Tissue Factor-initiated Thrombin Generation Research Articles

Andexanet versus prothrombin complex concentrates: Differences in reversal of factor Xa inhibitors in in vitro thrombin generation

BackgroundAndexanet alfa (andexanet) is a modified human factor Xa (FXa) approved for anticoagulation reversal in patients with life‐threatening bleeding treated with rivaroxaban or apixaban. Four‐factor prothrombin complex concentrates (4F‐PCCs) are approved for reversal of vitamin K antagonist–induced anticoagulation but not FXa inhibitors. The mechanism and effectiveness of 4F‐PCCs for FXa inhibitor reversal are unclear. ObjectiveTo investigate the mechanism and impact of 4F‐PCCs on reversal of rivaroxaban and apixaban in vitro compared to andexanet. MethodsThe effect of 4F‐PCCs (or individual factors) on tissue factor‐initiated thrombin generation (TF‐TG) was evaluated in human plasma, with or without rivaroxaban or apixaban, and compared with andexanet under the same conditions. ResultsIn the TF‐TG assay, 4F‐PCC completely reversed warfarin anticoagulation. Andexanet normalized TF‐TG over a wide range of apixaban and rivaroxaban concentrations tested (19‐2000 ng/mL). However, 4F‐PCC (or individual factors) was unable to normalize endogenous thrombin potential (ETP) or peak thrombin (Peak) in the presence of apixaban or rivaroxaban (75‐500 ng/mL). TF‐TG was only normalized by 4F‐PCC at inhibitor concentrations <75 ng/mL (ETP) or <37.5 ng/mL (Peak). These data can be explained by the estimated thresholds of FXa activity required to support normal TF‐TG based on the inhibitor:FXa ratios and levels of uninhibited FXa. The data are consistent with healthy volunteer studies where TF‐TG is not normalized until inhibitor levels are substantially decreased. ConclusionsBoth the theoretical calculations and experimental data demonstrated that 4F‐PCCs are only able to normalize TG over a low and narrow range of FXa inhibitor concentrations (<75 ng/mL).

Evaluation of a Blood Coagulation Factor IX Variant That Functions Independently of Factor VIII As an Alternative Treatment for Hemophilia A

The serine protease factor IXa (FIXa) serves an important role in coagulation by catalyzing the proteolytic activation of factor X (FX) together with its cofactor VIIIa (FVIIIa). Being a critical protease in coagulation, the FIXa structure has evolved to be subjected to strict regulatory mechanisms. While FIXa displays considerable structural homology with other coagulation serine proteases, its active site is uniquely controlled by the 99-loop that blocks access to the active site pocket. Cofactor-mediated interaction of FIXa with its substrate FX induces a conformational change that allows for active site engagement and substrate catalysis. Previously, the molecular constraints of the 99-loop were lifted due to specific modifications in both the 99-loop (K265A), the S1 active site subpocket (V181I, I383V), and the L6F substitution, thereby generating FIX-FIAV [Quade-Lyssy et al. J. Thromb. Haemost. 2014]. As a result, this variant is capable of functioning independently of factor VIII (FVIII). Moreover, FIX-FIAV was demonstrated to ameliorate the hemophilia A phenotype both in vitro and in vivo. To further evaluate its therapeutic potential, FIX-FIAV was stably expressed in HEK293 cells and purified by ion-exchange and hydrophobic interaction chromatography. Evaluation of the kinetics of tissue factor-factor VIIa (TF-FVIIa) activation of FIX-FIAV revealed kinetic parameters similar to those of human wild-type FIX(-WT). Analysis of FIX activation intermediates that are formed upon proteolysis by TF-FVIIa or factor XIa demonstrated prolonged formation of FIX-FIAVα, while no FIXa-WTα could be observed. This is consistent with delayed cleavage at position 180, likely resulting from the V181I substitution in FIX-FIAV. Given that the activation mechanism of FIX-FIAV is unperturbed, we next assessed the specific FVIII clotting activity and demonstrated that FIX-FIAV exhibited significant FVIII-like clotting activity (56 ± 4 U/mg) as opposed to FIX-WT (&lt;13 U/mg). These values correlate with up to 28% of FVIII-independent activity for FIX-FIAV at FIX plasma levels (5 ug/mL), confirming that FIX-FIAV has the potential to enhance thrombin generation in FVIII deficiency. To validate this, tissue factor-initiated (0.5 or 1.0 pM) thrombin generation was assessed in FVIII-immunodepleted plasma, leading to a severely reduced thrombin peak (88% or 81% reduction, respectively) relative to conditions with 100% FVIII. Addition of FIX-FIAV (5 ug/mL) partially restored thrombin generation, demonstrated by an up to ~30% increase in both thrombin peak and endogenous thrombin potential. Evaluation of the FVIII-independent activity of FIX-FIAV in severe hemophilia A patient plasma with or without an inhibitor resulted in an up to 18% or 32% FVIII-like activity, respectively, demonstrating efficacy of FIX-FIAV in the presence of FVIII inhibitors. Although unlikely, it remains to be determined whether specific FVIII-inhibitors may impact FIX-FIAV function. Adding 100% FVIII or low- to mid-range therapeutic concentrations of the bispecific antibody emicizumab to FVIII-deficient plasma incubations with FIX-FIAV resulted in a synergistic enhancement of thrombin generation, demonstrated by a 9-fold increase in thrombin peak. This is consistent with the previously demonstrated hyperactivity of FIX-FIAV in a cofactor-dependent system. In contrast, no synergistic effect on thrombin generation was observed when combining FIX-FIAV with physiologically relevant concentrations of FEIBA or NovoSeven. Summarizing, FIX-FIAV is characterized by a preserved mechanism of activation in addition to being capable of sustaining therapeutic levels of coagulation activity in FVIII deficiency. This provides support for the use of FIX-FIAV as an alternative treatment for hemophilia A. Disclosures Strijbis: uniQure Biopharma B.V.: Research Funding. Konstantinova:uniQure Biopharma B.V.: Employment. Liu:uniQure Biopharma B.V.: Employment. van Deventer:uniQure Biopharma B.V.: Employment. Bos:uniQure Biopharma B.V.: Membership on an entity's Board of Directors or advisory committees, Research Funding.

