Intrinsic Coagulation Pathway Research Articles

Biocompatible sulfenamide and sulfonamide derivatives of metformin can exert beneficial effects on plasma haemostasis

As the pharmacokinetic properties of metformin are unfavourable, several analogues and prodrugs have been synthesised to improve its bioavailability. The aim of this study was to assess the plasma stability of sulfenamide and sulfonamide derivatives of metformin and establish their effects on plasma haemostasis and integrity of red blood cells (RBCs).The overall haemostasis potential was evaluated spectrophotometrically by clot formation and lysis test (CL-test). PT (Prothrombin Time) and APTT (Activated Partial Tromboplastin Time) were used to evaluate the effects if the compounds on the extrinsic and intrinsic coagulation pathway. Haemolysis assay, microscopy and flow cytometry studies were conducted to determine the effect of the compounds on RBCs.Two sulfonamide and one sulfenamide derivatives of metformin were associated with a statistically significant decrease in the overall potential of clot formation and fibrinolysis (↓ CLAUC), suggesting that these compounds may exert beneficial effects regarding plasma haemostasis, which is frequently impaired in diabetic patients. p- and o-Nitrobenzene sulfonamides contributed to the beneficial change in kinetic parameters of clot formation and fibrinolysis. o-Nitrobenzene sulfonamide significantly increased thrombin generation time (↑ TGt) and was also found to prolong both APTT and PT. All compounds did not exert any effects on the integrity of RBCs over the concentration range 0.006–0.6 μmol/mL which constitutes the expected therapeutic concentration.In conclusion, sulfonamide derivatives of metformin present potentially beneficial properties in terms of plasma haemostasis which is frequently impaired in T2DM patients. Therefore, metformin sulfonamides may become a prototype for further design and synthesis of novel metformin analogues and prodrugs with improved pharmacokinetic properties.

Inhibition of intrinsic coagulation improves safety and tumor-targeted drug delivery of cationic solid lipid nanoparticles

Cationic solid lipid nanoparticles (cSLNs) are promising nanoparticles for controlled drug delivery. Increasing surface charge and/or reducing PEG density enhance cellular uptake of cSLNs in vitro, but for unknown reasons fail to improve drug delivery in vivo. Herein, we show that cSLNs present a risk for systemic platelet activation and aggregation in vivo, and this toxic effect can be significantly augmented by increasing the surface charge and reducing the PEG density. Furthermore, thrombotic toxicity significantly reduces blood circulation time and in vivo cellular uptake of cSLNs. Mechanistic studies revealed that the intrinsic coagulation pathway is responsible for cSLN-induced platelet activation. Importantly, pretreatment of the recipient mice with heparin, a clinically-approved intrinsic coagulation inhibitor, was highly effective in preventing toxicity, prolonging the circulation time of cSLNs, and improving cSLN-based antitumor drug delivery and therapeutic efficacy in tumor-bearing mice. This study offers a useful strategy for improving both the safety and efficacy of cSLN-based anticancer therapies.

Coagulation Testing in the Core Laboratory.

