FXI), FXII, and FXI in comparison to the kaolin group. In contrast, kaolin exhibited particular adsorption on antithrombin III (protease inhibitors, that inhibit serine-containing proteases, like thrombin and FXIIa, FXIa, FIXa, FXa, etc.). During the activation of the coagulation system, the surfaces of kaolin and BGs undergo distinct dynamic adsorption processes. Here, the adsorption capacity of 0B has not yet reached the activated coagulation threshold value obtained due to the complexity of blood detection and the sensitivity of blood activation. As selective protein adhesion did not necessarily have a stabilizing effect on thrombus generation. In addition to protein surface adhesion regulation, the activation of the coagulation system was also influenced by space collision, contact, solute, and other interactions. As has been observed in Figure 5-7, after coming into contact with the electronegative interface of 0B's surface and the ensuing ionic microenvironment in space, a microscopic amount of coagulation-related protease www.advancedsciencenews.com www.advhealthmat.de www.advancedsciencenews.com www.advhealthmat.de would immediately stimulate the occurrence of coagulation cascade reactions.

Unusual Generation of Thrombin upon Materials
The coagulation response comprises a succession of protease hydrolysis reactions, and the activation rates of FX and prothrombin have the greatest influence on the amplification impact of the coagulation cascade reactions. [5][6][7] As measured by chromogenic substrate reaction, the production rates of FXa and thrombin caused by Kaolin and BGs differed significantly in Figure 7e-h. After interacting with calcium plasma, 0B activates FX and prothrombin more rapidly than the control group and kaolin. With the consumption of coagulation factor response, 0B started to approach the plateau of the thrombin production curve in two minutes, while thrombin levels in normal physiological plasma surged explosively in around ten minutes. Intriguingly, while kaolin's thrombin production was higher than that of the normal physiological group at 2 min, the following thrombin platform activity was much lower. In the absence of activated platelets, the kaolin surface may have non-competitively adsorbed a considerable quantity of protease (Figure 6e, 7a), which may have had a blocking effect on the thrombin cascade reaction chain and led to a decrease in thrombin generation. The activation mode of thrombin production induced by 0B has two notable characteristics: 1) It significantly accelerates the activation of Xa, and the thrombin plateau stage is approximately four times faster than normal physiological conditions. 2) The plateau period of 0B is similar to that of normal physiology, indicating that 0B has no significant effect on the maintenance of thrombin activity. In addition, we discovered that 3B suppressed thrombin formation considerably; this will be the subject of future research.

Activity of Plasmin upon Materials
Massive hemorrhage hemostasis was especially susceptible to hyperfibrinolysis, resulting in significant rebleeding. However, the involvement of the fibrinolytic system is frequently overlooked in the hemostatic mechanisms of many current materials. Here, plasmin, which is the fundamental component of the fibrinolytic system, was measured using the PAP (plasmin--antiplasmin complexes) Elisa kit. After the plasmin interaction with BGs, the concentration of plasmin decreased significantly, which was different from MMT, which increased the concentration of plasmin. Based on the findings in Figure 1g-j, BGs improved the thrombus strength by inhibiting hyperfibrinolysis. In addition, MMT's involvement in promoting the fibrinolytic system ( Figure S16, Supporting Information) and fibrinogen adsorption (Figure 6e, 7b) might explain its rebleeding phenomena in vivo (Figure 8f), as well as the extremely low intensity of thrombus in vitro (Figure 1j).

Comprehensive Impact of Materials on Clotting
Although plasma coagulation played a vital role in the hemostatic mechanism, the reaction of blood cells was indispensable. Therefore, a thromboela-stogram (TEG) was used to explore the influence of materials (suspension and powder) on whole blood in vitro. TEG is an index of dynamic coagulation changes used to evaluate the aggregation and rigidity of RBCs, clotting rate, and fibrinolytic activity level. [54] The R, K, MA, and A30 values represent the coagulation time, coagulation rate, maximum thrombosis amplitude, thrombosis amplitude after 30 min of clotting, and percentage of fibrinolysis (Figure 6n,o). These values correspondingly reflect the functions of coagulation factor, fibrinogen, thrombus, and hyperfibrinolysis. The relative percentage between each group and the blank group in the same period was estimated from several blood coagulation interference variables. In Figure 6p-s, the percentages of R and K in the 0B, kaolin, and MMT groups were noticeably lower than the blank group, indicating that they might play an active role in interacting with coagulation factors and accelerating fibrinogen production. Although the relative value of increased for all groups (BGs, kaolin, and MMT), the 0B group showed the strongest function of fibrinogen. BGs, notably the 0B, showed the highest thrombus strength (MA) compared to kaolin and MMT (Figure 1i,j, 6s), consistent with the pro-coagulant action in vivo and in vitro but also the clotting behavior of the 0B in hemostasis (Figure 8f). Additionally, the analysis showed that, under these test conditions, the whole blood coagulation system was not very different whether BG was in the form of a suspension or a powder ( Figure S17, Supporting Information).
