Evidence for a Factor IX-Independent Role for Factor XI in Thrombin Generation

Abstract Abstract 2244 In the widely used activated partial thromboplastin time (aPTT) assay, fibrin formation is induced by a series of sequential activations of the plasma protease zymogens factor (f) XII, fXI, fIX, fX and prothrombin, in that order. Conversion of prothrombin to the protease α-thrombin results in fibrin formation. α-Thrombin also enhances its own generation through activation of the cofactors fV and fVIII. While the linear sequence of reactions in the aPTT implies that loss of any single protease should have a comparable deleterious effect on the system, it is recognized that complete deficiency of a protein near the start of the sequence (e.g. fXII or fXI) results in greater aPTT prolongation than deficiency of proteins further down the sequence (e.g. fIX). This implies that proteases activated early in the process have multiple plasma substrates. For example, fXIa was recently reported to activate fVIII and fV (JTH 8;1532:2010), in addition to its role in fIX activation. Here, we present evidence that fXIa contributes to α-thrombin generation in the absence of fIX through activation of fX and/or fV. We noted that an anti-fXI antibody (O1A6) prolonged the aPTT of plasma from a patient with severe hemophilia B (fIX antigen undetectable) or plasma immunodepleted of fIX. This observation held even when an anti-fIX antibody was added to neutralize potential traces of fIX. Addition of activated fXI (fXIa - 3 nM) directly to fIX-deficient recalcified plasmas induced clot formation, and the time to clot formation was prolonged by O1A6. To further exclude the possibility that traces of fIX were contributing to thrombin generation, we confirmed the results using plasma from mice with combined complete deficiencies of fXII, fXI, and fIX. We tested the capacity of fXIa to cleave/activate fX and fV, the protease zymogen and cofactor, respectively, immediately downstream of fIX in the coagulation cascade. FX, the zymogen of the protease fXa, is evolutionarily related to fIX. SDS-PAGE analysis confirmed that fXIa cleaves fX. FX cleaved by fXIa demonstrated fXa activity in a chromogenic substrate assay, and converted prothrombin to α-thrombin in the presence of fVa and phospholipid. As previously reported, fXIa readily cleaved fV. The cleavage pattern differed from that generated by α-thrombin, however, formation of the fVa light chain was clearly evident. In a plasma clotting assay designed to measure either fXa or fVa activity, fX or fV pre-incubated with fXIa significantly shortened the clotting time of fIX-deficient plasma, while fX or fV pre-incubated with vehicle did not. In thrombin generation assays, fXIa (1.25 to 15 nM) induced thrombin generation in fIX-deficient plasma supplemented with anti-fIX antibody in a concentration dependent manner. FXIa did not induce thrombin generation in plasma lacking fV, or in fIX-deficient plasma containing the fXa inhibitor apixaban. This indicates that fXIa is working at the level of fX/fV in this assay, and is not directly converting prothrombin to α-thrombin. A recombinant variant of fXIa lacking the major fIX-binding exosite (fXIaPKA3, J Biol Chem 1996;271:29023) demonstrated a marked defect, compared to wild type fXIa (fXIaWT), in its capacity to induce thrombin generation in normal plasma. However, in fIX-deficient plasma, fXIaPKA3 and fXIaWT are comparable in their ability to enhance thrombin generation, supporting the premise that fXIa is acting through activation of fX and/or fV in the absence of fIX. Previously, we observed that fXI deficient mice and fIX deficient mice are comparably resistant to carotid artery thrombosis induced by exposure of the vessel to ferric chloride, despite having very different propensities to bleed. The animals were uniformly resistant to thrombosis with 5% FeCl3, and some were resistant at 7.5% FeCl3. All experienced vessel occlusion with 10% FeCl3. This is consistent with fXIa contributing to thrombosis in this model through fIX activation. However, we observed that some mice with combined fIX and fXI deficiency were resistant to FeCl3 concentrations up to 12.5%, implying that fXIa was contributing to thrombosis in a fIX-independent manner, as well. These results are consistent with those from the in vitro assays described above, and support the hypothesis that fXIa contributes to thrombin generation through fIX-dependent and fIX-independent processes. Disclosures: Tucker: Aronora, LLC: Employment, Equity Ownership. Gruber:Aronora, LLC: Consultancy, Equity Ownership.

