Quality control, safety assessment and preparation approaches of low molecular weight heparin.

Low Molecular Weight Heparins (LMWHs) are well-established for use in the prevention and treatment of thrombotic diseases, and as a substitute for unfractionated heparin (UFH) due to their predictable pharmacokinetics and subcutaneous bioavailability. LMWHs are produced by various depolymerization methods from UFH, resulting in heterogeneous compounds with similar biochemical and pharmacological properties. However, the delicate supply chain of UFH and potential contamination from animal sources require new manufacturing approaches for LMWHs. Various LMWH preparation methods are emerging, such as chemical synthesis, enzymatic or chemical depolymerization and chemoenzymatic synthesis. To establish the sameness of active ingredients in both innovator and generic LMWH products, the Food and Drug Administration has implemented a stringent scientific method of equivalence based on physicochemical properties, heparin source material and depolymerization techniques, disaccharide composition and oligosaccharide mapping, biological and biochemical properties, and in vivo pharmacodynamic profiles. In this review, we discuss currently available LMWHs, potential manufacturing methods, and recent progress for manufacturing quality control of these LMWHs.

Engineering and evaluation of FXa bypassing agents that restore hemostasis following Apixaban associated bleeding.

Direct oral anticoagulants (DOACs) targeting activated factor Xa (FXa) are used to prevent or treat thromboembolic disorders. DOACs reversibly bind to FXa and inhibit its enzymatic activity. However, DOAC treatment carries the risk of anticoagulant-associated bleeding. Currently, only one specific agent, andexanet alfa, is approved to reverse the anticoagulant effects of FXa-targeting DOACs (FXaDOACs) and control life-threatening bleeding. However, because of its mechanism of action, andexanet alfa requires a cumbersome dosing schedule, and its use is associated with the risk of thrombosis. Here, we present the computational design, engineering, and evaluation of FXa-variants that exhibit anticoagulation reversal activity in the presence of FXaDOACs. Our designs demonstrate low DOAC binding affinity, retain FXa-enzymatic activity and reduce the DOAC-associated bleeding by restoring hemostasis in mice treated with apixaban. Importantly, the FXaDOACs reversal agents we designed, unlike andexanet alfa, do not inhibit TFPI, and consequently, may have a safer thrombogenic profile.

Factors Affecting the Discrepancy Between Coagulation Times on Extracorporeal Circulation Using Unfractionated Heparin in Children and Young Adults.

In unfractionated heparin (UFH) monitoring during extracorporeal circulation, the traditional measures of activated clotting time (ACT) or activated partial thromboplastin time (APTT) may diverge, confounding anticoagulant adjustments. We aimed to explore the factors explaining this discrepancy in children and young adults. This retrospective observational study, conducted at an urban regional tertiary hospital, included consecutive pediatric patients who received UFH during extracorporeal circulation (continuous kidney replacement therapy or extracorporeal membrane oxygenation) between April 2017 and March 2021. After patients whose ACT and APTT were not measured simultaneously or who were also taking other anticoagulants were excluded, we analyzed 94 samples from 23 patients. To explain the discrepancy between ACT and APTT, regression equations were created using a generalized linear model (family = gamma, link = logarithmic) with ACT as the response variable. Other explanatory variables included age, platelet count, and antithrombin. Compared to APTT alone as an explanatory variable, the Akaike information criterion and pseudo-coefficient of determination improved from 855 to 625 and from 0.01 to 0.42, respectively, when these explanatory variables were used. In conclusion, we identified several factors that may explain some of the discrepancy between ACT and APTT in the routinely measured tests. Evaluation of these factors may aid in appropriate adjustments in anticoagulation therapy.

Evaluating anthelmintic, anti-platelet, and anti-coagulant activities, and identifying the bioactive phytochemicals of Amaranthus blitum L.