Briefly, 0B immediately triggered the coagulation process, boosting fibrin synthesis, inhibiting hyperfibrinolysis, and eventually forming a stronger three-dimensional network thrombus to seal the wound site, producing successful hemostasis, and preventing rebleeding.

Biocompatibility
Biocompatibility is a prerequisite for the hemostat application. [9,35,41] Thus, in vitro hemolysis, cytotoxicity, skin irritation, organ status after hemostasis, and histocompatibility after BGs leakage were thoroughly conducted.

Hemolysis
The high hemolysis rate implied that materials might induce proximal or distal embolization. Herein, the hemolytic potentialities of BGs, kaolin, MMT, and HA were evaluated. The blank group without materials and the TX-100 (2%) group were set up as negative and positive controls, respectively. The hemolysis of 0B, kaolin, and HA was less than 5%, as illustrated in Figure 8a,b, but MMT, 1B, 1.5B, 2B, and 3B showed hemolysis higher than 5%. More intuitively, in contrast to the blank control, RBCs submerged in 0B, kaolin, and HA maintained normal morphology, whereas those in the MMT, 1B, 1.5B, 2B, and 3B groups experienced noxious deformation and rupture (Figure 8c, Figure S18, Supporting Information). In conclusion, 0B exhibited superior blood compatibility compared to the other BGs.

Cytotoxicity
The cytotoxicity of BGs, kaolin, MMT, and HA on L929 cells during a 24 h incubation period was investigated in Figure 8d  cell viability of the extracts of 0B, kaolin, MMT, and HA was comparable to that of the blank, while cell viability was only 25%-50% for the 1B, 1.5B, 2B, and 3B groups. The result suggests that boron-doped silicate BGs may induce cytotoxicity. Additionally, live/dead images of cells fluorescently labeled using calcein AM and propidium iodide (PI) clearly show that cells grown in 0B, kaolin, MMT, and HA extracts were healthy (well-spread and adhered to the surface), while cells cultured in 1B, 1.5B, 2B, and 3B extracts were balled-up, which is a sign of apoptosis (Figure 8e, Figure S19, Supporting Information).

Skin Stimulus
As shown in Figure S20a,b, Supporting Information, the 0B and 1.5B groups had almost no apparent redness and swelling compared to the positive control group (20% SDS). H&E staining also demonstrated that BGs exhibited no inflammatory response to skin tissue.

Histocompatibility
Another distinguishing feature of BG over other inorganic salts (zeolite, kaolin, and MMT) was that BGs could modulate macrophage polarization, encourage angiogenesis, and hence accelerate soft tissue healing throughout their degradation process. [36][37][38] Some amounts of 0B, kaolin, and MMT were left behind at the wound site after hemostasis to see whether the materials would induce any inflammation or tissue reaction, hindering wound healing. The wound was then sutured, and any signs of rebleeding, redness, swelling, or suppuration at the wound site were observed at 0, 1, 4, 14, and 21 days (Figure 8f,g). The wound site after the suture showed rebleeding and large thrombus formation for the gauze and MMT groups on days 0 and 1, compared to the kaolin and 0B groups. This result suggests that clots formed in the gauze and MMT groups are relatively weak. On the fourth day, severe swelling caused the sutures to break in the gauze, kaolin, and MMT groups; however, the suture remained intact for the 0B group. The external appearance showed that the wound healing of the 0B and kaolin groups was far superior to that of the gauze and MMT groups on 14 and 21 days. The anatomical regions had substantial residues enclosed in the fibrous tissues in both the kaolin and MMT groups. On the other hand, 0B almost degraded at the injury site, showing lower inflammation and better healing, as verified by the H&E and Masson staining (Figure 8g). In addition, blood biochemical indices of New Zealand rabbits in Figure S21, Supporting Information, revealed that all monocyte values were above the norm, whereas all other indices fell within the normal value range. It was suggested that all wound sites may continue to exhibit a mild inflammatory response. Also, on the 21st day, the HE staining of the heart, liver, spleen, lung, and kidney of New Zealand rabbits in group 0B did not differ substantially from that of the blank group, indicating the biocompatibility of 0B ( Figure S22, Supporting Information).