Primary hemostasis begins with endothelial injury. VWF, produced by endothelial cells, binds to platelets and links them to subendothelial collagen. Platelet-derived ADP and thromboxane activate non-adhered platelets via their GPIIb/IIIa receptors, allowing these platelets to participate in platelet aggregation. Secondary hemostasis is initiated with the binding of factor VII to extravascular tissue factor (TF). Factors II, VII, IX and X are vitamin K-dependent factors. The role of vitamin K is to assist in the addition of gamma carboxylate groups to glutamic acids in the "GLA" domains of these factors.In vitro the intrinsic pathway is initiated when fresh whole blood is placed in a glass tube. The negative charge of the glass initiates the "contact pathway" where FXII is activated and then FXIa cleaves FIX to FIXa. The extrinsic pathway is triggered when tissue factor, phospholipid and calcium are added to plasma anticoagulated with citrate. In vitro, FVII is activated to FVIIa, and TF-FVIIa preferentially converts FX to FXa activating the common pathway.The prothrombin time is commonly used to monitor warfarin anticoagulant therapy. To correct for differences in reagent and instrument, the international normalized ratio was developed to improve standardization of PT reporting globally. The activated partial thromboplastin time (aPTT) is used to evaluate the intrinsic and common pathways of coagulation. The aPTT is useful clinically as a screening test for inherited and acquired factor deficiencies as well as to monitor unfractionated heparin therapy although the anti-Xa assay is now the preferred measure of the effects of unfractionated heparin. The Clauss assay is the most commonly performed fibrinogen assay and uses diluted plasma where clotting is initiated with a high concentration of reagent thrombin.The mixing study assists in the assessment of an abnormally prolonged PT or aPTT. An equal volume of citrated patient plasma is mixed with normal pooled plasma and the PT or aPTT are repeated on the 1:1 mix. Factor activity assays are most commonly performed as a one-stage assay. The patient's citrated plasma is diluted and mixed 1-to-1 with a single factor-deficient substrate plasma. A PT or aPTT is performed on the above mix, depending on the factor being tested.Factor inhibitors are antibodies that are most commonly diagnosed in male patients with severe hemophilia A (FVIII deficiency) where they are induced by factor replacement therapy.Factor inhibitors can also appear in the form of spontaneous autoantibodies in both male and female individuals who were previously well. This is an autoimmune condition called "acquired hemophilia."Most coagulation laboratories can measure the plasma concentration of VWF protein (VWF antigen) by an immunoturbidimetric technique. Testing the functional activity of VWF, utilizes the drug ristocetin.The state of multimerization of VWF is important and is assessed by electrophoresis on agarose gels. Type 2a and 2b VWD are associated with the lack of intermediate- and high molecular weight multimers.The antiphospholipid syndrome (APLS) is an acquired autoimmune phenomenon associated with an increased incidence of both venous and arterial thromboses, as well as fetal loss. Typically, there is a paradoxical prolongation of the aPTT in the absence of any clinical features of bleeding. This is the so-called "lupus anticoagulant (LA) effect." The laboratory definition of the APLS requires the presence of either a "lupus anticoagulant" or a persistent titer of antiphospholipid antibodies.There are now 2 broad classes of direct-acting oral anticoagulants (DOACs): [1] The oral direct thrombin inhibitors (DTIs) such as dabigatran; and [2] The oral direct factor Xa inhibitors such as rivaroxaban and apixaban. The PT and aPTT are variably affected by the DOACs and are generally unhelpful in monitoring their concentrations. Most importantly, a normal PT or aPTT does NOT exclude the presence of any of the DOACs.

An immobilized perylene diimide derivative for fucoidan purification from a crude brown algae extract

Abstract A polycationic perylene diimide derivative was successfully immobilized to capture polysulphated fucoidan from a crude extract of F. vesiculosus. At a slightly acidic pH, immobilized perylene diimide derivative is positively charged and can form electrostatically-driven aggregates with more than 80% of fucoidan in only 16 h, compared to 44 h in recently developed immobilized toluidine blue. Purified fucoidan was identified by FTIR, which demonstrated typical IR bands in comparison with the commercial product. Moreover, it showed improved purity than crude type by a factor of 1.4 after purification. Pharmacologically, it prolonged APTT and TT, revealing a potential heparin-like anticoagulant activity by interfering with intrinsic and common blood coagulation pathway. Automated downstream and scale-up processes were further performed with developing a FPLC protocol. The new technique proved to be a competitor to immobilized toluidine blue for production of a high-grade fucoidan.

A semi-synthetic glycosaminoglycan analogue inhibits and reverses Plasmodium falciparum cytoadherence.

A feature of mature Plasmodium falciparum parasitized red blood cells is their ability to bind surface molecules of the microvascular endothelium via the parasite-derived surface protein Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). This ligand is associated with the cytoadherence pathology observed in severe malaria. As pRBC treated with effective anti-malarial drugs are still able to cytoadhere, there is therefore a need to find an adjunct treatment that can inhibit and reverse the adhesion process. One semi-synthetic, sulfated polysaccharide has been identified that is capable of inhibiting and reversing sequestration of pRBC on endothelial cells in vitro under physiological flow conditions. Furthermore, it exhibits low toxicity in the intrinsic (APTT assay) and extrinsic (PT assay) clotting pathways, as well as exhibiting minimal effects on cell (HUVEC) viability (MTT proliferation assay). These findings suggest that carbohydrate-based anti-adhesive candidates may provide potential leads for therapeutics for severe malaria.

