The present Research Topic is derived from some the presentations at the Factor XI and the Contact System Standardization Subcommittee (SSC) session of the 62nd annual meeting of the International Society on Thrombosis and Haemostasis (ISTH). The session was a part of the annual conference and it was devoted to the discussion of diagnostic standards, nomenclature and assays related to factor XI, factor XII, prekallikrein, and high molecular weight kininogen of the Contact Activation System. The meeting was held in Montpellier, France, on May 27, 2016. Conference presenters were requested to contribute to this research topic articles that focus on these topics.

Case Western Reserve University

University of Amsterdam

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Frontiers in Medicine

Hematology

Presentations at the Factor XI and the Contact System SSC Session of the ISTH, Montpellier, FR, May 27, 2016

Editorial on the Research Topic<a href="http://journal.frontiersin.org/researchtopic/5096">Introduction to the Presentations at the Factor XI and the Contact System SSC Session of the ISTH, Montpellier, France, May 27, 2016</a>The present volume comprises some of the oral presentations that took place in the FXI and the Contact Activation Subcommittee on Standards of Coagulation in the Montpellier, France meeting on May 27, 2016. The assembly of articles is from participants who chose to submit their work. The cohesive bond that brings these manuscripts together is the active participation in the subcommittee&#x2019;s program. There are six manuscripts that were assembled for this e-book.The first two presentations address the business of the subcommittee, standardization. The first manuscript presented was by <a href="https://doi.org/10.3389/fmed.2016.00058">Rosén</a> of Molndal, Sweden. Dr. Rosen applied a commercial assay for Factor XIa (FXIa) that employs an activated Factor X readout of hydrolysis of a chromogenic substrate. The assay provides Factors IX, VIII, and X. FXI as well as Factor XII (FXII) and prekallikrein activation in this assay are initiated by contact activation with a commercial aPTT reagent. Further, the stability of the assay may be improved by the use of MES-BSA buffer.The second presentation was by <a href="https://doi.org/10.3389/fmed.2017.00028">Wilmot et al.</a> of the National Institute for Biological Standards and Controls, Potters Bar, Hertfordshire, UK, on the establishment of an international standard for human plasma Factor XI. Twenty-nine laboratories participated in the functional assay; 11 laboratories participated in the antigen assay. The plasma standard was established to have a functional activity value (FXI:C) of 0.71 U/ml and an antigen value (FXI:Ag) of 0.78 IU/ml.The next series of presentations focused on mechanisms associated with contact proteins. <a href="https://doi.org/10.3389/fmed.2016.00052">Jukema et al.</a> of Utrecht, Netherlands, presented some work in progress on FXII processing in a non-canonical manner during inflammatory reactions. The authors raise the hypothesis that non-contact system enzymes, e.g., plasmin or elastase, prime zymogen FXII to produce a βFXII that then gets activated to βFXIIa or the so-called FXII fragment. These processes may be active during inflammatory processes such as Gram-negative sepsis, anaphylaxis, and neuroinflammatory disorders like multiple sclerosis.<a href="https://doi.org/10.3389/fmed.2016.00061">Schmaier</a> presented a review of his recent work characterizing mechanisms for reduced thrombosis observed in genetics deletions of proteins of the contact system. Although most agree that f12−/− mice have reduced contact activation for their mechanism for reduced thrombosis risk, investigations in the Schmaier laboratory indicate that klkb1−/− (prekallikrein deficient) and bdkrb2−/− (bradykinin B2 receptor deficient) mice have a non-contact activation-mediated mechanism. Studies show that both these animals have reduced vessel wall tissue factor arising from elevated prostacyclin levels to reduce their thrombosis risk. Prostacyclin levels in klkb1−/− mice are less than bdkrb2−/− who also have a selective platelet GPVI and spreading defect.<a href="https://doi.org/10.3389/fmed.2017.00019">Weitz and Fredenburgh</a> of Hamilton, ON, Canada presented their thoughts of targeting Factors XI or XII as safer choices to prevent thrombosis. In this perspective, rationales for inhibiting FXIa over FXIIa and vice versa are presented; advantages of one versus the other are discussed; and strategies for inhibition of either also are presented. Further, initial human trials of FXI knockdown are delineated. Last the authors discuss the potential unmet needs in anticoagulation and antithrombotic therapy by targeting FXII or FXI. These novel agents provide new opportunities for improved patient care.Finally, <a href="https://doi.org/10.3389/fmed.2017.00121">Simão and Feener</a> provide a detailed review of the contact system and their hypothesis how plasma kallikrein and its product of high-molecular-weight kininogen cleavage, bradykinin, may contribute to hemorrhage. The Feener lab has presented data to suggest that plasma kallikrein cleaves collagen to reduce platelet aggregation, and liberated bradykinin has potential to elevate prostacyclin and tissue plasminogen activator that could contribute to bleeding.In sum, these articles present in part some of the diverse and active research in the FXI, FXII, and contact activation system. This field has developed new research avenues that should lead to novel therapies for presently unrealized targets in this increasingly important area.Each author contributed an equal share to this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Editorial: Introduction to the Presentations at the Factor XI and the Contact System SSC Session of the ISTH, Montpellier, France, May 27, 2016

