Amidolytic Assay Research Articles

P1077PERSISTENCE OF CIRCULATING RESIDUAL HEPARIN IN ESRD PATIENTS UNDERGOING MAINTENANCE HEMODIALYSIS

Abstract Background and Aims ESRD patients who receive routine maintenance hemodialysis are administered with unfractionated heparin to prevent thrombotic complications. The hemostatic dysregulation along with detectable levels of circulating heparin may cause them to be in a hypocoagulable state. The purpose of this study is to determine the circulating levels of heparin in ESRD patients and its characterization using heparinase digestion methods. Method Blood plasma samples collected at routine pre-dialysis sessions from 95 ESRD patients undergoing maintenance hemodialysis were analyzed for the presence of residual heparin utilizing standard laboratory methods such as aPTT, Anti-Xa and Anti-IIa activities On a centrifugal analyzer (ACL-Elite: Instrumentation Laboratory, Bedford, MA). The levels of heparin were calculated in terms of units per ml relative to the USP reference standard. Heparinase digestion was used to confirm the presence of heparin. Results Blood plasma samples collected at routine pre-dialysis sessions from 95 ESRD patients undergoing maintenance hemodialysis were analyzed for the presence of residual heparin utilizing standard laboratory methods such as aPTT, Anti-Xa and Anti-IIa activities On a centrifugal analyzer (ACL-Elite: Instrumentation Laboratory, Bedford, MA). The levels of heparin were calculated in terms of units per ml relative to the USP reference standard. Heparinase digestion was used to confirm the presence of heparin. Conclusion The presence of residual heparin was demonstrated by both clot- based and amidolytic assays in the plasma samples collected from ESRD patients prior to their next-dialysis session. Since these samples were obtained 3 days following the last dialysis session the presence of significant levels of heparin was surprising. Upon heparinase treatment of these samples, the aPTT and the anti-Xa and IIa tests were restored to near normal levels. Our studies confirm the presence of residual heparin in pre-dialysis plasma samples obtained from ESRD patients. The Anti-IIa activity was greater pre-heparinase and it was not decreased to the same extent as Anti-Xa after heparinase digestion. These results suggest that heparin found in ESRD patient’s plasma is of high molecular weight origin with delayed clearance.

Tissue Factor Pathway Inhibitor (TFPI) release by bovine, ovine and porcine unfractionated heparins in primates. Comparative studies at mass and potency adjusted intravenous dosing

BackgroundUnfractionated heparin (UFH) is a highly sulfated glycosaminoglycan (GAG) that consists of repeating disaccharide units, containing iduronic acid (or glucuronic acid) and glucosamine, exhibiting variable degrees of sulfation. UFHs release tissue factor pathway inhibitor (TFPI) which inhibits extrinsic pathway of coagulation by rapidly inactivating of Factor Xa and the Factor VIIa/TF complex. Bovine mucosal heparins (BMH) and ovine mucosal heparin (OMH) are currently being developed for re‐introduction for both medical and surgical indications. In contrast to the porcine mucosal heparins (PMH), the active pharmaceutical ingredient (API) BMH exhibits a somewhat weaker anti‐coagulant activity as cross‐referenced against PMH. In this study, we determined the TFPI antigen release level by various dosages of UFHs using ELISA method. Also, we projected that potency adjusted BMH at equivalent dosage has the same TFPI release profile compared to OMH and PMH.Materials & MethodsBMH, OMH and PMH were obtained from various commercial sources. Potencies of each heparin were determined by an amidolytic anti‐Xa assay in relation to the USP heparin standard. Individual groups of primates (n=4) were administered 0.5 mg/kg or (50 or 100) U/kg dosage of bovine, ovine or porcine UFH via intravenous route. Blood samples were collected at baseline, 15, 30, 60 and 120 minutes post‐administration. Tissue Factor Pathway Inhibitor (TFPI) antigen levels were measured using a commercially available sandwich ELISA methods. All results were compiled as mean ± SD.ResultsAt a gravimetric level (0.5 mg/kg), BMH exhibited a lower TFPI level than OMH & PMH at all time points with a maximum value of (200 ng/ml) at 15 mins post drug administration. OMH and PMH showed comparable TFPI levels at all time points with maximum values of (250 ng/ml) and (265 ng/ml) respectively at 15 mins post drug administration. However, USP cross‐referenced anti‐Xa potency adjusted based dosing showed comparable TFPI antigen levels for all three UFHs at all time points with a maximum value of 250 ng/ml at 15 mins post drug administration. At 60 mins post drug administration, potency adjusted BMH showed slightly higher TFPI level of (200 ng/ml) compared to both OMH & PMH.ConclusionTissue factor pathway inhibitor (TFPI) levels at 15 minutes after IV administration of 0.5 mg/kg heparins to non‐human primates showed that both PMH and OMH release comparable TFPI antigen levels which were significantly higher than BMH. Potency equated dosing resulted in comparable TFPI release profiles for all three heparins. Therefore such dosing may provide uniform levels of anticoagulation for the parenteral indications for UFHs. These observations suggest that potency equated bovine heparin has comparable release of TFPI and therefore is biosimilar to the OMH and PMH.

The Pharmacokinetics of Fucoidan after Topical Application to Rats.

Fucoidan, a fucose-rich polysaccharide from brown algae, has been used for transdermal formulations targeting inflammatory skin conditions, for the treatment of thrombosis, vascular permeability diseases, subcutaneous wounds, and burns. However, the pharmacokinetics of fucoidan after topical application has not been described. In this study, an ointment (OF) containing 15% fucoidan was topically applied to rats at the doses of 50–150 mg/g. The anti-Xa activity was selected as the biomarker, and the amidolytic assay method was validated and applied for pharmacokinetic studies of fucoidan. Fucoidan in OF penetrated the skin and distributed into the skin, striated muscle, and plasma with AUC0–48 = 0.94 μg·h/g, 2.22 μg·h/g, and 1.92 µg·h/mL, respectively. The longest half-life for fucoidan was observed in plasma, then in striated muscle and skin. It was found that the pharmacokinetics of fucoidan after topical OF application was linear, in the range of 50–150 mg/kg. No accumulation of fucoidan in plasma was observed after repeated topical applications of 100 mg/kg during five days. Our results support the rationality of topical application of formulations with fucoidan.

