Aptameric hirudins as selective and reversible EXosite-ACTive site (EXACT) inhibitors.

Potent and selective inhibition of the structurally homologous proteases of coagulation poses challenges for drug development. Hematophagous organisms frequently accomplish this by fashioning peptide inhibitors combining exosite and active site binding motifs. Inspired by this biological strategy, we create several EXACT inhibitors targeting thrombin and factor Xa de novo by linking EXosite-binding aptamers with small molecule ACTive site inhibitors. The aptamer component within the EXACT inhibitor (1) synergizes with and enhances the potency of small-molecule active site inhibitors by many hundred-fold (2) can redirect an active site inhibitor's selectivity towards a different protease, and (3) enable efficient reversal of inhibition by an antidote that disrupts bivalent binding. One EXACT inhibitor, HD22-7A-DAB, demonstrates extraordinary anticoagulation activity, exhibiting great potential as a potent, rapid onset anticoagulant to support cardiovascular surgeries. Using this generalizable molecular engineering strategy, selective, potent, and rapidly reversible EXACT inhibitors can be created against many enzymes through simple oligonucleotide conjugation for numerous research and therapeutic applications.

Biosynthetic production of anticoagulant heparin polysaccharides through metabolic and sulfotransferases engineering strategies.

Heparin is an important anticoagulant drug, and microbial heparin biosynthesis is a potential alternative to animal-derived heparin production. However, effectively using heparin synthesis enzymes faces challenges, especially with microbial recombinant expression of active heparan sulfate N-deacetylase/N-sulfotransferase. Here, we introduce the monosaccharide N-trifluoroacetylglucosamine into Escherichia coli K5 to facilitate sulfation modification. The Protein Repair One-Stop Service-Focused Rational Iterative Site-specific Mutagenesis (PROSS-FRISM) platform is used to enhance sulfotransferase efficiency, resulting in the engineered NST-M8 enzyme with significantly improved stability (11.32-fold) and activity (2.53-fold) compared to the wild-type N-sulfotransferase. This approach can be applied to engineering various sulfotransferases. The multienzyme cascade reaction enables the production of active heparin from bioengineered heparosan, demonstrating anti-FXa (246.09 IU/mg) and anti-FIIa (48.62 IU/mg) activities. This study offers insights into overcoming challenges in heparin synthesis and modification, paving the way for the future development of animal-free heparins using a cellular system-based semisynthetic strategy.

Total Content and Composition of Phenolic Compounds from Filipendula Genus Plants and Their Potential Health-Promoting Properties.

This current article was dedicated to the determination of the composition of phenolic compounds in extracts of four species of the genus Filipendula in order to establish a connection between the composition of polyphenols and biological effects. A chemical analysis revealed that the composition of the extracts studied depended both on the plant species and its part (leaf or flower) and on the extractant used. All four species of Filipendula were rich sources of phenolic compounds and contained hydrolyzable tannins, condensed tannins, phenolic acids and their derivatives, and flavonoids. The activities included data on those that are most important for creating functional foods with Filipendula plant components: the influence on blood coagulation measured by prothrombin and activated partial thromboplastin time, and on the activity of the digestive enzymes (pancreatic amylase and lipase). It was established that plant species, their parts, and extraction methods contribute meaningfully to biological activity. The most prominent result is as follows: the plant organ determines the selective inhibition of either amylase or lipase; thus, the anticoagulant activities of F. camtschatica and F. stepposa hold promise for health-promoting food formulations associated with general metabolic disorders.

Mussel-Mimetic Hydrogel Coating with Anticoagulant and Antiinflammatory Properties on a Poly(lactic acid) Vascular Stent.

Biodegradable stents are the most promising alternatives for the treatment of cardiovascular disease nowadays, and the strategy of preparing functional coatings on the surface is highly anticipated for addressing adverse effects such as in-stent restenosis and stent thrombosis. Yet, inadequate mechanical stability and biomultifunctionality limit their clinical application. In this study, we developed a multicross-linking hydrogel on the polylactic acid substrates by dip coating that boasts impressive antithrombotic ability, antibacterial capability, mechanical stability, and self-healing ability. Gelatin methacryloyl, carboxymethyl chitosan, and oxidized sodium alginate construct a double-cross-linking hydrogel through the dynamic Schiff base chemical and in situ blue initiation reaction. Inspired by the adhesion mechanism employed by mussels, a triple-cross-linked hydrogel is formed with the addition of tannic acid to increase the adhesion and antibiofouling properties. The strength and hydrophilicity of hydrogel coating are regulated by changing the composition ratio and cross-linking degree. It has been demonstrated in tests in vitro that the hydrogel coating significantly reduces the adhesion of proteins, MC3T3-E1 cells, platelets, and bacteria by 85% and minimizes the formation of blood clots. The hydrogel coating also exhibits excellent antimicrobial in vitro and antiinflammatory properties in vivo, indicating its potential value in vascular intervention and other biomedical fields.