Влияние активности факторов внутреннего каскада коагуляции и гемостатических маркеров эндотелиальной дисфункции на тромботические осложнения у пациентов с атеросклеротическими заболеваниями периферических артерий

Влияние активности факторов внутреннего каскада коагуляции и гемостатических маркеров эндотелиальной дисфункции на тромботические осложнения у пациентов с атеросклеротическими заболеваниями периферических артерий

Selective factor VIII activation by the tissue factor–factor VIIa–factor Xa complex

Safe and effective antithrombotic therapy requires understanding of mechanisms that contribute to pathological thrombosis but have a lesser impact on hemostasis. We found that the extrinsic tissue factor (TF) coagulation initiation complex can selectively activate the antihemophilic cofactor, FVIII, triggering the hemostatic intrinsic coagulation pathway independently of thrombin feedback loops. In a mouse model with a relatively mild thrombogenic lesion, TF-dependent FVIII activation sets the threshold for thrombus formation through contact phase-generated FIXa. In vitro, FXa stably associated with TF-FVIIa activates FVIII, but not FV. Moreover, nascent FXa product of TF-FVIIa can transiently escape the slow kinetics of Kunitz-type inhibition by TF pathway inhibitor and preferentially activates FVIII over FV. Thus, TF synergistically primes FIXa-dependent thrombin generation independently of cofactor activation by thrombin. Accordingly, FVIIa mutants deficient in direct TF-dependent thrombin generation, but preserving FVIIIa generation by nascent FXa, can support intrinsic pathway coagulation. In ex vivo flowing blood, a TF-FVIIa mutant complex with impaired free FXa generation but activating both FVIII and FIX supports efficient FVIII-dependent thrombus formation. Thus, a previously unrecognized TF-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostatic before further coagulation amplification by thrombin-dependent feedback loops enhances the risk of thrombosis.

Chitosan-based hydrogels for developing a small-diameter vascular graft: in vitro and in vivo evaluation

Aims. Vascular grafts made of synthetic polymers perform poorly in small-diameter applications (cardiac and peripheral bypass). Chitosan is a biocompatible natural polymer that can provide a novel biological scaffold for tissue engineering development. The goal of this study was to demonstrate the biocompatibility of a novel chitosan preparation in vitro and in vivo, and to assess its potential as a scaffold for vascular applications. Methods and results. A series of experiments of increasing complexity, ranging from in vitro biocompatibility and hemocompatibility tests to in vivo studies in small and large animals (rats and sheep), was performed to provide a comprehensive analysis of chitosan hydrogels’ biological properties. In vitro studies established that: (i) chitosan supported human endothelial progenitor cells adhesion, proliferation and resistance to physiological shear stress; (ii) chitosan did not activate platelets, the complement system, or the intrinsic coagulation pathway. In vivo results showed: (iii) no resorption of chitosan and no chronic inflammation at 60 days in a rat heterotopic implantation model (magnetic resonance imaging and histology); (iv) no flow obstruction (Doppler ultrasound) and no thrombus formation (histology and scanning electron microscopy) at 2 h after a carotid arteriotomy repair with chitosan patches in sheep. Finally, two chitosan tubes were implanted as carotid interposition grafts for 3 days in sheep showing that chitosan was strong enough to be sutured, to withstand arterial pressure, and no flow obstruction was observed through this short period. Conclusion. Chitosan-based hydrogels displayed promising in vitro biocompatibility and hemocompatibility properties as well as in vivo short-term performance.

The Procoagulant Activity of Apoptotic Cells Is Mediated by Interaction with Factor XII

