Reversible High-Affinity Binding of Coagulation Factor Xa to Zeolites Induces Accelerated Blood Coagulation

Abstract

Zeolite is recognized as an essential hemostatic material for controlling massive bleeding. Elucidating the procoagulant mechanism of zeolite is critically important, as it will facilitate the rational design of more effective zeolite-based hemostatic materials. In this study, it is discovered an extremely strong, calcium-dependent interaction between coagulation factor Xa (FXa) and zeolite-termed target-specific biorecognition-that mimics the FXa/factor Va (FXa/FVa) interface formed during the natural coagulation cascade. This interaction alters the prothrombin activation pathway to a more efficient mechanism, significantly amplifying FXa activity. Notably, the complex structure and FXa activity can be reversibly modulated through Na⁺/Ca²⁺ ion exchange of zeolites, offering a novel strategy for dynamically tuning enzymatic activity. Furthermore, this protein-zeolite based biorecognition system, mediated by reversible interactions, represents a promising biomimetic platform for regulating protein bioactivity in cell-free applications, extending its utility beyond hemostatic material development.

Keywords: biorecognition; coagulation; factor Xa; protease; zeolite.

Figure 1

Covalently binding of FXa with CaY zeolites and the corresponding significant amplification and regulation of FXa enzymatic activity. a) Structure of coagulation FXa. b) Schematic representation of the conventional protein corona and covalent bonded protein corona, and demonstrating the bonding ability for short‐lived, long‐lived and permanent FXa. c) Bonding capacities of permanent FXa light chain with CaY‐HC‐90 zeolite via WB. d) Bonding capacities of CaY zeolites with FXa light chain via WB. e) Bonding capacities of CaY zeolites with FXa via BCA protein assay. Region I: FXa weakly bonded to CaY‐LC zeolite surface. Region II: FXa strong covalently bonded to CaY‐HC zeolite surface (n = 3). f) Specific enzymatic activity of FXa on CaY zeolites with several calcium exchange degree for prothrombin‐to‐thrombin conversion (n = 3). Pro: prothrombin; Thr: thrombin; ND: not detected. g) Enzymatic activity of FXa heavy chain, FXa, heavy chain/CaY‐HC‐90 and FXa/CaY‐HC‐90, respectively (n = 3). h) Schematic diagram of regulating FXa activity on zeolite surface via sodium/calcium ion exchange. i) Regulation of the interaction between FXa light chain and zeolite surface via sodium/calcium ion exchange. j) Enzymatic activity of FXa on zeolites via sodium/calcium ion exchange for prothrombin‐to‐thrombin conversion (n = 3). Data values correspond to mean ± SD.

Figure 2

EGF‐LD‐target covalent‐binding of FXa on the CaY‐HC zeolite identified by molecular dynamics (MD) simulation. a) Conformation of FXa confined on CaY‐HC zeolite and details of E142 and E91 interacting with CaY‐HC zeolite. FXa heavy chain is shown in pale yellow, EGF‐LD of FXa light chain is shown in salmon and GLA domain of FXa light chain is shown in slate blue. b) Schematic diagram of FXa on CaY‐HC zeolite. FXa bonding on the CaY‐HC zeolite via coordination interaction of light chain EGF‐LD. c) FXa/FVa complex on the activated platelet membrane surface. d) Distance of center mass between FXa chains and CaY‐HC zeolite (n = 3). e) Coulomb energy between FXa chains and CaY‐HC zeolite (n = 3). f) Coulomb energy between FXa chains and NaY zeolite (n = 3). g) Coulomb energy between FXa light chain and the components of CaY‐HC zeolite (n = 3). (h) Binding energy between the main amino acid residues of FXa and CaY‐HC zeolite. Data values correspond to mean ± SD. Each group contains three points at energy equilibration. ns: non‐significant, ***P < 0.001, one‐way analysis of variance (ANOVA).

Figure 3

EGF‐LD‐target covalent‐binding of FXa on the CaY‐HC zeolite identified by liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) analysis. a) Schematic diagram of benzoyl hydrazide (BHD) labeling for glutamate (E) and gamma‐carboxyglutamic acid (CGU) followed by mass spectrometry analysis. b) Color gradient diagram of labeling level for E and CGU residues in FXa light chain; increased labeling level is shown in green, decreased labeling level is shown in red. c) Distribution of the key amino acid residues in FXa light chain which labeling level decreased/increased in front/back view; the amino acid residues with increased labeling level are shown in green, the amino acid residues with decreased labeling level are shown in red. E91 and E142 from EGF‐LD, which strongly interact with CaY‐HC‐90 zeolite, are marked in purple. d) Target covalent‐binding conformation of FXa on CaY‐HC‐90 zeolite.

Figure 4

FXa/FVa‐like interface of FXa SP domain (heavy chain) on CaY‐HC zeolite and enzymatic activity of FXa/CaY‐HC for prothrombin‐to‐thrombin conversion pathway. a) Schematic diagram of lysine (K) dimethyl labeling followed by mass spectrometry analysis. Color gradient diagram of changes in amino acid residues labeling level of FXa heavy chain. Decreased labeling level is shown in red. b) Distribution of the key amino residues in FXa heavy chain of which labeling level is decreased in top/bottom view; the amino residues with decreased labeling level are shown in red. c) Distribution of the key amino residues in reported FXa/FVa interaction sites; the non‐lysine residues are colored in cyan. The lysine residues with reduced labeling in FXa/CaY‐HC‐zeolite are highlighted in purple. d) Schematic diagram of prothrombin‐to‐thrombin conversion pathway of FXa/FVa complex and free FXa. e) Prothrombin‐to‐thrombin conversion pathway activated by free FXa and FXa/CaY‐HC‐90 identified using WB. f) Prothrombin‐to‐thrombin conversion pathway activated by FXa/FVa‐like interface of FXa SP domain (heavy chain) on zeolites via sodium/calcium ion exchange. g) FXa/FVa complex on the activated platelet membrane surface with the labeled interaction domain (cyan color). h) Schematic diagram of FXa on CaY‐HC zeolite. FXa bonding on the CaY‐HC zeolite via coordination interaction of light chain EGF‐LD and FVa/FXa‐like interface of SP domain.