Andexanet Alpha Differentially Neutralizes the Anticoagulant, Antiprotease and Thrombin Generation Inhibitory Effects of Unfractionated Heparin, Enoxaparin and Fondaparinux

Introduction: Andexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh-zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the effects of apixaban and rivaroxaban and is approved for the control of bleeding complications associated with their use. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma-carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti-Xa agents AA is also reported to neutralize the biologic effects of heparin and related drugs. Assay dependent variations in the neutralization profile of various factor Xa inhibitors by andexanet has been recently reported https://doi.org/10.1177/1076029619847524. Since heparin and related drugs also mediate their biologic actions by inhibiting factor Xa via AT complexation, it is hypothesized that AA may also inhibit their biologic effects as measured in various laboratory assays. It is the purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin, enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA. Materials and Methods: API versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT) and thrombin time (TT) in a concentration range of 0 - 10 ug/ml in pooled human plasma. The anti-Xa and anti-IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti-protease and thrombin generation effects of each of these agents were studied by supplementing this agent at 100 ug/ml. The results are compared to determine the difference between pre and post AA neutralization settings. Results: All agents produce a concentration dependent effect in the anticoagulant and anti-protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti-IIa actions and strong anti-Xa activity. In the anti-Xa assay the IC-50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). However, there was no neutralization of the anti-Xa effects of the F by AA and the IC50 remained the same for both pre and post andexxa studies. The anticoagulant effects of UFH as measured by aPTT and TT was strongly neutralized whereas E was only partially neutralized in the aPTT assay and almost completely neutralized in the thrombin time assay. At concentrations of up to 10 ug/ml F did not produced any significant anticoagulant effects, both in the presence and absence of AA. In the thrombin generation inhibition assays, UFH produced a complete inhibition of thrombin generation which was completely reversed by AA. Although both E and F produced strong inhibition of thrombin generation, AA did not completely neutralize these effects. The results are tabulated on table 1 for the studies carried out at 10 ug/ml of UFH, E and F. Conclusion: These results indicate that AA is capable of differentially neutralizing anticoagulant and anti-protease effects of UFH in an assay dependent manner. However, AA is incapable of neutralizing the anti-Xa effects of E and F. This may be due to the relatively differential affinities of enoxaparin and fondaparinux AT complex to factor Xa rendering it inhibited in the presence of AA. These studies also demonstrate that the primary surrogate marker anti-Xa activity for measuring the activities of anti-Xa agents is not proportional to the anticoagulant and thrombin generation inhibitory effects of these agents. A global clotting assay may be a better indication of the biologic effects of these agents and their reversal by AA. Disclosures Tafur: Recovery Force: Consultancy; Janssen: Other: Educational Grants, Research Funding; BMS: Research Funding; Idorsia: Research Funding; Daichi Sanyo: Research Funding; Stago: Research Funding; Doasense: Research Funding.

USP Potency Adjusted Bovine Mucosal Heparins (BMH) Are Comparable to Porcine Mucosal Heparin (PMH) at Equivalent Levels

Introduction: Unfractionated heparin (UFH) remains to be the only parenteral anticoagulant used in the management of various thrombotic disorders such as deep vein thrombosis (DVT), pulmonary embolism (PE), and cardiovascular interventions. Most of the heparins used clinically are derived from porcine intestinal mucosa. There is likelihood of supply shortage of this important anticoagulant which is crucial for hemodialysis, cardiopulmonary bypass surgery and other vascular interventions. BMH are currently being developed for re-introduction for both medical and surgical indications. In contrast to the PMH, the active pharmaceutical ingredient (API) of BMH exhibit a somewhat weaker USP potency as cross-referenced against PMH. We hypothesized that at equivalent potencies as adjusted by using the USP reference, the BMH may exhibit comparable in vitro and in vivo effects. Therefore, in vitro and in vivo studies were used to compare the APIs of the bovine (140 U/mg) and the PMH (190 U/mg) to demonstrate their bioequivalence. Materials and Methods: API versions of PMH (190 U/mg) were obtained from Celsus Laboratories (Franklin, OH). API versions of BMH (140 U/mg) were obtained from KinMaster (Paso Fundo, Brazil). Each of these heparins was assayed for their molecular weight profile, AT affinity, USP potency, protamine and platelet factor 4 neutralization and anticoagulant/antiprotease profiles using standard laboratory methods. In the primate studies, potencies of each heparin were determined by amidolytic anti-Xa assay in relation to the USP heparin standard. Individual groups of primates (n=4) were administered 100 anti-Xa U/kg doses of bovine or porcine heparin via intravenous route. Blood samples were collected prior to dosing and at 15-, 30-, 60- and 120-minutes post-administration. Anti-Xa and anti-IIa activities were measured to determine circulating heparin concentrations using commercially available USP compliant kits (Aniara Diagnostica, West Chester, OH). These drug concentrations were used to determine pharmacokinetic parameters such as area under the curve (AUC), half-life (t1/2), clearance (Cl) and volume of distribution (Vd) using the PKSolver add-in for Excel. Results: BMH exhibited higher molecular weight profiles compared to PMH as determined by size exclusion chromatography (BMH (Mw) 18.6 ± 0.5 kDa and PMH 15.4 ± 0.4 kDa). BMH exhibited a potency of 140 U/mg and PMH had a potency of 195 U/mg. In the anticoagulant and antiprotease assays, the BMH exhibited lower functionality which was proportional to USP potency. In vitro, when the BMH was compared at a potency adjusted concentration with PMH, it showed identical calibration curves in the aPTT and anti-protease assays. However, in the protamine neutralization and platelet factor 4 studies, BMH required slightly higher amounts of the agents in contrast to PMH. The concentration vs. time curves for both heparins were almost superimposable. Peak drug levels of approximately 1.5 and 1.4 U/mL were measured using anti-Xa and anti-IIa assays, respectively. After 2 hours, circulating drug levels were decreased to approximately 0.4 U/mL for all heparins. Pharmacokinetic parameters calculated from plasma concentration-time curves indicated that both heparins behaved similarly. Mean half-life based on anti-Xa activity ranged from 54 ± 11 min for porcine heparin to 71 ± 18 min for bovine heparin. Slightly longer half-lives were observed using plasma concentrations determined using anti-IIa activity. Mean AUC values based on anti-Xa or anti-IIa activities were comparable for both heparins. Mean Vd (~60 ml/kg) and Cl (~0.75 ml/kg/min) were also comparable for both heparins. Conclusion: In vitro, BMH at adjusted biologic potency is comparable to PMH, however, it requires proportionally higher amount of protamine and platelet factor 4 due to the increased mass for adjusting to higher potency. In the non-human primates, USP cross-referenced anti-Xa potency adjusted based dosing results in comparable pharmacokinetic profiles for bovine and porcine heparins. Therefore, such dosing may provide uniform levels of anticoagulation for the parenteral indications for heparins. These observations warrant clinical validations in the specific indications. Disclosures No relevant conflicts of interest to declare.