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Kallikrein directly interacts with and activates Factor IX, resulting in thrombin generation and fibrin formation independent of Factor XI

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2014810118 ◽

2021 ◽

Vol 118 (3) ◽

pp. e2014810118

Author(s):

Katherine J. Kearney ◽

Juliet Butler ◽

Olga M. Posada ◽

Clare Wilson ◽

Samantha Heal ◽

...

Keyword(s):

Thrombin Generation ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Coagulation Factor ◽

Factor Ix ◽

Coagulation Cascade ◽

Contact Activation ◽

Kinin System ◽

Fibrin Formation ◽

Kallikrein Kinin System

Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.

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A Novel Use of Rehydrated, Lyophilized Platelets Increases Recombinant FVIIa Procoagulant Activity in a Model of Hemophilia.

Blood ◽

10.1182/blood.v108.11.1754.1754 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1754-1754

Author(s):

Robert A. Campbell ◽

Thomas H. Fischer ◽

Alisa S. Wolberg

Keyword(s):

Thrombin Generation ◽

Factor Viia ◽

Specific Interaction ◽

Annexin V ◽

Factor Ix ◽

Clot Formation ◽

Generation Rate ◽

Tissue Type ◽

Dependent Manner ◽

Platelet Surface

Abstract The anti-bleeding therapy recombinant factor VIIa (rFVIIa) is used to abrogate bleeding in hemophiliacs with inhibitors, bypassing the need for replacement factors. RFVIIa is hypothesized to work by increasing Xa generation on the platelet’s surface. However, high plasma levels of rFVIIa are required, in part due to the weak binding of rFVIIa to platelets. We hypothesized that the efficacy of the therapy could be improved by administering rFVIIa already bound to platelets. One platelet preparative that may be used in this application is rehydrated, lyophilized (RL) platelets. RL platelets are fixed with paraformaldehyde, which allows them to be frozen and lyophilized while retaining their hemostatic capabilities. Previously, we have shown RL platelets are capable of supporting rFVIIa-mediated thrombin generation and that thrombin generation is increased in a rFVIIa dose-dependent manner (Blood, 106:4057, 2005). In this current study, we have characterized the ability of RL platelets to modulate rFVIIa-mediated thrombin generation and fibrin clot formation in a cell-based complete model of hemophilia. The addition of RL platelets with 50 nM rFVIIa increased the thrombin generation rate in hemophilia 2.8-fold more than 50 nM rFVIIa, alone. Further, the addition of RL platelets with 50 nM rFVIIa normalized clot formation and stability in a fibrinolytic environment, which did not occur in the presence of rFVIIa, alone. In contrast, the addition of RL platelets, alone, to hemophilic conditions had minimal to no effect on thrombin generation rate or the onset of clot formation, suggesting that the effects were due to a specific interaction between rFVIIa and RL platelets. When rFVIIa plus RL platelets were added to platelet-rich plasma from patients with hemophilia A in the presence of tissue-type plasminogen activator, clot formation and stability were improved more than the addition of either agent alone. To examine the mechanism of RL platelets’ augmentation of rFVIIa activity, we titrated the phosphatidylserine (PS) binding protein, annexin V, into reactions with RL platelets in the presence of factors Xa, Va, and prothrombin and measured thrombin generation. The addition of annexin V reduced thrombin generation equally in reactions that contained RL platelets stimulated with or without SFLLRN. Further, thrombin generation was similar on RL platelets simulated with or without SFLLRN in the absence of annexin V. These data suggest that RL platelets already have PS exposed on their surface. We conclude that RL platelets can support rFVIIa-mediated thrombin generation in the absence of factor IX and may enhance rFVIIa activity in part due to PS exposure on the RL platelet surface. We hypothesize that co-administration of RL platelets with rFVIIa may increase the efficacy of rFVIIa, at lower doses of rFVIIa than are currently required to achieve hemostasis.

