Thrombin Generation In Human Plasma Research Articles

Rivaroxaban and apixaban are less effective than enoxaparin for the prevention of catheter-induced clotting in vitro

BackgroundCentral venous catheters are prone to clotting, particularly in patients with cancer. Although low-molecular-weight heparin and direct oral anticoagulants, such as apixaban and rivaroxaban, have been evaluated for the prevention of catheter thrombosis, their efficacy remains uncertain. ObjectivesCompare apixaban and rivaroxaban with enoxaparin for the prevention of catheter-induced clotting in vitro. MethodsTo address this uncertainty, we used a well-established microplate-based assay to compare the effects of enoxaparin, apixaban, and rivaroxaban on catheter-induced thrombosis and thrombin generation in human plasma. ResultsConsistent with our previous findings, catheter segments shortened the clotting time and promoted thrombin generation. When compared at concentrations with similar anti-factor Xa activity as enoxaparin, apixaban and rivaroxaban were >20-fold less potent than enoxaparin for the prevention of catheter-induced clotting and thrombin generation. ConclusionThe prevention of catheter thrombosis in patients with cancer is challenging. Clinical trials are needed to compare the efficacy of low-molecular-weight heparin with that of direct oral anticoagulants both for the prevention and treatment of catheter thrombosis.

Intrinsic coagulation pathway-mediated thrombin generation in mouse whole blood.

Calibrated Automated Thrombography (CAT) is a versatile and sensitive method for analyzing coagulation reactions culminating in thrombin generation (TG). Here, we present a CAT method for analyzing TG in murine whole blood by adapting the CAT assay used for measuring TG in human plasma. The diagnostically used artificial and physiologic factor XII (FXII) contact activators kaolin, ellagic acid and polyphosphate (polyP) stimulated TG in murine blood in a dose-dependent manner resulting in a gradual increase in endogenous thrombin potential and peak thrombin, with shortened lag times and times to peak. The activated FXII inhibitor rHA-Infestin-4 and direct oral anticoagulants (DOACs) interfered with TG triggered by kaolin, ellagic acid and polyP and TG was completely attenuated in blood of FXII- (F12 -/-) and FXI-deficient (F11 -/-) mice. Moreover, reconstitution of blood from F12 -/- mice with human FXII restored impaired contact-stimulated TG. HEK293 cell-purified polyP also initiated FXII-driven TG in mouse whole blood and addition of the selective inhibitor PPX_Δ12 ablated natural polyP-stimulated TG. In conclusion, the data provide a method for analysis of contact activation-mediated TG in murine whole blood. As the FXII-driven intrinsic pathway of coagulation has emerged as novel target for antithrombotic agents that are validated in mouse thrombosis and bleeding models, our novel assay could expedite therapeutic drug development.

Molecular Interaction Site on Procoagulant Skeletal Muscle Myosin for Factor Xa-Dependent Prothrombin Activation

Skeletal muscle myosin (SkM) is a muscle protein consisting of a dimer of heterotrimers, each trimer comprising a regulatory light chain (RLC), an essential light chain (ELC), and a heavy chain (HC). Recently it was discovered that SkM has potent procoagulant and prothrombotic activity (Deguchi H, et al, Blood. 2016;128:1870-1878). Mechanistic studies showed that SkM's potent prothrombotic activity involved enhancing thrombin generation due to SkM's ability to bind coagulation factors Xa and Va which accelerates prothrombin activation. However, detailed molecular mechanisms for SkM's binding of these coagulation factors have not been described. Since a well-known myosin inhibitor, trifluoperazine (TFP), inhibited SkM's procoagulant activity and since this inhibitor binds to the ELC in SkM's "neck" region which connects the HC head region to the HC tail (Figure, panel A), we hypothesized that SkM's TFP binding region on the ELC in the neck region directly contributes to SkM's procoagulant activity. To identify potential binding site(s) on SkM for factors Xa and Va, 22 peptides representing the neck region's RLC, ELC, and HC were screened for inhibition of SkM-supported prothrombin activation by purified factor Xa, factor Va, and calcium ions. These peptides contained 25-40 residues and overlapped by approximately 5-10 amino acids. Peptides ELC109-138 and ELC129-159, corresponding to amino acid residues 109-138 and 129-159 of the ELC, inhibited SkM-supported prothrombin activation at 100 μM, whereas their partially overlapping neighboring peptides, ELC99-122 and ELC149-173, did not. Three HC peptides (peptides HC781-810, HC796-835, HC815-854) and one RLC peptide (RLC133-162) inhibited SkM-supported prothrombin activation at 100 μM, and each was also inhibitory, to varying degrees, when assayed at 5 μM. Dose-dependency inhibition assays gave IC50 values (50% inhibition of activity) for the peptides HC781-810, HC796-835, HC815-854, and RLC133-162 of 64, 1.2, 2.3 and 26 μM. Peptides HC781-810 and HC815-854 also inhibited prothrombin activation in the absence of myosin but in the presence of phospholipid vesicles containing 20 % phosphatidylserine (IC50 = 7.5 and 104 μM, respectively). In contrast, the strong inhibitory effects of peptides HC796-835, RLC133-162, ELC109-138 and ELC129-159 seen in the presence of myosin were not at all apparent in the presence of phospholipid-supported prothrombin activation when myosin was absent. This suggests that peptides HC796-835, RLC133-162, ELC109-138 and ELC129-159 specifically inhibit SkM-supported prothrombin activation. The 19 synthetic peptides representing the SkM neck region were also screened at 25 µM (final) for their inhibition of recalcification-induced thrombin generation in human plasma which contains significant circulating levels of SkM. Among the 19 peptides tested, HC796-835 and HC815-854 significantly inhibited thrombin generation when screened at 25 µM in plasma. Immobilized peptide HC796-835 showed direct binding of purified factor Xa with apparent Kd of 1.4 μM. This very potent inhibitory peptide, HC796-835, exhibited 50% inhibition of SkM-enhanced prothrombin activation at 1.2 μM, indicating that this peptide's sequence provides a factor Xa binding site on SkM which contributes to its inhibitory action. More specifically, an overlapping peptide containing amino acid residues 815-835 inhibited SkM-enhanced prothrombin activation by factors Xa and Va while a peptide comprising residues 796-811 did not. These studies suggest that residues 815-835 of SkM's HC are responsible for directly binding factor Xa and implies that this binding is responsible for SkM's procoagulant activity (Figure, panel B). In summary, we identified human SkM peptides which specifically blocked SkM-enhanced thrombin generation but not phospholipid-stimulated prothrombin activation in purified reaction mixtures and which inhibited blood clotting in plasma. The most potent anticoagulant HC peptide also directly binds purified factor Xa. These findings strongly suggest that the neck region of SkM, as defined by these inhibitory peptides (Figure, panel B), provides a phospholipid-independent procoagulant surface for thrombin generation that, depending on the in vivo physiologic context, may contribute to either hemostasis or thrombosis. Figure Disclosures No relevant conflicts of interest to declare.

