Plasma Anticoagulant Activity Research Articles

Increased Antithrombotic Properties of Unfractionated Heparin in Rats Following Oral Administration of Multiple Doses and Its Correlation with Endothelial Heparin Concentration

In a rat model of venous thrombosis, a single oral dose of 7.5mg kg-1 of Unfractionated Heparin (UFH) lowers thrombosis by 50%. Since clinical long-term use is necessary, our goals were to investigate the antithrombotic effects of repeated oral UFH administration. Rats were given three doses of 7.5mg kg-1 every 12, 24, 48, and 72 hours apart—as well as three or fifteen doses of 1 mg kg—1 every 48 hours—by oral gavage of cow lung UFH. The final dosage was injecting 10% formalin in methanol into the jugular vein right after thrombus start. Four hours later, the vessel was checked for thrombosis. Heparin levels in endothelium and tissue as well as plasma anticoagulant activity were assessed. As 3×7.5 When heparin (mg kg-1) was administered, the incidence of thrombosis was notably lower during 48-hour dosing intervals than with a single dose. Endothelial heparin concentration and thrombotic incidence were negatively correlated, while anticoagulant activity was not. Treatment with three doses of 1mg kg-1 every 48 hours resulted in a thrombotic incidence comparable to that of a single dosage. The total thrombotic incidence after 15 doses was comparable to that following s.c. treatment and was lower than that of 3 doses. Oral UFH delivery resulted in increased antithrombotic activity and antithrombotic effects that were comparable to those of s.c. administration. Heparin’s antithrombotic effect on endothelium was linked to it.

Exploring antithrombin: insights into its physiological features, clinical implications and analytical techniques.

Antithrombin is an essential protein that acts as a natural anticoagulant in the human body. It is synthesized by the liver and belongs to the serine protease inhibitors, which are commonly referred to as the SERPINS superfamily. The antithrombin molecule comprises 432 amino acids and has a molecular weight of approximately 58 200 D. It consists of three domains, including an amino-terminal domain, a carbohydrate-rich domain, and a carboxyl-terminal domain. The amino-terminal domain binds with heparin, whereas the carboxyl-terminal domain binds with serine protease. Antithrombin is a crucial natural anticoagulant that contributes approximately 60-80% of plasma anticoagulant activities in the human body. Moreover, antithrombin has anti-inflammatory effects that can be divided into coagulation-dependent and coagulation-independent effects. Furthermore, it exhibits antitumor activity and possesses a broad range of antiviral properties. Inherited type I antithrombin deficiency is a quantitative disorder that is characterized by low antithrombin activity due to low plasma levels. On the other hand, inherited type II antithrombin deficiency is a qualitative disorder that is characterized by defects in the antithrombin molecule. Acquired antithrombin deficiencies are more common than hereditary deficiencies and are associated with various clinical conditions due to reduced synthesis, increased loss, or enhanced consumption. The purpose of this review was to provide an update on the structure, functions, clinical implications, and methods of detection of antithrombin.

Zn2+ Differentially Influences the Neutralisation of Heparins by HRG, Fibrinogen, and Fibronectin.

For coagulation to be initiated, anticoagulant glycosaminoglycans (GAGs) such as heparins need to be neutralised to allow fibrin clot formation. Platelet activation triggers the release of several proteins that bind GAGs, including histidine-rich glycoprotein (HRG), fibrinogen, and fibronectin. Zn2+ ions are also released and have been shown to enhance the binding of HRG to heparins of a high molecular weight (HMWH) but not to those of low molecular weight (LMWH). The effect of Zn2+ on fibrinogen and fibronectin binding to GAGs is unknown. Here, chromogenic assays were used to measure the anti-factor Xa and anti-thrombin activities of heparins of different molecular weights and to assess the effects of HRG, fibrinogen, fibronectin, and Zn2+. Surface plasmon resonance was also used to examine the influence of Zn2+ on the binding of fibrinogen to heparins of different molecular weights. Zn2+ had no effect on the neutralisation of anti-factor Xa (FXa) or anti-thrombin activities of heparin by fibronectin, whereas it enhanced the neutralisation of unfractionated heparin (UFH) and HMWH by both fibrinogen and HRG. Zn2+ also increased neutralisation of the anti-FXa activity of LMWH by fibrinogen but not HRG. SPR showed that Zn2+ increased fibrinogen binding to both UFH and LMWH in a concentration-dependent manner. The presented results reveal that an increase in Zn2+ concentration has differential effects upon anticoagulant GAG neutralisation by HRG and fibrinogen, with implications for modulating anti-coagulant activity in plasma.