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Factor XIII Contributes to Clot Formation and Thrombin Generation

Blood ◽

10.1182/blood.v128.22.208.208 ◽

2016 ◽

Vol 128 (22) ◽

pp. 208-208

Author(s):

M Rick Rollins ◽

Emily A Larson ◽

Hillary J Larson ◽

Jason A Taylor

Keyword(s):

Factor Viii ◽

Research Funding ◽

Factor Xiii ◽

Thrombin Generation ◽

Calcium Influx ◽

Factor Xa ◽

Factor Ix ◽

Clot Formation ◽

Α Angle ◽

Deficient Mice

Abstract Background Factor XIII (FXIII) stabilizes fibrin clots, minimizing the amount of thrombin generation required to stop bleeding. It also has a long half-life (>7 days) and supratherapeutic levels are not associated with thrombosis making it an attractive therapeutic agent to augment factor replacement in hemophilia. FXIII is a transglutaminase that cross-links fibrin and localizes alpha 2-antiplasmin to fibrin inhibiting fibrinolysis. It also interacts with numerous other proteins, most of which have not been well studied and their physiologic significance is unknown. FXIII is classically considered to be activated by thrombin, which may limit its utility in low thrombin states such as hemophilia. However, activation also occurs physiologically through other mechanisms including calpain, neutrophil elastase and calcium influx. In addition, low levels of thrombin are generated by Factor Xa in the absence of the Factor VIII (FVIII)/Factor IX (FIX) complex that may contribute to both FXIII activation and clot formation. Thus, it is not clear if thrombin generated through the participation of FVIII and FIX is necessary for FXIII activation. Hypothesis We hypothesize that FXIII (recombinant FXIII-A2) may contribute to both clot formation and thrombin generation in hemophilia. Methods Animal models include Exon 16-deleted FVIII-deficient mice (FVIII-KO), wild-type mice (WT), and exon 16-deleted Factor VIII deficient/GP1bα-FVIII knock-in mice (PF). The PF mice are FVIII-deficient mice expressing human FVIII driven from the glycoprotein 1bα promoter resulting in approximately 3% circulating FVIII. Various combinations of factors were given via tail-vein injection. Citrated blood was collected by cardiac puncture 1.5 - 4 hours post-injection, depending on the factor type. Clotting was characterized using thromboelastography (TEG). Thrombin generation was measured on a fluorescence reader using a reagent comprised of a low concentration of phospholipid micelles containing tissue factor in HEPES buffer. Results TEG characterization shows differences in clotting times (R), speed of clot formation (K), and the kinetics of the formation of the clot (α angle) between the various groups without changes in overall clot stability (MA) or degree of fibrinolysis (LY30). R, K, and α angle are all measurements of clot formation, with prolongation of R and K and reduced α angle characteristic of hemophilia. PF mice have similar R, K, and α angle compared to FVIII-KO (p = 0.5, 0.68, and 0.89, respectively), and elongated R and K, and reduced α angle compared to WT (p = 4.0E-3, 2.0E-3, and 1.37E-6, respectively). Giving supratherapeutic FXIII to PF mice results in normalization of these values compared to WT, with a trend towards elongated K and α angle (p = 0.21, 0.08, and 0.13, respectively), and differences compared to FVIII-KO (p = 8.1E-3, 7.4E-3, and 2.1E-3, respectively). Administering FXIII to FVIII-KO mice did not alter R, K, and α angle compared to untreated FVIII-KO mice (p = 0.25, 0.37, and 0.67, respectively). PF mice have similar peak thrombin generation compared to FVIII-KO (p = 0.56) and reduced peak thrombin generation compared to WT (p = 6.69E-5). Giving supratherapeutic FXIII to the PF mice results in peak thrombin generation similar to that of WT (p = 0.97). In contrast, giving supratherapeutic FXIII to FVIII-KO mice did not alter peak thrombin generation levels compared to untreated FVIII-KO mice (p = 0.72). The administration of a cocktail containing both FVIII (2.5 U/kg) and FXIII resulted in a trend for improved peak thrombin generation when compared to an injection of FVIII alone (p = 0.12). Conclusions The function of FXIII has classically been considered to be secondary to its transglutaminase activity. With a direct impact on early clot formation and thrombin generation, these data suggest that FXIII has other roles beyond its known activities. The implication of these findings is that FXIII may be an effective hemostatic agent in mild and moderate hemophilia. Disclosures Taylor: Novo Nordisk: Research Funding; Kedrion: Research Funding; Baxalta/Shire: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding.