High Molecular Weight Kininogen but Not Factor XII Deficiency Attenuates Acetaminophen-Induced Liver Injury in Mice

Acetaminophen (APAP) toxicity is a common cause of acute liver failure (ALF). Dysregulation of coagulation is well documented in ALF patients. Recent reports indicate that small subset of ALF patients (~10%) exhibit spontaneous bleeding, while thrombotic complications are more frequent. Animal studies demonstrated that the inhibition of tissue factor-initiated thrombin generation attenuates APAP-induced liver injury in mice via both fibrin- and protease activated receptor 1-dependent mechanisms. Activation of FXII leads to two events: propagation of coagulation by activation of FXI (intrinsic pathway) and activation of plasma prekallikrein to kallikrein, with subsequent cleavage of high molecular weight kininogen (HK) into bradykinin (BK) and cleaved HK fragments (contact pathway). There is a growing interest in therapeutic targeting of FXII or FXI to prevent thrombosis, primarily because this goal may be attainable without incurring a significant bleeding risk. In this study, we evaluated if the intrinsic coagulation pathway contributes to the activation of coagulation and liver injury in a mouse model of APAP-induced ALF. We postulated that targeting FXII would attenuate coagulation and reduce liver injury without affecting hemostasis. In addition, FXII deficiency could provide a further protection by preventing activation of contact pathway and subsequent reduction of inflammatory response. We used 12 weeks old FXII, FXI, prekallkrein and HK deficient male mice and their respective WT controls. Mice were fasted for 16 hours followed by a single intraperitoneal injection of APAP (400 mg/kg) or sterile saline. Livers and plasma samples were collected 6 and 24 hours after injections. Hepatocellular necrosis was determined on liver sections stained with H&amp;E. Plasma levels of alanine transaminase (ALT- marker of liver injury), thrombin-antithrombin (TAT) complexes, plasmin-α2 antiplasmin (PAP) complexes and interleukin-6 were analyzed using commercially available assays. HK cleavage was determined using BK ELISA, Western blot, and mass spectrometry analysis. Consistently with previously published data, plasma levels of ALT, TAT and IL-6, as well as liver injury scores were significantly elevated in APAP-challenged mice compared to saline-injected WT mice at both 6 and 24 hours. Surprisingly, neither FXII, FXI, nor prekallikrein deficiency had statistically significant effects on any of these parameters in APAP-challenged mice at either time point. Interestingly, however, plasma levels of ALT were significantly attenuated in HK-/- mice (n=23-34) compared to HK+/+ (n=19-31) controls at both 6 (1278 ± 184 vs. 1850 ± 235 U/L; p&amp;lt;0.05) and 24 hours (1546 ± 302 vs. 3857 ± 493 U/L; p&amp;lt;0.01) after injection of APAP. The attenuation of liver injury was observed despite the lack of a significant effect on APAP-induced coagulation activation (plasma TAT levels [mean±SEM]: 72.9 ± 8.2 μg/L in HK+/+ vs. 70.8 ± 4.3 μg/L in HK-/-). HK deficiency also reduced plasma levels of IL-6 (75.8%; p&amp;lt;0.001), number of infiltrating neutrophils in liver (59.8%; p&amp;lt;0.001) and liver necrotic area (28.1%; p&amp;lt;0.001) 24 hours after APAP challenge. Importantly, the protective effects of HK deficiency were completely reversed by the injection of HK protein into APAP-treated HK-/- mice. It has been previously shown that plasminogen deficiency protects against APAP-induced liver injury. Indeed, APAP administration activated the fibrinolytic system in WT mice as demonstrated by increased plasma levels of active tissue plasminogen activator (19.5 fold; p&amp;lt;0.01) and PAP complexes (64.4 fold; p&amp;lt;0.01). Using Western blotting analysis, BK ELISA, and mass spectrometry analysis we demonstrated that plasmin efficiently cleaves HK in a buffer system as well as in mouse and human plasma. Importantly, plasmin mediated cleavage of HK resulted in the release of BK and generation of cleaved HK fragments. In summary, our data indicate that the extrinsic but not the intrinsic coagulation pathway drives the coagulation-mediated pathologies associated with APAP-induced liver injury in mice. HK contributes to the liver injury independently of thrombin generation. We propose that in APAP-challenged mice, HK cleavage and downstream hepatotoxicity, are mediated by plasmin rather than FXIIa-dependent generation of kallikrein. Disclosures McCrae: Sanofi Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Pfizer Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Dova Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Rigel Pharmaceutical: Membership on an entity's Board of Directors or advisory committees. Key:Uniqure BV: Research Funding.

Utilizing Plasma Composition Data to Help Determine Procoagulant Dynamics in Patients with Thermal Injury: A Computational Assessment.

The development of methods that generate individualized assessments of the procoagulant potential of burn patients could improve their treatment. Beyond its role as an essential intermediate in the formation of thrombin, factor (F)Xa has systemic effects as an agonist to inflammatory processes. In this study, we use a computational model to study the FXa dynamics underlying tissue factor-initiated thrombin generation in a small cohort of burn patients. Plasma samples were collected upon admission (Hour 0) from nine subjects (five non-survivors) with major burn injuries and then at 48 hours. Coagulation factor concentrations (II, V, VII, VIII, IX, X, TFPI, antithrombin (AT), protein C (PC)) were measured and used in a computational model to generate time course profiles for thrombin (IIa), FXa, extrinsic tenase, intrinsic tenase and prothrombinase complexes upon a 5 pM tissue factor stimulus in the presence of 1 nM thrombomodulin. Parameters were extracted from the thrombin and FXa profiles (including max rate (MaxRIIa and MaxRFXa) and peak level (MaxLIIa and MaxLFXa)). Procoagulant potential was also evaluated by determining the concentration of the complexes at select times. Parameter values were compared between survivors and non-survivors in the burn cohort and between the burn cohort and a simulation based on the mean physiological (100%) concentration for all factor levels. Burn patients differed at Hour 0 (p < 0.05) from 100% mean physiological levels for all coagulation factor levels except FV and FVII. The concentration of FX, FII, TFPI, AT and PC was lower; FIX and FVIII were increased. The composition differences resulted in all nine burn patients at Hour 0 displaying a procoagulant phenotype relative to 100% mean physiological simulation (MaxLIIa (306 ± 90 nM vs. 52 nM), MaxRIIa (2.9 ± 1.1 nM/s vs. 0.3 nM/s), respectively p < 0.001); MaxRFXa and MaxLFXa were also an order of magnitude greater than 100% mean physiological simulation (p < 0.001). When grouped by survival status and compared at the time of admission, non-survivors had lower PC levels (56 ± 18% vs. 82 ± 9%, p < 0.05), and faster MaxRFXa (29 ± 6 pM/s vs. 18 ± 6 pM/s, p < 0.05) than those that survived; similar trends were observed for all other procoagulant parameters. At 48 hours when comparing non-survivors to survivors, TFPI levels were higher (108 ± 18% vs. 59 ± 18%, p < 0.05), and MaxRIIa (1.5 ± 1.4 nM/s vs. 3.6 ± 0.7 nM/s, p < 0.05) and MaxRFXa (13 ± 12 pM/s vs. 35 ± 4 pM/s, p < 0.05) were lower; similar trends were observed with all other procoagulant parameters. Overall, between admission and 48 hours, procoagulant potential, as represented by MaxR and MaxL parameters for thrombin and FXa, in non-survivors decreased while in survivors they increased (p < 0.05). In patients that survived, there was a positive correlation between FX levels and MaxLFXa (r = 0.96) and reversed in mortality (r= -0.91). Thrombin and FXa generation are increased in burn patients at admission compared to mean physiological simulations. Over the first 48 hours, burn survivors became more procoagulant while non-survivors became less procoagulant. Differences between survivors and non-survivors appear to be present in the underlying dynamics that contribute to FXa dynamics. Understanding how the individual specific balance of procoagulant and anticoagulant proteins contributes to thrombin and FXa generation could ultimately guide therapy and potentially reduce burn injury-related morbidity and mortality.