Anionic Ultrasmall Gold Nanoparticles Bind to Coagulation Factors and Disturb Normal Hemostatic Balance

Ultrasmall gold nanoparticles (usNPs) and nanoclusters are an emerging class of nanomaterials exhibiting distinctive physicochemical properties and in vivo behaviors. Although understanding the interactions of usNPs with blood components is of fundamental importance to advance their clinical translation, currently, little is known about the way that usNPs interact with the hemostatic system. This study describes the effects of a model anionic p-mercaptobenzoic acid-coated usNP on the coagulation cascade, with particular emphasis on the contact pathway. It is found that in a purified system, the anionic usNPs bind to and activate factor XII (FXII). The formed usNP-FXII complexes are short-lived (residence time of ∼10 s) and characterized by an affinity constant of ∼200 nM. In human plasma, the anionic usNPs activate the contact pathway and promote coagulation. The usNPs also exhibit anticoagulant activity in plasma by interfering with the thrombin-mediated cleavage of fibrinogen. Taken together, these findings establish that anionic usNPs can disturb the normal hemostatic balance, which in turn may hinder their clinical translation. Finally, it is shown that usNPs can be designed to be nearly inert in plasma by surface coating with the natural peptide glutathione.

Factor V Has Anticoagulant Activity in Plasma in the Presence of TFPIα: Difference between FV1 and FV2.

Activated factor V (FVa) is a potent procoagulant cofactor in the prothrombinase complex, whereas its precursor factor V (FV) stimulates the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-α (TFPIα), presumably by promoting TFPIα binding to phospholipids. Plasma FV comprises two glycosylation isoforms (FV1 and FV2) with low and high phospholipid-binding affinity, respectively. The FV1/FV2 ratio is increased in carriers of the FV R2 haplotype. This article demonstrates the TFPIα-cofactor function of FV in plasma and compares FV1 and FV2. Thrombin generation at low TF concentration was measured in FV-depleted plasma reconstituted with 0 to 100% FV, FV1 or FV2, and in 122 individuals genotyped for the R2 haplotype. The TFPIα-cofactor activities of FV1 and FV2 were also investigated in a model system of TFPIα-mediated FXa inhibition. In the FV titration, thrombin generation first increased (up to 5% FV) and then progressively decreased at higher FV concentrations. This anticoagulant effect of FV, which was also observed with FV2 but not with FV1, was largely abolished by anti-TFPIα antibodies, suggesting that it reflects TFPIα-cofactor activity of FV. In the model system of TFPIα-mediated FXa inhibition, FV2 was a more potent TFPIα-cofactor than FV1, in line with their respective phospholipid affinities. Accordingly, FV R2 carriers had higher thrombin generation than non-carriers, even after correction for demographics and plasma levels of coagulation factors and inhibitors. FV (and particularly its FV2 isoform) contributes to the TFPIα-dependent down-regulation of thrombin generation in plasma triggered with low TF.

The effect of increasing the sulfation level of chondroitin sulfate on anticoagulant specific activity and activation of the kinin system.

Oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in certain heparin preparations as the cause of adverse reactions in patients. OSCS was found to possess both plasma anticoagulant activity and the ability to activate prekallikrein to kallikrein. Differentially sulfated chondroitin sulfates were prepared by synthetic modification of chondroitin sulfate and were compared to the activity of OSCS purified from contaminated heparin. Whilst chondroitin sulfate was found to have minimal anticoagulant activity, increasing sulfation levels produced an anticoagulant response which we directly show for the first time is mediated through heparin cofactor II. However, the tetra-sulfated preparations did not possess any higher anticoagulant activity than several tri-sulfated variants, and also had lower heparin cofactor II mediated activity. Activation of prekallikrein was concentration dependent for all samples, and broadly increased with the degree of sulfation, though the di-sulfated preparation was able to form more kallikrein than some of the tri-sulfated preparations. The ability of the samples to activate the kinin system, as measured by bradykinin, was observed to be through kallikrein generation. These results show that whilst an increase in sulfation of chondroitin sulfate did cause an increase in anticoagulant activity and activation of the kinin system, there may be subtler structural interactions other than sulfation at play given the different responses observed.