BACKGROUND: Highlighting affordable alternative crops that are rich in bioactive phytoconstituents is essential for advancing nutrition and ensuring food security. Amaranthus blitum L. (AB) stands out as one such crop with a traditional history of being used to treat intestinal disorders, roundworm infections, and hemorrhage. This study aimed to evaluate the anthelmintic and hematologic activities across various extracts of AB and investigate the phytoconstituents responsible for these activities. METHODS: In vitro anthelmintic activity against Trichinella spiralis was evaluated in terms of larval viability reduction. The anti-platelet activities were assessed based on the inhibitory effect against induced platelet aggregation. Further, effects on the extrinsic pathway, the intrinsic pathway, and the ultimate common stage of blood coagulation, were monitored through measuring blood coagulation parameters: prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT), respectively. The structures of isolated compounds were elucidated by spectroscopic analysis. RESULTS: Interestingly, a previously undescribed compound (19), N-(cis-p-coumaroyl)-ʟ-tryptophan, was isolated and identified along with 21 known compounds. Significant in vitro larvicidal activities were demonstrated by the investigated AB extracts at 1 mg/mL. Among tested compounds, compound 18 (rutin) displayed the highest larvicidal activity. Moreover, compounds 19 and 20 (N-(trans-p-coumaroyl)-ʟ-tryptophan) induced complete larval death within 48 h. The crude extract exhibited the minimal platelet aggregation of 43.42 ± 11.69%, compared with 76.22 ± 14.34% in the control plasma. Additionally, the crude extract and two compounds 19 and 20 significantly inhibited the extrinsic coagulation pathway. CONCLUSIONS: These findings extend awareness about the nutritional value of AB as a food, with thrombosis-preventing capabilities and introducing a promising source for new anthelmintic and anticoagulant agents.

The effect of preoperative use of anticoagulants on the hemostatic effect of intravenous application of tranexamic acid in PLIF: a case control study.

Intravenous application of tranexamic acid (TXA) in posterior lumbar interbody fusion (PLIF) can effectively reduce blood loss without affecting coagulation function. However, it has not been reported whether preoperative use of anticoagulants may affect the efficacy of TXA in PLIF. The purpose of this study is to observe the effect of preoperative use of anticoagulants on coagulation indicators and blood loss after PLIF receiving intravenous unit dose TXA. A retrospective analysis was conducted on data from 53 patients with PLIF between 2020.11 and 2022.9, who received intravenous application of a unit dose of TXA (1 g/100 mL) 15 min before the skin incision after general anesthesia. Those who used anticoagulants within one week before surgery were recorded as the observation group, while those who did not use anticoagulants were recorded as the control group. The main observation indicators include surgical time, intraoperative blood loss, postoperative drainage volume, blood transfusion, and red blood cell (RBC), hemoglobin (HB), and hematocrit (HCT) measured on the 1st, 4th, 7th, and last-test postoperative days. Secondary observation indicators included postoperative incision healing, deep vein thrombosis of lower limbs, postoperative hospital stay, and activated partial thrombin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen (FIB), and platelets (PLT) on the 1st and 4th days after surgery. The operation was successfully completed in both groups, the incision healed well after operation, and no lower limb deep vein thrombosis occurred. There was no significant difference in surgical time, intraoperative blood loss, postoperative drainage volume, and blood transfusion between the two groups (p > 0.05). There was no significant difference in the RBC, HB, and HCT measured on the 1st, 4th, 7th, and last-test postoperative days between the two groups (p > 0.05). There was no statistically significant difference in APTT, PT, TT, FIB and PLT between the two groups on the 1st and 4th postoperative days (p > 0.05). There was no significant difference in postoperative hospital stay between the two groups (p > 0.05). The use of anticoagulants within one week before surgery does not affect the hemostatic effect of intravenous unit dose TXA in PLIF.

Interactions between antiepileptic drugs and direct oral anticoagulants for primary and secondary stroke prevention.