Plasma kallikrein contributes to ambient particulate matter-induced lung injury

Particulate matter (PM) is a key component of air pollutants and is associated with mortality of cardiovascular and respiratory diseases. PM-induced tissue injury involves inflammation and coagulation. Plasma prekallikrein (pKal), along with coagulation factor XII (FXII) and high-molecular-weight kininogen (HK), form the plasma kallikrein-kinin system (KKS), a component of the innate immune response that generates proinflammatory products in response to injury. When the KKS proteins contact with activation surface such as negatively charged molecules, this system becomes activated. Activated kallikrein (Kal) activates FXII to initiate the intrinsic coagulation pathway, and cleaves HK to release bradykinin to enhance vascular permeability and systemic inflammation. In his study we determined the role of plasma pKal in the PM2.5-induced lung injury. Using TALEN technology, we generated a new mouse strain lacking the gene for pKal. In PM2.5-induced lung injury model, Klkb1−/− mice exhibited a decrease in total protein, cells numbers in bronchoalveolar lavage fluid (BALF) and histologic lung injury score. The TNF-α and IL-6 levels in BALF were significantly decreased in PM2.5-treated Klkb1−/− mice. Plasma thrombin-antithrombin (TAT) complex levels were significantly decreased in PM2.5-treated Klkb1−/− mice. PM2.5 induces pKal activation, HK cleavage and bradykinin production. PM2.5-induced HK cleavage in plasma was completely blocked by a Kal inhibitor, as well as in pKal-deficient plasma. PM2.5 markedly induced thrombin generation in human plasma and wild-type mouse plasma, which was inhibited by both blockade and deficiency of pKal. Taken together, plasma pKal is activated by PM2.5 and the activated Kal plays an important role in PM2.5-induced lung injury.

Effect of Andexanet-TFPI Interaction on in Vitro Thrombin Formation and Coagulation Markers in the TF-Pathway