Thermal Effect
In addition, the temperature changes for 0 min, 1 min, and 3 min after soaking the material in SBF solution were tested in vitro ( Figure S23, Supporting Information). 0B hardly caused the temperature change, which differed from the crystalline zeolite hemostatic agent (Quickclot), increasing the temperature to 60°C and indicating a thermal effect. [55] Overall, soaking 0B in whole blood did not cause tissue burn or necrosis.
Although BGs showed good biocompatibility in cells and tissues in a short period of time, the long-term potential risk of powders entering the blood system could not be ignored. In order to avoid this risk, loading the BG powders on the carriers (such as sponge, gauze, hydrogel, or microspheres) by physical or chemical action was worth considering. In addition, the degradation mechanisms of BGs in vivo would provide a broader perspective for the creation of antimicrobial, hemostatic, and healingpromoting multifunctional materials.

Discussion
The hemostatic application of inorganic salts has attracted attention for decades, but our knowledge of the mechanism is only limited to the fundamental physical and chemical features. [1,10,18,56] Few investigations have been truly conducted on the interaction between inorganic surfaces and functional proteins. [18] In this work, the effects of surface roughness and hydration of crystalline kaolin and amorphous BG on proteins' adsorption are determined by proteomic analysis, and the activity of coagulation factors is monitored throughout the coagulation reactions. Moreover, the minute-by-minute rate at which kaolin and BGs stimulate thrombin generation is determined. Hence, we propose a novel model of hemostasis based on the interface of crystalline kaolin and amorphous BGs in light of these results.
In general, the affinity of fibrinogen and antithrombin III on the interface of kaolin results in a weaker thrombus and subsequent rebleeding after hemostasis (Figures 1, 6-8). As for the amorphous BG with a dynamic ion microenvironment (Figure 4 and Figure S14, Supporting Information), it breaks through the hydration shell barrier and selectively captures procoagulant components of kiniogen-1, plasma kallikrein, FXII, and FXI proteins on its interface (Figure 6), simultaneously providing a continuous biocatalytic interface to rapidly activate both intrinsic and extrinsic coagulation pathways ( Figure 6). Thus, prothrombin complexes are successfully hydrolyzed to thrombin in the absence of platelet membrane involvement (Figure 7), thereby speeding the development of high-strength blood clots ( Figure 6). While borate ions in borosilicate/borate bioactive glass will limit the action of extrinsic coagulation factors and diminish the interface's electronegativity of BGs (Figures 1, 7).
Excluding the action of kaolin on PLTs, RBCs, and other blood cells, the hemostatic model caused by the kaolin surface mostly consists of the three points below (Figure 9a): 1) Upon initial contact with blood, kaolin with a high interface electronegativity primarily initiates and activates the endogenous coagulation cascade by preferentially adsorbing kiniogen-1 and then activating FXII and FXI factors. However, kaolin inhibits the activation of the endogenous pathway's subsequent enzyme-linked reactions, FIX and FVIII complexes. 2) The kaolin surface progressively becomes wet and clings to blood coagulation-related proteins, particularly fibrinogen and antithrombin III. During the test of kaolin's ability to promote fibrinogen conversion to fibrin, neither kaolin nor MMT with a crystalline structure was able to achieve plasma coagulation, which implied that the strong adsorption of kaolin on fibrinogen on the surface would decrease the concentration of fibrin in the blood clot, thereby weakening the clot. This may be one reason why kaolin and MMT have low thrombosis intensity and induce post-hemostasis rebleeding. [17] 3) Following the formation of a hydration layer and protein adhesion on the surface of kaolin, its surface structure may reduce the secondary, tertiary, or continuous activation of the endogenous coagulation system.
