From Venom to Vein: Factor VII Activation as a Major Pathophysiological Target for Procoagulant Australian Elapid Snake Venoms.

Australian elapid snake venoms are uniquely procoagulant, utilizing blood clotting enzyme Factor Xa (FXa) as a toxin, which evolved as a basal trait in this clade. The subsequent recruitment of Factor Va (FVa) as a toxin occurred in the last common ancestor of taipans (Oxyuranus species) and brown snakes (Pseudonaja species). Factor II (prothrombin) activation has been stated as the primary mechanism for the lethal coagulopathy, but this hypothesis has never been tested. The additional activation of Factor VII (FVII) by Oxyuranus/Pseudonaja venoms has historically been considered as a minor, unimportant novelty. This study aimed to investigate the significance of toxic FVII activation relative to prothrombin activation by testing a wide taxonomical range of Australian elapid species with procoagulant venoms. The activation of FVII or prothrombin, with and without the Factor Va as a cofactor, was assessed, along with the structural changes involved in these processes. All procoagulant species could activate FVII, establishing this as a basal trait. In contrast, only some lineages could activate prothrombin, indicating that this is a derived trait. For species able to activate both zymogens, Factor VII was consistently more strongly activated than prothrombin. FVa was revealed as an essential cofactor for FVII activation, a mechanism previously undocumented. Species lacking FVa in their venom utilized endogenous plasma FVa to exert this activity. The ability of the human FXa:FVa complex to activate FVII was also revealed as a new feedback loop in the endogenous clotting cascade. Toxin sequence analyses identified structural changes essential for the derived trait of prothrombin activation. This study presents a paradigm shift in understanding how elapid venoms activate coagulation factors, highlighting the critical role of FVII activation in the pathophysiological effects upon the coagulation cascade produced by Australian elapid snake venoms. It also documented the novel use of Factor Va as a cofactor for FVII activation for both venom and endogenous forms of FXa. These findings are crucial for developing better antivenoms and treatments for snakebite victims and have broader implications for drug design and the treatment of coagulation disorders. The research also advances the evolutionary biology knowledge of snake venoms.

Kinetic analysis of prothrombinase assembly and substrate delivery mechanisms.

Prothrombinase complex, composed of coagulation factors Xa (FXa) and Va (FVa) is a major enzyme of the blood coagulation network that produces thrombin via activation of its inactive precursor prothrombin (FII) on the surface of phospholipid membranes. However, pathways and mechanisms of prothrombinase formation and substrate delivery are still discussed. Here we designed a novel mathematical model that considered different potential pathways of FXa or FII binding (from the membrane or from solution) and analyzed the kinetics of thrombin formation in the presence of a wide range of reactants concentrations. We observed the inhibitory effect of large FVa concentrations and this effect was phospholipid concentration-dependent. We predicted that efficient FII activation occurred via formation of the ternary complex, in which FVa, FXa and FII were in the membrane-bound state. Prothrombin delivery was mostly membrane-dependent, but delivery from solution was predominant under conditions of phospholipid deficiency or FXa/FVa excess. Likewise, FXa delivery from solution was predominant in the case of FVa excess, but high FII did not switch the FXa delivery to the solution-dependent one. Additionally, the FXa delivery pathway did not depend on the phospholipid concentration, being the membrane-dependent one even in case of the phospholipid deficiency. These results suggest a flexible mechanism of prothrombinase functioning which utilizes different complex formation and even inhibitory mechanisms depending on conditions.

Activated protein C, protein S, and tissue factor pathway inhibitor cooperate to inhibit thrombin activation.

INTRODUCTION: Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life-threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). However, prothrombinase is resistant to either of these inhibitory systems in isolation. MATERIALS AND METHODS: We hypothesized that these anticoagulants function best together, and tested this hypothesis using purified proteins and plasma-based systems. RESULTS: In plasma, TFPIα had greater anticoagulant activity in the presence of APC and PS, maximum PS activity required both TFPIα and APC, and antibodies against TFPI and APC had an additive procoagulant effect, which was mimicked by an antibody against PS alone. In purified protein systems, TFPIα dose-dependently inhibited thrombin activation by prothrombinase, but only in the presence of APC, and this activity was enhanced by PS. Conversely, FXa protected FVa from cleavage by APC, even in the presence of PS, and TFPIα reversed this protection. However, prothrombinase assembled on platelets was still protected from inhibition, even in the presence of TFPIα, APC, and PS. CONCLUSIONS: We propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. Platelets protect prothrombinase from inhibition, however, supporting a procoagulant environment within the clot.