Differential Neutralization of Unfractionated Heparin, Enoxaparin and Fondaparinux by Andexanet Alpha

IntroductionAndexanet Alpha (Coagulation factor Xa recombinant, inactivated Zh‐zo; AA, Portola Pharmaceuticals) is a recombinant factor Xa decoy protein which is designed to reverse the inhibition of factor Xa in humans to control bleeding associated with newer oral anti‐Xa agents. The molecular modification in this recombinant protein involves the substitution of serine active site by alanine and the removal of the gamma‐carboxyglutamic acid (GLA) domain to restrict its assemblage into prothrombinase complex. Beside the reversal of the effects of anti‐Xa agents AA is also reported to neutralize the effects of heparin and related drugs. The purpose of this study is to compare the relative neutralization profile of heparin (UFH), a low molecular weight heparin enoxaparin (E) and a chemically synthetic pentasaccharide, Fondaparinux (F) by AA.Materials and MethodsAPI versions of UFH, E and F were commercially obtained in powdered forms and dissolved in saline at a working dilution of 1mg/ml. AA was dissolved in saline to obtain a 10mg/ml working solution. The anticoagulant profile of UFH, E and F was studied using the activated partial thromboplastin time (APTT), thrombin time (TT) and prothrombinase activated clotting time (PICT) in a concentration range of 0 – 10 ug/ml in pooled human plasma. The anti‐Xa and anti‐IIa studies were carried out in amidolytic assays in the same concentration range. The thrombin generation inhibition was studied using calibrated automated thrombin generation systems (CAT, Diagnostica Stago). The effect of AA on the reversal of the anticoagulant and anti‐protease effects of each of these agents was studied by supplementing this agent at 100 ug/ml.ResultsAll agents produce a concentration dependent effect in the anticoagulant and anti‐protease assays with the exception of F which showed mild anticoagulant effects, and very weak anti‐IIa actions and strong anti‐Xa activity. In the anti‐Xa assay the IC‐50 for UFH was 2.1ug/ml (0.13 um), E 4.3 ug/ml (0.95 um) and F 0.7 ug/ml (0.41 um) upon supplementation of AA the IC50s for UFH was increased to 5 ug/ml (0.31 um) and for E 5 ug/ml (1.11 um). There was no change in the IC50 for F. The anticoagulant effects of UFH and E were neutralized at variable levels and were assay dependent. However there was no neutralization of the anti‐Xa effects of the F by AA. At high concentrations of greater than 2.5 ug/ml (1.47 um) the mild anticoagulant effects of F were neutralized in an assay dependent fashion.ConclusionThese results indicate that AA is capable of differentially neutralizing anticoagulant and antiprotease effects of UFH and E in an assay dependent manner. However AA is incapable of neutralizing the anti‐Xa effects of F. This may be due to the relatively strong affinity of F AT complex to factor Xa rendering it inhibited in the presence of AA.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Pharmacokinetic and Pharmacodynamic Profiles of Potency‐Adjusted Doses of Bovine, Ovine and Porcine Heparin are Comparable in a Primates Model

BackgroundUnfractionated heparin remains to be the only parenteral anticoagulant for various indications. Currently heparin is mainly derived from porcine mucosal heparin tissues. The USFDA and other regulatory agencies have considered resourcing heparin from alternate sources. Currently sheep (ovine) and cow (bovine) mucosal heparins are being developed for clinical use. The purpose of this study was to compare commercially available porcine heparin with the heparins obtained from bovine and ovine mucosal sources.Materials and MethodsBovine, ovine and porcine heparins were obtained from various commercial sources. Potencies of each heparin were determined by an amidolytic anti‐Xa assay in relation to the USP heparin standard. Individual groups of primates (n=4) were administered 100 anti‐Xa U/kg dosage of bovine, ovine or porcine heparin via intravenous route. Blood samples were collected at baseline, 15, 30, 60 and 120 minutes post‐administration. Anti‐Xa and anti‐IIa activities were measured to determine circulating heparin concentrations using commercially available USP compliant kits (Aniara Diagnostica, West Chester, OH). These drug concentrations were used to determine pharmacokinetic parameters such as area under the curve (AUC), half‐life (t1/2), clearance (Cl) and volume of distribution (Vd) using the PKSolver add‐in for Excel. The tissue factor pathway inhibitor (TFPI) levels were measured at 15 minutes.ResultsThe concentration vs. time curves for the three heparins were almost superimposable. Peak drug levels of approximately 1.5 and 1.4 U/mL were measured using anti‐Xa and anti‐IIa assays, respectively. After 2 hours, circulating drug levels were decreased to approximately 0.4 U/mL for all heparins. Pharmacokinetic parameters calculated from plasma concentration‐time curves indicated that all three heparins behaved similarly. Mean half‐life based on anti‐Xa activity ranged from 44 ± 13 min for ovine heparin to 71 ± 18 min for bovine heparin. Slightly longer half‐lives were observed using plasma concentrations determined using anti‐IIa activity. Mean AUC values based on anti‐Xa or anti‐IIa activities were comparable for all heparins. Mean Vd (~60 ml/kg) and Cl (~0.75 ml/kg/min) were also comparable for all heparins. The TFPI levels were similar for the porcine and ovine heparin whereas the bovine heparin exhibited lower levels.ConclusionAt a gravimetric level the bovine heparin exhibits a lower anticoagulant potency (140 U/mg) than ovine or porcine heparins (200 U/mg). USP cross‐referenced anti‐Xa potency adjusted based dosing results in comparable pharmacokinetic profiles for all three heparins. Therefore such dosing may provide uniform levels of anticoagulation for the parenteral indications for heparins. These observations warrant clinical validations in the specific indications.Support or Funding InformationNone.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BFPI: A Minimal Prothrombinase Inhibitor from the Saliva of Simulium Vittatum