Model of coagulation induced by the 0B surface (Figure 9b,c). There are four primary steps: 1) The blood quickly adheres to the hydrophilic negative charge surface, simultaneously absorbs and concentrates kiniogen-1, plasma kallikrein, FXII, and FXI, then magnificently initiates the internal (FXII, FXI) and external (FVII) coagulation pathways, accelerates the composite assembly of coagulation-promoting ion microenvironment and protease (FIV, FVII, FX), and lays the foundation for the production of large quantities of thrombin. 2) Under the catalysis of the coagulation-promoting composite assembly, a considerable quantity of thrombin is created in the dynamic ionic layer on the surface of 0B. 3) During the development of a fibrin clot, 0B may operate as a platelet-like agent to strengthen the thrombus clot due to its poor protein adhesion property. 4) Furthermore, the dynamic microenvironment formed by 0B may somewhat restrict fibrin activity. In massive hemorrhage, minimizing the hyperfibrinolysis produced by coagulation factor depletion is advantageous in lowering the risk of rebleeding. According to clinical report statistics, subsequent bleeding will exacerbate the difficulty of hospital treatment and increase death. The mortality rate of emergency bleeds is more than fifty percent of the overall mortality rate during the first twenty-four hours of hospital care. [4,7,14] Thus, stable thrombosis is crucial for major hemorrhage and may save more lives.
It could be seen that compared with kaolin, 0B might trigger the coagulation process faster under the electronegative interface, accelerate the production of thrombin, promote the production of fibrin under the dynamic ionic microenvironment with less adhesion to the fibrinogen interface, inhibit the occurrence of high fibrinolysis, and finally form a stronger threedimensional network thrombus to achieve efficient and rapid hemostasis.
In the initial design of amorphous BGs, several components (Si, Ca, Na, K, Mg, Sr, etc.) containing essential body trace elements (B) were used. These substances produce an ideal interface and ionic microenvironment that may stimulate the blood coagulation system and speed up fibrin production. Different forms Figure 9. a) Plasma coagulation mechanism at the interface of kaolin. b) Plasma coagulation mechanism at the interface of 0B. c) The whole blood hemostatic mechanism of 0B. of bioactive glass solidify to varying degrees, with silicate resulting in the quickest and most effective solidification, preventing major bleeding, followed by borosilicate and borate glass. Boron doping enhances the zeta potential of BGs surface, diminishes the vitrification impact of its surface structure, suppresses the activities of coagulation factors VII, V, and II associated with the extrinsic coagulation pathway, and partially inhibits the activity of thrombin (Figure 6).
Materials' hemostatic characteristics are intimately connected to their macro-micro scopic interface patterns with blood. Through chemical activation of coagulation factors (inorganic salts), [1,10,11] blood cell action (chitosan), [12,13] physical expansion and compression (bandage), [2] adsorption and concentration (sponge), [12][13][14] the coagulation mechanisms of these hemostats are mainly described from macroscopic perspectives, which lacks theoretical direction for the development of novel materials and occasionally overlooks the possible adverse consequences of materials. This research demonstrates the impact of the hydration layer, electronegativity, protein-specific binding, and microenvironment on the whole plasma coagulation process, as well as a full investigation of comprehensive hemostasis and its possible negative consequences. Particularly, the adsorption and activation of coagulation-related proteins, and their correlational influence on coagulation rate and thrombosis strength were uncovered, providing a theoretical basis and experimental methods for the design of new hemostatic materials for massive hemorrhage control.
