Contact Activation Inhibitor and Factor XI Antibody, AB023, Produces Safe, Dose-Dependent Anticoagulation in a Phase 1 First-In-Human Trial.

Objective- Factor XI (FXI) contributes to thrombotic disease while playing a limited role in normal hemostasis. We generated a unique, humanized anti-FXI antibody, AB023, which blocks factor XIIa-mediated FXI activation without inhibiting FXI activation by thrombin or the procoagulant function of FXIa. We sought to confirm the antithrombotic activity of AB023 in a baboon thrombosis model and to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics in healthy adult subjects. Approach and Results- In a primate model of acute vascular graft thrombosis, AB023 reduced platelet and fibrin accumulation within the grafts by >75%. To evaluate the safety of AB023, we performed a first-in-human study in healthy adult volunteers without any serious adverse events. Overall, 10 of 21 (48%) subjects experienced 20 treatment-emergent adverse events, with 7 of 16 (44%) subjects following active treatment and 3 of 5 (60%) subjects following placebo. AB023 did not increase bleeding or prothrombin times. Anticoagulation was verified by a saturable ≈2-fold prolongation of the partial thromboplastin time for over 1 month after the highest dose. Conclusions- AB023, which inhibits contact activation-initiated blood coagulation in vitro and experimental thrombus formation in primates, produced a dose-dependent duration of limited anticoagulation without drug-related adverse effects in a phase 1 trial. When put in context with earlier observations suggesting that FXI contributes to venous thromboembolism and cardiovascular disease, although contributing minimally to hemostasis, our data further justify clinical evaluation of AB023 in conditions where contact-initiated FXI activation is suspected to have a pathogenic role. Clinical Trial Registration- URL: http://www.clinicaltrials.gov . Unique identifier: NCT03097341.

Hereditary angioedema: the plasma contact system out of control.

The plasma contact system contributes to thrombosis in experimental models. Even though our standard blood coagulation tests are prolonged when plasma lacks contact factors, this enzyme system appears to have a minor (if any) role in hemostasis. In this review, we explore the clinical phenotype of C1 esterase inhibitor (C1-INH) deficiency. C1-INH is the key plasma inhibitor of the contact system enzymes, and its deficiency causes hereditary angioedema (HAE). This inflammatory disorder is characterized by recurrent aggressive attacks of tissue swelling that occur at unpredictable locations throughout the body. Bradykinin, which is considered to be a byproduct of the plasma contact system during in vitro coagulation, is the main disease mediator in HAE. Surprisingly, there is little evidence for thrombotic events in HAE patients, suggesting mechanistic uncoupling from the intrinsic pathway of coagulation. In addition, it is questionable whether a surface is responsible for contact system activation in HAE. In this review, we discuss the clinical phenotype, disease modifiers and diagnostic challenges of HAE. We subsequently describe the underlying biochemical mechanisms and contributing disease mediators. Furthermore, we review three types of HAE that are not caused by C1-INH inhibitor deficiency. Finally, we propose a central enzymatic axis that we hypothesize to be responsible for bradykinin production in health and disease.

Platelet- and erythrocyte-derived microparticles trigger thrombin generation via factor XIIa.

BACKGROUND: The procoagulant properties of microparticles (MPs) are due to the of the presence of phosphatidylserine (PS) and tissue factor (TF) on their surface. The latter has been demonstrated especially on MPs derived from monocytes. OBJECTIVES: To investigate the relative contribution of TF and factor (F)XII in initiating coagulation on MPs derived from monocytes, platelets and erythrocytes. METHODS: Microparticles were isolated from calcium ionophore-stimulated platelets, erythrocytes and monocytic THP-1 cells. MPs were quantified, characterized for cell-specific antigens and analyzed for TF, PS exposure and their thrombin-generating potential. RESULTS: The MP number was not proportional to PS exposure and the majority of the MPs exposed PS. TF activity was undetectable on platelet- and erythrocyte-derived MPs (< 1 fM nM(-1) PS), whereas monocyte-derived MPs exposed TF (32 fM nM(-1) PS). Platelet-, erythrocyte- and monocyte-derived MPs, but not purified phospholipids, initiated thrombin generation in normal plasma in the absence of an external trigger (lag time < 11 min). Deficiency or inhibition of FVII had no effect on thrombin generation induced by platelet- and erythrocyte-derived MPs, but interfered with monocyte MP-triggered coagulation. Platelet- and erythrocyte-derived MPs completely failed to induce thrombin generation in FXII-deficient plasma. In contrast, monocyte-derived MPs induced similar thrombin generation in normal vs. FXII-deficient plasma. CONCLUSION: MPs from platelets and erythrocytes not only propagate coagulation by exposing PS but also initiate thrombin generation independently of TF in a FXII-dependent manner. In contrast, monocyte-derived MPs trigger coagulation predominantly via TF.