INTRODUCTION: Direct oral anticoagulants (DOAC) are the guideline-recommended therapy for prevention of stroke in atrial fibrillation (AF) and venous thromboembolism. Since approximately 10% of patients using antiepileptic drugs (AED) also receive DOAC, aim of this review is to summarize data about drug-drug interactions (DDI) of DOAC with AED by using data from PubMed until December 2023. AREAS COVERED: Of 49 AED, only 16 have been investigated regarding DDI with DOAC by case reports or observational studies. No increased risk for stroke was reported only for topiramate, zonisamide, pregabalin, and gabapentin, whereas for the remaining 12 AED conflicting results regarding the risk for stroke and bleeding were found. Further 16 AED have the potential for pharmacodynamic or pharmacokinetic DDI, but no data regarding DOAC are available. For the remaining 17 AED it is unknown if they have DDI with DOAC. EXPERT OPINION: Knowledge about pharmacokinetic and pharmacodynamic DDI of AED and DOAC is limited and frequently restricted to in vitro and in vivo findings. Since no data about DDI with DOAC are available for 67% of AED and an increasing number of patients have a combined medication of DOAC and AED, there is an urgent need for research on this topic.

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.

Antidote-controlled DNA aptamer modulates human factor IXa activity.

Thrombosis leads to elevated mortality rates and substantial medical expenses worldwide. Human factor IXa (HFIXa) protease is pivotal in tissue factor (TF)-mediated thrombin generation, and represents a promising target for anticoagulant therapy. We herein isolated novel DNA aptamers that specifically bind to HFIXa through systematic evolution of ligands by exponential enrichment (SELEX) method. We identified two distinct aptamers, seq 5 and seq 11, which demonstrated high binding affinity to HFIXa (Kd = 74.07 ± 2.53 nM, and 4.93 ± 0.15 nM, respectively). Computer software was used for conformational simulation and kinetic analysis of DNA aptamers and HFIXa binding. These aptamers dose-dependently prolonged activated partial thromboplastin time (aPTT) in plasma. We further rationally optimized the aptamers by truncation and site-directed mutation, and generated the truncated forms (Seq 5-1t, Seq 11-1t) and truncated-mutated forms (Seq 5-2tm, Seq 11-2tm). They also showed good anticoagulant effects. The rationally and structurally designed antidotes (seq 5-2b and seq 11-2b) were competitively bound to the DNA aptamers and effectively reversed the anticoagulant effect. This strategy provides DNA aptamer drug-antidote pair with effective anticoagulation and rapid reversal, developing advanced therapies by safe, regulatable aptamer drug-antidote pair.

Intermediate molecular weight-fucosylated chondroitin sulfate from sea cucumber Cucumaria frondosa is a promising anticoagulant targeting intrinsic factor IXa.

Thromboembolic diseases pose a serious risk to human health worldwide. Fucosylated chondroitin sulfate (FCS) is reported to have good anticoagulant activity with a low bleeding risk. Molecular weight plays a significant role in the anticoagulant activity of FCS, and FCS smaller than octasaccharide in size has no anticoagulant activity. Therefore, identifying the best candidate for developing novel anticoagulant FCS drugs is crucial. Herein, native FCS was isolated from sea cucumber Cucumaria frondosa (FCScf) and depolymerized into a series of lower molecular weights (FCScfs). A comprehensive assessment of the in vitro anticoagulant activity and in vivo bleeding risk of FCScfs with different molecule weights demonstrated that 10 kDa FCScf (FCScf-10 K) had a greater intrinsic anticoagulant activity than low molecular weight heparin (LMWH) without any bleeding risk. Using molecular modeling combined with experimental validation, we revealed that FCScf-10 K can specifically inhibit the formation of the Xase complex by binding the negatively charged sulfate group of FCScf-10 K to the positively charged side chain of arginine residues on the specific surface of factor IXa. Thus, these data demonstrate that the intermediate molecular weight FCScf-10 K is a promising candidate for the development of novel anticoagulant drugs.

Carbonization of quercetin into nanogels: a leap in anticoagulant development.