Apoptotic cells, by externalizing phosphatidylserine (PS) as a hallmark feature, are procoagulant. However, the mechanism by which apoptotic cells activate coagulation system remains unknown. Intrinsic coagulation pathway is initiated by coagulation factor XII (FXII) of contact activation system. The purpose of this study was to determine whether FXII is involved in procoagulant activity of apoptotic cells. Using western blotting and chromogenic substrate assay, we found that incubation with apoptotic cells, but not with viable cells, resulted in rapid cleavage and activation of FXII in the presence of prekallikrein and high molecular weight kininogen (HK), other two components of contact activation system. As detected by flow cytometry, FXII bound to apoptotic cells in a concentration-dependent manner, which was inhibited by annexin V and PS liposome. Direct association of FXII with PS was confirmed in a surface plasmon resonance assay. Clotting time of FXII-deficient plasma induced by apoptotic cells was significantly prolonged, which was fully reversed by replenishment with FXII. Corn trypsin inhibitor, a FXII inhibitor, completely prevented apoptotic cells-induced intrinsic tenase complex formation. Consistently, apoptotic cells significantly increased thrombin production in normal plasma, which was not affected by an inhibitory anti-tissue factor antibody. However, blocking of PS by annexin V, inhibition of FXII, or the deficiency of FXII suppressed apoptotic cells-induced thrombin generation. Addition of purified FXII to FXII-deficient plasma recovered thrombin generation to the normal plasma level. In conclusion, FXII binds to apoptotic cells via PS and becomes activated, thereby constituting a novel mechanism mediating the procoagulant activity of apoptotic cells.

Anticoagulant properties of a high molecular weight polysaccharide fraction (1000RS) of the ascidian Microcosmus exasperatus

Aims: The anticoagulant effect and cytotoxicity of a high molecular weight polysaccharide fraction (1000RS) obtained from the tunic of the ascidia Microcosmus exasperatus were evaluated. Methods: Anticoagulant properties of 1000RS was evaluated by activated Partial Thromboplastin Time (aPTT), Thrombin Time (TT), Prothrombin Time (PT), anti-factor Xa and lupic anticoagulant (dRVVT) assays. Cytotoxicity was tested on murine hematopoietic cells using MTT assay. Results: This galactose rich fraction showed to be a potential anticoagulant due to its inhibitory effect on the intrinsic coagulation pathway. At the same time, anticoagulant doses of this fraction have no effect on cellular viability, which means that it can be used as a therapeutic agent. Conclusion: In vitro anticoagulant effect of 1000RS occurs at innocuous doses, however, it still need to be tested using in vivo models and its cytotoxicity evaluated in normal human cell lines.

Apixaban - Metabolism, Pharmacologic Properties and Drug Interactions.

Apixaban is an oral, potent, highly selective, reversible and direct inhibitor of activated coagulation factor X, that is the end point of the intrinsic and extrinsic coagulation pathway. Additionally, apixaban has the capacity to indirectly inhibit thrombin-induced platelet aggregation. This new oral anticoagulant represents an immediate-release form of peroral drug with quick dissolution, linear pharmacokinetics, good bioavailability and rapid onset and offset of action. No clinically relevant age- or sex-dependent difference in the apixaban pharmacokinetics and pharmacodynamics which would lead to the modification of the dose exists, apixaban may even be administered with or without food. Its elimination is mediated by metabolism, renal elimination of unmodified drug and excretion in the gastrointestinal tract. The authors aim to provide a review of currently available literature about apixaban. The authors summed-up the data from the scientific journals related to thrombosis and hemostasis and searched the available databases. Apixaban has many advantages including predictable pharmacokinetics and pharmacodynamics, low number of drug and food interactions, and relatively wide therapeutic window.

Different chemical groups modification on the surface of chitosan nonwoven dressing and the hemostatic properties

The hemostatic properties of surface modified chitosan nonwoven had been investigated. The succinyl groups, carboxymethyl groups and quaternary ammonium groups were introduced into the surface of chitosan nonwoven (obtained NSCS, CMCS and TMCS nonwoven, respectively). For blood clotting, absorbance value (0.105±0.03) of NSCS1 nonwoven was the smallest (CS 0.307±0.002, NSCS2 0.148±0.002, CMCS1 0.195±0.02, CMCS2 0.233±0.001, TMCS1 0.191±0.002, TMCS2 0.345±0.002), which indicated the stronger hemostatic potential. For platelet aggregation, adenosine diphosphate agonist was added to induce the nonwoven to adhered platelets. The aggregation of platelet with TMCS2 nonwoven was highest (10.97±0.16%). Further research of blood coagulation mechanism was discussed, which indicated NSCS and CMCS nonwoven could activate the intrinsic pathway of coagulation to accelerate blood coagulation. NSCS1 nonwoven showed the shortest hemostatic time (147±3.7s) and the lowest blood loss (0.23±0.05g) in a rabbit ear artery injury model. These results demonstrated that these surface modified chitosan nonwoven dressings could use as a promising hemostatic intervention, especially NSCS nonwoven dressing.