Phenolic fractions from nine Trifolium species modulate the coagulant properties of blood plasma in vitro without cytotoxicity towards blood cells.

The study covers an evaluation of the influence of extracts (1-50 μg/ml), isolated from aerial parts of nine Trifolium L. species (i.e. T. alexandrinum, T. fragiferum, T. hybridum, T. incarnatum, T. pallidum, T. pratense, T. resupinatum var. majus, T. resupinatum var. resupinatum and T. scabrum) on haemostatic properties of blood plasma. The clot formation and fibrinolysis assay (CFF), blood clotting times, the extrinsic and intrinsic coagulation pathway-dependent polymerization of plasma fibrin were measured. The effects of plant extracts on amidolytic activity of thrombin were also evaluated and compared with argatroban, an antithrombotic drug. Cytotoxicity was assessed in a model of blood platelets and as the viability of peripheral blood mononuclear cells. While no changes in blood clotting times or fibrinolytic properties of blood plasma were found, some fractions impaired the blood plasma coagulation induced by the intrinsic coagulation pathway. Reduction in the maximal velocity of fibrin polymerization was also observed in the clot formation and fibrinolysis assay. No cytotoxicity of Trifolium extracts towards the investigated cells was recorded. The most efficient anticoagulant activity in plasma was found for T. fragiferum and T. incarnatum extracts, while the T. alexandrinum fraction was the most effective inhibitor of thrombin amidolytic activity.

In vitro anti-thrombotic and anti-coagulant properties of blacklip abalone (Haliotis rubra) viscera hydrolysate.

Abalone viscera contain sulphated polysaccharides with anti-thrombotic and anti-coagulant activities. In this study, a hydrolysate was prepared from blacklip abalone (Haliotis rubra) viscera using papain and bromelain and fractionated using ion exchange and size exclusion chromatography. Hydrolysates and fractions were investigated for in vitro thrombin inhibition mediated through heparin cofactor II (HCII) as well as anti-coagulant activity in plasma and whole blood. On the basis of sulphated polysaccharide concentration, the hydrolysate inhibited thrombin through HCII with an inhibitor concentration at 50% (IC50) of 16.5μg/mL compared with 2.1μg/mL for standard heparin. Fractionation concentrated HCII-mediated thrombin inhibition down to an IC50 of 1.8μg/mL and improved anti-coagulant activities by significantly delaying clotting time. This study confirmed the presence of anti-thrombotic and anti-coagulant molecules in blacklip abalone viscera and demonstrated that these activities can be enriched with a simple chromatography regime. Blacklip abalone viscera warrant further investigation as a source of nutraceutical or functional food ingredients. Graphical abstract Schematic showing preparation of bioactive extracts and fractions from blacklip abalone.

In vitro Anti-Thrombotic Activity of Extracts from Blacklip Abalone (Haliotis rubra) Processing Waste.

Waste generated from the processing of marine organisms for food represents an underutilized resource that has the potential to provide bioactive molecules with pharmaceutical applications. Some of these molecules have known anti-thrombotic and anti-coagulant activities and are being investigated as alternatives to common anti-thrombotic drugs, like heparin and warfarin that have serious side effects. In the current study, extracts prepared from blacklip abalone (Haliotis rubra) processing waste, using food grade enzymes papain and bromelain, were found to contain sulphated polysaccharide with anti-thrombotic activity. Extracts were found to be enriched with sulphated polysaccharides and assessed for anti-thrombotic activity in vitro through heparin cofactor-II (HCII)-mediated inhibition of thrombin. More than 60% thrombin inhibition was observed in response to 100 μg/mL sulphated polysaccharides. Anti-thrombotic potential was further assessed as anti-coagulant activity in plasma and blood, using prothrombin time (PT), activated partial thromboplastin time (aPTT), and thromboelastography (TEG). All abalone extracts had significant activity compared with saline control. Anion exchange chromatography was used to separate extracts into fractions with enhanced anti-thrombotic activity, improving HCII-mediated thrombin inhibition, PT and aPTT almost 2-fold. Overall this study identifies an alternative source of anti-thrombotic molecules that can be easily processed offering alternatives to current anti-thrombotic agents like heparin.