Quercetin, a flavonoid abundantly found in onions, fruits, and vegetables, is recognized for its pharmacological potential, especially for its anticoagulant properties that work by inhibiting thrombin and coagulation factor Xa. However, its clinical application is limited due to poor water solubility and bioavailability. To address these limitations, we engineered carbonized nanogels derived from quercetin (CNGsQur) using controlled pyrolysis and polymerization techniques. This led to substantial improvements in its anticoagulation efficacy, water solubility, and biocompatibility. We generated a range of CNGsQur by subjecting quercetin to varying pyrolytic temperatures and then assessed their anticoagulation capacities both in vitro and in vivo. Coagulation metrics, including thrombin clotting time (TCT), activated partial thromboplastin time (aPTT), and prothrombin time (PT), along with a rat tail bleeding assay, were utilized to gauge the efficacy. CNGsQur showed a pronounced extension of coagulation time compared to uncarbonized quercetin. Specifically, CNGsQur synthesized at 270 °C (CNGsQur270) exhibited the most significant enhancement in TCT, with a binding affinity to thrombin exceeding 400 times that of quercetin. Moreover, variants synthesized at 310 °C (CNGsQur310) and 290 °C (CNGsQur290) showed the most substantial delays in PT and aPTT, respectively. Our findings indicate that the degree of carbonization significantly influences the transformation of quercetin into various CNGsQur forms, each affecting distinct coagulation pathways. Additionally, both intravenous and oral administrations of CNGsQur were found to extend rat tail bleeding times by up to fivefold. Our studies also demonstrate that CNGsQur270 effectively delays and even prevents FeCl3-induced vascular occlusion in a dose-dependent manner in mice. Thus, controlled pyrolysis offers an innovative approach for generating quercetin-derived CNGs with enhanced anticoagulation properties and water solubility, revealing the potential for synthesizing self-functional carbonized nanomaterials from other flavonoids for diverse biomedical applications.

Fangs and foliage: Unearthing the haemotoxic secrets of cannabis-dwelling rattlesnakes.

Despite a recent surge in high-throughput venom research that has enabled many species to be studied, some snake venoms remain understudied. The long-tailed rattlesnakes (Crotalus ericsmithi, C. lannomi, and C. stejnegeri) are one group where such research lags, largely owing to the rarity of these snakes and the hazardous areas, ripe with drug (marijuana and opium) production, they inhabit in Mexico. To fill this knowledge gap, we used multiple functional assays to examine the coagulotoxic (including across different plasma types), neurotoxic, and myotoxic activity of the venom of the long-tailed rattlesnakes. All crude venoms were shown to be potently anticoagulant on human plasma, which we discovered was not due to the destruction of fibrinogen, except for C. stejnegeri displaying minor fibrinogen destruction activity. All venoms exhibited anticoagulant activity on rat, avian, and amphibian plasmas, with C. ericsmithi being the most potent. We determined the mechanism of anticoagulant activity by C. ericsmithi and C. lannomi venoms to be phospholipid destruction and inhibition of multiple coagulation factors, leading to a net disruption of the clotting cascade. In the chick biventer assay, C. ericsmithi and C. lannomi did not exhibit neurotoxic activity but displayed potential weak myotoxic activity. BIRMEX® (Faboterápico Polivalente Antiviperino) antivenom was not effective in neutralising this venom effect. Overall, this study provides an in-depth investigation of venom function of understudied long-tailed rattlesnakes and provides a springboard for future venom and ecology research on the group.

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.

The natural anticoagulant protein S; hemostatic functions and deficiency.

Protein S (PS) is a vital endogenous anticoagulant. It plays a crucial role in regulating coagulation by acting as a cofactor for the activated protein C (APC) and tissue factor pathway inhibitor (TFPI) pathways. Additionally, it possesses direct anticoagulant properties by impeding the intrinsic tenase and prothrombinase complexes. Protein S oversees the coagulation process in both the initiation and propagation stages through these roles. The significance of protein S in regulating blood clotting can be inferred from the significant correlation between deficits in protein S and an elevated susceptibility to venous thrombosis. This is likely because activated protein C and tissue factor pathway inhibitor exhibit low efficacy as anticoagulants when no cofactors exist. The precise biochemical mechanisms underlying the roles of protein S cofactors have yet to be fully elucidated. Nevertheless, recent scientific breakthroughs have significantly enhanced comprehension findings for these functions. The diagnosis of protein S deficiency, both from a technical and genetic standpoint, is still a subject of debate due to the complex structural characteristics of the condition. This paper will provide an in-depth review of the molecular structure of protein S and its hemostatic effects. Furthermore, we shall address the insufficiency of protein S and its methods of diagnosis and treatment.