Background: Tissue factor pathway inhibitor (TFPI) plays a major role in regulation of tissue factor (TF)-initiated coagulation in a FXa-dependent manner. Andexanet alfa (AnXa) is a modified, recombinant, catalytically inactive form of human FXa being developed to reverse the anticoagulant activity of FXa inhibitors in patients during episodes of major bleeding. As a modified FXa, AnXa retained high binding affinity to fXa inhibitors, including TFPI. Our previous in vitro studies demonstrated that AnXa reverses rivaroxaban-induced inhibition of thrombin generation (TG) initiated by either the extrinsic or intrinsic pathways. However, the thromboelastography (TEG) results showed that AnXa-TFPI interaction enhanced thrombin formation at low TF conditions, which suggested potential increase in the fibrin degradation product in the presence of recombinant tissue plasminogen activator (rtPA) (Lu, G., et al, ASH2016). In the current study, we further characterized the differential effect of AnXa on thrombin formation and clot lysis via the two different pathways, and correlated the TG and TEG parameters with the coagulation markers in the TF-initiated reaction in plasma, including prothrombin fragment (F1+2), thrombin-antithrombin complex (TAT), and fibrin degradation product (D-dimer).Methods: TF-initiated TG in human plasma was measured using a calibrated automated thrombogram (TF-CAT) and the PPP-reagent (5 pM TF, Diagnostica Stago). Non-TF-initiated TG was measured using CAT and an aPTT reagent, Actin FS (Siemens Healthcare) as the activator (Actin FS-CAT). Clot formation was measured in plasma using a TEG 5000 analyzer (Haemonetics). TF (1 pM) and Actin FS (1:240 final dilution) were used as the activators with or without rtPA (150 ng/mL). Time-dependent thrombin formation and clot lysis were monitored by quenching samples at various times (0, 2.5, 5, 10, 15, 30, 60 min) following initiation of the reaction by TF (1 pM) in plasma with or without rtPA (150 ng/mL) and AnXa (4.0 µM). Corn trypsin inhibitor (CTI, 50 µg/mL) was added to block contact activation. F1+2, TAT and D-dimer were quantified according to manufacturers' recommendations.Results: The effect of AnXa on TF-CAT and Actin FS-CAT were compared side-by-side with AnXa at low (0 - 20 nM) and high (0 - 4.0 µM) concentration ranges. AnXa (5 nM) increased TF-CAT parameters sensitive to TFPI activity (i.e., peak thrombin), with less effect on endogenous thrombin potential (ETP). No further increase in these parameters was observed at AnXa therapeutic concentrations (e.g., 2.0 and 4.0 µM), consistent with the expected effect of AnXa on the activity of TFPI, which is present at low plasma concentration (~2.4 nM). In contrast, AnXa did not significantly affect the Actin FS-CAT parameters.The effect of AnXa-TFPI interaction on coagulation and fibrinolytic pathways in human plasma were compared using TEG, with or without rtPA. AnXa (4.0 µM) reduced the TF-TEG-R parameter (lag time, equivalent to clotting time) and resulted in an increased area under the fibrinolytic profile with rtPA as previously shown. In contrast, AnXa and the buffer controls had similar profiles in the Actin FS-TEG assay under similar conditions, with or without rtPA (150 ng/mL) in the current studies, suggesting lack of an AnXa-TFPI effect.We further investigated the effect of AnXa-TFPI interaction on coagulation markers following TF (1pM)-initiated thrombin formation in plasma. AnXa (4 µM) increased F1+2 and TAT levels at early time points following initiation (2.5, 5, 10, 15 min), but had no difference at or after 30 min, compared to buffer control. A similar pattern was observed for D-dimer level when exogenous rtPA (150 ng/mL) was added. Compared to buffer control, AnXa caused elevation of D-dimer level at early time points but had no difference at or after 30 min following initiation of the reaction.Conclusions: AnXa-TFPI interaction can modulate the TF/TFPI activity in the initiation phase of the extrinsic coagulation pathway and increase parameters sensitive to TFPI activity in the TF-CAT and TF-TEG assays, but has minimal effect on the later phase of the reactions and the overall thrombin formation potential. Consistent with clinical observations, AnXa-TFPI interaction increased the F1+2 and TAT levels, as well as D-dimer level in the presence of rtPA in the early phase of the reaction following initiation of the reaction by TF. DisclosuresLu:Portola Pharmaceuticals, Inc.: Employment. Lin:Portola Pharmaceuticals, Inc.: Employment. Curnutte:3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy; Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties. Conley:Portola Pharmaceuticals, Inc.: Employment, Equity Ownership, Patents & Royalties.

Pig tissue factor pathway inhibitor α fusion immunoglobulin inhibits pig tissue factor activity in human plasma moderately more efficiently than the human counterpart.

To determine the efficacy of soluble pig tissue factor pathway inhibitor fusion immunoglobulin (TFPI-Ig) in blocking pig to human xenogeneic blood coagulation. To generate pig TFPI-Ig or human TFPI-Ig, expression vector containing cDNA encoding pig TFPIα or human TFPIα combined with human constant Ig heavy chain region was cloned and introduced into CHO cells. After purification of pig TFPI-Ig and human TFPI-Ig, the inhibition of each recombinant protein on pig tissue factor (TF)-mediated blood coagulation was examined in human plasma. Compared to human TFPI-Ig, pig TFPI-Ig inhibited pig TF activity and thrombin generation in human plasma more efficiently at certain concentrations. Pig TFPI-Ig will be be useful as a therapeutic protein to treat pig to human xenogeneic blood coagulation.

Andexanet Alfa, a Universal Antidote Under Development for Factor Xa Inhibitors, Reverses Rivaroxaban-Induced Inhibition of Thrombin Generation Initiated By the Intrinsic Coagulation Pathway Independent of TFPI