Silencing anticoagulant protein c causes spontaneous atherothrombosis in ApoE knockout mice

Objectives: Studies on atherothrombosis, a major cause of cardiovascular events, are hampered by the lack of animal models spontaneously developing atherothrombosis. Major underlying problems are the stability of the atherosclerotic plaques, combined with the strong plasma anticoagulant activity. We hypothesized that inhibition of the anticoagulant system by silencing of protein C (Proc) may cause murine atherosclerotic plaques to become prone to atherothrombosis.

Dose Dependence of the Anticoagulant Effect of Intravenously Administered Cellulose Sulfate.

Experiments on rabbits showed that increasing the dose of intravenously administered cellulose sulfate from wheat straw (dynamic viscosity 3.4 cP, sulfur content 14.1%) increased plasma clotting time in some coagulation tests and plasma anticoagulant activity. When cellulose sulfate was administered in the dose of 1 mg/kg, plasma clotting time in the presence of the anticoagulant (5 min after administration) was ~3-fold higher than after saline administration.

Thrombin-Inhibiting Anticoagulant Liposomes: Development and Characterization.

Many peptides and peptidomimetic drugs suffer from rapid clearance in vivo; this can be reduced by increasing their size through oligomerization or covalent conjugation with polymers. As proof of principle, an alternative strategy for drug oligomerization is described, in which peptidomimetic thrombin inhibitors are incorporated into the liposome surface. For this purpose, the inhibitor moieties were covalently coupled to a palmitic acid residue through a short bifunctionalized ethylene glycol spacer. These molecules were directly added to the lipid mixture used for liposome preparation. The obtained liposomes possess strong thrombin inhibitory potency in enzyme kinetic measurements and anticoagulant activity in plasma. Their strong potency and positive ζ potential indicate that large amounts of the benzamidine-derived inhibitors are located on the surface of the liposomes. This concept should be applicable to other drug molecules that suffer from rapid elimination and allow covalent modification with a suitable fatty acid residue.

Orally bioavailable factor Xa inhibitors containing alpha-substituted gem-dimethyl P4 moieties

In an effort to identify a potential back-up to apixaban (Eliquis®), we explored a series of diversified P4 moieties. Several analogs with substituted gem-dimethyl moieties replacing the terminal lactam of apixaban were identified which demonstrated potent FXa binding affinity (FXa Ki), good human plasma anticoagulant activity (PT EC2x), cell permeability, and oral bioavailability.