Background: The black fly, Simulium Vittatum, has an anticoagulant protein in its saliva that allows it to feed on mammalian blood (black fly protease inhibitor; BFPI). Remarkably, BFPI is similar to the human anticoagulant tissue factor pathway inhibitor alpha (TFPIα). TFPIα contains three Kunitz-type protease inhibitor domains (K1, K2, K3), which inhibit factor VIIa (FVIIa) and factor Xa (FXa) and bind the co-factor protein S (PS), respectively; BFPI contains a single Kunitz domain that inhibits FXa. In addition, TFPIα and BFPI contain homologous basic regions (BRs) near their C-termini (252LIKTKRKRKK261 in human TFPIα, LIKTRKRKPKK in BFPI). The TFPIα BR binds a regulatory acidic region (AR) in factor Va (FVa). The AR is present in forms of FVa released by collagen-activated platelets and generated through limited proteolysis by FXa (FVaXa), and is removed by thrombin (FVaIIa). We hypothesized that BFPI, through its Kunitz domain and basic C-terminus, inhibits early forms of the prothrombinase complex, but does not possess the other inhibitory functions of TFPIα: (1) K1-dependent inhibition of the tissue factor (TF)-FVIIa complex; and (2) PS/K3-dependent FXa inhibition.Results: Recombinant BFPI inhibited FXa in an amidolytic activity assay, and PS did not promote this inhibition. BFPI did not inhibit TF-FVIIa-mediated FX activation. As described with TFPIα, FV promoted FXa inhibition by BFPI but FVaIIa did not, suggesting that the BFPI BR is capable of binding the FVa AR. In a purified protein assay, BFPI inhibited prothrombinase assembled with FVaXa (IC50=4.9nM), but not FVaIIa. Similarly, 5nM BFPI increased the lag time for FXa-initiated plasma thrombin generation by 10.4±1.5%.We next used BFPI as a backbone to evaluate a reported human mutation in the TFPIα BR, K254E. Every mammalian, avian, or reptilian TFPIα sequence available contains either a Lys or Arg residue at this position, suggesting that this residue is functionally important. In purified protein assays, BFPI-K254E inhibited FXa amidolytic activity identically to BFPI, but FV did not promote this inhibition, suggesting that BFPI-K254E has a specific defect in its interaction with FV. Consistent with this, BFPI-K254E was a weaker inhibitor of prothrombinase assembled with FVaXa (IC50 = 15.8nM) and FXa-initiated plasma thrombin generation.The results obtained with BFPI-K254E were confirmed using peptides and full-length TFPIα proteins. First, a peptide mimicking the wild type TFPIα BR (LIKTKRKRKK) inhibited prothrombinase assembled with FVaXa (IC50 = 1.0 µM), while the substituted peptide (LIETKRKRKK) was substantially weaker (20% inhibition observed with 340 µM peptide). Second, full-length TFPIα-K254E was a weaker inhibitor of prothrombinase containing FXa-activated FVa (IC50 = 14.8 nM, vs. 1.8 nM for TFPIα) and had greatly reduced anticoagulant activity in plasma-based thrombin generation assays.Conclusions: In summary, the anticoagulant effect of BFPI is mediated through inhibition of early forms of prothrombinase, independent of TF-FVIIa inhibition or PS-dependent FXa inhibition. The natural mutation TFPIα K254E disrupts prothrombinase inhibition, despite the presence of six other conserved basic residues, and is thus procoagulant in human plasma. The absolute conservation of the TFPIα BR, and its usurpation to allow feeding by black flies, point to formation of the initial prothrombinase complex as a key regulatory step in blood coagulation. DisclosuresMast:Novo Nordisk: Research Funding.

Pharmacokinetic and Tissue Distribution of Fucoidan from Fucus vesiculosus after Oral Administration to Rats.

Fucus vesiculosus L., known as bladderwrack, belongs to the brown seaweeds, which are widely distributed throughout northern Russia, Atlantic shores of Europe, the Baltic Sea, Greenland, the Azores, the Canary Islands, and shores of the Pacific Ocean. Fucoidan is a major fucose-rich sulfated polysaccharide found in Fucus (F.) vesiculosus. The pharmacokinetic profiling of active compounds is essential for drug development and approval. The aim of the study was to evaluate the pharmacokinetics and tissue distribution of fucoidan in rats after a single-dose oral administration. Fucoidan was isolated from F. vesiculosus. The method of measuring anti-activated factor X (anti-Xa) activity by amidolytic assay was used to analyze the plasma and tissue concentrations of fucoidan. The tissue distribution of fucoidan after intragastric administration to the rats was characterized, and it exhibited considerable heterogeneity. Fucoidan preferentially accumulates in the kidneys (AUC0–t = 10.74 µg·h/g; Cmax = 1.23 µg/g after 5 h), spleen (AUC0–t = 6.89 µg·h/g; Cmax = 0.78 µg/g after 3 h), and liver (AUC0–t = 3.26 µg·h/g; Cmax = 0.53 µg/g after 2 h) and shows a relatively long absorption time and extended circulation in the blood, with a mean residence time (MRT) = 6.79 h. The outcome of this study provides additional scientific data for traditional use of fucoidan-containing plants and offers tangible support for the continued development of new effective pharmaceuticals using fucoidan.