Background: Andexanet alfa (AnXa) is a modified, recombinant human fXa molecule being developed to reverse the anticoagulant activity of fXa inhibitors in patients during episodes of major bleeding. As a modified fXa, AnXa retained high binding affinity to fXa inhibitors and had no significant interaction with major plasma coagulation proteins, except for tissue factor pathway inhibitor (TFPI), an endogenous fXa inhibitor with sub-nanomolar affinity to both fXa and AnXa. In previous in vitro and clinical studies in healthy volunteers, AnXa has shown dose-dependent and complete reversal of both direct and indirect fXa inhibitors in tissue factor (TF)-initiated thrombin generation (TG). To delineate the contribution of AnXa-TFPI interactions to TG, we compared rivaroxaban (Riva)-induced inhibition of TG initiated via the extrinsic pathway (TF) versus the intrinsic pathway (non-TF). The differential effect of AnXa on TG and clot formation via the two pathways was further studied in the context of recombinant tissue plasminogen activator (rtPA)-induced fibrinolysis using thromboelastography (TEG).Methods: TF-initiated TG in human plasma was measured using a calibrated automated thrombogram (CAT) and the PPP-reagent (5 pM TF). Non-TF-initiated TG was measured using CAT and Actin FS, an aPTT reagent. Anti-fXa activity was measured using a modified anti-fXa chromogenic assay with reagents from the Coamatic Heparin kit (DiaPharma). Riva was used as the standard. Pooled human plasma was spiked with Riva (0 - 2 µM) or Riva (1 µM)+AnXa (0 - 4 µM), and TG and anti-fXa activity were measured. Clot formation with or without AnXa was measured in plasma using a TEG 5000 analyzer. The functional fibrinogen (TF) and Kaolin reagents (both from Haemonetics) were used according to the manufacturer’s instruction. For low TF-initiated clot formation, the PPP-reagent (5 pM TF) was diluted in phospholipid (4.0 µM) followed by measuring the TEG profiles with 0.25, 0.5 and 1.0 pM TF.Results: The potential contribution of AnXa-TFPI interaction to TG was studied under similar conditions using TF or Actin FS in human plasma containing AnXa, Riva or Riva+AnXa. AnXa alone had minimal effect on the endogenous thrombin potential (ETP) in either assay. AnXa was able to fully reverse Riva-induced anticoagulation in the Actin FS TG assay, independent of AnXa-TFPI interaction that primarily modulates TF activity. The modulation of TF activity could be assessed by correlating ETP vs. the anti-fXa activity in samples containing Riva or Riva+AnXa. Riva dose-dependently inhibited TF-initiated TG as anti-fXa activity increased. At similar anti-fXa levels (i.e., similar free Riva concentration), Riva+AnXa had higher ETP than Riva alone. As expected, this difference was not observed in the Actin FS TG assay.To further investigate the role of AnXa-TFPI interaction on coagulation and fibrinolytic pathways, the profile of clot formation was studied in human plasma using TEG without Riva. AnXa (4 µM) had no effect on the TEG parameters in the kaolin assay or the functional fibrinogen assay containing high TF, with or without rtPA (150 ng/mL). When low TF (0.25, 0.5, and 1.0 pM) was used to initiate clot formation in the absence of rtPA, AnXa reduced the TEG-R parameter (lag time equivalent to clotting time), but had no effect on maximum amplitude (MA). The fibrin clot formed under each condition (±AnXa) was lysed slowly at low rtPA (75 ng/mL, ~1 nM), resulting in well-segregated processes of coagulation and fibrinolysis. However, rtPA at 150 ng/mL (~2 nM) dramatically changed the lysis profiles that overlapped the fibrin formation, possibly due to the higher rtPA activity ratio, relative to the major plasma inhibitor PAI-1 (<1 nM). With the optimal rtPA, fibrin clot formed at each TF concentration (±AnXa) was compensated by the fibrinolytic activity of rtPA resulting in an increased fibrin degradation product.Conclusions: In the absence of a fXa inhibitor, AnXa had minimal effect on TF or Actin FS-initiated TG with no direct interaction or effect on rtPA function. AnXa dose-dependently and completely reversed Riva-induced inhibition of TG initiated by either the intrinsic or extrinsic pathway, but had different effect on ETP due to the AnXa-TFPI interaction that mainly modulates TF function. AnXa-TFPI interaction may enhance TF-initiated TG at low TF conditions with increased fibrin degradation product in the presence of rtPA. DisclosuresLu:Portola Pharmaceuticals, Inc.: Employment. Lin:Portola Pharmaceuticals, Inc.: Employment. Curnutte:3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy; Portola Pharmaceuticals: Employment, Equity Ownership, Patents & Royalties, Research Funding. Conley:Portola Pharmaceuticals, Inc.: Employment.

The Alzheimer's disease peptide β‐amyloid promotes thrombin generation through activation of coagulation factor XII

Essentials How the Alzheimer's disease (AD) peptide β-amyloid (Aβ) disrupts neuronal function in the disease is unclear. Factor (F) XII initiates blood clotting via FXI, and thrombosis has been implicated in AD. Aβ triggers FXII-dependent FXI and thrombin activation, evidence of which is seen in AD plasma. Aβ-triggered clotting could contribute to neuronal dysfunction in AD and be a novel therapeutic target. Background β-Amyloid (Aβ) is a key pathologic element in Alzheimer's disease (AD), but the mechanisms by which it disrupts neuronal function in vivo are not completely understood. AD is characterized by a prothrombotic state, which could contribute to neuronal dysfunction by affecting cerebral blood flow and inducing inflammation. The plasma protein factor XII triggers clot formation via the intrinsic coagulation cascade, and has been implicated in thrombosis. Objectives To investigate the potential for Aβ to contribute to a prothrombotic state. Methods and results We show that Aβ activates FXII, resulting in FXI activation and thrombin generation in human plasma, thereby establishing Aβ as a possible driver of prothrombotic states. We provide evidence for this process in AD by demonstrating decreased levels of FXI and its inhibitor C1 esterase inhibitor in AD patient plasma, suggesting chronic activation, inhibition and clearance of FXI in AD. Activation of the intrinsic coagulation pathway in AD is further supported by elevated fibrin levels in AD patient plasma. Conclusions The ability of Aβ to promote coagulation via the FXII-driven contact system identifies new mechanisms by which it could contribute to neuronal dysfunction and suggests potential new therapeutic targets in AD.