Generalized Multifunctional Coating Strategies Based on Polyphenol-Amine-Inspired Chemistry and Layer-by-Layer Deposition for Blood Contact Catheters.

Blood-contacting catheters play a pivotal role in contemporary medical treatments, particularly in the management of cardiovascular diseases. However, these catheters exhibit inappropriate wettability and lack antimicrobial characteristics, which often lead to catheter-related infections and thrombosis. Therefore, there is an urgent need for blood contact catheters with antimicrobial and anticoagulant properties. In this study, we employed tannic acid (TA) and 3-aminopropyltriethoxysilane (APTES) to create a stable hydrophilic coating under mild conditions. Heparin (Hep) and poly(lysine) (PL) were then modified on the TA-APTES coating surface using the layer-by-layer (LBL) technique to create a superhydrophilic TA/APTES/(LBL)4 coating on silicone rubber (SR) catheters. Leveraging the superhydrophilic nature of this coating, it can be effectively applied to blood-contacting catheters to impart antibacterial, antiprotein adsorption, and anticoagulant properties. Due to Hep's anticoagulant attributes, the activated partial thromboplastin time and thrombin time tests conducted on SR/TA-APTES/(LBL)4 catheters revealed remarkable extensions of 276 and 103%, respectively, when compared to uncoated commercial SR catheters. Furthermore, the synergistic interaction between PL and TA serves to enhance the resistance of SR/TA-APTES/(LBL)4 catheters against bacterial adherence, reducing it by up to 99.9% compared to uncoated commercial SR catheters. Remarkably, the SR/TA-APTES/(LBL)4 catheter exhibits good biocompatibility with human umbilical vein endothelial cells in culture, positioning it as a promising solution to address the current challenges associated with blood-contact catheters.

Semisynthesis of new sulfated heterorhamnan derivatives obtained from green seaweed Gayralia brasiliensis and evaluation of their anticoagulant activity.

Marine green algae produce sulfated polysaccharides with diverse structures and a wide range of biological activities. This study aimed to enhance the biotechnological potential of sulfated heterorhamnan (Gb1) from Gayralia brasiliensis by chemically modifying it for improved or new biological functions. Using controlled Smith Degradation (GBS) and O-alkylation with 3-chloropropylamine, we synthesized partially water-soluble amine derivatives. GBS modification increase sulfate groups (29.3 to 37.5 %) and α-l-rhamnose units (69.9 to 81.2 mol%), reducing xylose and glucose, compared to Gb1. The backbone featured predominantly 3- and 2-linked α-l-rhamnosyl and 2,3- linked α-l-rhamnosyl units as branching points. Infrared and NMR analyses confirmed the substitution of hydroxyl groups with aminoalkyl groups. The modified compounds, GBS-AHCs and GBS-AHK, exhibited altered anticoagulant properties. GBS-AHCs showed reduced effectiveness in the APTT assay, while GBS-AHK maintained a similar anticoagulant activity level to Gb1 and GBS. Increased nitrogen content and N-alkylation in GBS-AHCs compared to GBS-AHK may explain their structural differences. The chemical modification proposed did not enhance its anticoagulant activity, possibly due to the introduction of amino groups and a positive charge to the polymer. This characteristic presents new opportunities for investigating the potential of these polysaccharides in various biological applications, such as antimicrobial and antitumoral activities.

Chemical Synthesis of Fucosylated Chondroitin Sulfate Tetrasaccharide with Fucosyl Branch at the 6-OH of GalNAc Residue.