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Genetic Duplication of Tissue Factor Leads to Partial Specialization of Function in Zebrafish

Blood ◽

10.1182/blood-2019-131980 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 486-486

Author(s):

Steven Grzegorski ◽

Divyani Paul ◽

James H. Morrissey ◽

Jordan A. Shavit

Keyword(s):

Tissue Factor ◽

Complete Loss ◽

Clot Formation ◽

Low Flow ◽

Coagulation Cascade ◽

Null Alleles ◽

Prolonged Treatment ◽

External Fertilization ◽

Equity Ownership

Tissue factor (TF) is a critical factor for hemostasis in response to tissue injury. Among mouse knockouts of procoagulant factors, those lacking TF have the most severe phenotype, with complete lethality by midgestation. Furthermore, complete loss of TF has never been described in humans. Together, these suggest additional roles in embryonic development beyond coagulation. Zebrafish are a small freshwater teleost fish with a well described hemostatic system, including conservation of the coagulation cascade. Zebrafish are prolific breeders that reproduce through external fertilization, with subsequent rapid and transparent development, allowing studies not possible in mammals. Due to an ancient genomic event, 30-40% of the teleost genome is duplicated, resulting in two TF paralogs (TFa and TFb) with unknown functions. Here we use CRISPR/Cas9 to produce null alleles of TFa and TFb and uncover partial subspecialization of these duplicates. It has been shown previously that both TFs are expressed before the initiation of blood circulation, between 24 and 48 hours post fertilization, yet complete loss of TFa and TFb yielded no gross abnormalities. Embryos and larvae were able to develop normally through juvenile stages but succumbed to hemorrhage by early adulthood at 9 weeks of age. Surprisingly, a single allele of either TFa or TFb was able to rescue survival in the context of complete loss of the other gene. To evaluate for hemostatic effects of TF deficiency, laser-mediated endothelial injury was used in the venous and arterial systems at 3 and 5 days post fertilization (dpf), respectively. Loss of TFb alone at 3 dpf resulted in no observable hemostatic defects. Conversely, loss of TFa led to a 50% increase in the time to venous occlusion (TTO), which was exacerbated by concomitant loss of one allele of TFb. Total TF deficiency led to a complete inability to form occlusive venous thrombi, indicating that both TFs can trigger coagulation but TFa is able to completely compensate for the loss of TFb. Concordant with these data, loss of TFb resulted in transcriptional upregulation of TFa but not vice versa. Interestingly, the roles are reversed in the arterial vasculature. Loss of TFa had no effect, loss of TFb lead to a 60% reduction in the number of occlusive thrombi, and complete deficiency resulted in no arterial occlusion. Combined with the venous results, these data point to differentiated roles of TFa and TFb in the venous and arterial systems. In order to test whether these differences were functional, recombinant TF (rTF) molecules were expressed in E. coli, purified, and incorporated into 80% phosphatidylcholine/20% phosphatidylserine liposomes. Ex vivo tube-tilt clotting assays were performed by using each rTF to activate citrated plasma from lake trout. rTFa triggered stable clot formation within 1-2 minutes of recalcification. rTFb usually failed to induce clot formation, with occasional delayed fibrin thrombi that appeared to be grossly disorganized and were easily disrupted following agitation. Taken with the in vivo data, this hints at an altered kinetic profile, with TFa being a more potent cofactor for factor VIIa in low flow (venous) settings. The laboratory is an artificially safe environment, so a synthetic chemical stress test was performed on 3 dpf larvae. Prolonged treatment with cortisol and epinephrine led to the development of cardiac tamponade in larvae with complete TF deficiency (61%), but similar results were only found at low levels in wild type siblings (2-5%). The same assay in prothrombin mutants also revealed a high rate of tamponade (75%), but lower levels in fibrinogen-deficient larvae (20%). These data suggest an extrahemostatic risk factor for tamponade that is modified by prothrombin and tissue factor levels and is independent of fibrin formation. Our results intimate that TFa and TFb have overlapping procoagulant functions but differential kinetic profiles in venous vs arterial systems. We also find that the duplication provides a layer of quantitative regulation and creates a titratable level for regulation of hemostatic and extrahemostatic roles of TF. Overall, this novel model provides new structural and physiologic information about TF function in vivo, including potential previously unknown roles in perivascular development, cardiovascular stability, remodeling and/or regeneration. Disclosures Morrissey: PrevThro Pharmaceuticals: Equity Ownership; Cayuga Pharmaceuticals: Equity Ownership; Kerafast, Inc: Research Funding; Issued and pending patent applications relating to medical uses of polyphosphate and polyphosphate inhibitors: Patents & Royalties. Shavit:Bayer: Consultancy; Sanofi: Consultancy; Shire/Takeda: Consultancy; Spark Therapeutics: Consultancy; CSL-Behring: Consultancy; Novo Nordisk: Consultancy.