Assessment of the protein interaction between coagulation factor XII and corn trypsin inhibitor by molecular docking and biochemical validation

Essentials Corn Trypsin Inhibitor (CTI) is a selective inhibitor of coagulation Factor XII (FXII).Molecular modelling of the CTI‐FXIIa complex suggested a canonical inhibitor binding mode.Mutagenesis revealed the CTI inhibitory loop and helices α1 and α2 mediate the interaction.This confirms that CTI inhibits FXII in canonical fashion and validates the molecular model. SummaryBackgroundCorn trypsin inhibitor (CTI) has selectivity for the serine proteases coagulation factor XII and trypsin. CTI is in widespread use as a reagent that specifically inhibits the intrinsic pathway of blood coagulation but not the extrinsic pathway.ObjectivesTo investigate the molecular basis of FXII inhibition by CTI.MethodsWe performed molecular docking of CTI, using its known crystal structure, with a model of the activated FXII (FXIIa) protease domain. The interaction model was verified by use of a panel of recombinant CTI variants tested for their ability to inhibit FXIIa enzymatic activity in a substrate cleavage assay.ResultsThe docking predicted that: (i) the CTI central inhibitory loop P1 Arg34 side chain forms a salt bridge with the FXIIa S1 pocket Asp189 side chain; (ii) Trp22 from CTI helix α1 interacts with the FXIIa S3 pocket; and (iii) Arg43 from CTI helix α2 forms a salt bridge with FXIIa H1 pocket Asp60A. CTI amino acid substitution R34A negated all inhibitory activity, whereas the G32W, L35A, W22A and R42A/R43A substitutions reduced activity by large degrees of 108‐fold, 41‐fold, 158‐fold, and 100‐fold, respectively; the R27A, W37A, W39A and R42A substitutions had no effect. Synthetic peptides spanning CTI residues 20–44 had inhibitory activity that was three‐fold to 4000‐fold less than that of full‐length CTI.ConclusionsThe data confirm the validity of a canonical model of the FXIIa–CTI interaction, with helix α1 (Trp22), central inhibitory loop (Arg34) and helix α2 (Arg43) of CTI being required for effective binding by contacting the S1, S3 and H1 pockets of FXIIa, respectively.

Factor XI‐deficient mice exhibit increased bleeding after injury to the saphenous vein

Background Factor XI (FXI) is a key component of the intrinsic pathway of coagulation. It can be activated by either FXIIa or thrombin and amplifies thrombin generation during clot formation. Congenital FXI deficiency in humans (known as hemophilia C) is associated with bleeding after hemostatic challenge. However, to date there are no reports of excess bleeding in FXI-deficient mice. Objectives To determine if the absence of FXI in mice prolongs bleeding in different models. Methods We assessed the hemostatic capacity of FXI-/- mice in three different bleeding models: tail bleeding, surgical bleeding and saphenous vein bleeding. Results We found that tail bleeding and surgical bleeding of FXI-/- mice were similar to wild-type mice. However, FXI-/- mice had an impaired hemostatic capacity in the saphenous vein bleeding model compared with wild-type controls. Conclusions Our results indicate that FXI-/- mice have a mild hemostatic defect after injury to the saphenous vein but not after transection of the tail or vessels in the abdominal wall.

A kallikrein‐targeting RNA aptamer inhibits the intrinsic pathway of coagulation and reduces bradykinin release

Background Plasma kallikrein is a serine protease that plays an integral role in many biological processes, including coagulation, inflammation, and fibrinolysis. The main function of kallikrein in coagulation is the amplification of activated factor XII (FXIIa) production, which ultimately leads to thrombin generation and fibrin clot formation. Kallikrein is generated by FXIIa-mediated cleavage of the zymogen prekallikrein, which is usually complexed with the non-enzymatic cofactor high molecular weight kininogen (HK). HK also serves as a substrate for kallikrein to generate the proinflammatory peptide bradykinin (BK). Interestingly, prekallikrein-deficient mice are protected from thrombotic events while retaining normal hemostatic capacity. Therefore, therapeutic targeting of kallikrein may provide a safer alternative to traditional anticoagulants with anti-inflammatory benefits. Objectives To isolate and characterize an RNA aptamer that binds to and inhibits plasma kallikrein, and to elucidate its mechanism of action. Methods and Results Using convergent Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated an RNA aptamer that targets kallikrein. This aptamer, Kall1-T4, specifically binds to both prekallikrein and kallikrein with similar subnanomolar binding affinities, and dose-dependently prolongs fibrin clot formation in an activated partial thromboplastin time (APTT) coagulation assay. In a purified in vitro system, Kall1-T4 inhibits the reciprocal activation of prekallikrein and FXII primarily by reducing the rate of FXIIa-mediated prekallikrein activation. Additionally, Kall1-T4 significantly reduces kallikrein-mediated HK cleavage and subsequent BK release. Conclusions We have isolated a specific and potent inhibitor of prekallikrein/kallikrein activity that serves as a powerful tool for further elucidating the role of kallikrein in thrombosis and inflammation.