TFPI Expresses Anticoagulant Activity Independent Of TF-FVIIa

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine protease inhibitor that down-regulates the extrinsic coagulation pathway by inhibiting the tissue factor-factor VIIa complex (TF-FVIIa). Inhibition of TF-FVIIa by TFPI is greatly enhanced by FXa. In order to inhibit TF-FVIIa, TFPI first binds to and inhibits Factor Xa (FXa) thus forming a binary TFPI-FXa complex in a reaction that is stimulated by protein S. The TFPI-FXa complex subsequently forms a quaternary complex with TF-FVIIa and blocks the initiation of coagulation. Although TFPI can directly inhibit FXa, this inhibition is generally considered to be of limited physiological significance because the plasma TFPI concentration (0.25-0.5 nM) is several orders of magnitude lower than the plasma concentration of other FXa inhibitors e.g. antithrombin (AT = 2.5-5 μM).We investigated whether TFPI expresses anticoagulant activity independent of TF-FVIIa i.e. to test whether direct inhibition of FXa by TFPI contributes to the down-regulation of coagulation.In model systems, the inhibition of FXa by TFPI and AT was determined in the presence of calcium chloride, phospholipids and protein S using a chromogenic substrate to monitor FXa activity. FXa inhibition by TFPI in plasma was determined by measuring thrombin generation curves triggered with FXa, with the FXa activator from Russell's viper venom (RVV-X) and with FXIa or FIXa in normal pooled plasma. These experiments were performed in the presence of a cocktail of anti TF/anti FVIIa antibodies to inhibit any contribution of traces of TF that might be present in plasma to initiate coagulation. The TF-independent contributions of TFPI and/or protein S to the down-regulation of thrombin generation were quantified 1) after neutralization of TFPI and protein S present in plasma with an anti-TFPI antibody cocktail or anti-protein S antibodies and 2) via TFPI and protein S titrations in TFPI- and in protein S-depleted plasma.In model systems containing purified proteins, physiological concentrations TFPI (0.25 nM) appeared to be more effective in FXa inhibition (t1/2 = 2.5 min) than physiological concentrations AT (2.5 μM) which inhibited FXa with t1/2 = 3.2 min. Both anti-TFPI and anti-protein S antibodies enhanced thrombin generation that was triggered in plasma with RVV-X, FXa, FIXa or FXIa. Depending on the trigger and trigger concentration used anti-TFPI and anti-protein S antibodies decreased the lag time and/or increased the peak height of thrombin generation. At high trigger concentrations neither anti-TFPI nor anti-protein S antibodies enhanced thrombin generation. TFPI and protein S titrations in TFPI- and protein S-depleted plasma in which thrombin formation was initiated with triggers other than TF confirmed that both TFPI and protein S express TF-independent anticoagulant activity. TFPI titrations in protein S-depleted plasma triggered with RVV-X or FXIa showed that the TF-independent anticoagulant activity of TFPI was greatly reduced in the absence of protein S. Supplementation of the protein S-depleted plasma with protein S restored the anticoagulant effect of TFPI.We demonstrated that TFPI, despite its low plasma concentration, expresses anticoagulant activity in the absence of TF. Kinetic experiments in model systems at plasma TFPI concentrations show that direct inhibition of FXa by TFPI can account for the TF-independent anticoagulant effect of TFPI on thrombin generation. Blocking the anticoagulant activity of protein S with anti-protein S antibodies also enhances thrombin generation with triggers other than TF. The fact that neutralization of TFPI and of protein S with specific antibodies resulted in a similar stimulation of thrombin generation suggests that TFPI is strongly dependent on the presence of protein S for expression of TF-independent anticoagulant activity in plasma. The ability of TFPI to inhibit thrombin generation initiated via the intrinsic coagulation pathway is an important observation particularly in the light of recent reports which indicate that the intrinsic coagulation pathway plays a relevant role in thrombus formation and in cardiovascular disease. Disclosures:Rosing:Baxter Innovations GmbH, Vienna, Austria: Consultancy, Research Funding. Hartmann:Baxter Innovations GmbH, Vienna, Austria: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment. Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment.

Novel anticoagulants and laboratory testing

The introduction of several oral direct anticoagulants within the past 2-3years has dramatically changed clinical practice and has also impacted on utilization and interpretation of coagulation laboratory testing. This article reviews the effects of the oral thrombin inhibitor, dabigatran, and the oral factor Xa inhibitors, rivaroxaban and apixaban, on screening and diagnostic coagulation tests, and describes methods for measuring the their anticoagulant activity in plasma. Currently, there are evidence gaps regarding the role of laboratory testing for surveillance and management of adverse events associated with these new anticoagulants which do not require routine therapeutic drug monitoring. This is a rapidly changing field, and coagulation laboratory experts have a major role in ensuring patients receive appropriate testing and accurate interpretations of results.

Characterization of a novel variant of the second domain of bikunin with increased leukocyte elastase inhibitory activity

The light chain of inter-α inhibitor, also known as bikunin or urinary trypsin inhibitor, is composed of two tandemly arranged Kunitz-type protease inhibitor domains. The second domain of bikunin has factor Xa inhibitory activity which previously was enhanced by mutating two amino acids, glutamine 19 and tyrosine 46 to lysine and aspartate, respectively. In this study, we tried to potentiate its inhibitory activity against leukocyte elastase. A molecular docking model of the second domain of bikunin with leukocyte elastase revealed that P5 arginine 11 was a candidate residue for a third substitution. We generated six triple point mutants using site-directed mutagenesis, compared their leukocyte elastase-inhibitory activities, and selected the most potent variant with arginine 11 substituted to serine. The IC50 values for factor XIa, factor Xa, and leukocyte elastase were 182, 302, and 273 nM, respectively. Moreover, this triple point mutant prolonged the activated partial thromboplastin time and moderately reduced leukocyte elastase-induced endothelial injury. Additionally, favorable conformations created by these mutations were speculated using the structure of the Kunitz protease inhibitor domain of protease nexin 2 complexed with factor XIa as a reference. We discovered a novel triple point mutant of the second domain of bikunin that has potent inhibitory activities against factor XIa, factor Xa, and leukocyte elastase. This variant exhibited anticoagulant activity in plasma and suppressed endothelial cell injury.