Fucosylated chondroitin sulfate is a unique glycosaminoglycan isolated from sea cucumbers, with excellent anticoagulant activity. The fucosyl branch in FCS is generally located at the 3-OH of D-glucuronic acid but, recently, a novel structure with α-L-fucose linked to the 6-OH of N-acetyl-galactosamine has been found. Here, using functionalized monosaccharide building blocks, we prepared novel FCS tetrasaccharides with fucosyl branches both at the 6-OH of GalNAc and 3-OH of GlcA. In the synthesis, the protective group strategy of selective O-sulfation, as well as stereoselective glycosylation, was established, which enabled the efficient synthesis of the specific tetrasaccharide compounds. This research enriches knowledge on the structural types of FCS oligosaccharides and facilitates the exploration of the structure-activity relationship in the future.

Changes in Internal Structure and Dynamics upon Binding Stabilise the Nematode Anticoagulant NAPc2.

Abnormal blood coagulation is a major health problem and natural anticoagulants from blood-feeding organisms have been investigated as novel therapeutics. NAPc2, a potent nematode-derived inhibitor of coagulation, has an unusual mode of action that requires coagulation factor Xa but does not inhibit it. Molecular dynamics simulations of NAPc2 and factor Xa were generated to better understand NAPc2. The simulations suggest that parts of NAPc2 become more rigid upon binding factor Xa and reveal that two highly conserved residues form an internal salt bridge that stabilises the bound conformation. Clotting time assays with mutants confirmed the utility of the salt bridge and suggested that it is a conserved mechanism for stabilising the bound conformation of secondary structure-poor protease inhibitors.

Genetically encoded biocompatible anti-coagulant protein-coated coronary artery stents drive endothelialization.

In response to the critical demand for advancements in coronary artery stents, this study addresses the challenges associated with arterial recoil and restenosis post-angioplasty and the imperative to encourage rapid re-endothelialization for minimizing thrombosis risks. We employed an innovative approach inspired by mussel adhesion, incorporating placental anticoagulant protein (AnnexinV) on stent design. The introduction of a post-translationally modified catecholic amino acid L-3,4-dihydroxyphenylalanine (L-Dopa), mimicking mussel characteristics, allowed for effective surface modification of Stainless steel stents through genetic code engineering in AnnexinV (AnxDopa). The efficacy of AnxDopa was analyzed through microscale thermophoresis and flow cytometry, confirming AnxDopa's exceptional binding with phosphatidylserine and activated platelets. AnxDopa coated stainless steel demonstrates remarkable bio-, hemo-, and immuno-compatibility, preventing smooth muscle cell proliferation, platelet adhesion, and fibrin formation. It acts as an interface between the stent and biological fluid, which facilitates the anticoagulation and rapid endothelialization. Surface modification of SS verified through XPS analysis and contact angle measurement attests to the efficacy of AnxDopa mediated surface modification. The hydrophilic nature of the AnxDopa-coated surface enhanced the endothelialization through increased protein absorption. This approach represents a significant stride in developing coronary stents with improved biocompatibility and reduced restenosis risks, offering valuable contributions to scientific and clinical realms alike.

Antiplatelet and Anticoagulant Effects of Two New Phenylpropanoid Sucrose Esters and Other Secondary Metabolites from the Aerial Part of Canna edulis.

This study isolated pure compounds from Canna edulis aerial parts and assessed their antiplatelet and anticoagulant potential. Structural elucidation resulted in the identification of two new compounds: caneduloside A (1) and caneduloside B (2), and eleven known compounds: 6'-acetyl-3,6,2'-tri-p-coumaroyl sucrose (3), 6'-acetyl-3,6,2'-triferuloyl sucrose (4), tiliroside (5), afzelin (6), quercitrin (7), 2-hydroxycinnamaldehyde (8), cinnamic acid (9), 3,4-dimethoxycinnamic acid (10), dehydrovomifoliol (11), 4-hydroxy-3,5-dimethoxybenzaldehyde (12), and (S)-(-)-rosmarinic acid (13). Compounds 3, 4, 6-9, 13 were previously reported for antithrombotic properties. Hence, antithrombotic tests were conducted for 1, 2, 5, 10-12. All tested compounds demonstrated a dose-dependent antiaggregatory effect, and 10 and 12 were the most potent for both ADP and collagen activators. Additionally, 10 and 12 showed anticoagulant effects, with prolonged prothrombin time and activated partial thromboplastin time. The new compound 1 displayed antiplatelet and anticoagulant activity, while 2 mildly inhibited platelet aggregation. C. edulis is a potential source for developing antithrombotic agents.