A role for factor XIIa-mediated factor XI activation in thrombus formation in vivo.

Mice lacking factor XII (fXII) or factor XI (fXI) are resistant to experimentally-induced thrombosis, suggesting fXIIa activation of fXI contributes to thrombus formation in vivo. It is not clear whether this reaction has relevance for thrombosis in pri mates. In 2 carotid artery injury models (FeCl(3) and Rose Bengal/laser), fXII-deficient mice are more resistant to thrombosis than fXI- or factor IX (fIX)-deficient mice, raising the possibility that fXII and fXI function in distinct pathways. Antibody 14E11 binds fXI from a variety of mammals and interferes with fXI activation by fXIIa in vitro. In mice, 14E11 prevented arterial occlusion induced by FeCl(3) to a similar degree to total fXI deficiency. 14E11 also had a modest beneficial effect in a tissue factor-induced pulmonary embolism model, indicating fXI and fXII contribute to thrombus formation even when factor VIIa/tissue factor initiates thrombosis. In baboons, 14E11 reduced platelet-rich thrombus growth in collagen-coated grafts inserted into an arteriovenous shunt. These data support the hypothesis that fXIIa-mediated fXI activation contributes to thrombus formation in rodents and primates. Since fXII deficiency does not impair hemostasis, targeted inhibition of fXI activation by fXIIa may be a useful antithrombotic strategy associated with a low risk of bleeding complications.

Inactivation of plasminogen activator inhibitor type 1 by activated factor XII plays a role in the enhancement of fibrinolysis by contact factors in-vitro.

AIMS: Several activated coagulation factors have been reported to enhance fibrinolysis by inactivating plasminogen activator inhibitor type 1 (PAI-1), a serine protease inhibitor. We analyzed the interaction between PAI-1 and the three serine proteases generated during contact activation of plasma, activated factor XII (FXIIa), FXIa, and kallikrein, and evaluated their effects on fibrinolysis in-vitro. MAIN METHODS: Effects of kaolin on euglobulin clot lysis time (ECLT) and behavior of PAI-1 in factor-depleted plasma were analyzed. KEY FINDINGS: The ECLT of pooled plasma obtained from normal volunteers (designated as 100%) was shortened to 62.1+/-3.1% by Ca(2+) (5 mM) and 29.9+/-3.1% by kaolin. Activated protein C reversed the ECLT shortened by Ca(2+)-supplementation (86.3+/-17.4%), but did not affect the ECLT shortened by kaolin (31.4+/-2.1%). Thus, in contrary to Ca(2+)-supplementation, kaolin appeared to shorten the ECLT by a mechanism independent of thrombin generation. In three kinds of contact factor-depleted plasma, kaolin did not shorten ECLT only in FXII-depleted plasma. PAI-1 was cleaved to its inactive form in the Ca(2+) as well as the kaolin-supplemented euglobulin fraction in normal plasma, the latter of which, however, was not observed in FXII-depleted plasma. Similarly, a high molecular weight complex between FXIIa and PAI-1, as well as a cleaved form of PAI-1, was observed in kaolin-supplemented normal plasma, but neither was found in kaolin-supplemented FXII-depleted plasma. SIGNIFICANCE: PAI-1 inactivation by FXIIa appears to be a mechanism by which contact phase coagulation factors enhance fibrinolysis independently of thrombin generation.

Kallikrein-kinin system and fibrinolysis in hereditary angioedema due to factor XII gene mutation Thr309Lys.

In a subgroup of hereditary angioedema (HAE) patients with normal C1-esterase inhibitor levels, HAE is caused by a Thr309Lys mutation in the coagulation factor XII (F12) gene. The aim of this study was to examine elements of the kallikrein-kinin system ('contact system') and the downstream-linked coagulation, complement and fibrinolytic systems in the plasma of six patients with HAE caused by the Thr309Lys mutation and healthy probands. Blood samples were taken from participants during the symptom-free interval between attacks. Samples were analyzed for activity and concentrations of components of the kallikrein-kinin system and linked enzyme systems. The mean FXII clotting activity was 90% in patients with the FXII mutation, and the concentration of FXIIa was 4.1 ng/ml; this did not differ from healthy probands. Mean prekallikrein amidolytic activity and high-molecular-weight kininogen clotting activity were 130 and 144%, respectively, both higher than in healthy probands. The mean kallikrein-like activity of the HAE patients was 11.4 U/l and did not differ from healthy probands. There was no difference in FXII surface activation by silicon dioxide or in kallikrein-like activity with and without activation by dextran sulfate. Contrary to the results of a recently published study, no indication that the Thr309Lys mutation causes a 'gain-of-function' of FXIIa was observed in this investigation.