Modeling time-delayed concentration-QT effects with ACT-1014-6470, a novel oral complement factor 5a receptor 1 (C5a1 receptor) antagonist.

The novel oral complement factor 5a receptor 1 antagonist ACT-1014-6470 was well tolerated in single- and multiple-ascending dose studies, including 24 h Holter electrocardiogram (ECG) recordings evaluating its cardiodynamics based on data from single doses of 30-200 mg and twice-daily (b.i.d.) dosing of 30-120 mg for 4.5 days. By-time point, categorical, and morphological analyses as well as concentration-QT modeling and simulations were performed. No relevant effect of ACT-1014-6470 on ECG parameters was observed in the categorical and morphological analyses. After single-dose administration, the by-time point analysis indicated a delayed dose-dependent increase in placebo-corrected change from baseline in QT interval corrected with Fridericia's formula (ΔΔQTcF) at >6 h postdose. After b.i.d. dosing, ΔΔQTcF remained elevated during the 24-h recording period, suggesting that the effect was not directly related to ACT-1014-6470 plasma concentration. The concentration-QT model described change from baseline in QTcF (ΔQTcF)-time profiles best with a 1-oscillator model of 24 h for circadian rhythm, an effect compartment, and a sigmoidal maximum effect model. Model-predicted ΔΔQTcF was derived for lower doses and less-frequent dosing than assessed clinically. Median and 90% prediction intervals of ΔΔQTcF for once-daily doses of 30 mg and b.i.d. doses of 10 mg did not exceed the regulatory threshold of 10 ms but would achieve ACT-1014-6470 plasma concentrations enabling adequate target engagement. Results from cardiodynamic assessments identified dose levels and dosing regimens that could be considered for future clinical trials, attempting to reduce QT liability.

Evaluation of the cytochrome P450 2C19 and 3A4 inhibition potential of the complement factor 5a receptor 1 antagonist ACT-1014-6470 in vitro and in vivo.

ACT-1014-6470 is an orally available complement factor 5a receptor 1 antagonist and a novel treatment option in auto-inflammatory diseases. The in vitro inhibition potential of ACT-1014-6470 on cytochrome P450 isozymes (CYPs) and its effect on the pharmacokinetics (PK) of the CYP2C19 and CYP3A4 substrates omeprazole and midazolam, respectively, in humans were assessed. In vitro assays were conducted with isoform-specific substrates in human liver microsomes. In an open-label, two-period, fixed-sequence cocktail study, single doses of 20 mg omeprazole and 2 mg midazolam were administered concomitantly to 20 healthy male subjects alone (treatment A) and after a single dose of 100 mg ACT-1014-6470 (treatment B) under fed conditions. Safety and PK assessments were performed. Geometric mean ratios (GMRs) and 90% confidence intervals (CIs) of noncompartmental PK parameters of treatment B versus treatment A were calculated. In vitro, no time-dependent inhibition was observed and the lowest inhibition constant of 4.3 µM ACT-1014-6470 was recorded for CYP2C19. In humans, GMRs (90% CI) of omeprazole PK were 1.9 (1.5-2.5) for maximum plasma concentration (Cmax ) and 1.9 (1.5-2.3) for area under the plasma concentration-time curve from 0 to 12 h (AUC0-12 h ). Midazolam PK showed GMRs (90% CI) of 1.1 (1.1-1.2) for Cmax and 1.5 (1.4-1.6) for AUC0-24 h . All treatments were well-tolerated. In line with in vitro results and regulatory risk factor calculation, the increased exposure to omeprazole and midazolam in humans after concomitant administration with a single dose of 100 mg ACT-1014-6470 reflected a weak inhibition of CYP2C19 and CYP3A4.

Cryo-EM structure of coagulation factor V short.