What is the purpose of this summary? To provide an in-depth review of the molecular structure of protein S and its hemostatic effects.To address the deficiency of protein S and its methods of diagnosis and treatment.What is known? Protein S operates as an anticoagulant through its roles as a cofactor for APC, TFPI, and an inhibitor of FIXa.Protein S deficiency can be either inherited or acquired.What is new? Plasma protein S and platelet-derived protein S contribute to regulating coagulation and maintaining hemostasis. Protein S can be used as a potential promising treatment target for persons diagnosed with hemophilia.

Storage of plasma-derived exosomes: evaluation of anticoagulant use and preserving temperatures.

Exosomes carry large cargo of proteins, lipids, and nucleic acids, serving as versatile biomarkers for disease diagnosis and vehicles for drug delivery. However, up to date, no well recognized standard procedures for exosome storage were available for clinical application. This study aimed to determine the optimal storage conditions and the anticoagulants for plasma-derived exosome isolation. Fresh whole blood samples were collected from healthy participants and preserved in four different anticoagulants including sodium citrate (SC1/4), sodium citrate (SC1/9), lithium heparin (LH), or Ethylenediamine tetraacetic acid (EDTA), respectively. Exosomes were extracted from the plasma by differential ultracentrifugation and stored at three different temperatures, 4°C, -20°C or - 80°C for a duration ranging from one week to six months. All plasma samples for storage conditions comparison were pretreated with LH anticoagulant. Exosome features including morphological characteristics, pariticles size diameter, and surface protein profiles (TSG101, CD63, CD81, CD9, CALNEXIN) were assessed by transmission electron microscopy, Nanoparticle Tracking Analysis, and Western Blotting, respectively. Exosomes preserved in LH and SC1/4 group tended to remain intact microstructure with highly abundant protein biomarkers. Exosomes stored at 4°C for short time were prone to be more stable compared to thos at -80°C. Exosomes stored in plasma were superior in terms of ultrastructure, size diameter and surface protein expression to those stored in PBS. In conclusion, plasma-dervied exosome characteristics strictly depend on the anticoagulants and storage temperature and duration.

What is the context? Effective isolation of exosomes is a prerequisite for subsequent investigation into its involvemnt in disease development as well as potentialtherapeutic applications.Anticoagulants, storage temperature and durations might change the microscopical structure, integrity and also the stability of plasma-derived exosomes. However, no internationally recognized standard of exosome storage procedure was available for clinical use.What is new? Our finding evaluated the effect of anticoagulants and storage on plasma exosome characteristics.Exosomes isolated from plasma preserved with Li-heparin and sodium citrate (1/4) showed better physical properties and surface marker protein expression.Isolated exosomes appeared more stable in a short time for 4°C compared to −80°C. Storage of exosomes in plasma showed better physical properties and surface marker protein expression than in PBS.What is the impact? Our findings inform the significance of standardizing procedure of exosome isolation and preservation.

[New anticoagulants in 2024: Development of factor XI and XIa inhibitors]. / Les nouveaux anticoagulants en 2024 : développement des inhibiteurs du facteur XI et du XIa.