Ponatinib and Cardiovascular Complications

Patients with T315I positive CML are resistant to most tyrosine kinase inhibitors (TKIs). Ponatinib (Iclusig) is approved for CML patients with the T315I ABL kinase polymorphism. However, ponatinib treatment is associated with vascular events (myocardial infarction, stroke, coronary artery stenosis, limb ischemia and occlusion, and venous thrombosis) in~29% of patients. The mechanism(s) for these events has not been characterized. We developed a murine model to examine TKIs influence on arterial thrombosis risk. C57BL/6 mice, 18-22 weeks of age and treated with ponatinib by gavage for 14 days at 15 mg/kg PO BID, had significantly shorter carotid artery occlusion times induced by photochemical activation of Rose-Bengal compared to vehicle-treated mice (10.4 ± 2.9 min versus 32.3 ± 4.8 min, p < 0.0001). Mice were treated with ponatinib for 14 days at the 3 mg/kg PO BID, a dose that yields plasma concentrations similar to patients at 45 mg po daily, also had significantly shorter vessel occlusion times compared to control (18.7 ± 3.7 min versus 32.3 ± 4.8 min, p<0.0001). No difference in time to carotid artery occlusion was observed between imatinib at 180 mg/kg PO BID treatment compared to control (32.7 ± 5.6 min versus 32.3 ± 4.8 min, p = 0.85) or nilotinib at 29 mg/kg PO BID treatment compared to vehicle-treated mice (32.8 ± 5.5 min versus 33.8 ± 5.1 min, p = 0.71). These studies show that uniquely ponatinib treatment is prothrombotic. Plasma of ponatinib-treated animals had normal PT, aPTT, and complete blood counts (WBC, RBC, Hgb, Hct, MCV, MCH, MCHC and platelet counts) assays. Also contact activation- and tissue factor-initiated thrombin generation times (TGT) showed no difference between treated and untreated mouse plasma.We next examined the mechanism(s) of ponatinib-induced thrombosis. Ponatinib at 3 mg/kg PO BID daily inhibited p-LynY396 in murine platelets. Lyn is a negative regulator of platelet GPVI. Collagen-related peptide (CRP)-induced expression on murine platelets of the activated heterodimeric complex of α2bβ3 integrins (the JON/A epitope) and the alpha granule constituent P-selectin (CD62) when examined by flow cytometry ex vivo were significantly higher at 3 μg/ml in ponatinib-treated versus untreated mice (p< 0.03). The CRP concentration needed to induce platelet activation in ponatinib-treated mice was significantly lower than untreated mice (p<0.0001, 2-way ANOVA). These data suggested that platelets from ponatinib-treated mice are more GPVI actable. Additional studies with α-thrombin also show ponatinib-treatment makes more active platelets. The threshold for α-thrombin-induced expression of the JON/A epitope also was significantly lower (p<0.0125) at 0.075 and 0.1 nM in ponatinib-treated platelets versus untreated platelets. Likewise, α-thrombin-induced platelet membrane expression of P-selectin also was significantly lower (p<0.025) at 0.075 and 0.1 nM in ex vivo studies of ponatinib-treated platelets. Next, we examined vessel wall for changes in ponatinib-treated mice. Aortic sections showed increased caspase 3 staining in vessel adventitia and surrounding adipose tissue in treated mice, a sign of apoptosis. Also genes involved in vessel anti-thrombosis were altered in 3 mg/kg PO BID ponatinib-treated mice. Expression of mRNA of both COX2 and eNOS and their vasculo-protective transcriptional regulators, Sirt1 and KLF4, respectively, were significantly decreased (p<0.05) in the vessel wall of ponatinib-treated mice.We then sought agents to blunt the prothrombotic changes with ponatinib treatment. Since PPAR-γ agonism elevates Sirt1, and vessel wall Sirt1 is reduced in treated mice, we determined if pioglitazone treatment, a PPAR-γ agonist thiazolidinedione, corrects thrombosis risk after ponatinib in vivo. When ponatinib-treated mice were given oral pioglitazone (10 mg/kg/day po), their short times to thrombosis significantly lengthened (49±6.9 min, p<0.0251) to values like untreated mice. Additionally, neither lisinopril nor atorvastatin ameliorated the ponatinib’s prothrombotic effect in vivo. In sum, ponatinib uniquely induces a prothrombotic state due increased platelet activation and reduced vessel wall anti-thrombosis. The effect of ponatinib on platelets may arise in part from its inhibition of p-Lyn. In a murine model of arterial thrombosis, ponatinib’s prothrombotic effect is ameliorated by PPAR-γ agonism with pioglitazone. DisclosuresNo relevant conflicts of interest to declare.

Unfractionated and Low-Molecular-Weight Heparin and the Phosphodiesterase Inhibitors, IBMX and Cilostazol, Block Ex Vivo Equid Herpesvirus Type-1-Induced Platelet Activation.

Equid herpes virus type-1 (EHV-1) is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins) or platelet inhibitors that impede post-receptor thrombin signaling [phosphodiesterase (PDE) antagonists] would inhibit EHV-1-induced platelet activation ex vivo. We exposed platelet-rich plasma (PRP) collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque-forming unit/cell in the presence or absence of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX), and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1–0.2 U/mL anti-factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM, but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX) or isoenzyme-3 selective (cilostazol) PDE antagonists inhibit ex vivo EHV-1-induced platelet activation. These drugs have potential as adjunctive therapy to reduce the serious complications associated with EHV-1-induced thrombosis. Treatment trials are warranted to determine whether these drugs yield clinical benefit when administered to horses infected with EHV-1.

Ancrod revisited: viscoelastic analyses of the effects of Calloselasma rhodostoma venom on plasma coagulation and fibrinolysis.

Fibrinogen depletion via catalysis by snake venom enzymes as a therapeutic strategy to prevent or treat thrombotic disorders was utilized for over four decades, with ancrod being the quintessential agent. However, ancrod eventually was found to not be of clinical utility in large scale stroke trial, resulting in the eventual discontinuation of the administration of the drug for any indication. It was hypothesized that ancrod, possessing thrombin-like activity, may have unappreciated robust coagulation kinetics. Using thrombelastographic methods, a comparison of equivalent tissue factor initiated thrombin generation and Calloselasma rhodostoma venom (rich in ancrod activity) on plasmatic coagulation kinetics was performed. The venom resulted in thrombi that formed nearly twice as fast compared to thrombin formed clots, and there was no difference in fibrinolytic kinetics initiated by tissue-type plasminogen activator. In plasma containing iron and carbon monoxide modified fibrinogen, which may be found in patients at risk of stroke, the coagulation kinetic differences observed with venom was still more vigorous than that seen with thrombin. These phenomena may provide insight into the clinical failure of ancrod, and may serve as an impetus to revisit the concept of fibrinogen depletion via fibrinogenolytic enzymes, not those with thrombin-like activity.

Activation, activity and inactivation of factor VIII in factor VIII products.