Last, due to the complexity of the blood environment, we share some of the experiences gained throughout this investigation of hemostatic performance and its mechanism. First, hemostatic agents were evaluated in vitro using standard coagulation techniques, such as calcium recovery time, coagulation index (BCI), and a rheometer measurement. In contrast to the other two procedures, the calcium recovery time method is straightforward. However, this approach cannot demonstrate the change in thrombus strength and needs a substantial quantity of blood for detection. Materials that destroy RBCs, such as chitosan and Surgicel, are incapable of detecting BCI, leading to false-negative or positive readings. The rheometer approach requires 100-200 μL of blood to determine the precise gel transition point and thrombus strength. It is hoped that the correlation between thrombus strength and shear force may partly replace animal testing to assess the rebleeding issue induced by material movement. However, this in vitro approach has a significant drawback because in vitro models cannot fully mimic the room-temperature coagulation process. Before adding Ca 2+ , the blood sample should be cooled to between 2 and 8°C; otherwise, the gel's conversion point would be lost. Therefore, establishing a hemostatic animal model for the study is a better alternative. The most popular animal models are rats with severed tails, livers, or arteries. However, these standard models are not appropriate for screening hemostatic substances for pre-hospital first aid. As of now, there is a need for animal models that can mimic normal high blood pressure and vigorous blood flow following catastrophic bleeding. Therefore, this study investigated and designed animal models with major hemorrhages. In addition, semi-automated and automatic coagulation equipment, thromboelastography, and rheometer were used to examine the coagulation process. Magnetic bead-based coagulation equipment was better than optical technology in investigating the impact of materials on four coagulation species (i.e., the activity of the coagulation systems, including PT, APTT, TT, and FIB tests). Using fresh plasma that has not been exposed to glass containers or procoagulant chemicals was preferred. Furthermore, TEG was advanced because it showed the whole deformation of the coagulation and fibrinolysis system. However, TEG needed blood samples with stringent requirements. Because evaluating each sample takes 1-2 hours, the additional blood will degrade with time. Due to the difficulty in obtaining fresh human blood, we utilized rabbit blood anticoagulated with sodium citrate as a blood sample to determine the stability of rabbit blood in a thromboelastography apparatus. Nevertheless, ensuring that only one variable was modified at a time was difficult. To resolve this issue, we simultaneously evaluated two channels, one blank and the other experimental group. The degree of impact on blood parameters of the materials was determined by calculating the relative changes from the control group. In addition, the MA analysis in TEG was explored. Because only a small quantity of blood from the test cup reached the revolving needle tip, defining the interface between the substance and the thrombus was challenging. For a comprehensive understanding of the hemostatic process, instrument settings and sample manipulation must be chosen with care.

Conclusions
In conclusion, we explore a technique to manufacture amorphous bioactive glasses on a large scale and demonstrate their effectiveness and safety in reducing severe hemorrhages compared to standard hemostatic treatments such as kaolin. The unique coagulation mechanisms at the interface of biominerals are proposed to guide the way to novel hemostats for hemorrhage control in first aid.

Experimental Section
Materials: Various inorganic salts used to prepare bioactive glass were provided by Aladdin Reagent Co., Ltd., Shanghai, China. In addition, kaolin and montmorillonite (98%) were purchased from JiuDing Co., Ltd., Shanghai, China. Pig blood and plasma were purchased from Chuzhou shinuoda Biological Technology Co., Ltd.. Thrombin chromogenic substrate solution (2238, HYPHEN BioMed). Coagulation factor kits were purchased from Siemens. PT, APTT, TT, and FIB reagents were bought from Shanghai Sun Biotech Co. Ltd. Thromboelastogram test kits (ordinary cups) were provided by CFMS.
Detailed information on other reagents could be found in the Supporting Information.
ICP Determination: 1.0000 g of material was weight and put into a 50 mL centrifuge tube. 20.00 mL of deionized water was added into the above tube, and then was fixed into a rotary mixer to mix for 1 min and 5 min respectively. Immediately all their samples were filtered through a 0.22 μm filter membrane to obtain the extracts. Finally, the extracts were subjected to ICP determination according to the procedures.
Hemostasis In Vitro and In Vivo -Experiments with Animal Samples: All animals were raised in pathogen-free environments and were fed freely. The Ethics Committee of the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, authorized the procedures for animal care and use, and we followed all applicable legislation and institutional standards governing the ethical use of animals (SIAT-IACUC-200821-YYS-LCY-A1389).
SD rats (≈700 g) and New Zealand white rabbits (≈ 2.5 kg) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. and Guangzhou Huateng Biomedical Technology Co.,c Ltd. All animals were anesthetized with isoflurane gas after 24 h of fasting. All items used in surgical procedures were sterilized under UV irradiation for 1 h. All animals were sacrificed in accordance with NIH guidelines.
Hemostasis In Vitro: Recalcification time: The recalcification coagulation of whole blood (pig blood anticoagulated with sodium citrate, Chuzhoushinuoda Biological Technology Co., Ltd., Hubei, China) was performed using the previously reported method. [31,35] The clotting time was recorded when a blood clot formed and no visible blood flowed. Rheometer experiment: At 4°C, 200 μL sodium citrate-anticoagulated rabbit blood was combined with 0.5 mg BG and 5 μL 0.25 M CaCl 2 solution. The mixture was then dropped onto the sampling table at a volume of 140 μL. The scan with the time sweep (37°) rheometer program (Anton Paar MCR302) was set in advance, which was used to keep track of the recalcification time.