A direct thrombin inhibitor suppresses protein C activation and factor Va degradation in human plasma: Possible mechanisms of paradoxical enhancement of thrombin generation

We have demonstrated that antithrombin (AT)-independent thrombin inhibitors paradoxically increase thrombin generation (TG) in human plasma in a thrombomodulin (TM)- and protein C (PC)-dependent manner. We determined the effects of AT-independent thrombin inhibitors on the negative-feedback system, activation of PC and production and degradation of factor Va (FVa), as possible mechanisms underlying the paradoxical enhancement of TG. TG in human plasma containing 10nM TM was assayed by means of the calibrated automated thrombography. As an index of PC activation, plasma concentration of activated PC-PC inhibitor complex (aPC-PCI) was measured. The amounts of FVa heavy chain and its degradation product (FVa307–506) were examined by western blotting. AT-independent thrombin inhibitors, melagatran and dabigatran (both at 25–600nM) and 3–30μg/ml active site-blocked thrombin (IIai), increased peak levels of TG. Melagatran, dabigatran and IIai significantly decreased plasma concentration of aPC-PCI complex at 25nM or more, 75nM or more, and 10 and 30μg/ml, respectively. Melagatran (300nM) significantly increased FVa and decreased FVa307–506. In contrast, a direct factor Xa inhibitor edoxaban preferentially inhibited thrombin generation (≥25nM), and higher concentrations were required to inhibit PC activation (≥150nM) and FVa degradation (300nM). The present study suggests that the inhibitions of protein C activation and subsequent degradation of FVa and increase in FVa by antithrombin-independent thrombin inhibitors may contribute to the paradoxical TG enhancement, and edoxaban may inhibit PC activation and FVa degradation as a result of TG suppression.

The Application of Thrombin Generation Test Is Compromised By Antithrombin III Deficiency

Background: Antithrombin III (ATIII) is a stoichiometric inhibitor of factor Xa and thrombin and a major physiological regulator of coagulation. Reduction of ATIII levels, for example in patients with congenital ATIII deficiency, causes a rise in active thrombin which is associated with risk of venous and arterial thrombosis (1, 2). The calibrated automated thrombogram (CAT) assay is widely used to measure thrombin generation in human plasma. Therewith, the fluorogenic substrate Z-G-G-R-AMC is cleaved by the thrombin emerging over time in the plasma sample. Thus, an increase in fluorescence signal reflects the amount of thrombin provided that fluorogenic substrate is in excess over thrombin. To account for substrate consumption, plasma color, and 'inner filter' effect, the fluorescence signal is compared to alpha-2-macroglobulin thrombin complex, the thrombin calibrator. In this complex, thrombin is protected from its physiological inhibitors, but retains its ability to cleave small substrates (3).Aim: We assessed the technical feasibility of measuring the effect of ATIII reduction on thrombin generation in normal and FVIII-inhibited human plasma in the tissue factor (TF) triggered CAT assay.Methods: CAT was performed in ATIII deficient plasma with 1 pM of TF to trigger thrombin generation. The plasma was supplemented with 0.125 - 2.5 µM ATIII to achieve ATIII plasma levels of 5 - 100% of normal. The ATIII titration was also done in ATIII deficient plasma inhibited with a goat polyclonal anti-human FVIII antibody to simulate hemophilia A conditions. Raw data analysis focused on the velocity of fluorescence signal increase, which is usually automatically converted to nM of thrombin by the Thrombinoscope software.Results: Reduced ATIII levels resulted in an apparent concentration dependent increase in thrombin generation in both normal and FVIII inhibited human plasma. For example, a reduction from 2.5 to 1.25 µM ATIII resulted in an apparent increase of 45 - 55% in thrombin peak and ETP values. Most importantly, analysis of the raw data showed that the fluorescence signal reached a plateau after 30 min (normal plasma) and 50 min (FVIII-inhibited plasma) in samples with <1.25 µM ATIII. The plateau (at ~1700 FU) is due to complete depletion of fluorescent substrate. In comparison, samples with ³1.25 µM ATIII did not reach a plateau and show substrate conversion until the end of the assay. In summary, a plasma level of at least 1.25 µM ATIII (50% of normal) was required to obtain thrombin generation profiles which were not compromised by fluorogenic substrate depletion. Below 1.25 µM ATIII, it is not possible to distinguish between inhibition of thrombin and excessive consumption of fluorescence substrate, and therefore, data may be misinterpreted.Conclusion: Thrombin generation at low ATIII plasma levels is difficult to interpret as the physiological inhibition of the generated thrombin is reduced. Consequently, the thrombin substrate is consumed, resulting in substrate depletion. Therefore, we conclude that the CAT assay is technically not suitable to assess the effect of ATIII levels < 1.25 µM ATIII corresponding to 50% of normal plasma. DisclosuresScheiflinger:Baxalta Innovations GmbH: Employment.