Factor VIII (FVIII) products used in haemophilia A treatment show inter-and intra-product and inter-assay differences in specific activity. The mechanistic basis of these differences remains unclear. The aim of this study was to mechanistically compare the functional properties of an in-house excipient-free full-length FVIII standard and pharmacologic recombinant products containing full-length (products A and B) or B-domainless (C and D) FVIII. Factor VIII protein concentration was quantitated by ELISA. Product potency determinations (APTT, intrinsic tenase assays) and kinetic analyses detailing these products' activations by thrombin and FXa, their spontaneous and activated protein C (APC) catalysed inactivation and their performances in coagulation proteome reconstructions were studied +/- von Willebrand factor (VWF). Computational models were developed to facilitate interpretation of empirical data. Factor VIII protein content per manufacturer activity unit was highest for product C with the other three products similar to the standard. Potency estimates, done five different ways, varied 20-30% in inter- and intra-assay comparisons, with product B consistently showing lower specific activity. Kinetic analyses showed the five FVIII species to differ somewhat in maximum rate of activation, the maximum level of activity achieved, the rate of spontaneous or APC catalysed inactivation and the magnitude of the effect of VWF on these parameters. When evaluated both computationally and empirically in the context of tissue factor initiated thrombin generation, product C appears the most dissimilar. Assessments of FVIII activation/inactivation dynamics report larger differences between FVIII products than standard functional assays. However, all FVIII products promote a 'normal' thrombin generation response to TF.

Engineered Factor Xa Variants Retain Procoagulant Activity Independent of Direct Factor Xa-Inhibitors

The venom of the Australian Elapid snake Pseudonaja textilis contains a prothrombin-activating complex that consists of factor Xa (FXa) and factor Va (FVa) homologs which are evolutionary adapted to derail the hemostatic system of its prey, leading to runaway coagulation. These adaptations include functional resistance to inactivation by the main inhibitors of coagulation, antithrombin and activated protein C. Further studies revealed that venom FXa, unlike other FXa species, is also resistant to inhibition by direct oral FXa-inhibitors such as rivaroxaban and apixaban (Ki >1000 nM for venom FXa vs. 1 nM for human FXa). Accordingly, venom FXa is able to support thrombin generation (TG) in FX-depleted plasma spiked with pharmacological concentrations (0.4-2 μM) of these FXa-inhibitors. While human FXa-initiated TG resulted in a 8-fold prolonged lag time and a 70% reduced thrombin peak, those parameters were within normal range in venom FXa-triggered TG.Venom FX homologs produced by Elapid snakes comprise a heterogeneous insertion between His91-Tyr99 (chymotrypsin numbering) in the serine protease domain. A recent crystal structure of one of these homologs shows that this insertion is in close proximity of the active site pocket. In contrast, P. textilis liver-derived plasma FX, which, when activated, is fully inhibited by the FXa-inhibitors (Ki10 nM), lacks this structural feature. We investigated whether the His91-Tyr99 insertion is at the basis of the reduced sensitivity of venom FXa towards FXa-inhibitors. To do so, we constructed and stably expressed human-snake FX chimeras (FX-A, -B, -C) that incorporate His91-Tyr99 insertions from three venom FXa homologs. The chimeric FX variants were purified by successive ion-exchange and hydrophobic interaction chromatography steps, and FXa was generated following RVV-X-activation and size-exclusion chromatography.Evaluation of the kinetic parameters of prothrombin conversion in the presence of saturating amounts of FVa and anionic phospholipids revealed that the chimeric FXa variants exhibit an up to ~4-fold enhanced affinity for prothrombin (Km 0.11-0.29 μM) as compared to recombinant human FXa (rhFXa; Km 0.41 μM). The rate of prothrombin activation was 3-10-fold reduced (kcat 118-370 min-1 vs. 1243 min-1 for rhFXa), which may be indicative of a modified active site conformation. Consistent with this, the rate of chimeric FXa inhibition by antithrombin was impaired (kapp 0.12-0.95 x 103 M-1 s-1 vs. 4.07 x 103 M-1 s-1 for rhFXa). Furthermore, the variant that was most poorly inhibited by antithrombin (variant A) also exhibited the lowest catalytic rate of prothrombin conversion and vice versa (variant C). Conversely, apixaban or edoxaban inhibition of the FXa variants assembled into prothrombinase led to the highest Ki for chimeric variant C (2.3 or 0.3 µM), followed by variants B (1.4 or 0.2 µM), and A (0.2 or 0.006 µM) compared to rhFXa (0.004 or 0.0005 µM). Evaluation of the inhibition of uncomplexed FXa variants employing peptidyl substrate conversion revealed a similar decrease in sensitivity to the FXa-inhibitors (≤500-fold). These data suggest that insertion of the snake venom His91-Tyr99 regions indeed results in impaired engagement of the FXa active site pocket.We next assessed whether chimeric variant C, which is most resistant to inhibition by the direct FXa-inhibitors, is able to restore thrombin generation in a plasma system in the presence of apixaban or edoxaban. While rhFXa-triggered (5 nM) thrombin formation in FX-depleted plasma was inhibited by 2 μM apixaban, initiation with FXa-C (5 nM) resulted in normal thrombin generation parameters (peak thrombin 98%). In addition, the zymogen form of variant C also supported tissue factor-initiated (2 pM) thrombin generation in FX-depleted plasma with inhibitor concentrations up to 6 μM (apixaban) or 2 μM (edoxaban). Under these conditions, little if any thrombin was formed with rhFX present (peak thrombin 5%). We obtained similar results when performing these experiments in normal pooled plasma.Taken together, these results show that chimeric FX is able to restore hemostasis in plasma inhibited by the direct FXa-inhibitors, both in the zymogen as well as protease state. As such, these variants have the potential to serve as rescue therapeutic agents to overcome the effect of FXa-inhibitors in case of potential life-threatening bleeding events or emergency surgical interventions. DisclosuresBos:Bayer Hemophilia Awards: Research Funding.

In Vitro Characterization of a Homogeneous Low-Molecular Weight Heparin with Reversible Anticoagulant Activity