Tail Amputation of SD Rats: Using surgical scissors, a tail amputation was performed five centimeters from the tail's base. The bleeding site was then treated with either kaolin or 0B. Record the duration of bleeding. In order to record the blood loss, filter paper was placed beneath the injured tail to absorb the blood. In the control group, no hemostatic materials were applied. Repeated six times for each group. [12] Inguinal Hemorrhage in SD Rats: To imitate major inguinal bleeding, the femoral artery, vein, and nerve at the root of the right leg of SD rat was all cut at the same time. The wounded area was dusted with 0B and kaolin, followed by gauze and 100 g gravity for 30 s. The entire hemostasis process in SD rats was monitored. [31] Abdominal Hemorrhage in SD Rats: The liver injury model was created to evaluate the hemostatic effect of materials on bleeding or leakage of parenchymatous organs. The abdomens of SD rats were dissected, disclosing the liver. The left lobe of the liver was removed, with tissue measuring 3 cm in length and 1 cm in breadth. Then either 0B or kaolin components were sprinkled on the wound site. The gauze group was designated as the blank control group. Both the clotting time and the amount of blood loss were observed.
Abdominal Hemorrhage in New Zealand White Rabbits: An X-shaped wound about 2 cm (length) x 2 cm (width) x 1 cm (depth) on the left lobe of the liver was created using a scalpel. A layer of gauze is put beneath the liver to prevent bodily fluids from affecting the experiment's accuracy. One gram of 0B, kaolin, or MMT was applied to the injured site without providing any pressure. Then, apply a layer of gauze to absorb the blood. The amount of blood lost was documented. The evaluation of the clotting time was based on observing whether the wound site was completely stopping bleeding after 10 min. In addition, following hemostasis, the damaged portion of the liver was incised for Masson staining. [35] Inguinal Hemorrhage in New Zealand White Rabbits: The groin soft tissue was exposed using surgical scissors and tissue forceps, and then the femoral artery was freed using blood vessel cell glass separation needles. The distal end of the femoral artery was initially tied up, and then both ends of the femoral artery were flattened horizontally with tissue forceps before a 1.6 mm syringe needle inserted was vertical to increase the repeatability and consistency of the method. 4 g of 0B, kaolin, or MMT were applied to the groin right away, followed by gauze and 150 g of gravity compression for 30 s. The control group only used gauze. The clotting time of hemostatic process was recorded.
Hemostatic Mechanisms of BGs and Kaolin -Experiments With Clinical Samples: The collection of human blood samples was conducted in compliance with the Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences' ethical guidelines (SIAT-IRB-221015-H0617). All subjects gave their informed consent.
Contact Angle: Five hundred mg of 0B, kaolin, and MMT were laid out on the glass plate. One attention apparatus (Attention Theta Lite, Biolin Scientific) examined the contact angle of materials using deionized water as the liquid phase.
Absorption: The water-absorbing capacities of BG, kaolin, and MMT were determined by the previous study. [31] The water-absorbing capacity of each material was calculated by measuring the maximum quantity of water absorbed per gram of dry material in a given immersion period.
Platelet Activation: As a platelet activation-dependent granule surface protein, sP-selection was considered one of the indicators of platelet activation. Here, 2 mg of materials were incubated for 15 min at 37°C with PRP (New Zealand white rabbit, 500 μL). After that, all of the samples were centrifuged for 10 min at 1500 g. After that, all samples underwent a 10 min centrifugation at 1500 g to obtain the supernatant. The concentration of sP-selection in the samples was then analyzed using an ELISA kit for rabbit sP-selection (Shanghai Yuduo Biotechnology Co., Ltd, China).
RBCs Aggregation and Morphology: In a 24-well plate, a certain quantity of 0B-1, 0B-2, kaolin, and MMT was placed, and 50 μL of rabbit red blood cells (RBCs) were progressively added to incubate at 37°C for a period of time. The unaggregated RBCs were washed five times using PBS to remove them. Three mL of deionized water were added to induce the aggregated RBCs to rupture and release hemoglobin. Finally, using a multiskan spectrum (Multiskan GO, USA), the absorbance of hemoglobin was determined at 540 nm. The absorbance of hemoglobin was positively linked with the relative aggregation of RBCs. The control group consisted of only RBCs and no other components. In addition, RBCs were also exposed to materials for 10 min to examine their morphology. All samples were fixed for 2 h with 2.5% glutaraldehyde, then for another 2 h with a 2% tannin solution (PBS buffer, pH 7.4) before being dehydrated using an ethanol gradient. The morphology of RBCs with the material intertwined was observed by SEM (Zeiss Sigma 300). [31,35] Concentration of Plasmin: The entire volume of blood from New Zealand rabbits was anticoagulated with EDTA and centrifuged at 1000 g for 15 min to obtain platelet-poor plasma (platelet-poor plasma was abbreviated as PPP.). Ten μL of BG, kaolin, or MMT solution (10 mg mL −1 ) were mixed with 100 μL PPP in a 1.5 mL centrifuge tube. The Elisa kit of plasma thrombomodulin and plasmin-2-antiplasmin complexes (PAP, Shanghai Jianglai Biotechnology Co., Ltd, China) was used to assess the concentration of plasmin.