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Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

International Journal of Toxicology ◽

10.1177/1091581820907324 ◽

2020 ◽

Vol 39 (3) ◽

pp. 207-217

Author(s):

F. Poitout-Belissent ◽

D. Culang ◽

D. Poulin ◽

R. Samadfan ◽

S. Cotton ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

In Vivo Studies ◽

Coagulation Cascade ◽

Dependent Manner ◽

Kinetic Assay ◽

Thrombin Generation Assay ◽

Coefficients Of Variation

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

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Recombinant Thrombomodulin Inhibits Tissue Factor Mediated Thrombin Generation in Blood Plasma and Is Modulated By Prothrombin Complex Concentrates

Blood ◽

10.1182/blood.v126.23.2303.2303 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2303-2303

Author(s):

Jawed Fareed ◽

Debra Hoppensteadt ◽

Daneyal Syed ◽

Daniel Kahn ◽

Michael Mosier ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Platelet Rich Plasma ◽

Anticoagulant Effect ◽

Dependent Manner ◽

Prothrombin Complex Concentrates ◽

Complex Disorders ◽

Fibrin Formation ◽

Recombinant Thrombomodulin ◽

Inhibition Of Thrombin

Abstract Introduction: Thrombomodulin plays an important role in the regulation of serine proteases and endothelial function. Because of its multiple modulatory action it has a central role in inhibiting thrombogenesis and inflammatory processes in such complex disorders as sepsis associated coagulopathies. A recombinant thrombomodulin (Recomodulin, Asahai Kasei, Tokyo, Japan) is currently used for the management of disseminated intravascular coagulation (DIC) and related disorders in Japan and is currently undergoing a global phase 3 trial in sepsis-induced coagulopathy. Recomodulin (RM) is capable of forming complexes with circulating thrombin generated in sepsis and not only inhibits its coagulant function but also inhibits thrombin generation. The purpose of this study is to determine the effect of RM on thrombin generation in plasma systems and its modulation by both activated and non-activated prothrombin complex concentrates (PCCs). PCCs may have the potential to overcome the anticoagulant effects of RM and may be useful as an antidote. Materials and Methods: RM 12,800 IU (2.02 mg) ampules were commercially obtained. Working solutions of buffered RM were prepared at 100 µg/ml. Tissue factor mediated generation of thrombin and its inhibition in normal human plasma (NHP) (n=50), platelet rich plasma (PRP) (n=50), plasma obtained from patients with DIC (n=25) and various PCCs such as Beriplex, Cofact, Feiba, Konyne, Octaplex, Preconativ, Profilnine, Prothromplex at 1 - 5 IU/ml were investigated. A fluorometric thrombokinetics method (Technoclone, Vienna, Austria) was used to determine thrombin generation and its modulation by RM. In addition such thrombin generation markers as prothrombin fragment (F1.2) and thrombin-antithrombin complex (TAT) were measured. The effect of RM on thrombin mediated fibrinokinetics was also measured using an optical method. Results: RM produced a concentration dependent inhibition of thrombin generation in the plasma based systems. In NHP the IC50 was 0.29±0.06 µg/ml in contrast to PRP where the IC50 was 1.99±0.31 µg/ml. The inhibition of thrombin generation in various PCCs was also concentration and product dependent and only four factor concentrates were found to generate thrombin.. At concentrations of 1 IU/ml marked inhibition of thrombin generation was noted in Preconativ, Prothromplex and Beriplex. RM decreased the generation of F1.2 and TAT in a concentration dependent manner. However, at concentrations >2.5 IU/ml the thrombin generated in such PCCs as Prothromplex and Beriplex, markedly higher thrombin was generated overcoming the anticoagulant effect of RM. Proportionately higher levels of thrombin generation markers were formed and were dependant on PCC concentrations. RM also inhibited the thrombin induced formation of fibrin in both PRP and PPP systems in a differential manner. However, at higher concentration of thrombin the inhibitory effects of RM on fibrin formation were overcome. Conclusions: These results suggest that in plasma based systems and in the PCCs at concentrations <1 IU/ml, RM is capable of inhibiting tissue factor mediated thrombin generation in addition to the direct inhibition of pre-formed thrombin. However, such PCCs as Beriplex and Prothromplex are capable of overcoming the thrombin generation inhibitory actions of RM in a concentration dependent fashion. Moreover, RM is also capable of modulating fibrin formation as evident by fibrinokinetic studies where increased thrombin levels may also overcome its effect on fibrin formation. These observations suggest that PCCs may have the potential to reverse the anticoagulant effect of RM and may be considered as a potential antidote for this agent. Disclosures Williams: Asahi Kasei Pharma America: Employment.