Coagulation factor XII (FXII) activity, activated FXII, distribution of FXII C46T gene polymorphism and coronary risk.

BACKGROUND: Whether factor XII (FXII) activity, its 46C>T polymorphism and activated FXII (FXIIa) are associated with coronary heart disease (CHD) remains to be determined. METHODS: FXII, FXIIa and the FXII 46C>T polymorphism were determined in a hospital-based cohort of 2615 patients undergoing coronary angiography. RESULTS: Fifty-seven per cent of the participants were identified as wild-type (46CC), 38% as heterozygous (46CT) and 5% as homozygous (46TT) for FXII 46C>T. FXII and FXIIa levels were significantly lower in carriers of the T-allele: 132 (97-151) U dL(-1) FXII in 46CC, 87 (77-99) U dL(-1) FXII in 46CT and 53 (42-67) U dL(-1) FXII in 46TT carriers (P < 0.001), and 2.8 (2.3-3.5) microg L(-1) FXIIa in CC, 2.1 (1.6-2.6) microg L(-1) FXIIa in CT and 1.2 (0.9-1.5) microg L(-1) FXIIa in TT carriers (P < 0.001; medians, lower and upper quartiles). Patients with stable CHD (n = 935), a history of myocardial infarction (n = 785) or who were suffering from acute coronary syndromes (ACS; n = 323) had significantly lower FXII levels than controls (n = 572). The differences remained statistically significant after adjustments for age, sex, diabetes mellitus, smoking, hypercholesterolemia and hypertension. Significantly reduced FXIIa levels in ACS patients lost significance once adjusted for covariates. FXII genotype was not associated with any clinical phenotype. CONCLUSION: Lower FXII activity represents an independent risk for CHD and ACS. This is not the case for FXIIa levels or the FXII 46C>T variation.

Impaired factor XIIa-dependent activation of fibrinolysis in treated antiphospholipid syndrome gestations developing late-pregnancy complications.

OBJECTIVE: The objective of the study was to investigate the potential role of impaired factor XII-dependent activation of fibrinolysis in treated antiphospholipid syndrome gestations developing late-pregnancy complications. STUDY DESIGN: This was a prospective study in a third-level teaching hospital, including 75 patients: 25 pregnant patients having the antiphospholipid syndrome and carrying their pregnancies until 26 weeks' gestation or later (group 1); 25 pregnant patients having normal term pregnancies and delivery and no previous miscarriage (group 2); and 25 pregnant patients being diagnosed as having severe pre-eclampsia and/or intrauterine growth restriction but testing negative for antiphospholipid antibodies (group 3). Hemostatic evaluation was carried out from patients in groups 1 and 2 between 6 and 10 weeks, between 18 and 22 weeks, and between 28 and 32 weeks' gestation. Patients in group 3 were sampled between 28 and 32 weeks. An additional blood sample was obtained 4 to 6 months after delivery (baseline). The Mann-Whitney U test, the Friedman test, and the chi2 test were used. RESULTS: Patients in group 1 were characterized by increased factor VIIa levels, increased prothrombin fragment 1+2 levels, reduced factor XIIa levels, diminished functional urokinase-type plasminogen activator levels, and decreased levels of plasmin/alpha-2-plasmin inhibitor complexes. These abnormalities were more evident in patients in group 1 developing pre-eclampsia and/or intrauterine growth restriction. CONCLUSIONS: Impaired factor XIIa-dependent activation of fibrinolysis seems to be a key mechanism related to late-pregnancy complications in patients with the antiphospholipid syndrome.

Factor XIa dimer in the activation of factor IX.