Effect on Intrinsic and Extrinsic Coagulation Pathways: Prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), and fibrinogen (FIB) were measured with a semi-automatic coagulation analyzer. [31,35] Two mg of material was added to the test cup with one magnetic bead inside. Then a certain amount of fresh pig platelet-poor plasma anticoagulated with sodium citrate (Chuzhoushinuoda Biological Technology Co., Ltd., Hubei, China) was dropped into the test cup, and the mixed samples were conducted after incubation at 37°C for 30 s. All the tests would be carried out in strict accordance with the instructions for the PT, APTT, TT, and FIB reagents (Shanghai Sun Biotechnology Co., Ltd, Shanghai, China).
Effect on Coagulation Factors: The test samples, consisting of 1 mg of material (BGs, kaolin, or MMT), were mixed with 50-100 μL of human plasma (the amount of human plasma was added according to the requirements of the kits). Then, according to the guidelines of the coagulation factors, the activity for the coagulation factors (FII, FV, FVII, FVIII, FIX, FX, FXI, and FXII) was detected using the semi-automatic coagulation instrument (URIT-600, China). The saline solution was used as a blank control. The test was repeated three times (n = 3). [57] Thromboelastograph Analysis In Vitro: The coagulation condition of whole blood following contact with materials was monitored and analyzed using thromboelastography (CFMSLEPU-8800, China). The device ran two experiments simultaneously 340 μL of fresh anticoagulant blood harvested from New Zealand rabbits was incubated in a test cup for 5 min at 37°C. Then, 10 μL of BG extract (50 mg mL −1 ) or 0.5 mg of BG powder were added. After 3 min of incubation, 20 μL of 0.2 M CaCl 2 solution was added to initiate the coagulation. Different thromboelastography variables were measured, including reaction time (R per min), angle ( per degree), setting time (K), maximum amplitude (MA/mm), and amplitude after 30 min (A30/mm). Normal saline was used as the blank control group. Due to the complexity of blood, experimental and blank groups were tested simultaneously to acquire relatively accurate data. The relative percentage (n = 3) of each parameter was measured by comparing the experimental sample to the blank group.
Unusual Activation Patterns of Thrombin in BGs and Kaolin: Assay for the production of thrombin without the tissue factor. CaCl 2 was the only trigger employed in this experiment. Five mg of material (BG or kaolin) was incubated with 30 μL of human plasma, 3 μL of CaCl 2 solution (0.2 M), and 70 μL of HEPES buffer for 2-30 min at 37°C on a thermo shaker. After incubation, 270 μL of HEPES buffer and 30 μL of thrombin chromogenic substrate solution (4 mg mL −1 , S2238, HYPHEN BioMed) were added to the mixture. [18] The reaction was carried out on a thermo shaker at 37°C for 30 min, and then 50 μL of acetic acid was added rapidly to halt the process. At 405 nm, the absorbance of the supernatant was measured. Human thrombin was employed as a standard. The group that did not receive any materials served as the control group. Repeat three times per group. During the development of this process, it is essential to examine the material-to-plasma weight-to-volume ratio, the reaction duration, the concentration of the reaction substrate, and whether or not the reaction substrate will interact with the material.
Statistical Analyses: A one-way ANOVA of Multiple Comparisons (GraphPad Prism 8.0) was used to assess the statistical differences. The value of *p < 0.05, **p < 0.01, ***p < 0.005 and ****p < 0.001, # p < 0.05, ## p < 0.01, ### p < 0.005 and #### p < 0.001 were deemed statistically different, significantly statistically different, extremely significantly statistically different, respectively. The * represented the statistical difference between the experimental group and the blank control group; while the # represented a statistical difference between other experimental groups and group 0B.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.