Coagulation factor V (fV) is the precursor of activated fV (fVa), an essential component of the prothrombinase complex required for the rapid activation of prothrombin in the penultimate step of the coagulation cascade. In addition, fV regulates the tissue factor pathway inhibitor α (TFPIα) and protein C pathways that inhibit the coagulation response. A recent cryogenic electron microscopy (cryo-EM) structure of fV has revealed the architecture of its A1-A2-B-A3-C1-C2 assembly but left the mechanism that keeps fV in its inactive state unresolved because of an intrinsic disorder in the B domain. A splice variant of fV, fV short, carries a large deletion of the B domain that produces constitutive fVa-like activity and unmasks epitopes for the binding of TFPIα. The cryo-EM structure of fV short was solved at 3.2 Å resolution and revealed the arrangement of the entire A1-A2-B-A3-C1-C2 assembly. The shorter B domain stretches across the entire width of the protein, making contacts with the A1, A2, and A3 domains but suspended over the C1 and C2 domains. In the portion distal to the splice site, several hydrophobic clusters and acidic residues provide a potential binding site for the basic C-terminal end of TFPIα. In fV, these epitopes may bind intramolecularly to the basic region of the B domain. The cryo-EM structure reported in this study advances our understanding of the mechanism that keeps fV in its inactive state, provides new targets for mutagenesis and facilitates future structural analysis of fV short in complex with TFPIα, protein S, and fXa.

Membrane curvature and PS localize coagulation proteins to filopodia and retraction fibers of endothelial cells.

Prior reports indicate that the convex membrane curvature of phosphatidylserine (PS)-containing vesicles enhances formation of binding sites for factor Va and lactadherin. Yet, the relationship of convex curvature to localization of these proteins on cells remains unknown. We developed a membrane topology model, using phospholipid bilayers supported by nano-etched silica substrates, to further explore the relationship between curvature and localization of coagulation proteins. Ridge convexity corresponded to maximal curvature of physiologic membranes (radii of 10 or 30 nm) and the troughs had a variable concave curvature. The benchmark PS probe lactadherin exhibited strong differential binding to the ridges, on membranes with 4% to 15% PS. Factor Va, with a PS-binding motif homologous to lactadherin, also bound selectively to the ridges. Bound factor Va supported coincident binding of factor Xa, localizing prothrombinase complexes to the ridges. Endothelial cells responded to prothrombotic stressors and stimuli (staurosporine, tumor necrosis factor-α [TNF- α]) by retracting cell margins and forming filaments and filopodia. These had a high positive curvature similar to supported membrane ridges and selectively bound lactadherin. Likewise, the retraction filaments and filopodia bound factor Va and supported assembly of prothrombinase, whereas the cell body did not. The perfusion of plasma over TNF-α-stimulated endothelia in culture dishes and engineered 3-dimensional microvessels led to fibrin deposition at cell margins, inhibited by lactadherin, without clotting of bulk plasma. Our results indicate that stressed or stimulated endothelial cells support prothrombinase activity localized to convex topological features at cell margins. These findings may relate to perivascular fibrin deposition in sepsis and inflammation.

Effect of Severe Renal Impairment on Pharmacokinetics, Safety, and Tolerability of ACT-1014-6470, a Novel Oral Complement Factor 5a Receptor 1 Antagonist.

The aim of this study was to examine the safety and the effect of severe renal impairment (RI) on the pharmacokinetics of ACT-1014-6470, a novel oral complement factor 5a receptor 1 antagonist. A phase 1 single-center, open-label, single-dose, parallel-group study was conducted in subjects with severe RI (n = 8) compared to demographically pairwise matched subjects with normal renal function (n = 8). Plasma levels of ACT-1014-6470 were measured up to 120 hours following an oral 40-mg dose. Safety evaluations included adverse events (AEs), vital signs, hematology, coagulation, clinical chemistry tests, and electrocardiograms. All 16 subjects completed the study. Relative to subjects with normal renal function, ACT-1014-6470 time to maximum plasma concentration was delayed with a median of differences of 3 hours. The maximum plasma concentration and the area under the plasma concentration-time profile from time zero to infinity were comparable indicated by geometric mean ratios (90%CI) of 0.85 (0.53-1.37) and 1.17 (0.73-1.85), respectively. Four transient and mild AEs in three subjects with severe RI were reported; three AEs were considered not related to ACT-1014-6470. These results support the use of ACT-1014-6470 in subjects with mild to severe RI without the need of dose adjustment.