Nutrition and Cardiovascular Disease: The Potential Role of Marine Bioactive Proteins and Peptides in Thrombosis Prevention.

Thrombus and cardiovascular diseases pose a significant health threat, and dietary interventions have shown promising potential in reducing the incidence of these diseases. Marine bioactive proteins and peptides have been extensively studied for their antithrombotic properties. They can inhibit platelet activation and aggregation by binding to key receptors on the platelet surface. Additionally, they can competitively anchor to critical enzyme sites, leading to the inhibition of coagulation factors. Marine microorganisms also offer alternative sources for the development of novel fibrinolytic proteins, which can help dissolve blood clots. The advancements in technologies, such as targeted hydrolysis, specific purification, and encapsulation, have provided a solid foundation for the industrialization of bioactive peptides. These techniques enable precise control over the production and delivery of bioactive peptides, enhancing their efficacy and safety. However, it is important to note that further research and clinical studies are needed to fully understand the mechanisms of action and therapeutic potential of marine bioactive proteins and peptides in mitigating thrombotic events. The challenges and future application perspectives of these bioactive peptides also need to be explored.

A synergistic strategy based on active hydroxymethyl amine compounds and fucoidan for bioprosthetic heart valves with enhancing anti-coagulation and anti-calcification properties.

With an aging population, the patients with valvular heart disease (VHD) are growing worldwide, and valve replacement is a primary choice for these patients with severe valvular disease. Among them, bioprosthetic heart valves (BHVs), especially BHVs trough transcatheter aortic valve replacement, are widely accepted by patients on account of their good hemodynamics and biocompatibility. Commercial BHVs in clinic are prepared by glutaraldehyde cross-linked pericardial tissue with the risk of calcification and thrombotic complications. In the present study, a strategy combines improved hemocompatibility and anti-calcification properties for BHVs has been developed based on a novel non-glutaraldehyde BHV crosslinker hexakis(hydroxymethyl)melamine (HMM) and the anticoagulant fucoidan. Besides the similar mechanical properties and enhanced component stability compared to glutaraldehyde crosslinked PP (G-PP), the fucoidan modified HMM-crosslinked PPs (HMM-Fu-PPs) also exhibit significantly enhanced anticoagulation performance with a 72 % decrease in thrombus weight compared with G-PP in ex-vivo shunt assay, along with the superior biocompatibility, satisfactory anti-calcification properties confirmed by subcutaneous implantation. Owing to good comprehensive performance of these HMM-Fu-PPs, this simple and feasible strategy may offer a great potential for BHV fabrication in the future, and open a new avenue to explore more N-hydroxymethyl compound based crosslinker with excellent performance in the field of biomaterials.

Synthesis of bioengineered heparin chemically and biologically similar to porcine-derived products and convertible to low MW heparin.

Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases. Chemoenzymatic synthesis of animal-free heparin products would offer several advantages, including reliable and scalable production processes, improved purity and consistency, and the ability to produce heparin polysaccharides with molecular weight, structural, and functional properties equivalent to those of the United States Pharmacopeia (USP) heparin, currently only sourced from porcine intestinal mucosa. We report a scalable process for the production of bioengineered heparin that is biologically and compositionally similar to USP heparin. This process relies on enzymes from the heparin biosynthetic pathway, immobilized on an inert support and requires a tailored N-sulfoheparosan with N-sulfo levels similar to those of porcine heparins. We also report the conversion of our bioengineered heparin into a LMWH that is biologically and compositionally similar to USP enoxaparin. Ultimately, we demonstrate major advances to a process to provide a potential clinical and sustainable alternative to porcine-derived heparin products.

Recent advances in the design and immobilization of heparin for biomedical application: A review.

