Andy T. Long

Contact system revisited: an interface between inflammation, coagulation, and innate immunity.

The contact system is a plasma protease cascade initiated by factor XII (FXII) that activates the proinflammatory kallikrein‐kinin system and the procoagulant intrinsic coagulation pathway. Anionic surfaces induce FXII zymogen activation to form proteolytically active FXIIa. Bacterial surfaces also have the ability to activate contact system proteins, indicating an important role for host defense using the cooperation of the inflammatory and coagulation pathways. Recent research has shown that inorganic polyphosphate found in platelets activates FXII in vivo and can induce coagulation in pathological thrombus formation. Experimental studies have shown that interference with FXII provides thromboprotection without a therapy‐associated increase in bleeding, renewing interest in the FXIIa‐driven intrinsic pathway of coagulation as a therapeutic target. This review summarizes how the contact system acts as the cross‐road of inflammation, coagulation, and innate immunity.

Host DNases prevent vascular occlusion by neutrophil extracellular traps

Blood DNases hack the NET Neutrophil extracellular traps (NETs) are lattices of processed chromatin decorated with select secreted and cytoplasmic proteins that trap and neutralize microbes. However, their inappropriate release may do more harm than good by promoting inflammation and thrombosis. Jiménez-Alcázar et al. report that two deoxyribonucleases (DNases), DNASE1 and DNASE1L3, have partially redundant roles in degrading NETs in the circulation (see the Perspective by Gunzer). Knockout mice lacking these enzymes were unable to tolerate chronic neutrophilia, quickly dying after blood vessels were occluded by NET clots. Furthermore, the damage unleashed by clots during septicemia was enhanced when these DNases were absent. Science, this issue p. 1202; see also p. 1126 Deoxyribonucleases work together to control vascular occlusion by neutrophil-induced blood clots. Platelet and fibrin clots occlude blood vessels in hemostasis and thrombosis. Here we report a noncanonical mechanism for vascular occlusion based on neutrophil extracellular traps (NETs), DNA fibers released by neutrophils during inflammation. We investigated which host factors control NETs in vivo and found that two deoxyribonucleases (DNases), DNase1 and DNase1-like 3, degraded NETs in circulation during sterile neutrophilia and septicemia. In the absence of both DNases, intravascular NETs formed clots that obstructed blood vessels and caused organ damage. Vascular occlusions in patients with severe bacterial infections were associated with a defect to degrade NETs ex vivo and the formation of intravascular NET clots. DNase1 and DNase1-like 3 are independently expressed and thus provide dual host protection against deleterious effects of intravascular NETs.

Neutralizing blood-borne polyphosphate in vivo provides safe thromboprotection

Polyphosphate is an inorganic procoagulant polymer. Here we develop specific inhibitors of polyphosphate and show that this strategy confers thromboprotection in a factor XII-dependent manner. Recombinant Escherichia coli exopolyphosphatase (PPX) specifically degrades polyphosphate, while a PPX variant lacking domains 1 and 2 (PPX_Δ12) binds to the polymer without degrading it. Both PPX and PPX_Δ12 interfere with polyphosphate- but not tissue factor- or nucleic acid-driven thrombin formation. Targeting polyphosphate abolishes procoagulant platelet activity in a factor XII-dependent manner, reduces fibrin accumulation and impedes thrombus formation in blood under flow. PPX and PPX_Δ12 infusions in wild-type mice interfere with arterial thrombosis and protect animals from activated platelet-induced venous thromboembolism without increasing bleeding from injury sites. In contrast, targeting polyphosphate does not provide additional protection from thrombosis in factor XII-deficient animals. Our data provide a proof-of-concept approach for combating thrombotic diseases without increased bleeding risk, indicating that polyphosphate drives thrombosis via factor XII.