Antithrombin Debrecen (p.Leu205Pro) – Clinical and molecular characterization of a novel mutation associated with severe thrombotic tendency

IntroductionHereditary antithrombin (AT) deficiency is a rare thrombophilic disorder with heterogeneous genetic background and various clinical presentations. In this study we identified a novel AT mutation. Genotype-phenotype correlations, molecular characteristics and thrombotic manifestations of the mutation were investigated. Materials and methodsThirty-one members of a single family were included. Clinical data was collected regarding thrombotic history. The mutation was identified by direct sequencing of the SERPINC1 gene. HEK293 cells were transfected with wild type and mutant SERPINC1 plasmids. Western blotting, ELISA and functional amidolytic assay were used to detect wild type and mutant AT. After double immunostaining, confocal laser scanning microscopy was used to localize mutant AT in the cells. Molecular modeling was carried out to study the structural-functional consequences of the mutation. ResultsUnprovoked venous thrombotic events at early age, fatal first episodes and recurrences were observed in the affected individuals. The median AT activity was 59%. Genetic analysis revealed heterozygous form of the novel mutation p.Leu205Pro (AT Debrecen). The mutant AT was expressed and synthesized in HEK293 cells but only a small amount was secreted. The majority was trapped intracellularly in the trans‑Golgi and 26S proteasome. The mutation is suspected to cause considerable structural distortion of the protein. The low specific activity of the mutant AT suggested functional abnormality. ConclusionsAT Debrecen was associated with very severe thrombotic tendency. The mutation led to misfolded AT, impaired secretion and altered function. Detailed clinical and molecular characterization of a pathogenic mutation might provide valuable information for individualized management.

In vitro characterization of jellyfish venom fibrin(ogen)olytic enzymes from Nemopilema nomurai

BackgroundBecause jellyfish are capable of provoking envenomation in humans, they are considered hazardous organisms. Although the effects of their toxins are a matter of concern, information on the venom components, biological activity and pathological mechanisms are still scarce. Therefore, the aim of the present study was to investigate a serine protease component of Nemopilema nomurai jellyfish venom (NnV) and unveil its characteristics.MethodsTo determine the relationship between fibrinolytic activity of NnV and the serine protease, fibrin zymography was performed using metalloprotease and serine protease inhibitors. The biochemical characterization of serine proteases of NnV were determined by the amidolytic assay. Fractions with fibrinolytic activity were obtained by DEAE cation exchange column.ResultsNnV displayed fibrinolytic activities with molecular masses of approximately 70, 35, 30, and 28 kDa. The fibrinolytic activity of NnV was completely obliterated by phenylmethylsulfonyl fluoride, a prototype serine protease inhibitor. Based on amidolytic assays using chromogenic substrates specific for various kinds of serine proteases, NnV predominantly manifested a chymotrypsin-like feature. Its activity was completely eliminated at low pH (< 6) and high temperatures (> 37 °C). Some metal ions (Co2+, Cu2+, Zn2+ and Ni2+) strongly suppressed its fibrinolytic activity, while others (Ca2+ and Mg2+) failed to do so. Isolation of a serine protease with fibrionolytic activity from NnV revealed that only p3 showed the fibrinolytic activity, which was completely inhibited by PMSF.ConclusionThe present study showed that N. nomurai jellyfish venom has a chymotrypsin-like serine protease with fibrinolytic activity. Such information might be useful for developing clinical management of jellyfish envenomation and pharmacological agents with therapeutic potential for thrombotic diseases in the future.

Browplasminin, a condensed tannin with anti-plasmin activity isolated from an aqueous extract of Brownea grandiceps Jacq. flowers

Ethnopharmacological relevanceFollowing Venezuelan traditional medicine, females with heavy menstrual blood loss (menorrhagia) drink Brownea grandiceps Jacq. flowers (BG) decoctions to reduce the bleeding. In a previous study, we demonstrated that BG aqueous extract (E) possesses a potent anti-fibrinolytic activity capable of inhibiting plasmin, the main serine-protease that degrades fibrin. It is widely known that plasmin inhibitors are often used as anti-fibrinolytics to reduce bleeding during surgeries with high risk of blood loss such as cardiac, liver, vascular, tooth extraction and large orthopedic procedures, as well as for menorrhagia treatments. The aim of this work was to isolate and characterize from BGE the compound responsible for the reported anti-fibrinolytic activity. Materials and methodsA decoction of BG was prepared; then it was homogenized, centrifuged and lyophilized to obtain BGE. Subsequently the extract was fractionated by gel filtration and reverse phase using HPLC and the active compound was characterized by MALDI-ToF MS. The kinetic parameters of anti-plasmin activity were evaluated by an amidolytic assay using a chromogenic substrate; also the anti-plasmin activity was estimated by fibrin plate method. Data were analyzed by nonparametric statistics. ResultsThe active compound was a condensed tannin denominated Browplasminin, which is capable of inhibiting the plasmin activity in a dose-dependent manner when measured in fibrin plates or by the amidolytic activity method; it also has a minor effect on the FXa activity. However, it does not affect the activity of other serine-proteases such as trypsin, t-PA or u-PA. Browplasminin consists predominately of heteroflavan-3-ols of catechin with B-type linkages, and extents up to heptadecamers (~ 5000Da), with hexose residues attached to the polymer that presents a high degree of galloylation. Its IC50 for plasmin was 47.80μg/mL and for FXa was 237.08μg/mL, while the Ki were 0.76 and 61.61μg/mL for plasmin and FXa, respectively. ConclusionsThe overall outcome of this study suggests that Browplasminin could be responsible for reducing heavy menstrual bleeding in women because its kinetic parameters showed that is a good plasmin inhibitor.