Multiple-ascending doses of ACT-1014-6470, an oral complement factor 5a receptor 1 (C5a1 receptor) antagonist: Tolerability, pharmacokinetics and target engagement.

AIMS: Targeting the complement factor 5a receptor 1 (C5a1 receptor) offers potential to treat various autoimmune diseases. The C5a1 receptor antagonist ACT-1014-6470 was well tolerated in a single-ascending dose study in healthy subjects. This double-blind, randomized, placebo-controlled study aimed to investigate the safety, tolerability, pharmacokinetics (PK) and target engagement of multiple-ascending doses of ACT-1014-6470. METHODS: Per dose level, 10 healthy male and female subjects of nonchildbearing potential (1:1 sex ratio) were enrolled to assess 30, 60 and 120 mg ACT-1014-6470 administered twice daily for 4.5 days under fed conditions. Adverse events, clinical laboratory data, vital signs, electrocardiogram and PK blood samples were collected up to 120 h post last dose and ex vivo stimulated matrix metalloproteinase 9 was quantified as target engagement biomarker. At the 60-mg dose level, PK samples were collected until 8 weeks post last dose. RESULTS: The total adverse event number was 57 and no treatment-related safety pattern was apparent. At steady state, ACT-1014-6470 reached maximum plasma concentrations after 2-3 h and the half-life estimated up to Day 10 was 115-146 h across dose levels. Exposure parameters increased dose-proportionally, steady state was attained between Day 3-5, and ACT-1014-6470 accumulated 2-fold. At the 60-mg dose level, ACT-1014-6470 was quantifiable until 8 weeks after the last dose. Matrix metalloproteinase 9 release was suppressed to endogenous background concentrations up to the last sampling time point, confirming sustained target engagement of ACT-1014-6470. CONCLUSION: The compound was generally safe and well tolerated at all dose levels, warranting further clinical investigations.

Regulation of factor V by the anticoagulant protease activated protein C: Influence of the B-domain and TFPIα.

Activated protein C (APC) is an important anticoagulant protein that regulates thrombin generation through inactivation of factor V (FV) and activated factor V (FVa). The rate of APC inactivation of FV is slower compared to FVa, although proteolysis occurs at the same sites (Arg306, Arg506, and Arg679). The molecular basis for FV resistance to APC is unknown. Further, there is no information about how FV-short, a physiologically relevant isoform of FV with a shortened B-domain, is regulated by APC. Here, we identify the molecular determinants which differentially regulate APC recognition of FV versus FVa and uncover how FV-short can be protected from this anticoagulant pathway. Using recombinant FV derivatives and B-domain fragments, we show that the conserved basic region (BR; 963-1008) within the central portion of the B-domain plays a major role in limiting APC cleavage at Arg506. Derivatives of FV lacking the BR, including FV-short, were subject to rapid cleavage at Arg506 and were inactivated like FVa. The addition of a FV-BR fragment reversed this effect and delayed APC inactivation. We also found that anticoagulant glycoprotein TFPIα, which has a C-terminal BR homologous to FV-BR, protects FV-short from APC inactivation by delaying cleavage at Arg506. We conclude that the FV-BR plays a major role in protecting FV from APC inactivation. Using a similar mechanistic strategy, TFPIα also shields FV-short from APC. These findings clarify the resistance of FV to APC, advance our understanding of FV/FVa regulation, and establish a mechanistic framework for manipulating this reaction to alter coagulation.

Antiphospholipid syndrome: Reversal of antiphosphatidylserine/prothrombin-induced activated protein C resistance.