The contact activation system (CAS) exerts effects on coagulation via multiple mechanisms, which modulate both the intrinsic and extrinsic coagulation cascades as well as fibrinolysis and platelet activation. While the effects of the CAS on blood coagulation measured as activated partial thromboplastin time shortening are well documented, genetic mutations that result in deficiencies in the expression of either plasma prekallikrein (PPK) or factor XII (FXII) are not associated with spontaneous bleeding or increased bleeding risk during surgery. Deficiencies in these proteins are often undiagnosed for decades and detected later in life during routine coagulation assays without an apparent clinical phenotype. Increased interest in the CAS as a potentially safe target for antithrombotic therapies has emerged, in large part, from studies on animal models with provoked thrombosis, which have shown that deficiencies in PPK or FXII can reduce thrombus formation without increasing bleeding. Gene targeting and pharmacological studies in healthy animals have confirmed that PPK and FXII blockade does not cause coagulopathies. These findings support the conclusion that CAS is not required for hemostasis. However, while deficiencies in FXII and PPK do not significantly affect bleeding associated with peripheral wounds, recent reports have demonstrated that these proteins can promote hemorrhage in the retina and brain. Intravitreal injection of plasma kallikrein (PKal) induces retinal hemorrhage and intracerebral injection of PKal increases intracranial bleeding. PPK deficiency and PKal inhibition ameliorates hematoma formation following cerebrovascular injury in diabetic animals. Moreover, both PPK and FXII deficiency are protective against intracerebral hemorrhage caused by tissue plasminogen activator-mediated thrombolytic therapy in mice with thrombotic middle cerebral artery occlusion. Thus, while the CAS is not required for hemostasis, its inhibition may provide an opportunity to reduce hemorrhage in the retina and brain. Characterization of the mechanisms and potential clinical implications associated with the effects of the CAS on hemorrhage requires further consideration of the effects of PPK and FXII on hemorrhage beyond their putative effects on coagulation cascades. Here, we review the experimental and clinical evidence on the effects of the CAS on bleeding and hemostatic mechanisms.

The contact activation system (CAS) exerts effects on coagulation via multiple mechanisms, which modulate both the intrinsic and extrinsic coagulation cascades as well as fibrinolysis and platelet activation. While the effects of the CAS on blood coagulation measured as activated partial thromboplastin time shortening are well documented, genetic mutations that result in deficiencies in the expression of either plasma prekallikrein (PPK) or factor XII (FXII) are not associated with spontaneous bleeding or increased bleeding risk during surgery. Deficiencies in these proteins are often undiagnosed for decades and detected later in life during routine coagulation assays without an apparent clinical phenotype. Increased interest in the CAS as a potentially safe target for antithrombotic therapies has emerged, in large part, from studies on animal models with provoked thrombosis, which have shown that deficiencies in PPK or FXII can reduce thrombus formation without increasing bleeding. Gene targeting and pharmacological studies in healthy animals have confirmed that PPK and FXII blockade does not cause coagulopathies. These findings support the conclusion that CAS is not required for hemostasis. However, while deficiencies in FXII and PPK do not significantly affect bleeding associated with peripheral wounds, recent reports have demonstrated that these proteins can promote hemorrhage in the retina and brain. Intravitreal injection of plasma kallikrein (PKal) induces retinal hemorrhage and intracerebral injection of PKal increases intracranial bleeding. PPK deficiency and PKal inhibition ameliorates hematoma formation following cerebrovascular injury in diabetic animals. Moreover, both PPK and FXII deficiency are protective against intracerebral hemorrhage caused by tissue plasminogen activator-mediated thrombolytic therapy in mice with thrombotic middle cerebral artery occlusion. Thus, while the CAS is not required for hemostasis, its inhibition may provide an opportunity to reduce hemorrhage in the retina and brain. Characterization of the mechanisms and potential clinical implications associated with the effects of the CAS on hemorrhage requires further consideration of the effects of PPK and FXII on hemorrhage beyond their putative effects on coagulation cascades. Here, we review the experimental and clinical evidence on the effects of the CAS on bleeding and hemostatic mechanisms.