Introduction. Due to their increased half-life over unfractionated heparin (UFH) and marked decrease in the incidence of heparin induced thrombocytopenia (HIT), low molecular weight heparins (LMWH) are the most widely prescribed heparin in the US. However owing to their incomplete reversibility with protamine, LMWHs (such as Enoxaparin) carry the risk of bleeding. The synthetic pentasaccharide, Fondaparinux, also lacks a specific antidote. We recently published (Xu et al. Nat. Chem. Biol. 2014) on a new class of synthetic LMWH that is not renal-excreted and offers the benefit of reversal by protamine. The new compound, dubbed “Super 12-mer”, is a 3,483 Da dodecasaccharide consisting of an antithrombin (AT) binding moiety with repeating units of IdoA2S-GlcNS6S (S is sulfate) and two 3-O-sulfate groups which afford the ability to bind protamine. We sought to characterize this new compound in a series of biochemical and global coagulation assays to better characterize its efficacy as a new reversible anticoagulant.Methods. Factor (F) Xa-AT inhibition assays were performed in both purified and plasma-based systems. The Super 12-mer was further tested in a purified prothrombinase system, as well as by tissue factor-initiated thrombin generation assays in contact pathway inhibited citrated plasma.Results. In vitro FXa inhibition studies indicated the IC50 to be 2-fold higher (49 ng/mL, 24 nM) than was previously reported. Nevertheless, the Super 12-mer anti FXa activity was approximately 2-fold greater than Enoxaparin at identical concentrations. However, the anti FXa activities of the Super 12-mer and Enoxaparin in plasma-based systems were roughly equivalent. Prothrombinase experiments indicated that both the Super 12-mer and Enoxaparin were equivalent in their ability to inhibit FXa in complex with FVa. When the two heparinoids were compared in a plasma-based thrombin generation assay (TGA), their effects on thrombin generation were nearly identical with a 50% reduction in peak thrombin generation occurring at approximately 325 nM heparinoid. When protamine is titrated against a fixed concentration of Super 12-mer (625 nM), the Super 12-mer displays a complete reconstitution of thrombin generation.Conclusions. In plasma and purified systems, the Super 12-mer displayed virtually identical efficacy in FXa inhibition compared to Enoxaparin. In buffered systems, the Super 12-mer was approximately 2-fold more active than Enoxaparin against FXa suggesting the Super 12-mer may have other binding partners in plasma. Interestingly, FXa inhibition in prothrombinase was essentially identical between the two heparinoids. Unlike Enoxaparin however, the Super 12-mer displayed near complete reversibility with protamine in TGAs. A significant lag in thrombin generation was observed when protamine was added, consistent with a previous report (Ni Ainle et al. Blood 2009) that protamine itself can act as an anticoagulant by interfering with FV activation. These data show that the Super 12-mer has almost identical efficacy to Enoxaparin in terms of FXa inhibition, while displaying significant reversibility with protamine. Taken together with the fact that this compound can be safely used in renal-impaired patients, the Super 12-mer is a promising new heparanoid anticoagulant with a potentially enhanced safety profile. DisclosuresNo relevant conflicts of interest to declare.

Active Site-labeled Prothrombin Inhibits Prothrombinase in Vitro and Thrombosis in Vivo

Mouse and human prothrombin (ProT) active site specifically labeled with D-Phe-Pro-Arg-CH(2)Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating the requirement for all three ProT domains. Kinetics of inhibition of ProT activation by the inactive ProT(S195A) mutant were compatible with competitive inhibition as an alternate nonproductive substrate, although FPR-ProT deviated from this mechanism, implicating a more complex process. FPR-ProT exhibited ∼10-fold more potent anticoagulant activity compared with ProT(S195A) as a result of conformational changes in the ProT catalytic domain that induce a more proteinase-like conformation upon FPR labeling. Unlike ProT and ProT(S195A), the pathway of FPR-ProT cleavage by prothrombinase was redirected from meizothrombin toward formation of the FPR-prethrombin 2 (Pre 2)·F1.2 inhibitory intermediate. Localization of ProT labeled with Alexa Fluor® 660 tethered through FPR-CH(2)Cl ([AF660]FPR-ProT) during laser-induced thrombus formation in vivo in murine arterioles was examined in real time wide-field and confocal fluorescence microscopy. [AF660]FPR-ProT bound rapidly to the vessel wall at the site of injury, preceding platelet accumulation, and subsequently to the thrombus proximal, but not distal, to the vessel wall. [AF660]FPR-ProT inhibited thrombus growth, whereas [AF660]FPR-Pre 1, lacking the F1 membrane-binding domain did not bind or inhibit. Labeled F1.2 localized similarly to [AF660]FPR-ProT, indicating binding to phosphatidylserine-rich membranes, but did not inhibit thrombosis. The studies provide new insight into the mechanism of ProT activation in vivo and in vitro, and the properties of a unique exosite-directed prothrombinase inhibitor.

Thrombin generation in chronic obstructive pulmonary disease: Dependence on plasma factor composition

BackgroundChronic obstructive pulmonary disease (COPD) is associated with an increased risk for thromboembolic events. We investigated thrombin generation profiles in COPD patients and their dependence on plasma factor/inhibitor composition. MethodsFactors (f) (fII, fV, fVII, fVIII, fIX, fX), antithrombin, protein C (PC) and free tissue factor pathway inhibitor (fTFPI) from 60 COPD patients (aged 64.2±10.1years; a mean forced expiratory volume in 1 second [FEV1], 55.6±15.8% of predicted values) were compared with those for 43 controls matched for age, sex, weight and smoking. Patients receiving anticoagulation were excluded. Using each individual's plasma coagulation protein composition, tissue factor-initiated thrombin generation was assessed computationally. ResultsCOPD patients had higher fII (115±16 vs 102±10%, p<0.0001), fV (114±19 vs 102±12%, p=0.0002), fVII (111±15 vs 102±17%, p=0.002), fVIII (170±34 vs 115±27%, p<0.0001), and fIX (119±21 vs 107±17%, p=0.003), and lower fTFPI (17.7±3.2 vs 18.9±3.2ng/ml, p=0.047) compared with controls, while fX, antithrombin, and PC were similar in both groups. Computational thrombin generation profiles showed that compared with controls, COPD patients had higher maximum thrombin levels (+28.3%, p<0.0001), rates of thrombin generation (+46.1%, p<0.0001) and total thrombin formation (+14.4%, p<0.001), together with shorter initiation phase of thrombin generation (p<0.0001) and the time to maximum thrombin levels (p<0.0001). Thrombin generation profiles in COPD patients can be normalized via correction of fII, fVIII , fIX and TFPI. The severity of COPD and inflammatory markers were not associated with thrombin generation profiles. ConclusionsProthrombotic phenotype in COPD patients is largely driven by increased prothrombin, fVIII, fIX, and lower fTFPI.

Mouse recombinant protein C variants with enhanced membrane affinity and hyper-anticoagulant activity in mouse plasma

Mouse anticoagulant protein C (461 residues) shares 69% sequence identity with its human ortholog. Interspecies experiments suggest that there is an incompatibility between mouse and human protein C, such that human protein C does not function efficiently in mouse plasma, nor does mouse protein C function efficiently in human plasma. Previously, we described a series of human activated protein C (APC) Gla domain mutants (e.g. QGNSEDY-APC), with enhanced membrane affinity that also served as superior anticoagulants. To characterize these Gla mutants further in mouse models of diseases, the analogous mutations were now made in mouse protein C. In total, seven mutants (mutated at one or more of positions P(10)S(12)D(23)Q(32)N(33)) and wild-type protein C were expressed and purified to homogeneity. In a surface plasmon resonance-based membrane-binding assay, several high affinity protein C mutants were identified. In Ca(2+) titration experiments, the high affinity variants had a significantly reduced (four-fold) Ca(2+) requirement for half-maximum binding. In a tissue factor-initiated thrombin generation assay using mouse plasma, all mouse APC variants, including wild-type, could completely inhibit thrombin generation; however, one of the variants denoted mutant III (P10Q/S12N/D23S/Q32E/N33D) was found to be a 30- to 50-fold better anticoagulant compared to the wild-type protein. This mouse APC variant will be attractive to use in mouse models aiming to elucidate the in vivo effects of APC variants with enhanced anticoagulant activity.

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.

Predicting Thrombosis in Factor VLeiden Heterozygotes.