Discovery of N-[2-Hydroxy-6-(4-methoxybenzamido)phenyl]-4- (4-methyl-1,4-diazepan-1-yl)benzamide (Darexaban, YM150) as a Potent and Orally Available Factor Xa Inhibitor

Inhibitors of factor Xa (FXa), a crucial serine protease in the coagulation cascade, have attracted a great deal of attention as a target for developing antithrombotic agents. We previously reported findings from our optimization study of a high-throughput screening (HTS) derived lead compound 1a that resulted in the discovery of potent amidine-containing FXa inhibitors represented by compound 2. We also conducted an alternative optimization study of 1a without incorporating a strong basic amidine group, which generally has an adverse effect on the pharmacokinetic profile after oral administration. Replacement of 4-methoxybenzene with a 1,4-benzodiazepine structure and introduction of a hydroxy group at the central benzene led to the discovery of the potent and orally effective factor Xa inhibitor 14i (darexaban, YM150). Subsequent extensive study revealed a unique aspect to the pharmacokinetic profile of this compound, wherein the hydroxy moiety of 14i is rapidly transformed into its glucuronide conjugate 16 (YM-222714) as an active metabolite after oral administration and it plays a major role in expression of potent anticoagulant activity in plasma. The distinctive, potent activity of inhibitor 14i after oral dosing was explained by this unique pharmacokinetic profile and its favorable membrane permeability. Compound 14i is currently undergoing clinical development for prevention and treatment of thromboembolic diseases.

Increased levels of citrullinated antithrombin in plasma of patients with rheumatoid arthritis and colorectal adenocarcinoma determined by a newly developed ELISA using a specific monoclonal antibody

Citrullination is a post-translational modification that plays essential roles in both physiological processes and disease. Recent studies have found increased levels of citrullinated antithrombin in patients with rheumatoid arthritis and in different malignant tumours. Antithrombin, the main haemostatic serpin, loses its anticoagulant function via citrullination, which might contribute to the pathogenesis or thrombotic side effects of these disorders. We have developed a specific monoclonal antibody against citrullinated antithrombin. We determined the levels of citrullinated antithrombin and anti-FXa activity in plasma from 66 donors, 17 patients with rheumatoid arthritis and 77 patients with colorectal adenocarcinoma (42 suffering from venous thrombosis). Healthy subjects had negligible amounts of citrullinated antithrombin in plasma (7.9 ± 22.1 ng/ml), while it significantly increased in patients with rheumatoid arthritis or adenocarcinoma (159.7 ± 237.6 ng/ml and 36.8 ± 66.1 ng/ml), levels that, however, did not modify the plasma anticoagulant activity. Moreover, we did not find association between citrullinated antithrombin and the thrombotic risk in patients with adenocarcinoma. In conclusion, we have developed an antibody specific for citrullinated antithrombin that allows its quantification in biological samples, offering a new tool for the analysis of citrullination in different diseases. We confirm increased levels of citrullinated antithrombin in plasma of patients with rheumatoid arthritis and adenocarcinoma. This modification, probably local, could have pathological consequences in both disorders, but only affects a minor fraction of plasma antithrombin, resulting in no significant reduction of global anticoagulant activity. This result explains the absence of association of this marker with an increased risk of thrombosis in patients with colorectal adenocarcinoma.

Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation

AbstractProtamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% ± 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by down-regulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation.

Highly efficacious factor Xa inhibitors containing α-substituted phenylcycloalkyl P4 moieties

We previously disclosed a series of highly potent FXa inhibitors bearing α-substituted (CH 2NR 1R 2) phenylcyclopropyl P4 moieties in the pyrazolodihydropyridone core system. Herein, we describe our continuous SAR efforts in this series. Effects of the C-3 substitution of the pyrazolodihydropyridone core and the α -substitution (R group) of the cyclopropyl ring on FXa binding affinity (FXa K i), human plasma anticoagulant activity (PT EC 2×) and permeability are discussed. A set of compounds obtained from optimization of the R group and the C-3 substituent were orally bioavailable in dogs. Furthermore, representative compounds were highly efficacious in the rabbit arterio-venous shunt thrombosis model (EC 50s = 29–81 nM).