Factor XI, unlike other coagulation proteins, is a homodimer of two identical subunits linked by a single disulfide bond formed by Cys321. The present study was undertaken to understand the physiological significance of the dimeric nature of factor XI. We have expressed a mutant FXI/G326C in which the Gly326 residue of factor XI has been mutated to Cys326, reasoning that Cys321 would form an intrachain disulfide bond with Cys326 as in prekallikrein, a plasma protein that exists as a monomer even with 58% amino acid sequence identity and a domain structure very similar to factor XI. No free thiol could be detected in the expressed protein, and it migrated as a monomer on nonreduced SDS-PAGE. In physiological buffer, however, the protein was found to exist in a state of monomer-dimer equilibrium as assessed by gel-filtration chromatography and ultracentrifugation studies (K(d) approximately 36 nM). Functional studies revealed that FXI/G326C was indistinguishable from plasma factor XI in a plasma-clotting assay and in a factor IX activation assay both in the presence and absence of activated platelets even at concentrations at which less than 5% of the mutant exists as dimers. We conclude that, for optimal function in the presence of activated platelets, a preformed dimer of factor XI is not required.

[Revision of the biological significance of the contact system].

Current concept of blood coagulation is divided into two stages: an "initiation" stage which is handled by tissue factor pathway, and an "augmentation" stage handled by intrinsic pathway beginning in factor XI. Recent studies have demonstrated that the contact system is a modulator for vascular biology with vascular tone regulation, anticoagulant, profibrinolytic, antiadhesive and proinflammatory functions. Changes of contact system are associated with sepsis, thrombosis, etc.

Contact activation of blood coagulation: trigger properties and hysteresis. Kinetic recognition of foreign surfaces upon contact activation of blood coagulation: a hypothesis.

A mathematical model of contact activation of blood coagulation was developed and analysed. The model variables are concentrations of factor XIIa, kallikrein and activated high-molecular-weight kininogen. Concentrations of active factors were shown to depend on the activating signal value in a hysteretic manner. Within a range of relatively small signals, two (activated and non-activated) stable states coexist (bistability). Signals of the natural environment (surfaces of endothelial and blood cells) seem to be in the range of bistability; therefore, contact activation that persists for a short time can induce a transition of the system to the activated state, and, correspondingly, the formation of a clot. The system cannot return to the initial state, which is characterized by low activation levels, until the activating signals decrease significantly below those present in the circulation.

Direct activation of factor X by monocytes occurs during cardiopulmonary bypass.

We hypothesized that monocyte procoagulant activity, which includes up-regulation of tissue factor and direct activation of factor X by CD11b. is an activator of coagulation during cardiopulmonary bypass (CPB), because recent studies have cast doubt on the presumption that the surfaces of CPB activate the intrinsic pathway. Sequential samples were taken from 17 patients undergoing cardiac surgery. During CPB a significant increase in thrombin-antithrombin complexes occurred (P < 0.0005). Factor XIIa levels increased (P < 0.005) but remained within the normal range. Total monocyte procoagulant activity was measured and a functional assay was developed to detect direct activation of factor X alone. There was a significant increase in total procoagulant activity on circulating monocytes from the start of CPB (P < 0.005) to which direct factor X activation was a major contributor (P < 0.005). Direct activation of factor X was inhibited by CD11b blocking peptides. Using flow cytometry, up-regulation of monocyte CD11b (P < 0.0005). but not up-regulation of tissue factor, was found on circulating monocytes. Monocytes adherent on the oxygenator fibres showed increased CD11b expression (P < 0.0001), but no tissue factor when assessed by fluorescent image analysis. In conclusion, direct activation of factor X through monocyte CD11b occurs during CPB and appears to contribute to thrombin generation during CPB.

Fibrinolytic and factor XIII activity in subarachnoid hemorrhagge

The balance between fibrinolytic activity and coagulation mechanisms seems to play an important role in the rebleeding of a subarachnoid hemorrhage (SAH) due to aneurysmatic rupture. In the present paper we describe our findings in a group of patients (n10)with SAH. The plasmatic levels of fibrinogen and their degradation products (FDP), APTT, prothrombin activity and factor XIII were determined within 72 hours of initial bleeding, or of eventual rebleeding. Factor XIII activity in the first bleeding was 82.1+4 percent, while the levels of FDP were 3.8+1mug/ml. In patients presenting rebleeding (n4), Factor XIII activity was 67.3+4.5 percent the day it manifested, which is significantly less than the values previously observed (p<0.01), while the FDP level was 4.1+2mug/ml. The decrease of factor XIII activity suggests an important role as regards clot stability in rupture location. It is also possible to attribute a rebleeding predictive value to its activity reduction.