Factor VLeiden (G1691A;R506Q) is an autosomal dominant allele displaying high prevalence (3–7%) in the United States Caucasian population and a high incidence of venous thrombosis in homozygotes (50% lifetime risk) but reduced penetrance in heterozygote carriers (<10% lifetime risk). Factors that precipitate or suppress the expression of the thrombotic phenotype in factor VLeiden heterozygotes are not well defined except for relatively infrequent instances of coexpression of other thrombophilic risk factors, e.g. the prothrombin G20210A mutant. Our goal is to mathematically evaluate the effect of factor VLeiden on tissue factor-initiated thrombin generation and determine whether concentration variations within the normal range of antithrombin (86–128%), prothrombin (60–140%) or protein C (77–183%) modulate the factor VLeiden effect when all other factor levels are at mean physiologic concentrations. Our mathematical model (Hockin et al. (2002) JBC 277:18322) was extended to incorporate the protein C pathway by including descriptions of thrombin binding to thrombomodulin, activation of protein C and factor Va inactivation by activated protein C. Simulations were conducted with 100% factor V, 50% factor V/50% factor VLeiden (heterozygote), and 100% factor VLeiden (homozygote). Heterozygous expression of factor VLeiden increases the maximum level of thrombin by 25% and the maximum rate of thrombin generation by a factor of 1.6 over that seen with 100% factor V. Homozygous factor VLeiden yields a 2.6-fold increase in maximum rate and a 60% increase in maximum thrombin level compared to 100% factor V. Decreasing the protein C concentration to 77% in a factor VLeiden heterozygote results in a thrombin generation profile with maximum levels and rates comparable to the factor VLeiden homozygote. At 150% protein C, thrombin generation is suppressed to levels similar to those seen with 100% factor V. When the effect of variable prothrombin levels in a factor VLeiden heterozygote is examined, a 60% level of prothrombin decreases the maximum level of thrombin generation below that seen with 100% factor V. When prothrombin levels are at 140% in the factor VLeiden heterozygote, the thrombin generation profile is similar to that observed with 100% factor VLeiden . When the effect of variable antithrombin levels was tested in a factor VLeiden heterozygote, a 14% decrease in antithrombin resulted in maximum thrombin levels approaching those of the factor VLeiden homozygote. In contrast, 128% antithrombin in a factor VLeiden heterozygote resulted in the suppression of thrombin generation to levels consistent with those seen with 100% factor V. Our results predict that the thrombotic effect of heterogenous expression of factor VLeiden can be reduced when prothrombin levels are at the low extreme of the normal range or when antithrombin or protein C levels are at the high extreme of the normal range. Conversely, the thrombotic effect of the heterozygous expression of factor VLeiden is intensified when prothrombin levels are at the high extreme of normal values or when antithrombin or protein C levels at the low extreme of normal. These results may contribute to the understanding of phenotypic variation within factor VLeiden heterozygotes.

Enhancement of the Anticoagulant Properties of Protamine Sulphate by Activated Protein C

Protamine sulphate is a positively-charged polypeptide widely used to reverse heparin-induced anticoagulation. Paradoxically, protamine also possesses intrinsic anticoagulant properties. Furthermore, administration of excess protamine in the neutralization of UFH is associated with increased bleeding, particularly following cardiothoracic surgery. In this study we have investigated the molecular mechanisms underlying the anticoagulant properties of protamine. In pooled normal plasma, we observed a dose-dependent prolongation of both PT and APTT assays with increasing protamine concentrations (0–30μg/ml). The anticoagulant effects of protamine in normal plasma were also examined using a tissue factor-initiated thrombin generation assay. 30μg/ml protamine resulted in a two-fold prolongation of lag-time, a two-fold reduction in peak thrombin generation, and a 41±17% (p=0.047) decrease in endogenous thrombin potential (ETP). In heparinised plasma (0.3U/ml), addition of increasing protamine concentration initially reversed the anticoagulant effect of heparin, resulting in a progressive increase in ETP to that of normal plasma. However, further increases in protamine resulted in a dose-dependent reduction in ETP to a minimum of 61±16%. Recent studies have shown that platelet factor 4 (PF4), another cationic protein used to reverse heparin, can bind to the anionic Gla domain of protein C, thereby enhancing APC generation (up to 25-fold). Consequently, we investigated potential interaction(s) between protamine and the protein C anticoagulant pathway. As expected, in normal plasma, APC (0–20nM) caused a concentration-dependent prolongation in APTT to 180±8%, and a parallel reduction in ETP. However in the presence of 30μg/ml protamine, the effects of APC on both the APTT and ETP were markedly enhanced compared to the effect of either substance alone. As APC down-regulates thrombin generation by inactivating FVa and FVIIIa, we used a phospholipid-dependent FVa proteolysis assay to elucidate the mechanism responsible for the synergistic interaction between APC and protamine. The ability of APC to reduce FVa cofactor activity in this assay (in the presence or absence of protein S) was not significantly affected by the presence of protamine. However, a potent synergistic anticoagulant interaction between APC and protamine was also observed in plasma from patients with homozygous FV Leiden, suggesting protamine enhances APC cleavage of FVa at position Arg-306. To determine whether protamine influences the rate of FVIIIa proteolysis by APC, we expressed and purified an APC-resistant FVIII variant (R336Q/R562Q). FVIII-deficient plasma was spiked with physiological concentrations of wild type or variant FVIII, and the anticoagulant effects of protamine ± APC studied using plasma thrombin generation assays. Similar synergistic anticoagulant effects of APC in combination with protamine were observed for both wildtype and variant FVIII. To assess whether protamine also influences procoagulant processes, the rate of factor V activation by thrombin was analysed by SDS-PAGE. In the presence of protamine (30μg/ml), FVa generation was significantly reduced. In addition to inhibiting the rate of FVa generation, we also observed that protamine significantly impaired the functional activity of the prothrombinase complex in a concentration dependent manner. In contrast, cationic polybrene had no significant effect on either rate of FVa generation or prothrombinase complex activity. In conclusion, we have shown a novel and profound anticoagulant synergy between protamine sulphate and APC. Moreover, we demonstrate that this synergistic effect is mediated by independent effects on FVa generation and proteolysis respectively. These novel findings provide further insights into the molecular mechanism underlying the anticoagulant effect of excess protamine in human plasma.