The Effects of the Contact Activation System on Hemorrhage

The 1st International Standard (IS) for blood coagulation factor XI (FXI), plasma, has been successfully used for potency labeling of FXI therapeutics and for diagnosis of FXI deficiency in patients. With stocks of the 1st IS near depletion, a replacement is required. In addition to the functional activity value, assignment of an antigen value to the 2nd IS would allow harmonization of antigen assay methods and differentiation of patients who have low functional activity but normal antigen FXI levels from patients who have both low functional and antigen FXI levels. The aims of this study were, therefore, to assign FXI functional activity to the 2nd IS for FXI, plasma, and to additionally assign a new analyte, FXI antigen, to the same International Standard. The candidate material was prepared from double-spun, virology negative, normal plasma, which was pooled and filled into siliconized glass ampoules and subsequently freeze-dried. Assignment of the functional activity (FXI:C) value in International Units (IUs) was performed by one-stage clotting assay by 29 laboratories, relative to the 1st IS. The overall geometric mean (GM) was 0.71 IU/amp with extremely low inter-laboratory variability (expressed as geometric coefficient of variation) of 1.8%. The antigen value assignment was performed by 11 laboratories and was calculated relative to normal plasma pools, as is customary with new coagulation factor analytes. The amount of antigen present in 1 ml of normal plasma was taken to be 1 U. The overall GM for the antigen assays was 0.78 IU/amp with an inter-laboratory variation of 10%. The candidate (National Institute for Biological Standards and Control code, 15/180) was established by the World Health Organization (WHO) Expert Committee on Biological Standardization in 2016 as the WHO 2nd IS for blood coagulation FXI, plasma, with a functional activity value (FXI:C) of 0.71 IU/amp and an antigen value (FXI:Ag) of 0.78 IU/amp.

The 1st International Standard (IS) for blood coagulation factor XI (FXI), plasma, has been successfully used for potency labeling of FXI therapeutics and for diagnosis of FXI deficiency in patients. With stocks of the 1st IS near depletion, a replacement is required. In addition to the functional activity value, assignment of an antigen value to the 2nd IS would allow harmonization of antigen assay methods and differentiation of patients who have low functional activity but normal antigen FXI levels from patients who have both low functional and antigen FXI levels. The aims of this study were, therefore, to assign FXI functional activity to the 2nd IS for FXI, plasma, and to additionally assign a new analyte, FXI antigen, to the same International Standard. The candidate material was prepared from double-spun, virology negative, normal plasma, which was pooled and filled into siliconized glass ampoules and subsequently freeze-dried. Assignment of the functional activity (FXI:C) value in International Units (IUs) was performed by one-stage clotting assay by 29 laboratories, relative to the 1st IS. The overall geometric mean (GM) was 0.71 IU/amp with extremely low inter-laboratory variability (expressed as geometric coefficient of variation) of 1.8%. The antigen value assignment was performed by 11 laboratories and was calculated relative to normal plasma pools, as is customary with new coagulation factor analytes. The amount of antigen present in 1 ml of normal plasma was taken to be 1 U. The overall GM for the antigen assays was 0.78 IU/amp with an inter-laboratory variation of 10%. The candidate (National Institute for Biological Standards and Control code, 15/180) was established by the World Health Organization (WHO) Expert Committee on Biological Standardization in 2016 as the WHO 2nd IS for blood coagulation FXI, plasma, with a functional activity value (FXI:C) of 0.71 IU/amp and an antigen value (FXI:Ag) of 0.78 IU/amp.