Porcine and Ovine Mucosal Heparins and Their Depolymerized Derivatives Are Comparable in Contrast to Their Bovine Equivalents

Introduction:The currently used unfractionated heparin (UFH) and low molecular weight heparins (LMWH) are mostly derived from Porcine mucosal tissue. Since the technology to manufacture heparin has advanced and the quality assurance practices are in place, improved products with high potency and purity are now available. Considering these factors the resourcing of heparins utilizing bovine (cow) and ovine (sheep) tissues is discussed at regulatory and pharmaceutical levels. The purpose of this study is to compare multiple individual batches of UFH obtained from Bovine, Ovine, and Porcine origin and their depolymerized product obtained by benzylation followed by alkaline hydrolysis representing enoxaparins.Materials and Methods:The molecular profile of the heparins and enoxaparins from various sources were determined using the size exclusion chromographic method. The anticoagulant potency was measured using clot based methods such as aPTT and Thrombin Time. The AT mediated anti-Xa and anti-IIa activities were also measured in defined biochemical sysytems. A comercially obtained chromogenic substrate based methods were used to determine the USP potency in terms of anti-Xa and anti-IIa activities (Hyphen Biomedical, Ohio, USA). The relative interaction of the heparins and enoxaparins with heparin induced thrombocytopenia (HIT) antibody induced aggregation of platelets were investigated using serum pool obtained from clinically confirmed HIT cases using aggregometry.Results:The molecular profile of multiple batches of the Bovine, ovine, and porcine heparins and enoxaparin were almost comparable and ranged from 15-18 kDa. The global anticoagulant and amidolytic protease assays for the bovine heparin were consistently lower than porcine and ovine samples. In the purified system the Porcine and Ovine preparations consistently showed lower IC50 values for both the thrombin and Xa inhibition in contrast to the bovine heparin. Similar trends were observed in the anti IIa assays. The USP potency of 28 batches of porcine ranged from 170-210 U/mg, whereas the 21 batches of ovine heparins exhibited comparable potencies in the range of 160-210 U/mL. In contrast the USP potency of 30 batches of bovine mucosal heparin was much lower and ranged from 110-140 U/mg. The anti-Xa - IIa ratio for the porcine and ovine heparins were comparable. However, the anti-Xa - IIa ratio of bovine heparin was somewhat lower. The ovine and porcine enoxaparin exhibited comparable potencies which ranged from 94-110 U/mg whereas bovine enoxaparin was slightly lower, ranging from 80-87 U/mg. However the antiXa and anti-IIa ratios of the enoxaparins derived from various species were comparable. In the HIT screening, there was no difference between the HIT responses in the heparins from different species. Similar results were obtained amongst enoxaparins of different origins.Conclusions:These studies show that heparins from bovine, ovine, and porcine origin exhibit comparable molecular profiles. While the porcine and ovine heparins exhibit similar biological potencies, the bovine heparin was found to be weaker. The enoxaparins derived from these species also exhibit similar molecular profiles, however, in functional assays, they exhibited similar trends where the bovine derived products were weaker. In the HIT screening profile, the heparins from all of these species produced stronger effects in comparison to their enoxaparin counterparts. These results suggest that heparin and enoxaparin derived from ovine mucosal tissue exhibit comparable biosimilar profiles. However, the bovine heparins and enoxaparin derived from bovine mucosal tissues are somewhat weaker. DisclosuresYao:Ronnsi Pharma: Employment.

Amblyomin-X having a Kunitz-type homologous domain, is a noncompetitive inhibitor of FXa and induces anticoagulation in vitro and in vivo

BackgroundCancer has long been associated with thrombosis and many of the standard chemotherapeutics used to treat cancer are pro-thrombotic. Thus, the identification of novel selective anticancer drugs that also have antithrombotic properties is of enormous significance. Amblyomin-X is an anticancer protein derived from the salivary glands of the Amblyomma cajennense tick. MethodsIn this work, we determined the inhibition profile of Amblyomin-X and its effect on activated partial thromboplastin time (aPTT) and prothrombin time (PT), using various approaches such as, kinetic analyses, amidolytic assays, SDS-PAGE, and mass spectrometry. ResultsAmblyomin-X inhibited factor Xa, prothrombinase and tenase activities. It was hydrolyzed by trypsin and plasmin. MS/MS data of tryptic hydrolysate of Amblyomin-X suggested the presence of Cys8-Cys59 and Cys19-Cys42 but not Cys34-Cys55 disulfide bond. Instead of Cys34-Cys55, two noncanonical Cys34-Cys74 and Cys55-Cys74 disulfide bonds were identified. Furthermore, when Amblyomin-X (1mg/kg) injected in rabbits, it prolonged aPTT and PT. ConclusionAmblyomin-X is a noncompetitive inhibitor (Ki=3.9μM) of factor Xa. It is a substrate for plasmin and trypsin, but not for factor Xa and thrombin. The disulfide Cys34-Cys55 bond probably scrambles with interchain seventh free cysteine residues (Cys74) of Amblyomin-X. The prolongation of PT and aPTT is reversible.General Significance.In term of anticoagulant property, this is structural and functional characterization of Amblyomin-X. All together, these results and previous findings suggest that Amblyomin-X has a potential to become an anticancer drug with antithrombotic property.