A Single Amino Acid Substitution (I18V) in the Gla Domain of Activated Protein C Markedly Impairs Ability of PE and GlyCer to Enhance Anticoagulant Activity

Phosphatidylethanolamine (PE) and glucosylceramide (GlyCer) are cell surface lipids that preferentially enhance anticoagulant, rather than procoagulant pathways. In particular, both PE and GlyCer enhance the anticoagulant activity of activated protein C (APC). Previous studies have indicated that specific APC Gla domain residues may mediate APC interaction with PE and GlyCer. To investigate whether specific APC residues mediate PE and GlyCer enhanced APC anticoagulant activity, we expressed a series of APC variants in which APC Gla domain residues not shared with the human prothrombin Gla domain were substituted with their prothrombin amino acid equivalent. The anticoagulant activity of each APC Gla domain variant was assessed in a tissue factor-initiated thrombin generation assay containing phospholipid vesicles of differing composition (80% PC/20% PS); or PC/PS/PE (60%/20%/20%); or PC/PS/GlyCer (60%/20%/20%). For each of these lipid mixtures, thrombin generation (endogenous thrombin potential, ETP) was not significantly different in the absence of APC. In the presence of PC/PS vesicles, APC reduced thrombin generation by 63±3% at the highest APC concentration tested (6nM). However, APC impairment of thrombin generation was enhanced 3-fold in the presence of PC/PS/PE compared with vesicles containing PC/PS alone, and in the presence of PC/PS/GlyCer was enhanced 4.3-fold. Enhancement of anticoagulant function by PE and GlyCer was similar for the majority of the APC Gla domain variants tested. Interestingly, one APC variant (APC-I18V) exhibited similar anticoagulant activity to that of wild type APC with PC/PS vesicles, but was not enhanced by the presence of PE- or GlyCer-containing vesicles. Phospholipid vesicles containing PE or GlyCer have been previously described to enhance protein S cofactor enhancement of APC. Therefore, to further characterize APC-I18V, we assessed the ability of wild type APC and APC-I18V to be enhanced by protein S in the presence of PC/PS, PC/PS/PE or PC/PS/GlyCer using a protein S-sensitive thrombin generation assay. In the presence of PC/PS, increasing protein S concentration in protein S-deficient plasma resulted in an APC-mediated slow decrease in thrombin generation, irrespective of whether wild type or APC-I18V was used (IC50 for protein S-mediated APC inhibition of thrombin generation with PC/PS, 130nM). However, in the presence of PC/PS/PE or PC/PS/GlyCer, thrombin generation was impaired by wild type APC at 3–4-fold lower protein S concentration than that observed when PC/PS vesicles alone were used (IC50, PC/PS/PE=31.5nM and PC/PS/GlyCer=37.5nM). APC-I18V, however, did not exhibit a similarly increased sensitivity to protein S in the presence of PE or GlyCer, as the anticoagulant activity of this variant was the same as when only PC/PS was included. To investigate whether the loss of specific neutral lipid enhancement in APC-I18V affected its ability to initiate cytoprotective signaling via EPCR-PAR-1 on endothelial cells, the capacity of APC-I18V to inhibit thrombin-induced endothelial cell barrier permeability was assessed. When cells were pre-treated with either wild type APC or APC-I18V, there was a significant enhancement in barrier integrity and attenuation of thrombin-induced permeability (P<0.05), demonstrating that loss of PE/GlyCer enhancement of APC anticoagulant activity does not adversely affect EPCR binding and EPCR/PAR-1 cytoprotective signaling. Collectively, these results suggest PE and GlyCer enhancement of APC anticoagulant activity is mediated by increased sensitivity to protein S, and that Ile-18 in the APC Gla domain is critical for mediating APC-specific functional enhancement by PE/GlyCer.

Dissociation of Activated Protein C Functions by Elimination of Protein S Cofactor Enhancement

Activated protein C (APC) plays a critical anticoagulant role by inactivating factor Va (FVa) and factor VIIIa (FVIIIa) and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor (EPCR) can initiate PAR-1 mediated cytoprotective signalling. Although protein S constitutes a critical cofactor for APC anticoagulant function, the molecular basis through which protein S interacts with APC is not fully understood. In this study, we employed a site-directed mutagenesis strategy to characterise the effects of four single amino acid substitutions (D35T, D36A, L38D and A39V) within a region of the APC Gla domain important for protein S cofactor enhancement. To maintain Gla domain structural integrity, each residue was substituted with the corresponding residue of the human prothrombin Gla domain. Protein C variants were expressed in HEK 293 cells and purified by ion-exchange chromatography. Upon activation, the amidolytic activity of each recombinant APC variant was identical to that of wild type APC. The anticoagulant function of recombinant wild type and variant APC was compared in a tissue factor-initiated thrombin generation assay using protein C-deficient plasma. Wild type APC diminished thrombin generation in a concentration-dependent manner as expected. Variants APC-D35T, APC-D36A and APC-A39V exhibited only mildly impaired (<2-fold) anticoagulant activity compared to wild type APC. The anticoagulant activity of APC-L38D, however, was severely impaired. APC-L38D was unable to achieve half-maximal inhibition of endogenous thrombin potential (ETP) at APC concentrations as high as 150nM, compared to wild type APC, which achieved half-maximal inhibition at 7.2nM APC. To clarify the role of Leu-38 in facilitating APC anticoagulant function, we further studied the ability of APC-L38D to be stimulated in protein S-deficient plasma reconstituted with plasma-purified protein S. Co-incubation of wild type APC with increasing protein S concentration (12.5–200nM) caused a corresponding reduction in ETP (IC50 = 24nM protein S). In contrast, APC-L38D was unresponsive to protein S. In the presence of APC-L38D, ETP was reduced only 22% at 1.5μM protein S (10-fold higher than plasma free protein S). In a phospholipid-dependent FVa proteolysis time course assay, both wild type APC and APC-L38D rapidly reduced FVa cofactor activity, indicating that the observed impaired plasma anticoagulant activity of APC-L38D is not mediated by impaired interaction with anionic phospholipids or FVa. In a modified version of this assay, wild type APC-mediated FVa proteolysis was rapidly enhanced by added protein S, with half-maximal inhibition observed at 5nM protein S. In contrast, APC-L38D exhibited no protein S-enhanced FVa proteolysis. Cumulatively, these data confirm that Leu-38 mediates APC anticoagulant function in plasma by facilitating critical protein S cofactor enhancement of FVa proteolysis. Previous studies have shown that APC Gla domain mutations can influence EPCR binding, a pre-requisite for PAR-1 mediated cytoprotective signalling. Consequently, we assessed APC binding to sEPCR using surface plasmon resonance. Binding affinities of APC-L38D and wild type APC were very similar (KD 112±25nM versus 117±36nM). Furthermore, the ability of APC-L38D to protect EAhy926 cells from staurosporine-induced apoptosis was also investigated using RT-PCR quantification of pro- (bax) and anti- (bcl-2) apoptotic gene expression. Pre-incubation with APC-L38D significantly reduced the bax/bcl-2 ratio to the same extent as wild type APC. The EPCR-dependence of these anti-apoptotic activities was confirmed using RCR-252, (an inhibitory anti-EPCR antibody) which ablated the cytoprotective effect of both APC species. In conclusion, we demonstrate that a single amino acid substitution (L38D) can significantly impair APC anticoagulant activity due to elimination of protein S cofactor enhancement. However, despite the location of Leu-38 in the Gla domain, APC-L38D retains its ability to bind EPCR, and trigger PAR-1 mediated cytoprotective signalling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to dissociate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

Recombinant Antidote for Reversal of Anticoagulation by Factor Xa Inhibitors.