BACKGROUND: Anti-phosphatidylserine/prothrombin (aPS/PT) antibodies are the major contributor to activated Protein C resistance (APC-R) in tetra-positive thrombotic high-risk patients with Antiphospholipid Syndrome (APS). OBJECTIVES: To evaluate the role of phospholipids (PL) on aPS/PT mediated APC-R. PATIENTS/METHODS: Total IgG were purified from plasma of 6 tetra-positive patients and IgG aPS/PT were affinity-purified from 3 of these patients. Purified material was spiked into Normal Pooled Plasma (NPP) and tested for APC-R in thrombin generation assay and in Factor Va inactivation assay using increasing amounts of phospholipids. RESULTS AND CONCLUSIONS: Total IgG showed APC-R at low PL concentration (1.5 µg/mL) that disappeared at increasing PL concentrations (5.8, 11.6 and 46.6 µg/mL). In the same way, affinity purified aPS/PT showed a robust (59 %, 52 %, 36 %) APC-R in patients #4, #5 and #6, respectively at low PL concentration (1.5 µg/mL) that was completely reversed at higher concentration (11.6 µg/mL). The inactivation of FVa by activated Protein C (aPC) was impaired in the presence of aPS/PT at low aPL concentration and reversed by increasing amounts of PL. These data point out the relevance of PL in reversing APC-R in this 'in vitro' system. The mechanism for reversal might be explained by loss of PL availability for aPC. These results may give some insight into the pathogenesis of thrombosis or suggestions for alternative treatments.

First-in-human study with ACT-1014-6470, a novel oral complement factor 5a receptor 1 (C5aR1) antagonist, supported by pharmacokinetic predictions from animals to patients.

Aberrantly controlled activation of the complement system contributes to inflammatory diseases. Safety, tolerability, and pharmacokinetics of single-ascending doses of ACT-1014-6470, a novel orally available complement factor 5a receptor 1 antagonist, were assessed in a randomized, double-blind, placebo-controlled Phase 1 study. Six ACT-1014-6470 doses (0.5-200 mg) were selected after predictions from a Complex Dedrick plot. In each group, ACT-1014-6470 or matching placebo was administered to six and two healthy male individuals under fed conditions, respectively, including a cross-over part with 10 mg administered also under fasted conditions. Pharmacokinetic blood sampling and safety assessments (adverse events, clinical laboratory, vital signs, 12-lead electrocardiogram, and QT telemetry) were performed. ACT-1014-6470 was absorbed with a time to maximum plasma concentration (tmax ) of 3 h across dose levels and eliminated with a terminal half-life of 30-46 h at doses ≥ 2.5 mg. Exposure increased approximately dose proportionally. Under fed compared to fasted conditions, ACT-1014-6470 exposure was 2.2-fold higher and tmax delayed by 1.5 h. Pharmacokinetic modelling predicted that twice-daily oral administration is warranted in a subsequent multiple-dose study. No clinically relevant findings were observed in safety assessments. ACT-1014-6470 was well tolerated at all doses and could provide a novel therapy with more patient-friendly administration route compared to biologicals.

Cryo-EM structure of the prothrombin-prothrombinase complex.

The intrinsic and extrinsic pathways of the coagulation cascade converge to a common step where the prothrombinase complex, comprising the enzyme factor Xa (fXa), the cofactor fVa, Ca2+ and phospholipids, activates the zymogen prothrombin to the protease thrombin. The reaction entails cleavage at 2 sites, R271 and R320, generating the intermediates prethrombin 2 and meizothrombin, respectively. The molecular basis of these interactions that are central to hemostasis remains elusive. We solved 2 cryogenic electron microscopy (cryo-EM) structures of the fVa-fXa complex, 1 free on nanodiscs at 5.3-Å resolution and the other bound to prothrombin at near atomic 4.1-Å resolution. In the prothrombin-fVa-fXa complex, the Gla domains of fXa and prothrombin align on a plane with the C1 and C2 domains of fVa for interaction with membranes. Prothrombin and fXa emerge from this plane in curved conformations that bring their protease domains in contact with each other against the A2 domain of fVa. The 672ESTVMATRKMHDRLEPEDEE691 segment of the A2 domain closes on the protease domain of fXa like a lid to fix orientation of the active site. The 696YDYQNRL702 segment binds to prothrombin and establishes the pathway of activation by sequestering R271 against D697 and directing R320 toward the active site of fXa. The cryo-EM structure provides a molecular view of prothrombin activation along the meizothrombin pathway and suggests a mechanism for cleavage at the alternative R271 site. The findings advance our basic knowledge of a key step of coagulation and bear broad relevance to other interactions in the blood.