Cloning a Chymotrypsin-Like 1 (CTRL-1) Protease cDNA from the Jellyfish Nemopilema nomurai

An enzyme in a nematocyst extract of the Nemopilema nomurai jellyfish, caught off the coast of the Republic of Korea, catalyzed the cleavage of chymotrypsin substrate in an amidolytic kinetic assay, and this activity was inhibited by the serine protease inhibitor, phenylmethanesulfonyl fluoride. We isolated the full-length cDNA sequence of this enzyme, which contains 850 nucleotides, with an open reading frame of 801 encoding 266 amino acids. A blast analysis of the deduced amino acid sequence showed 41% identity with human chymotrypsin-like (CTRL) and the CTRL-1 precursor. Therefore, we designated this enzyme N. nomurai CTRL-1. The primary structure of N. nomurai CTRL-1 includes a leader peptide and a highly conserved catalytic triad of His69, Asp117, and Ser216. The disulfide bonds of chymotrypsin and the substrate-binding sites are highly conserved compared with the CTRLs of other species, including mammalian species. Nemopilema nomurai CTRL-1 is evolutionarily more closely related to Actinopterygii than to Scyphozoan (Aurelia aurita) or Hydrozoan (Hydra vulgaris). The N. nomurai CTRL1 was amplified from the genomic DNA with PCR using specific primers designed based on the full-length cDNA, and then sequenced. The N. nomurai CTRL1 gene contains 2434 nucleotides and four distinct exons. The 5′ donor splice (GT) and 3′ acceptor splice sequences (AG) are wholly conserved. This is the first report of the CTRL1 gene and cDNA structures in the jellyfish N. nomurai.

The susceptibility of plasma coagulation factor XI to nitration and peroxynitrite action

Coagulation factor XI is present in blood plasma as the zymogen, like other serine proteases of hemostatic system, but as the only coagulation factor forms 140⿿160kDa homodimers. Its activation is induced by thrombin, and a positive feedback increases the generation of the extra thrombin. Experimental and clinical observations confirm protective roles of factor XI deficiencies in certain types of thromboembolic disorders. Thromboembolism still causes serious problems for modern civilization. Diseases associated with the blood coagulation system are often associated with inflammation and oxidative stress. Peroxynitrite is produced from nitric oxide and superoxide in inflammatory diseases. The aim of the current study is to evaluate effects of nitrative stress triggered by peroxynitrite on coagulation factor XI in human plasma employing biochemical and bioinformatic methods. The amidolytic assay shows increase in factor XI activity triggered by peroxynitrite. Peroxynitrite interferes factor XI by nitration and fragmentation, which is demonstrated by immunoprecipitation followed by western blotting. Nitrated factor XI is even present in control blood plasma. The results suggest possible modifications of factor XI on the molecular level. Computer simulations show tyrosine residues as targets of peroxynitrite action. The modifications induced by peroxynitrite in factor XI might be important in thrombotic disorders.

In Vitro Mode of Action and Anti-thrombotic Activity of Boophilin, a Multifunctional Kunitz Protease Inhibitor from the Midgut of a Tick Vector of Babesiosis, Rhipicephalus microplus.

BackgroundHematophagous mosquitos and ticks avoid host hemostatic system through expression of enzyme inhibitors targeting proteolytic reactions of the coagulation and complement cascades. While most inhibitors characterized to date were found in the salivary glands, relatively few others have been identified in the midgut. Among those, Boophilin is a 2-Kunitz multifunctional inhibitor targeting thrombin, elastase, and kallikrein. However, the kinetics of Boophilin interaction with these enzymes, how it modulates platelet function, and whether it inhibits thrombosis in vivo have not been determined.Methodology/Principal FindingsBoophilin was expressed in HEK293 cells and purified to homogeneity. Using amidolytic assays and surface plasmon resonance experiments, we have demonstrated that Boophilin behaves as a classical, non-competitive inhibitor of thrombin with respect to small chromogenic substrates by a mechanism dependent on both exosite-1 and catalytic site. Inhibition is accompanied by blockade of platelet aggregation, fibrin formation, and clot-bound thrombin in vitro. Notably, we also identified Boophilin as a non-competitive inhibitor of FXIa, preventing FIX activation. In addition, Boophilin inhibits kallikrein activity and the reciprocal activation, indicating that it targets the contact pathway. Furthermore, Boophilin abrogates cathepsin G- and plasmin-induced platelet aggregation and partially affects elastase-mediated cleavage of Tissue Factor Pathway Inhibitor (TFPI). Finally, Boophilin inhibits carotid artery occlusion in vivo triggered by FeCl3, and promotes bleeding according to the mice tail transection method.Conclusion/SignificanceThrough inhibition of several enzymes involved in proteolytic cascades and cell activation, Boophilin plays a major role in keeping the midgut microenvironment at low hemostatic and inflammatory tonus. This response allows ticks to successfully digest a blood meal which is critical for metabolism and egg development. Boophilin is the first tick midgut FXIa anticoagulant also found to inhibit thrombosis.

Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis.

Infusion of the heme-binding protein hemopexin has been proposed as a novel approach to decrease heme-induced inflammation in settings of red blood cell breakdown, but questions have been raised as to possible side effects related to protease activity and inhibition of chemotaxis. We evaluated protease activity and effects on chemotaxis of purified plasma hemopexin obtained from multiple sources as well as a novel recombinant fusion protein Fc-hemopexin. Amidolytic assay was performed to measure the protease activity of several plasma-derived hemopexin and recombinant Fc-hemopexin. Hemopexin was added to the human monocyte culture in the presence of lipopolysaccharides (LPS), and also injected into mice intravenously (i.v.) 30 min before inducing neutrophil migration via intraperitoneal (i.p.) injection of thioglycolate. Control groups received the same amount of albumin. Protease activity varied widely between hemopexins. Recombinant Fc-hemopexin bound heme, inhibited the synergy of heme with LPS on tumor necrosis factor (TNF) production from monocytes, and had minor but detectable protease activity. There was no effect of any hemopexin preparation on chemotaxis, and purified hemopexin did not alter the migration of neutrophils into the peritoneal cavity of mice. Heme and LPS synergistically induced the release of LTB4 from human monocytes, and hemopexin blocked this release, as well as chemotaxis of neutrophils in response to activated monocyte supernatants. These results suggest that hemopexin does not directly affect chemotaxis through protease activity, but may decrease heme-driven chemotaxis and secondary inflammation by attenuating the induction of chemoattractants from monocytes. This property could be beneficial in some settings to control potentially damaging inflammation induced by heme.