Factor XII as a Therapeutic Target in Thromboembolic and Inflammatory Diseases

Coagulation factor XII (FXII, Hageman factor) is a plasma protease that in its active form (FXIIa) initiates the procoagulant and proinflammatory contact system. This name arises from FXII’s unique mechanism of activation that is induced by binding (contact) to negatively charged surfaces. Various substances have the capacity to trigger FXII contact-activation in vivo including mast cell–derived heparin, misfolded protein aggregates, collagen, nucleic acids, and polyphosphate. FXII deficiency is not associated with bleeding, and for decades, the factor was considered to be dispensable for coagulation in vivo. However, despite the fact that humans and animals with deficiency in FXII have a normal hemostatic capacity, animal models revealed a critical role of FXIIa-driven coagulation in thromboembolic diseases. In addition to its role in thrombosis, FXIIa contributes to inflammation through the activation of the inflammatory bradykinin-producing kallikrein-kinin system. Pharmacological inhibition of FXII/FXIIa interferes with thrombosis and inflammation in animal models. Thus, targeting the FXIIa-driven contact system seems to be a promising and safe therapeutic anticoagulation treatment strategy, with additional anti-inflammatory effects. Here, we discuss novel functions of FXIIa in cardiovascular thrombotic and inflammatory disorders.

The factor XIIa blocking antibody 3F7: a safe anticoagulant with anti-inflammatory activities

The plasma protein factor XII (FXII) is the initiating protease of the procoagulant and proinflammatory contact system. FXII activates both the bradykinin (BK) producing kallikrein-kinin system and the intrinsic pathway of coagulation. Contact with negatively charged surfaces induces auto-activation of zymogen FXII that results in activated FXII (FXIIa). Various in vivo activators of FXII have been identified including heparin, misfolded protein aggregates, nucleic acids and polyphosphate. Murine models have established a central role of FXII in arterial and venous thromboembolic diseases. Despite the central function of FXII in pathologic thrombosis, its deficiency does not impair hemostasis in animals or humans. The selective role of FXIIa in thrombosis, but not hemostasis, offers an exciting novel strategy for safe anticoagulation based on interference with FXIIa. We have generated the recombinant fully human FXIIa-blocking antibody 3F7, which abolished FXIIa enzymatic activity and prevented thrombosis in a cardiopulmonary bypass system in large animals, in the absence of increased therapy-associated bleeding. Furthermore, 3F7 also interfered with BK-driven edema in the severe swelling disorder hereditary angioedema (HAE) type III. Taken together, targeting FXIIa with 3F7 appears to be a promising approach to treat edema disorders and thrombosis.

Factor XII and uPAR upregulate neutrophil functions to influence wound healing

Coagulation factor XII (FXII) deficiency is associated with decreased neutrophil migration, but the mechanisms remain uncharacterized. Here, we examine how FXII contributes to the inflammatory response. In 2 models of sterile inflammation, FXII-deficient mice (F12–/–) had fewer neutrophils recruited than WT mice. We discovered that neutrophils produced a pool of FXII that is functionally distinct from hepatic-derived FXII and contributes to neutrophil trafficking at sites of inflammation. FXII signals in neutrophils through urokinase plasminogen activator receptor–mediated (uPAR-mediated) Akt2 phosphorylation at S474 (pAktS474). Downstream of pAkt2S474, FXII stimulation of neutrophils upregulated surface expression of &agr;M&bgr;2 integrin, increased intracellular calcium, and promoted extracellular DNA release. The sum of these activities contributed to neutrophil cell adhesion, migration, and release of neutrophil extracellular traps in a process called NETosis. Decreased neutrophil signaling in F12–/– mice resulted in less inflammation and faster wound healing. Targeting hepatic F12 with siRNA did not affect neutrophil migration, whereas WT BM transplanted into F12–/– hosts was sufficient to correct the neutrophil migration defect in F12–/– mice and restore wound inflammation. Importantly, these activities were a zymogen FXII function and independent of FXIIa and contact activation, highlighting that FXII has a sophisticated role in vivo that has not been previously appreciated.