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Polyphosphate as a haemostatic modulator

Biochemical Society Transactions ◽

10.1042/bst20150207 ◽

2016 ◽

Vol 44 (1) ◽

pp. 18-24 ◽

Cited By ~ 6

Author(s):

Nicola J. Mutch

Keyword(s):

Wound Healing ◽

Thrombin Generation ◽

Plasminogen Activators ◽

Contact System ◽

Clot Formation ◽

Coagulation Cascade ◽

Linear Polymers ◽

Dense Granules ◽

Fibrin Network ◽

Clot Stability

Platelets are small anuclear cells that play a central role in haemostasis. Platelets become activated in response to various stimuli triggering release of their granular contents into the surrounding milieu. One of these types of granules, termed dense granules, have been found to contain polyphosphate (polyP) in addition to other inorganic biomolecules, such as serotonin, ADP, ATP, PPi. Individuals deficient in dense granules exhibit bleeding tendencies, emphasizing their importance in haemostasis. Platelet polyP is of a relatively defined size, approximately 60–100 phosphate monomers in length. These linear polymers act at various points in the coagulation and fibrinolytic systems thereby modulating the haemostatic response. Due to its highly anionic nature, polyP lends itself to being a natural activator of the contact system. The contact system functions in multiple pathways including coagulation, fibrinolysis, inflammation and complement. Activation of the contact system accelerates thrombin generation, the terminal enzyme in the coagulation cascade. PolyP also modulates factors further downstream in the coagulation cascade to augment thrombin generation. The net effect is increased fibrin formation and platelet activation resulting in faster clot formation. PolyP is incorporated into the forming clot thereby modifying the structure of the resulting fibrin network and its susceptibility to degradation by certain plasminogen activators. In conclusion, release of platelet polyP at the site of injury may facilitate clot formation and augment clot stability thereby promoting wound healing.

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Platelet-Targeted rFVIIa-Xten Improves Thrombin Generation and Fibrin Formation Compared to Recombinant FVIIa

Blood ◽

10.1182/blood.v126.23.2269.2269 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2269-2269 ◽

Cited By ~ 2

Author(s):

Maria M. Aleman ◽

Elena N. Kistanova ◽

Nancy Moore ◽

Volker Schellenberger ◽

Robert Peters ◽

...

Keyword(s):