Inhibition of thrombin generation in human plasma by phospholipid transfer protein

BackgroundPlasma phospholipid transfer protein (PLTP) transfers lipids between donors and acceptors (e.g., from HDL to VLDL) and modulates lipoprotein composition, size, and levels. No study has reported an assessment of the effects of PLTP on blood clotting reactions, such as reflected in thrombin generation assays, or on the association of venous thrombosis (VTE) risk with PLTP activity.MethodsThe in vitro effects of PLTP on blood coagulation reactions and the correlations between plasma PLTP activity levels and VTE were studied.ResultsRecombinant (r) PLTP concentration-dependently inhibited plasma thrombin generation and factor XII-dependent kallikrein generation when sulfatide was used to stimulate factor XII autoactivation in plasma. However, rPLTP did not inhibit thrombin generation in plasma induced by factor XIa or tissue factor, implicating an effect of PLTP on contact activation reactions. In purified systems, rPLTP inhibited factor XII autoactivation stimulated by sulfatide in the presence of VLDL. In surface plasmon resonance studies, purified factor XII bound to immobilized rPLTP, implying that rPLTP inhibits factor XII-dependent contact activation by binding factor XII in the presence of lipoproteins. Analysis of plasmas from 40 male patients with unprovoked VTE and 40 matched controls indicated that low PLTP lipid transfer activity (≤25th percentile) was associated with an increased risk of VTE after adjustment for body mass index, plasma lipids, and two known thrombophilic genetic risk factors.ConclusionThese data imply that PLTP may be an antithrombotic plasma protein by inhibiting generation of prothrombotic factor XIIa in the presence of VLDL. This newly discovered anticoagulant activity of PLTP merits further clinical and biochemical studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12959-015-0054-0) contains supplementary material, which is available to authorized users.

Paradoxical enhancement of the intrinsic pathway-induced thrombin generation in human plasma by melagatran, a direct thrombin inhibitor, but not edoxaban, a direct factor Xa inhibitor, or heparin

IntroductionThe blood coagulation cascade consists of two pathways, the tissue factor (TF)-dependent extrinsic pathway and the contact factor-dependent intrinsic pathway. We have previously shown that a direct thrombin inhibitor, melagatran, paradoxically increased TF-induced thrombin generation (TG) in thrombomodulin (TM)-containing human plasma in vitro. However, the effect of melagatran on the intrinsic pathway-induced TG remains to be investigated. We investigated whether melagatran enhances the intrinsic pathway-induced TG. Methods and resultsTG was induced by kaolin in human plasma and assayed by the calibrated automated thrombography method. Melagatran at 150 and 300 nM significantly increased the peak level (2.40-fold) and endogenous thrombin potential of TG in normal plasma in the presence of 5 nM TM. In the absence of TM or in protein C (PC)-deficient plasma, the paradoxical enhancement of TG by melagatran disappeared. A direct FXa inhibitor, edoxaban, and an antithrombin-dependent anticoagulant, unfractionated heparin (UFH), did not increase, but simply decreased TG under each condition in a concentration dependent manner. ConclusionMelagatran enhanced the intrinsic pathway-induced TG as well as the extrinsic pathway-induced TG in human plasma under the condition where PC system is active. In contrast, edoxaban and UFH showed concentration-dependent decrease of TG, but no enhancement. These results indicated that edoxaban and UFH may have a low risk of the paradoxical enhancement of TG by both the extrinsic and intrinsic pathway activation.

Tick Spit Shines a Light on the Initiation of Coagulation

Mammalian blood has numerous essential and well-known functions, including oxygen and nutrient delivery. This elixir is recognized by blood-feeding species of mosquitos, ticks, fleas, lice, leeches, and bats that rely on blood meals for nutrition, life cycle progression, and survival. To obtain these blood meals that require minutes to a week or longer to complete,1 these blood-sucking creatures must thwart endogenous defense systems contained within blood—immune and procoagulant cells and plasma proteins that rapidly clot (within 3–4 minutes) to provide first-line defense against breaches in vascular integrity. In a fascinating display of evolutionary agility, hemovores have adapted elegant mechanisms to evade detection and to prevent blood coagulation by synthesizing an extensive armament of molecules with anesthetic, immunosuppressive, vasodilatory, anticoagulant, and profibrinolytic properties in mammals.1–3 Research characterizing the molecules generated by hemovores to bypass mammalian defense pathways has revealed exciting new mechanisms and, in some cases, novel therapeutic approaches for anticoagulation. Article see p 254 In particular, ticks have received considerable attention for their remarkable evolutionary adaptations to life as obligate hemovores. Briefly, the typical tick life cycle includes 4 stages: egg, 6-legged larva, 8-legged nymph, and adult (Figure). It takes 1 to 3 years to complete this full cycle. Ticks must consume blood at the larval, nymph, and adult stages to survive, and they die if they do not find a host. Interestingly, although neither larva nor nymphs have overt sexual differentiation, adults are fully differentiated into males and females, and compared with males, longer nymph feeding is required for the expression of female characteristics.4 Consequently, tick saliva contains multiple proteins that maintain blood fluidity to enable feeding and therefore tick survival. Figure. Life cycle of a typical tick. Larvae, nymphs, and adults require blood meals for survival and secrete anticomplement (tick salivary lectin pathway inhibitor …