Establishment of the World Health Organization 2nd International Standard for Factor XI, Plasma, Human

Compared with vitamin K antagonists, the direct oral anticoagulants (DOACs) are simpler to administer and are associated with less intracranial bleeding. Nonetheless, even with the DOACs, bleeding still occurs and many patients with atrial fibrillation fail to receive anticoagulant thromboprophylaxis because of the fear of bleeding. Therefore, there is an urgent need for safer anticoagulants. Recent investigations into the biochemistry of hemostasis and thrombosis have identified new targets for development of novel anticoagulants. Using data from complementary sources, including epidemiological studies and investigations in various animal models, the contact pathway has emerged as a potential mediator of thrombosis that plays a minor part in hemostasis. Consequently, factor (F) XII of the contact system and FXI in the intrinsic pathway have been identified as potentially safer targets of anticoagulation than thrombin or FXa. However, further studies are needed to identify which is the better target for the appropriate indication. This review highlights the evidence for focusing on FXI and FXII and examines the novel approaches directed at these new targets. These emerging strategies should address current unmet medical needs and provide new avenues by which to improve anticoagulant therapy by reducing the risk of bleeding.

Compared with vitamin K antagonists, the direct oral anticoagulants (DOACs) are simpler to administer and are associated with less intracranial bleeding. Nonetheless, even with the DOACs, bleeding still occurs and many patients with atrial fibrillation fail to receive anticoagulant thromboprophylaxis because of the fear of bleeding. Therefore, there is an urgent need for safer anticoagulants. Recent investigations into the biochemistry of hemostasis and thrombosis have identified new targets for development of novel anticoagulants. Using data from complementary sources, including epidemiological studies and investigations in various animal models, the contact pathway has emerged as a potential mediator of thrombosis that plays a minor part in hemostasis. Consequently, factor (F) XII of the contact system and FXI in the intrinsic pathway have been identified as potentially safer targets of anticoagulation than thrombin or FXa. However, further studies are needed to identify which is the better target for the appropriate indication. This review highlights the evidence for focusing on FXI and FXII and examines the novel approaches directed at these new targets. These emerging strategies should address current unmet medical needs and provide new avenues by which to improve anticoagulant therapy by reducing the risk of bleeding.

Factors XI and XII as Targets for New Anticoagulants

The contact activation (CAS) and kallikrein/kinin (KKS) systems regulate thrombosis risk in two ways. First, the CAS influences contact activation-induced factor XI activation and thrombin formation through the hemostatic cascade. Second, prekallikrein (PK) and bradykinin of the KKS regulate expression of three vessel wall G-protein-coupled receptors, the bradykinin B2 receptor (B2R), angiotensin receptor 2, and Mas to influence prostacyclin formation. The degree of intravascular prostacyclin formation inversely regulates intravascular thrombosis risk. A 1.5- to 2-fold increase in prostacyclin, as seen in PK deficiency, increases vessel wall Sirt1 and KLF4 to downregulate vessel wall tissue factor which alone is sufficient to lengthen induced thrombosis times. A twofold to threefold increase in prostacyclin, as seen the B2R-deficient mouse, delays thrombosis and produces a selective platelet function defect of reduced GPVI activation and platelet spreading. Regulation of CAS and KKS protein expression has a profound influence on thrombosis-generating mechanisms in the intravascular compartment.

The contact activation (CAS) and kallikrein/kinin (KKS) systems regulate thrombosis risk in two ways. First, the CAS influences contact activation-induced factor XI activation and thrombin formation through the hemostatic cascade. Second, prekallikrein (PK) and bradykinin of the KKS regulate expression of three vessel wall G-protein-coupled receptors, the bradykinin B2 receptor (B2R), angiotensin receptor 2, and Mas to influence prostacyclin formation. The degree of intravascular prostacyclin formation inversely regulates intravascular thrombosis risk. A 1.5- to 2-fold increase in prostacyclin, as seen in PK deficiency, increases vessel wall Sirt1 and KLF4 to downregulate vessel wall tissue factor which alone is sufficient to lengthen induced thrombosis times. A twofold to threefold increase in prostacyclin, as seen the B2R-deficient mouse, delays thrombosis and produces a selective platelet function defect of reduced GPVI activation and platelet spreading. Regulation of CAS and KKS protein expression has a profound influence on thrombosis-generating mechanisms in the intravascular compartment.