Individuals anticoagulated with warfarin or heparin are typically treated with specific antidotes such as vitamin K or protamine, respectively, if they bleed or require surgery. In contrast, specific and effective antidotes are not available for the reversal of the anticoagulant effects of the low molecular weight heparins (LMWH) or the new oral anticoagulants targeting factor Xa (fXa) which are predictably only marginally affected by standard treatments using rfVIIa or fresh frozen plasma. The present study was designed to test the hypothesis that plasma-derived or recombinant fXa, modified to lack catalytic and membrane binding activities, could neutralize the anticoagulant activities of small molecule fXa inhibitors and LMWH. Plasma derived antidote (pd-Antidote) was prepared by chemical modification of the active site serine of fXa followed by chymotryptic removal of the Gla domain. Preliminary experiments showed that pd-Antidote dose dependently reversed the activity of rivaroxaban, apixaban or betrixaban, three small molecule fXa inhibitors currently in clinical trials. In a fXa amidolytic assay with 3nM enzyme, half maximal reversal of inhibitory activities (EC50) was attained at the following pd-Antidote concentrations: rivaroxaban =49 nM, apixaban = 122 nM, betrixaban = 41 nM. The pd-Antidote had no effect on the activity of fXa in the absence of inhibitors. Thus, active site inactivated pd-Antidote retained the ability to bind small molecule inhibitors of fXa. The activity of pd-Antidote was not limited to reversal of purified fXa catalytic activity. In a tissue factor-initiated thrombin generation assay in plasma, while pd-Antidote did not interfere with the normal function of prothrombinase complexes, it dose-dependently and completely reversed the inhibition produced by the small molecule fXa inhibitors. Pd-Antidote also demonstrated reversal of the in-vitro anticoagulant activity of the LMWH, enoxaparin. Addition of pd-Antidote (515nM) produced a 37% reduction of the clotting activity of enoxaparin (1.25Units/ml). In an activated partial thromboplastin time assay (aPTT), pd-Antidote also dose-dependently reversed the inhibitory effects of the fXa inhibitors. For example, aPTT prolongation by betrixaban (400nM) was reversed with an estimated EC50=650nM. Baseline aPTT was not altered upon addition of pd-Antidote up to a concentration of 2.5 μM, the highest concentration studied. Recombinant antidote (r-Antidote) was expressed in mammalian cells (Chinese hamster ovary) using a construct for human fXa with the S195A mutation and lacking the Gla-domain. The EC50's for purified r-Antidote in reversing 7.5 nM fXa inhibitors in the fXa catalytic activity assay were: rivaroxaban =17 nM, apixaban = 45 nM, betrixaban = 15 nM. As expected, there was no inhibition of fXa activity by r-Antidote alone. Purified r-Antidote also reversed anticoagulant activity in plasma clotting assays. Prothrombin time (PT) extensions achieved by supratherapeutic concentrations of rivaroxaban (1 μM) or apixaban (1 μM) were completely normalized upon addition of r-Antidote (1.5 μM). Ex vivo clotting prolongation by an excess of betrixaban (300 nM) was essentially reversed (88% correction) by pre-incubation with r-Antidote (570 nM) prior to addition of aPTT reagents. The concentration of r-Antidote required for corrective activity was related to the inhibitory potency of the oral fXa inhibitors. PT or aPTT baselines did not change upon addition of r-Antidote alone at 1.9 μM, the highest concentration tested. We also examined the ability of both pd-Antidote and r-Antidote to alleviate inhibitory activities produced upon oral dosing of fXa inhibitors in mice. Perturbation of whole blood PT/INR in dosed animals was followed as a marker of anticoagulation. The effect following dosing of a fXa inhibitor could be reversed by a single intravenous injection of antidote. INR measurements in blood samples of treated animals showed a >50% reduction of observed inhibitory activity compared to control animals. Our results suggest that these plasma derived or recombinant proteins have the potential to act as universal antidotes for reversal of anticoagulation of all current fXa inhibitors, both small molecule and antithrombin dependent, in patients with bleeding related medical emergencies or those requiring cessation of anticoagulation prior to surgery.

Platelet Factor 4 Mediates Activated Protein C Resistance by Impairment of Protein S Cofactor Enhancement

Platelet factor 4 (PF4) is an abundant platelet α-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the γ-carboxyglutamic acid (Gla) domain of protein C to significantly enhance activated protein C (APC) generation by the thrombin-thrombomodulin complex on the surface of endothelial cells. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. The functional consequences of the interaction between the APC Gla domain and PF4 in relation to APC anticoagulant, anti-inflammatory and anti-apoptotic functions have not previously been fully defined. In a tissue factor-initiated thrombin generation assay, APC impaired thrombin generation as previously described. However PF4 inhibited APC anticoagulant activity in a concentration-dependent manner (IC50 for PF4 inhibition of APC anticoagulant function, 11μg/ml). In contrast, addition of two other cationic polypeptides protamine and polybrene, both significantly enhanced APC anticoagulant activity in plasma. To elucidate the mechanism through which PF4 inhibits APC anticoagulant activity, we utilized a phospholipid-dependent FVa proteolysis time course assay. In the absence of protein S, PF4 had no effect upon FVa proteolysis by APC, indicating that PF4 does not influence the ability of APC to interact with either anionic phospholipids or FVa. However, in the presence of protein S, PF4 significantly inhibited APC-mediated FVa proteolysis (3–5 fold). Collectively, these findings demonstrate that in addition to enhancing APC generation, PF4 also significantly attenuates APC anticoagulant activity in plasma by impairing critical protein S cofactor enhancement of FVa proteolysis, and suggest that PF4 contributes to the poorly-understood APC resistance phenotype associated with activated platelets. APC bound to the endothelial cell protein C receptor (EPCR) via its Gla domain can activate PAR-1 on endothelial cells, triggering complex intracellular signaling that result in anti-inflammatory and anti-apoptotic cellular responses. To ascertain whether PF4 interaction with the protein C/APC Gla domain might impair APC-EPCR-PAR-1 cytoprotective signaling, APC protection against thrombin-induced endothelial barrier permeability and staurosporine-induced apoptosis in the presence of PF4 was determined. APC significantly attenuated thrombin-induced endothelial cell barrier permeability, as expected. PF4 alone (up to 1μM) had no independent effect upon endothelial barrier permeability, and did not protect against thrombin-mediated increased permeability. In contrast to its inhibition of APC anticoagulant activity, PF4 did not significantly inhibit the endothelial barrier protective properties of APC. To determine whether PF4 might interfere with APC-mediated cytoprotection, staurosporine-induced apoptosis in EAhy926 cells was assessed by RT-PCR quantification of pro-apoptotic (Bax) to anti-apoptotic (Bcl-2) gene expression. Pre-treatment of EAhy926 cells with APC decreased the Bax/Bcl-2 ratio close to that determined for untreated EAhy926 cells. PF4 alone, or in combination with APC, had no effect upon apoptosis-related gene expression as determined by alteration of Bax/Bcl-2 expression ratios in response to staurosporine. In summary, PF4 inhibits APC anticoagulant function via inhibition of essential protein S cofactor enhancement in plasma, whilst retaining EPCR/PAR-1 mediated cytoprotective signalling on endothelial cells. This provides a rationale for how PF4 can exert prothrombotic effects in vivo, but also mediate enhanced APC generation on the surface of endothelial cells to induce both anti-inflammatory and anti-apoptotic events. Based on these observations, we propose that PF4 acts as a critical regulator of APC generation in vivo, but also targets APC towards cytoprotective, rather than anticoagulant functions at sites of vascular injury with concurrent platelet activation.