Inhibitory Effect of Apixaban Compared With Rivaroxaban and Dabigatran on Thrombin Generation Assay

The effect of the oral direct activated factor X (factor Xa) inhibitor apixaban on tissue factor–induced thrombin generation in human plasma was investigated in vitro using the calibrated automated thrombogram (CAT) method and compared with the oral direct factor Xa inhibitor rivaroxaban and the direct thrombin inhibitor dabigatran. Pooled citrated, anticoagulated, platelet-poor human plasma was spiked with apixaban, rivaroxaban, or dabigatran at concentrations of 0.01 to 10 μM. The inhibitory potencies of the compounds were quantified by 5 CAT parameters: the control thrombin lag time (LT) and time to thrombin peak (TTP) for the doubling of inhibitor concentration (IC2x); and the control endogenous thrombin potential (ETP), thrombin peak, and maximum rate of thrombin generation (Vmax) for the inhibitor concentration, which inhibited 50% (IC50). The inhibitors modified CAT concentration dependently. Their inhibitory potencies, expressed as IC2x LT, IC2x TTP, IC50 ETP, IC50 peak thrombin, and IC50 Vmax, were as follows: 0.10 ± 0.01, 0.19 ± 0.02, 0.65 ± 0.11, 0.089 ± 0.019, and 0.049 ± 0.007 μM for apixaban; 0.049 ± 0.007, 0.070 ± 0.009, 0.43 ± 0.07, 0.048 ± 0.008, and 0.022 ± 0.005 μM for rivaroxaban; and 0.063 ± 0.019, 0.18 ± 0.06, 0.50 ± 0.08, 0.55 ± 0.06, and 0.57 ± 0.27 μM for dabigatran. In summary, apixaban, rivaroxaban, and dabigatran have similar potencies in the prolongation of LT and TTP. The CAT parameters that are related to the rate of thrombin generation during the propagation phase (ie, peak thrombin and Vmax) are more sensitive to activities of apixaban and rivaroxaban than dabigatran. The ETP is the least sensitive parameter for measuring the activities of these inhibitors. Recombinant activated factor VII at 5 and 50 μg/mL reversed the anticoagulant effects of apixaban more at 0.2 μM than at 2 μM. Our study suggests that the CAT method is a sensitive assay to monitor the pharmacodynamic and pharmacokinetic properties of apixaban, rivaroxaban, and dabigatran, and may provide insight into the mechanism of action of these inhibitors. Recombinant activated factor VII may have some potential to reverse the anticoagulant effects of apixaban in vitro.

A microarray-based resonance light scattering assay for detecting thrombin generation in human plasma by gold nanoparticle probes

A peptide microarray-based resonance light scattering (RLS) assay with gold nanoparticle probes has been developed for monitoring thrombin generation in human plasma. Thrombophilia, hypocoagulability and normal plasma have been successfully differentiated by the assay.

Antithrombin-independent thrombin inhibitors, but not direct factor Xa inhibitors, enhance thrombin generation in plasma through inhibition of thrombin-thrombomodulin-protein C system

There is increasing concern that some anticoagulants can paradoxically increase thrombogenesis under certain circumstances. Previously, we demonstrated that at certain doses a direct thrombin inhibitor, melagatran, worsens the coagulation status induced by tissue factor (TF) injection in a rat model. We utilised an in vitro thrombin generation (TG) assay to determine if direct thrombin inhibitors could enhance TG in human plasma, and whether inhibition of the negative-feedback system [thrombin-thrombomodulin (TM)-protein C] contributed to the TG enhancement. TG in human plasma was assayed by means of the calibrated automated thrombography. In this assay, direct factor Xa (FXa) inhibitors such as edoxaban and antithrombin (AT)-dependent anticoagulants such as heparin did not increase, but simply suppressed TG. AT-independent thrombin inhibitors (melagatran, lepirudin, and active site blocked thrombin (IIai)) increased peak levels of TG (2.0, 1.6, and 2.2-fold, respectively) in the presence of 12 nM recombinant human soluble TM (rhsTM). Melagatran and lepirudin at higher concentrations began to suppress TG. In the absence of rhsTM, the enhancement of peak TG by melagatran decreased to 1.2-fold. Furthermore, in protein C-deficient plasma, AT-independent thrombin inhibitors failed to enhance TG. In addition, a human protein C neutralising antibody increased the peak height of TG in the presence of rhsTM. These results suggest that AT-independent thrombin inhibitors may activate thrombogenesis by suppression of the thrombin-induced negative-feedback system through inhibition of protein C activation. In contrast, direct FXa inhibitors are more useful than AT-independent thrombin inhibitors in terms of lower possibility of activation of the coagulation pathway.

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.

Antithrombin-Independent Thrombin Inhibitors, but Not Factor Xa Inhibitors, Enhance Thrombin Generation in Human Plasma Via Inhibition of Thrombin-Thrombomodulin-Protein C System.