Platelet-bound prekallikrein promotes pro-urokinase-induced clot lysis: a mechanism for targeting the factor XII dependent intrinsic pathway of fibrinolysis.

Clots formed from platelet rich plasma were found to be lysed more readily by low concentrations of pro-urokinase (pro-UK) than clots formed from platelet poor plasma. This was not a non-specific effect since the reverse occurred with tissue plasminogen activator. A mechanical explanation due to platelet-mediated clot retraction was excluded by experiments in which retraction was inhibited with cytochalasin B. Therefore, a platelet-mediated enzymatic mechanism was postulated to explain the promotion of fibrinolysis. Casein autography of isolated platelets revealed a approximately 90 kDa band of activity which comigrated with plasma prekallikrein (PK)/kallikrein, a known activator of pro-UK. Furthermore, treatment of platelets with plasma PK activator (PPA), consisting essentially of factor XIIa, induced activation of pro-UK and of chromogenic substrate for kallikrein (S-2302). This activity corresponded to approximately 40-200 pM kallikrein per 10(8) washed and gel filtered platelets per ml. The activation of pro-UK by PPA-pretreated platelets was dose-dependent and inhibited by soybean trypsin inhibitor but not by bdellin, a specific inhibitor of plasmin, nor by the corn inhibitor of factor XIIa. Kinetic analysis of pro-UK activation by kallikrein showed promotion of the reaction by platelets. The KM of the reaction was reduced by platelets by approximately 7-fold, while the kcat was essentially unchanged. In conclusion, PK was shown to be tightly associated with platelets where it can be activated by factor XIIa during clotting. The activation of pro-UK by platelet-bound kallikrein provides an explanation for the observed platelet mediated promotion of pro-UK-induced clot lysis.(ABSTRACT TRUNCATED AT 250 WORDS)

[Contact factors in severe sepsis]. / Facteurs de contact au cours des états septiques graves.

Coagulation and fibrinolysis are not activated in an isolated system but it involves numerous interrelations with the kininogen-kinin pathway and the complement. In severe sepsis, substances released by microorganisms, notably lipopolysaccharides, can activate the contact system, and particularly in such circumstances, contact activation probably plays a role in the occurrence of haemodynamic changes and consumption coagulopathy. Evidence of kininogen-kinin pathway activation as assessed by biological investigations in patients with severe sepsis, could lead to the therapeutical use of natural or synthetic protease inhibitors.

FXII.

The plasma protein FXII (Hageman factor) has been shown to be linked with the plasma defence systems of coagulation, fibrinolysis, kallikrein-kinin and complement. It can be activated by surface contact activation and in solution. Surface contact activation is a complex phenomenon involving negatively charged surfaces, FXII, high molecular weight kininogen and plasma kallikrein. Fluid-phase activation can be effected by a variety of serine proteases. In both types of activation the FXII zymogen is converted to active enzymes. FXII levels in plasma are low or undetectable in both inherited deficiencies and in a variety of clinical conditions. FXII levels can also be elevated in some clinical conditions. Although discovered as a clotting protein FXII appears to play an important role in the kallikrein-kinin and fibrinolytic systems and also has effects on cells. Recent studies suggest that therapeutic blockade of activation of FXII can be of benefit in certain clinical conditions.

The inositol-phospholipid-accelerated activation of prekallikrein by activated factor XII at physiological ionic strength requires zinc ions and high-Mr kininogen.

In a system consisting of purified proteins inositol-phospholipid-accelerated activation of prekallikrein by alpha-factor XIIa was determined by measuring the appearance of kallikrein amidolytic activity towards the chromogenic substrate, H-D-Pro-Phe-Arg-NH-PhNO2 (PhNO2, 4-nitrophenyl). The activation reaction was ionic-strength dependent. In the absence of high-Mr kininogen optimal activity was recorded at I = 50 mM. Searching for conditions, which could change this optimum towards physiological values, high-Mr kininogen was added. This resulted in an inhibition of the activity, with no change in ionic strength optimum. If, however, Zn2+ were added concomitant with high-Mr kininogen, the inhibition was abolished and optimal activity recorded at physiological ionic strength. The optimal Zn2+ concentration was found to be 0.1 mM. Kinetic analysis of the reaction demonstrated that the kcat/Km was 1.2 x 10(5) M-1 s-1 in the absence and 1.1 x 10(6) M-1 s-1 in the presence of Zn2+. Zn2+ were also required for inositol-phospholipid-accelerated initiation of the contact activation in whole plasma.