Heparin, a member of the glycosaminoglycan family, is renowned as the most negatively charged biomolecule discovered within the realm of human biology. This polysaccharide serves a vital role as a regulator for various proteins, cells, and tissues within the human body, positioning itself as a pivotal macromolecule of significance. The domain of biology has witnessed substantial interest in the intricate design of heparin and its derivatives, particularly focusing on heparin-based polymers and hydrogels. This intrigue spans a wide spectrum of applications, encompassing diverse areas such as protein adsorption, anticoagulant properties, controlled drug release, development of implants, stent innovation, enhancement of blood compatibility, acceleration of wound healing, and pioneering strides in tissue engineering. This comprehensive overview delves into a multitude of developed heparin conjugates, employing various methods, and explores their functions in both the biomedicine and electronics fields. The efficacy of materials derived from heparin is also thoroughly investigated, encompassing considerations such as thrombogenicity, drug release kinetics, affinity for growth factors (GFs), biocompatibility, and electrochemical analyses. We firmly believe that by redirecting focus towards research and advancements in heparin-related polymers/hydrogels, this study will ignite further research and accelerate potential breakthroughs in this promising and evolving field of discovery.

Structural and biofunctional diversity of sulfated polysaccharides from the genus Codium (Bryopsidales, Chlorophyta): A review.

It is believed that polysaccharides will become a focal point for future production of food, pharmaceuticals, and materials due to their ubiquitous and renewable nature, as well as their exceptional properties that have been extensively validated in the fields of nutrition, healthcare, and materials. Sulfated polysaccharides derived from seaweed sources have attracted considerable attention owing to their distinctive structures and properties. The genus Codium, represented by the species C. fragile, holds significance as a vital economic green seaweed and serves as a traditional Chinese medicinal herb. To date, the cell walls of the genus Codium have been found to contain at least four types of sulfated polysaccharides, specifically pyruvylated ß-d-galactan sulfates, sulfated arabinogalactans, sulfated ß-l-arabinans, and sulfated ß-d-mannans. These sulfated polysaccharides exhibit diverse biofunctions, including anticoagulant, immune-enhancing, anticancer, antioxidant activities, and drug-carrying capacity. This review explores the structural and biofunctional diversity of sulfated polysaccharides derived from the genus Codium. Additionally, in addressing the impending challenges within the industrialization of these polysaccharides, encompassing concerns regarding scale-up production and quality control, we outline potential strategies to address these challenges from the perspectives of raw materials, extraction processes, purification technologies, and methods for quality control.

Crystal structure of tick tyrosylprotein sulfotransferase reveals the activation mechanism of the tick anticoagulant protein madanin.

Ticks pose a substantial public health risk as they transmit various pathogens. This concern is related to the adept blood-sucking strategy of ticks, underscored by the action of the anticoagulant, madanin, which is known to exhibit an approximately 1000-fold increase in anticoagulant activity following sulfation of its two tyrosine residues, Tyr51 and Tyr54. Despite this knowledge, the molecular mechanism underlying sulfation by tick tyrosylprotein sulfotransferase (TPST) remains unclear. In this study, we successfully prepared tick TPST as a soluble recombinant enzyme. We clarified the method by which this enzyme proficiently sulfates tyrosine residues in madanin. Biochemical analysis using a substrate peptide based on madanin and tick TPST, along with the analysis of the crystal structure of the complex and docking simulations, revealed a sequential sulfation process. Initial sulfation at the Tyr51 site augments binding, thereby facilitating efficient sulfation at Tyr54. Beyond direct biochemical implications, these findings considerably improve our understanding of tick blood-sucking strategies. Furthermore, combined with the utility of modified tick TPST, our findings may lead to the development of novel anticoagulants, promising avenues for thrombotic disease intervention and advancements in the field of public health.

Structure and Binding Properties to Blood Co-Factors of the Least Sulfated Galactan Found in the Cell Wall of the Red Alga Botryocladia occidentalis.

Three different populations of sulfated polysaccharides can be found in the cell wall of the red alga Botryocladia occidentalis. In a previous work, the structures of the two more sulfated polysaccharides were revised. In this work, NMR-based structural analysis was performed on the least sulfated polysaccharide and its chemically modified derivatives. Results have revealed the presence of both 4-linked α- and 3-linked ß-galactose units having the following chemical features: more than half of the total galactose units are not sulfated, the α-units occur primarily as 3,6-anhydrogalactose units either 2-O-methylated or 2-O-sulfated, and the ß-galactose units can be 4-O-sulfated or 2,4-O-disulfated. SPR-based results indicated weaker binding of the least sulfated galactan to thrombin, factor Xa, and antithrombin, but stronger binding to heparin cofactor II than unfractionated heparin. This report together with our previous publication completes the structural characterization of the three polysaccharides found in the cell wall of the red alga B. occidentalis and correlates the impact of their composing chemical groups with the levels of interaction with the blood co-factors.