Rebound like recurrent thrombotic events are concerns about anticoagulant therapies. Withdrawal of heparins and a direct thrombin inhibitor is reported to be associated with evidence of rebound coagulation phenomenon in patients with coronary artery diseases (Ref 1). Previously we have shown that low-dose administration of a direct thrombin inhibitor, melagatran, enhances coagulation induced by tissue factor (TF) in rats (Ref 2).Objectives: To determine whether anticoagulants enhance thrombin generation in human plasma, and whether the negative-feedback system [thrombin-thrombomodulin (TM)-protein C] contributes to the enhancement.Methods: Thrombin generation in pooled human plasma was assayed by means of the calibrated automated thrombography (CAT) with the thrombinoscope software in vitro. Thrombin generation was induced by 2.5 pM tissue factor (TF) and 4 μM phospholipids. The effects of following anticoagulants were assessed: antithrombin (AT)-independent thrombin inhibitors [melagatran, recombinant hirudin (lepirudin), and active site blocked thrombin (IIai)], AT-dependent anticoagulants (heparin, dalteparin, and fondaparinux), and AT-independent FXa inhibitors (DU-176b and DX-9065a).Results: Melagatran, lepirudin, and IIai increased peak levels of thrombin generation in the presence of 8 nM recombinant human soluble TM. The effects reached maximal at 200 nM of melagatran (2.3-fold), 8.95 nM of lepirudin (1.6-fold), and 405 nM of IIai (2.2-fold). At higher concentrations, melagatran and lepirudin turned to suppress thrombin generation. Heparin, dalteparin, fondaparinux, DU-176b, and DX-9065a did not enhance thrombin generation, just exerted inhibitory effects. In the absence of TM, the enhancement by melagatran of peak thrombin generation was only 1.2-fold, suggesting the significant role of the negative-feedback system in this aggravation of thrombin generation. Since thrombin acts both the pro- and anti-coagulant, the inhibition of the negative-feedback system by these thrombin inhibitors may cause enhancement of thrombin generation. To test this hypothesis, we examined thrombin generation in protein C-deficient plasma. AT-independent thrombin inhibitors failed to enhance thrombin generation in protein C-deficient plasma.Conclusions: These results indicate that AT-independent thrombin inhibitors at low concentrations enhance thrombin generation probably due to suppression of the negative feedback system by inhibiting protein C activation. This in vitro aggravation of thrombin generation may be a possible explanation of hypercoagulation by melagatran in a rat model of TF-induced intravascular coagulation. Furthermore this phenomenon would contribute to clinical rebound like recurrent thrombotic events associated with anticoagulant therapies with these inhibitors. In contrast, AT-independent FXa inhibitors like DU-176b are less prone to induce the rebound because of lack of increase in thrombin generation.

The respective and combined anticoagulant effects of recombinant human activated protein C, melagatran and heparins using CAT

Introduction Combinations of anticoagulants might be beneficial in some patients with sepsis, but most anticoagulants require specific clotting assays for monitoring. Thrombin generation assay, however, is a global function test of hemostasis. Materials and methods We performed an in vitro investigation of the respective effects of recombinant human activated protein C (rhAPC) alone and in combination with either melagatran (a new direct thrombin inhibitor), unfractionated heparin (UH) or low molecular weight heparin (LMWH) in varying concentrations on the thrombin generation (TG) using the calibrated automated thrombography. Results RhAPC, UH, LMWH and melagatran dose-dependently prolonged the lag time and the time to peak, and significantly suppressed the endogenous thrombin potential (ETP). Combined application of rhAPC with either melagatran, UH or LMWH induced an additive prolongation of the lag time; this effect was more pronounced in a combination of rhAPC with UH or LMWH. Conclusion In our in vitro study adding either melagatran, UH or LMWH augmented the capacity of rhAPC to suppress thrombin generation in human plasma. These findings suggest that patients with severe sepsis might benefit from a treatment with combinations of anticoagulant agents.

Drotrecogin Alfa (activated, Xigris�) in Combination with Heparin or Melagatran: An In vitro Investigation

The present in vitro study of human plasma investigated the anticoagulant effects of recombinant human activated protein C (rhAPC; drotrecogin alfa [activated, Xigris]), combined with either unfractionated heparin (UH) or the direct thrombin inhibitor melagatran. Prolongation of clotting time and generation of prothrombin fragments 1 and 2 (F1 + 2) and of thrombin-antithrombin (TAT) complex were measured in vitro. Clot formation was induced by adding low levels (final concentration, 20 pmol/L) of lipidated tissue factor (TF) to citrated venous plasma samples from healthy human volunteers (n = 16). It has been suggested that experimental activation of plasma with low levels of TF more closely simulates conditions in vivo. rhAPC, melagatran, or UH alone concentration-dependently prolonged the clotting time and suppressed F1 + 2 and TAT generation. rhAPC-mediated prolongation of clotting time concentration-dependently increased with the addition of either UH or melagatran, the effect being more pronounced with the addition of melagatran. Similarly, rhAPC-mediated generation of F1 + 2 and TAT also increased concentration-dependently with the addition of either UH or melagatran; the effect being, in this case, more pronounced with the addition of UH. This study demonstrated the effects of rhAPC, alone and combined with either UH or melagatran, on clotting time and markers of thrombin generation in human plasma. These results may guide facilitate estimation of appropriate doses of rhAPC and melagatran in future clinical trials.