Generation of an anticoagulant aptamer that targets factor V/Va and disrupts the FVa-membrane interaction in normal and COVID-19 patient samples.

Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions.

Assessing Factor V Antigen and Degradation Products in Burn and Trauma Patients.

INTRODUCTION: Proposed mechanisms of acute traumatic coagulopathy (ATC) include decreased clotting potential due to factor consumption and proteolytic inactivation of factor V (FV) and activated factor V (FVa) by activated protein C (aPC). The role of FV/FVa depletion or inactivation in burn-induced coagulopathy is not well characterized. This study evaluates FV dynamics following burn and nonburn trauma. METHODS: Burn and trauma patients were prospectively enrolled. Western blotting was performed on admission plasma to quantitate levels of FV antigen and to assess for aPC or other proteolytically derived FV/FVa degradation products. Statistical analysis was performed with Spearman's, Chi-square, Mann-Whitney U test, and logistic regression. RESULTS: Burn (n = 60) and trauma (n = 136) cohorts showed similar degrees of FV consumption with median FV levels of 76% versus 73% (P = 0.65) of normal, respectively. Percent total body surface area (TBSA) was not correlated with FV, nor were significant differences in median FV levels observed between low and high TBSA groups. The injury severity score (ISS) in trauma patients was inversely correlated with FV (ρ = -0.26; P = 0.01) and ISS ≥ 25 was associated with a lower FV antigen level (64% versus. 93%; P = 0.009). The proportion of samples showing proteolysis-derived FV was greater in trauma than burn patients (42% versus. 16%; P = 0.0006). CONCLUSIONS: Increasing traumatic injury severity is associated with decreased FV antigen levels, and a greater proportion of trauma patient samples exhibit proteolytically degraded FV fragments. These associations are not present in burns, suggesting that mechanisms underlying FV depletion in burn and nonburn trauma are not identical.

Mapping the prothrombin-binding site of pseutarin C by site-directed PEGylation.

The prothrombinase complex processes prothrombin to thrombin through sequential cleavage at Arg320 followed by Arg271 when cofactor, factor (f) Va, protease, fXa, and substrate, prothrombin, are all bound to the same membrane surface. In the absence of the membrane or cofactor, cleavage occurs in the opposite order. For the less favorable cleavage site at Arg320 to be cleaved first, it is thought that prothrombin docks on fVa in a way that presents Arg320 and hides Arg271 from the active site of fXa. Based on the crystal structure of the prothrombinase complex from the venom of the Australian eastern brown snake, pseutarin C, we modeled an initial prothrombin docking mode, which involved an interaction with discrete portions of the A1 and A2 domains of fV and the loop connecting the 2 domains, known as the a1-loop. We interrogated the proposed interface by site-directed PEGylation and by swapping the a1-loop in pseutarin C with that of human fV and fVIII and measuring the effect on rate and pathway of thrombin generation. PEGylation of residues within our proposed binding site greatly reduced the rate of thrombin generation, without affecting the pathway, whereas those outside the proposed interface had no effect. PEGylation of residues within the a1-loop also reduced the rate of thrombin generation. The sequence of the a1-loop was found to play a critical role in prothrombin binding and in the presentation of Arg320 for initial cleavage.

The functional role of the autolysis loop in the regulation of factor X upon hemostatic response.