Thrombosis remains one of the leading causes of death in the world. The history of anticoagulation has evolved considerably from non-specific drugs (i.e., heparins and vitamin K antagonists, VKA) to agents that directly target specific coagulation factors (i.e., argatroban, fondaparinux and direct oral anticoagulants, DOAC). Since the last decade, DOAC are widely used in clinical practice because of their ease to use, their favorable pharmacological profile and the fact that they do not require monitoring. However, despite having a better safety profile than vitamin K antagonist, their bleeding risk is not negligible. New anticoagulants targeting the contact phase of coagulation are currently being developed and could make it possible to prevent the risk of thrombosis without impairing hemostasis. Epidemiological and preclinical data on FXI deficiency make FXI a promising therapeutic target. The aim of this review is to summarize the results of the various clinical trials available that focus on FXI/FXIa inhibition, and to highlight the challenges that this new therapeutic class of anticoagulants will face.

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.

A Biodegradable Tissue Adhesive for Post-Extraction Alveolar Bone Regeneration under Ongoing Anticoagulation-A Microstructural Volumetric Analysis in a Rodent Model.

In addition to post-extraction bleeding, pronounced alveolar bone resorption is a very common complication after tooth extraction in patients undergoing anticoagulation therapy. The novel, biodegenerative, polyurethane adhesive VIVO has shown a positive effect on soft tissue regeneration and hemostasis. However, the regenerative potential of VIVO in terms of bone regeneration has not yet been explored. The present rodent study compared the post-extraction bone healing of a collagen sponge (COSP) and VIVO in the context of ongoing anticoagulation therapy. According to a split-mouth design, a total of 178 extraction sockets were generated under rivaroxaban treatment, of which 89 extraction sockets were treated with VIVO and 89 with COSP. Post-extraction bone analysis was conducted via in vivo micro-computed tomography (µCT), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX) after 5, 10, and 90 days. During the observation time of 90 days, µCT analysis revealed that VIVO and COSP led to significant increases in both bone volume and bone density (p ≤ 0.001). SEM images of the extraction sockets treated with either VIVO or COSP showed bone regeneration in the form of lamellar bone mass. Ratios of Ca/C and Ca/P observed via EDX indicated newly formed bone matrixes in both treatments after 90 days. There were no statistical differences between treatment with VIVO or COSP. The hemostatic agents VIVO and COSP were both able to prevent pronounced bone loss, and both demonstrated a strong positive influence on the bone regeneration of the alveolar ridge post-extraction.

Andexanet Alfa Neutralizes the Anticoagulant Effects of Unfractionated Heparin of Bovine, Ovine and Porcine Origin Almost as Protamine Sulfate.

INTRODUCTION: Andexanet alfa (AA) - zhzo, recombinant coagulation factor Xa, is an approved antidote for oral Xa inhibitors (apixaban and rivaroxaban). Unfractionated heparin (UFH) is commonly used for therapeutic, interventional, and surgical indications. Protamine sulfate (PrSO4) is frequently used to neutralize UFH. This study aimed to investigate the comparative neutralization profiles of AA and PrSO4 for heparins of bovine, ovine, and porcine origin. MATERIALS AND METHODS: The neutralization effect of PrSO4 at 25 µg/ml and AA at 100 µg/ml was studied on an approximate surgical/interventional concentration of heparin by supplementing whole blood with each of the heparins at 25 µg/ml. For the clotting profile (activated partial thromboplastin time: aPTT), amidolytic (anti-Xa and anti-IIa), and thrombin generation assay each of the heparin were supplemented from -10-0.62 µg/ml. RESULTS: In the whole blood ACT studies, all three heparins produced strong anti-coagulant effects (400-450 seconds) compared to saline (130-150 seconds). Both AA and PrSO4 almost fully neutralized the anti-coagulant effects of heparins (140-160 seconds). Both antidotes completely reversed the anticoagulant effects of all three heparins in the aPTT and thrombin generation assay. However, PrSO4 was more effective in neutralizing the anti-Xa, and anti-IIa effects than AA, which only partially neutralized these effects. CONCLUSION: Andexanet alfa at 100 µg/ml effectively neutralizes the therapeutic and surgical/interventional concentrations of heparins in in-vitro settings. While differences in the anti-Xa, and anti-IIa effects between heparins were noted, anti-coagulant effect of these agents in the aPTT assay were comparable. A similar neutralization profile was observed in the ACT and thrombin generation assays by both agents.

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.