Structural characterization of a distinct fucan sulfate from Pattalus mollis through an oligosaccharide mapping approach.

The elucidation of the precise structure of fucan sulfate is essential for understanding the structure-activity relationship and promoting potential biomedical applications. In this work, the structure of a distinct fucan sulfate fraction V (PmFS in Ref 15 and FSV in Ref 16 → PFV) from Pattalus mollis was investigated using an oligosaccharide mapping approach. Six size-homogeneous fractions were purified from the mild acid hydrolyzed PFV and identified as fucitols, disaccharides and trisaccharides by 1D/2D NMR and MS analysis. Significantly, the sulfation pattern, glycosidic linkages, and sequences of all the oligosaccharides were unambiguously identified. The common 2-desulfation of the reducing end residue of the oligosaccharides was observed. Overall, the backbone of PFV was composed of L-Fuc2S (major) and L-Fuc3S (minor) linked by α1,4 glycosidic bonds. Importantly, the branches contain both monosaccharide and disaccharide linked to the backbone by α1,3 glycosidic linkages. Thus, the tentative structure of natural PFV was shown to be {-(R-α1,3)-L-Fuc2S-α1,4-(L-Fuc2S/3S-α1,4)x-}n, where R is L-Fuc(2S)4S-α1,3/4-L-Fuc4S(0S)- or L-Fuc(2S)4S-. Our results provide insight into the heterogeneous structure of the fucan sulfate found in sea cucumbers. Additionally, PFV and its fractions showed strong anticoagulant and anti-iXase activities, which may be related to the distinct structure of PFV.

Daboialipase, a phospholipase A2 from Vipera russelli russelli venom posesses anti-platelet, anti-thrombin and anti-cancer properties.

Snake venoms are known to contain toxins capable of interfering with normal physiological processes of victims. Specificity of toxins from snake venoms give scope to identify new molecules with therapeutic action and/or help to understand different cellular mechanisms. Russell's viper venom (RVV) is a mixture of many bioactive molecules with enzymatic and non-enzymatic proteins. The present article describes Daboialipase (DLP), an enzymatic phospholipase A2 with molecular mass of 14.3 kDa isolated from RVV. DLP was obtained after cation exchange chromatography followed by size-exclusion high performance liquid chromatography (SE-HPLC). The isolated DLP presented strong inhibition of adenosine di-phosphate (ADP) and collagen induced platelet aggregation. It also showed anti-thrombin properties by significantly extending thrombin time in human blood samples. Trypan blue and resazurin cell viability assays confirmed time-dependent cytotoxic and cytostatic activities of DLP on MCF7 breast cancer cells, in vitro. DLP caused morphological changes and nuclear damage in MCF7 cells. However, DLP did not cause cytotoxic effects on non-cancer HaCaT cells. Peptide sequences of DLP obtained by O-HRLCMS analysis showed similarity with a previously reported PLA2 (Uniprot ID: PA2B_DABRR/PDB ID: 1VIP_A). An active Asp at 49th position, calcium ion binding site and anticoagulant activity sites were identified in 1 VIP_A. These findings are expected to contribute to designing new anti-platelet, anticoagulant and anti-cancer molecules.

TFPIα anticoagulant function is highly dependent on protein S in vivo.

Tissue factor pathway inhibitor α (TFPIα) is the major physiological regulator of the initiation of blood coagulation. In vitro, TFPIα anticoagulant function is enhanced by its cofactor, protein S. To define the role of protein S enhancement in TFPIα anticoagulant function in vivo, we blocked endogenous TFPI in mice using a monoclonal antibody (14D1). This caused a profound increase in fibrin deposition using the laser injury thrombosis model. To explore the role of plasma TFPIα in regulating thrombus formation, increasing concentrations of human TFPIα were coinjected with 14D1, which dose-dependently reduced fibrin deposition. Inhibition of protein S cofactor function using recombinant C4b-binding protein ß chain significantly reduced the anticoagulant function of human TFPIα in controlling fibrin deposition. We report an in vivo model that is sensitive to the anticoagulant properties of the TFPIα-protein S pathway and show the importance of protein S as a cofactor in the anticoagulant function of TFPIα in vivo.