BACKGROUND: The regulation of factor X (FX) is critical to maintain the balance between blood coagulation and fluidity. OBJECTIVES: To functionally characterize the role of the FX autolysis loop in the regulation of the zymogen and active form of FX. METHODS: We introduced novel N-linked glycosylations on the surface-exposed loop spanning residues 143-150 (chymotrypsin numbering) of FX. The activity and inhibition of recombinant FX variants was quantified in pure component assays. The in vitro thrombin generation potential of the FX variants was evaluated in FX-depleted plasma. RESULTS: The factor VIIa (FVIIa)-mediated activation and prothrombin activation was reduced, presumably through steric hinderance. Prothrombin activation was, however, recovered in presence of cofactor factor Va (FVa) despite a reduced prothrombinase assembly. The introduced N-glycans exhibited position-specific effects on the interaction with two FXa inhibitors: tissue factor pathway inhibitor (TFPI) and antithrombin (ATIII). Ki for the inhibition by full-length TFPI of these FXa variants was increased by 7- to 1150-fold, whereas ATIII inhibition in the presence of the heparin-analog Fondaparinux was modestly increased by 2- to 15-fold compared with wild-type. When supplemented in zymogen form, the FX variants exhibited reduced thrombin generation activity relative to wild-type FX, whereas enhanced procoagulant activity was measured for activated FXa variants. CONCLUSION: The autolysis loop participates in all aspects of FX regulation. In plasma-based assays, a modest decrease in FX activation rate appeared to knock down the procoagulant response even when down regulation of FXa activity by inhibitors was reduced.

An engineered activated factor V for the prevention and treatment of acute traumatic coagulopathy and bleeding in mice.

Acute traumatic coagulopathy (ATC) occurs in approximately 30% of patients with trauma and is associated with increased mortality. Excessive generation of activated protein C (APC) and hyperfibrinolysis are believed to be driving forces for ATC. Two mouse models were used to investigate whether an engineered activated FV variant (superFVa) that is resistant to inactivation by APC and contains a stabilizing A2-A3 domain disulfide bond can reduce traumatic bleeding and normalize hemostasis parameters in ATC. First, ATC was induced by the combination of trauma and shock. ATC was characterized by activated partial thromboplastin time (APTT) prolongation and reductions of factor V (FV), factor VIII (FVIII), and fibrinogen but not factor II and factor X. Administration of superFVa normalized the APTT, returned FV and FVIII clotting activity levels to their normal range, and reduced APC and thrombin-antithrombin (TAT) levels, indicating improved hemostasis. Next, a liver laceration model was used where ATC develops as a consequence of severe bleeding. superFVa prophylaxis before liver laceration reduced bleeding and prevented APTT prolongation, depletion of FV and FVIII, and excessive generation of APC. Thus, prophylactic administration of superFVa prevented the development of ATC. superFVa intervention started after the development of ATC stabilized bleeding, reversed prolonged APTT, returned FV and FVIII levels to their normal range, and reduced TAT levels that were increased by ATC. In summary, superFVa prevented ATC and traumatic bleeding when administered prophylactically, and superFVa stabilized bleeding and reversed abnormal hemostasis parameters when administered while ATC was in progress. Thus, superFVa may be an attractive strategy to intercept ATC and mitigate traumatic bleeding.

Understanding the anchoring interaction of coagulation factor Va light chain on zeolites: A molecular dynamics study.

HYPOTHESIS: Factor Va (FXa) and Xa (FVa) can assemble on the phosphatidylserine (PS) membrane (of platelet) to form prothrombinase complex and contribute to blood clotting. Very recently, we discovered that Ca-zeoliteacts as a type of reinforced activated inorganic platelet to enable assembly of prothrombinase complex and display an unusual zymogen (prothrombin) activation pattern. Inspired but not constrained by nature, it is of great interest to understand how FVa and FXa assembly on the inorganic surface (e.g., zeolites) and perform their biocatalytic function. EXPERIMENTS: Given the important role of FVa C1-C2 domains in the assembly and activity of the prothrombinase complex, in this work, molecular dynamics simulations were performed to investigate the binding details of FVa A3-C1-C2 domains on the PS membranes and Ca2+-LTA-type (CaA) zeolite surface. FINDINGS: We found that different from the natural PS membrane, FVa light chain repeatedly exhibits a strong C2 domain anchoring interaction on the CaA zeolite. It mainly arises from the porous surface structure of CaA zeolite and local highly dense solvation water clusters on the CaA zeolite surface restrict the movement of some lysine residues on the C2 domain. The anchoring interaction can be suppressed by reducing the surface negative charge density, so that FVa light chain can change from single-foot (only C2 domain) to double-foot (both C1-C2 domain) adsorption states on the zeolite surface. This double-foot adsorption state is similar to natural PS membrane systems, which may make FVa have higher cofactor activity.