Structural analysis of a sulfated galactan from the tunic of the ascidian Microcosmus exasperatus and its inhibitory effect of the intrinsic coagulation pathway

Several bioactive sulfated galactans have been isolated from the tunic of different species of ascidians. The biological activity of this kind of polysaccharides has been related with the presence and position of sulfate groups, and by the chemical composition of this kind of polysaccharides. A sulfated galactan (1000RS) was isolated from the tunic of the Brazilian ascidia Microcosmus exasperatus through proteolytic digestion, ethanol precipitation, dialysis and freeze-thaw cycles. Homogeneity and molecular weight were estimated by using size exclusion chromatography. Monosaccharide composition and type of linkage were assessed by Gas chromatography coupled to mass spectrometry and the sulfate content was quantified through gelatin/BaCl2 method. These experiments along with NMR and FTIR analysis allowed to claim that the galactan backbone is mainly composed of 4-linked α-l-Galp units. In addition, they permitted to establish that some of the galactose residues are sulfated at the 3-position. This sulfated polysaccharide, which has an average molecular mass of 439.5kDa, presents anticoagulant effect in a dose-dependent manner through the inhibition of the intrinsic coagulation pathway.

P3620The extrinsic and intrinsic pathways of coagulation are considerably activated in patients with paroxysmal atrial fibrillation

P3620The extrinsic and intrinsic pathways of coagulation are considerably activated in patients with paroxysmal atrial fibrillation

New prodrugs of metformin do not influence the overall haemostasis potential and integrity of the erythrocyte membrane

Although metformin, an oral anti-diabetic drug, has been found to have multidirectional effects over the past decade, it is characterised by unfavourable pharmacokinetic properties. This study discusses the effects of metformin, phenformin and three prodrugs of metformin on the haemostasis and integrity of Red Blood Cells (RBCs).The influence of examined biguanide derivatives on haemostasis was evaluated spectrophotometrically by clot formation and lysis test (CL-test) at 405nm. The extrinsic and intrinsic coagulation pathway were examined by measuring the PT (Prothrombin Time) and aPTT (Activated Partial Tromboplastin Time). Haemolysis assay, microscopy and flow cytometry studies were used to assess the effect of the tested compounds on RBCs.Although none of the tested biguanide derivatives significantly influenced the overall potential of clot formation and fibrinolysis (CLAUC constants), statistically significant changes were seen in the values of the kinetic parameters of fibrinolysis. Furthermore, only prodrug 2, with an 8-carbon alkyl chain, unfavourably affected RBCs by interaction with the erythrocyte membrane leading to significant haemolysis.Our results provide a further insight into the effects of metformin and its prodrugs on haemostasis and RBCs and underscore the necessity for further research.

Platelet-derived microparticles regulates thrombin generation via phophatidylserine in abdominal sepsis.

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis.

In silico Discovery of Novel FXa Inhibitors by Pharmacophore Modeling and Molecular Docking

Coagulation Factor Xa (FXa) is the crucial enzyme at the convergent point of the intrinsic and extrinsic coagulation pathways. The inhibition of FXa is an effective approach against thrombotic diseases. In the present study, a specific strategy is reported to discover 10 novel FXa inhibitors based on ligand-based (pharmacophore) virtual screening and molecular docking analysis from a dataset of specs(containing 220000 molecules). The binding modes analysis provide insights into the contribution of particular structural moieties of the compounds towards their activity against FXa, and 10 novel structural compounds were discovered as potent candidate molecules. This work could be helpful in further design and development of FXa inhibitors.Graphical