A Novel Antithrombotic Mechanism Mediated by the Receptors of the Kallikrein/Kinin and Renin–Angiotensin Systems

The recent availability of a sensitive chromogenic method approach for determination of FXIa activity has been explored for designing sensitive methods for FXIIa and kallikrein, both using FXa formation as the read-out. For both enzymes the assay range 1–10 nmol/L provides a resolution of about 0.8 absorbance units with a total assay time of about 20 min. For studies on activation kinetics, subsampling and extensive dilution can be performed in MES–bovine serum albumin (BSA) buffer pH 5.7 for quenching of enzyme activity and with ensuing determination of FXa generation in a chromogenic FXIa method. Optionally, suitable inhibitors such as aprotinin and/or corn trypsin inhibitor may be included. The stability of FXIa, FXIIa, and kallikrein in MES–BSA buffer was shown to be at least 5 h on ice. In conclusion, the use of a sensitive chromogenic FXIa method either per se or in combination with MES–BSA buffer pH 5.7 are new and potentially valuable tools for the study of contact factor enzymes and their inhibitors. So far, dose–response studies of FXIIa and kallikrein have been limited to purified systems, and hence more data are required to learn whether these new methods might or might not be applicable to the determination of FXIIa and kallikrein activities in plasma.

The recent availability of a sensitive chromogenic method approach for determination of FXIa activity has been explored for designing sensitive methods for FXIIa and kallikrein, both using FXa formation as the read-out. For both enzymes the assay range 1&#x2013;10 nmol/L provides a resolution of about 0.8 absorbance units with a total assay time of about 20 min. For studies on activation kinetics, subsampling and extensive dilution can be performed in MES&#x2013;bovine serum albumin (BSA) buffer pH 5.7 for quenching of enzyme activity and with ensuing determination of FXa generation in a chromogenic FXIa method. Optionally, suitable inhibitors such as aprotinin and/or corn trypsin inhibitor may be included. The stability of FXIa, FXIIa, and kallikrein in MES&#x2013;BSA buffer was shown to be at least 5 h on ice. In conclusion, the use of a sensitive chromogenic FXIa method either per se or in combination with MES&#x2013;BSA buffer pH 5.7 are new and potentially valuable tools for the study of contact factor enzymes and their inhibitors. So far, dose&#x2013;response studies of FXIIa and kallikrein have been limited to purified systems, and hence more data are required to learn whether these new methods might or might not be applicable to the determination of FXIIa and kallikrein activities in plasma.

New Tools to Study Contact Activation

The contact system was originally identified as an obsolete part of the coagulation system, but it has been repeatedly implicated in inflammatory states, such as infection, as well as in allergic- and chronic inflammatory disease. Under these conditions, there is surprisingly little evidence that factor XII (FXII) acts as a coagulation factor, and its activity appears to be mainly directed toward activation of the kallikrein–kinin system. The contact system factors interact with pathogens as well as cells of the (innate) immune system on several levels. Among others, these cells may provide negatively charged surfaces that contribute to contact activation as well as release enzymes that feed into this system. Furthermore, cellular receptors have been identified that bind contact factors at sites of inflammation. Based on the accumulated evidence, we propose a model for enzymatic crosstalk between inflammatory cells and the plasma contact system. During these reactions, FXII is enzymatically cleaved by non-contact system enzymes. This generates unactivated FXII fragments that can subsequently be rapidly activated in the fluid phase. The resulting enzyme lacks procoagulant properties, but retains its pro-inflammatory characteristic as a prekallikrein activator.

The contact system was originally identified as an obsolete part of the coagulation system, but it has been repeatedly implicated in inflammatory states, such as infection, as well as in allergic- and chronic inflammatory disease. Under these conditions, there is surprisingly little evidence that factor XII (FXII) acts as a coagulation factor, and its activity appears to be mainly directed toward activation of the kallikrein&#x2013;kinin system. The contact system factors interact with pathogens as well as cells of the (innate) immune system on several levels. Among others, these cells may provide negatively charged surfaces that contribute to contact activation as well as release enzymes that feed into this system. Furthermore, cellular receptors have been identified that bind contact factors at sites of inflammation. Based on the accumulated evidence, we propose a model for enzymatic crosstalk between inflammatory cells and the plasma contact system. During these reactions, FXII is enzymatically cleaved by non-contact system enzymes. This generates unactivated FXII fragments that can subsequently be rapidly activated in the fluid phase. The resulting enzyme lacks procoagulant properties, but retains its pro-inflammatory characteristic as a prekallikrein activator.