Half Life ◽

Thrombin Generation ◽

Hemophilia A ◽

Platelet Rich Plasma ◽

Life Extension ◽

Employment Equity ◽

On Demand ◽

Fibrin Formation ◽

Fibrin Network ◽

Equity Ownership

Abstract The development of inhibitors to replacement plasma factors in hemophilia is an ongoing clinical complication. Bypass therapies, such as recombinant factor VIIa (rFVIIa), have emerged as important alternative on-demand strategies for hemophilic patients with inhibitors to treat spontaneous bleeds and prevent bleeding during surgery. However, general prophylaxis strategies for hemophilia inhibitor patients are lacking. An attractive approach for effective prophylaxis and on-demand treatment includes engineering potent rFVIIa variants with prolonged pharmacokinetics. Because the primary mechanism of action of rFVIIa is thought to occur on the platelet surface, we combined rFVIIa with platelet-targeting and XTEN half-life extension technologies to improve its pharmacokinetic profile and procoagulant activity. Platelet-targeting was achieved by recombinant fusion of an antibody fragment which binds the human alpha IIb integrin. Half-life extension was achieved by fusion of an XTEN polypeptide which increases the hydrodynamic radius, and therefore half-life, of rFVIIa. We have shown that these rFVIIa modifications improve the pharmacokinetics and efficacy of rFVIIa in vivo in humanized alpha IIb transgenic hemophilia A mice. The goal of the current study was to evaluate and compare thrombin generation and fibrin formation kinetics in hemophilic platelet-rich plasma in the presence of platelet-targeted rFVIIa-XTEN or rFVIIa. To achieve this, platelet-rich plasma from normal human donors was treated with an inhibitory factor VIII antibody to model hemophilia A and spiked with doses of platelet-targeted rFVIIa-XTEN or rFVIIa. Reactions were triggered with low tissue factor and recalcification. Thrombin generation (n=9) was monitored by calibrated automated thrombography and fibrin formation (n=7) was monitored optically on a plate reader. Non-linear regression analysis of dose responses was used to determine EC50 values for each parameter for each donor. Platelet-targeted rFVIIa-XTEN increases the rate and peak of thrombin generation with 2- to 6-fold lower EC50 values (peak and rate, respectively) than rFVIIa. These data were confirmed by thrombin generation in platelet-rich plasma from 1 hemophilia A donor and 1 hemophilia B donor which demonstrated similar responses to platelet-targeted rFVIIa-XTEN with 2.5- to 5-fold lower EC50 values (peak and rate, respectively) than rFVIIa. Notably, platelet count-dependent changes in thrombin generation activity were similar between platelet-targeted rFVIIa-XTEN and rFVIIa. Fibrin formation analysis indicated platelet-targeted rFVIIa-XTEN increases the rate and time to plateau of fibrin formation with 5- to 13-fold lower EC50 values (rate and time to plateau, respectively) than rFVIIa. Analysis of fibrin network structure by confocal microscopy indicated platelet-targeted rFVIIa-XTEN increases fibrin network density in platelet-rich plasma clots. Together, these data show platelet-targeted rFVIIa-XTEN has more procoagulant activity than rFVIIa by supporting more thrombin generation and faster fibrin formation and suggest our approach has the potential to be an effective alternative for the treatment and prevention of bleeds in hemophilia patients with inhibitors. Disclosures Aleman: Biogen: Employment, Equity Ownership. Kistanova:Biogen: Employment, Equity Ownership. Moore:Biogen: Employment, Equity Ownership. Schellenberger:Amunix Operating Inc: Employment. Peters:Biogen: Employment. Salas:Biogen: Employment, Equity Ownership.

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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

Blood ◽

10.1182/blood.v128.22.3831.3831 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3831-3831 ◽

Cited By ~ 2

Author(s):

Genmin Lu ◽

Joyce Lin ◽

John T. Curnutte ◽

Pamela B. Conley

Keyword(s):

Human Plasma ◽

Degradation Product ◽

Thrombin Generation ◽

Fibrin Clot ◽

Clot Formation ◽

Minimal Effect ◽

Fibrin Degradation Product ◽

Extrinsic Pathway ◽

Equity Ownership ◽

Fxa Inhibitor

Abstract 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. Disclosures Lu: 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.

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Polyphosphate-induced Thrombosis in Mice is Factor XII-Dependent and is Attenuated by Histidine-rich Glycoprotein

Blood Advances ◽

10.1182/bloodadvances.2021004567 ◽

2021 ◽

Author(s):

Rida Asad Malik ◽

Ji Zhou ◽

James Fredenburgh ◽

Tammy K. Truong ◽

Jeffrey R Crosby ◽

...

Keyword(s):

Thrombin Generation ◽

Lung Perfusion ◽

Activated Partial Thromboplastin Time ◽

Contact System ◽

Factor Xii ◽

Dependent Manner ◽

Fibrin Deposition ◽

Wild Type ◽

Activated Platelets ◽

Deficient Mice

Histidine-rich glycoprotein (HRG) is an abundant plasma protein that binds factor (F) XIIa and inhibits FXII autoactivation and FXIIa-mediated activation of FXI. Polyphosphate (polyP), a potent procoagulant released from activated platelets, may serve as a physiological activator of the contact system. Previously, we showed that HRG binds DNA and neutralizes its procoagulant activity. Consequently, we set out to determine whether the capacity of HRG to bind polyanions enables it to regulate polyP-induced thrombosis. In a plate-based assay, immobilized polyP bound HRG, FXII, and FXIIa in a zinc-dependent manner. Basal and polyP-induced thrombin generation were greater in plasma from HRG-deficient mice than in plasma from wild-type mice. Intraperitoneal injection of polyP shortened the activated partial thromboplastin time, enhanced thrombin generation, increased thrombin-antithrombin (TAT) levels, reduced lung perfusion, and promoted pulmonary fibrin deposition to a greater extent in HRG-deficient mice than in wild-type mice, effects that were abrogated with FXII knockdown. Therefore, HRG attenuates the procoagulant and prothrombotic effects of polyP in a FXII dependent manner by modulating the contact system.

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