Resourcing of Heparin and Low Molecular Weight Heparins from Bovine, Ovine, and Porcine Origin. Studies to Demonstrate the Biosimilarities

Introduction:The currently used unfractionated heparin (UFH) and low molecular weight heparins (LMWH) are mostly derived from Porcine mucosal tissue. Since the technology to manufacture heparin has advanced and the quality assurance practices are in place, improved products with high potency and purity are now available. At the same time the demand for heparins has increased requiring alternate sources to obtain heparin and LMWH. Considering these factors the resourcing of heparins utilizing bovine (cow) and ovine (sheep) tissues is discussed at regulatory and pharmaceutical levels. The pharmaceutical industry has already initiated programs to manufacture Bovine, Ovine, and Porcine heparins and depolymerized enoxaparin from these products some of which are currently in various phases of development. The purpose of this study is to compare 5 individual batches of UFH obtained from Bovine, Ovine, and Porcine origin and their depolymerized product obtained by benzylation followed by alkaline hydrolysis representing enoxaparins.Methods:The molecular profile of the heparins and enoxaparins from various sources were determined using the size exclusion method. A narrow range calibration method was used for comparing the molecular weight of heparin, whereas the EP method was used to cross-reference the molecular weight of enoxaparins. The anticoagulant potency was measured use clot based methods such as aPTT and Thrombin Time. Chromogenic substrate based methods were used to determine the USP potency in terms of anti-Xa and anti-IIa activities (Hyphen Biomedical, Ohio, USA). The interaction between AT and heparins and enoxaparin were investigated in a purified biochemical system, using AT supplemented buffered assay. Thrombin Generation inhibition studies were carried out using a flourometric method (Technoclone, Vienna, Austria). The relative interaction of the heparins and enoxaparins with heparin induced thrombocytopenia (HIT) antibody induced aggregation of platelets were investigated using serum pool obtained from clinically confirmed HIT cases using aggregometry.Results:The molecular profile of the Bovine, ovine, and porcine heparins and enoxaparin were almost identical. In the clot based assays, such as PT and PTT, Porcine and Ovine heparin produce consistently comparable anticoagulant effects, which were stronger in comparison to the bovine derived heparins. In contrast, the enoxaparins derived from these three sources showed minimal differences. In the amidolytic anti Xa and IIa assays both ovine and porcine heparins produced similar inhibitory effects, whereas the bovine heparin exhibited lower activity. In the purified system the Porcine and Ovine preparations consistently showed lower IC50 values for both the thrombin and Xa inhibition in contrast to the bovine heparin. Similar trends were observed in the anti IIa assays. The USP potency of the Porcine and Ovine heparins ranged from 180 to 190u/mg, whereas the Bovine was found to be 130-140 u/mg. The anti-Xa - IIa ratio for the heparin were comparable. The ovine and porcine enoxaparin exhibited comparable potencies which ranged 94-110 u/mg whereas bovine enoxaparin was slightly lower 80-87 u/mg. However the antiXa and anti-IIa ratios were comparable. The AT mediated inhibition of factor Xa and anti-IIa was stronger with heparins in comparison to the enoxaparins. Similarly heparins produced stronger inhibition of thrombin generation in comparison to the enoxaparin. In the HIT screening there was no difference between the HIT responses in the heparins from different species. Similar results were obtained with enoxaparins.Conclusions:These studies show that while bovine, ovine and porcine heparins and enoxaparins exhibit comparable molecular profiles however in some of the functional assays bovine heparin and enoxaparin exhibited somewhat lesser potencies especially in the pharmacopeial assays. No differences were noted in the HIT antibody interactions among heparins and enoxaparins from different species. These studies demonstrate that ovine and porcine heparins are biosimilar and can be developed as such for clinical purposes. The bovine derived heparins exhibit slightly weaker potencies in functional assays despite comparable molecular profile. Potency adjustment for in vivo usage may be required to obtain comparable anticoagulant responses for the bovine heparin and enoxaparin. DisclosuresNo relevant conflicts of interest to declare.

Phospholipid Binding Protein C Inhibitor (PCI) Is Present on Microparticles Generated In Vitro and In Vivo.

Protein C inhibitor is a secreted, non-specific serine protease inhibitor with broad protease reactivity. It binds glycosaminoglycans and anionic phospholipids, which can modulate its activity. Anionic phospholipids, such as phosphatidylserine are normally localized to the inner leaflet of the plasma membrane, but are exposed on activated and apoptotic cells and on plasma membrane-derived microparticles. In this report we show by flow cytometry that microparticles derived from cultured cells and activated platelets incorporated protein C inhibitor during membrane blebbing. Moreover, protein C inhibitor is present in/on microparticles circulating in normal human plasma as judged from Western blots, ELISAs, flow cytometry, and mass spectrometry. These plasma microparticles are mainly derived from megakaryocytes. They seem to be saturated with protein C inhibitor, since they do not bind added fluorescence-labeled protein C inhibitor. Heparin partially removed microparticle-bound protein C inhibitor, supporting our assumption that protein C inhibitor is bound via phospholipids. To assess the biological role of microparticle-bound protein C inhibitor we performed protease inhibition assays and co-precipitated putative binding partners on microparticles with anti-protein C inhibitor IgG. As judged from amidolytic assays microparticle-bound protein C inhibitor did not inhibit activated protein C or thrombin, nor did microparticles modulate the activity of exogenous protein C inhibitor. Among the proteins co-precipitating with protein C inhibitor, complement factors, especially complement factor 3, were most striking. Taken together, our data do not support a major role of microparticle-associated protein C inhibitor in coagulation, but rather suggest an interaction with proteins of the complement system present on these phospholipid vesicles.