The polyphosphate/factor XII pathway in cancer-associated thrombosis: novel perspectives for safe anticoagulation in patients with malignancies

Cancer is an established risk factor for venous thromboembolism (VTE) and VTE is the second leading cause of death in patients with cancer. The incidence of cancer-related thrombosis is rising and is associated with worse outcomes. Despite our growing understanding on tumor-driven procoagulant mechanisms including cancer-released procoagulant proteases, expression of tissue factor on cancer cells and derived microvesicles, as well as alterations in the extracellular matrix of the cancer cell milieu, anticoagulation therapy in cancer patients has remained challenging. This review comments on a newly discovered cancer-associated procoagulant pathway. Experimental VTE models in mice and studies on patient cancer material revealed that prostate cancer cells and associated exosomes display the inorganic polymer polyphosphate on their plasma membrane. Polyphosphate activates blood coagulation factor XII and initiates thrombus formation via the intrinsic pathway of coagulation. Pharmacologic inhibition of factor XII activity protects mice from VTE and reduces thrombin coagulant activity in plasma of prostate cancer patients. Factor XII inhibitors provide thrombo-protection without impairing hemostatic mechanisms and thus, unlike currently used anticoagulants, do not increase bleeding risk. Interference with the polyphosphate/factor XII pathway may provide the novel opportunity for safe anticoagulation therapy in patients with malignancies.

A flow cytometry-based assay for procoagulant platelet polyphosphate

Platelet polyphosphate is an inorganic procoagulant polymer of orthophosphate units that is stored in dense granules and is released upon platelet activation. Here, we describe an assay to measure polyphosphate on the surface of procoagulant human platelets.

Factor XII-Driven Inflammatory Reactions with Implications for Anaphylaxis

Anaphylaxis is a life-threatening allergic reaction. It is triggered by the release of pro-inflammatory cytokines and mediators from mast cells and basophils in response to immunologic or non-immunologic mechanisms. Mediators that are released upon mast cell activation include the highly sulfated polysaccharide and inorganic polymer heparin and polyphosphate (polyP), respectively. Heparin and polyP supply a negative surface for factor XII (FXII) activation, a serine protease that drives contact system-mediated coagulation and inflammation. Activation of the FXII substrate plasma kallikrein leads to further activation of zymogen FXII and triggers the pro-inflammatory kallikrein–kinin system that results in the release of the mediator bradykinin (BK). The severity of anaphylaxis is correlated with the intensity of contact system activation, the magnitude of mast cell activation, and BK formation. The main inhibitor of the complement system, C1 esterase inhibitor, potently interferes with FXII activity, indicating a meaningful cross-link between complement and kallikrein–kinin systems. Deficiency in a functional C1 esterase inhibitor leads to a severe swelling disorder called hereditary angioedema (HAE). The significance of FXII in these disorders highlights the importance of studying how these processes are integrated and can be therapeutically targeted. In this review, we focus on how FXII integrates with inflammation and the complement system to cause anaphylaxis and HAE as well as highlight current diagnosis and treatments of BK-related diseases.

Eliciting Dose and Safety Outcomes From a Large Dataset of Standardized Multiple Food Challenges

Background: Food allergy prevalence has continued to rise over the past decade. While studies have reported threshold doses for multiple foods, large-scale multi-food allergen studies are lacking. Our goal was to identify threshold dose distributions and predictors of severe reactions during blinded oral food challenges (OFCs) in multi-food allergic patients. Methods: A retrospective chart review was performed on all Stanford-initiated clinical protocols involving standardized screening OFCs to any of 11 food allergens at 7 sites. Interval-censoring survival analysis was used to calculate eliciting dose (ED) curves for each food. Changes in severity and ED were also analyzed among participants who had repeated challenges to the same food. Results: Of 428 participants, 410 (96%) had at least one positive challenge (1445 standardized OFCs with 1054 total positive challenges). Participants undergoing peanut challenges had the highest ED50 (29.9 mg), while those challenged with egg or pistachio had the lowest (7.07 or 1.7 mg, respectively). The most common adverse event was skin related (54%), followed by gastrointestinal (GI) events (33%). A history of asthma was associated with a significantly higher risk of a severe reaction (hazard ratio [HR]: 2.37, 95% confidence interval [CI]: 1.36, 4.13). Higher values of allergen-specific IgE (sIgE) and sIgE to total IgE ratio (sIgEr) were also associated with higher risk of a severe reaction (1.49 [1.19, 1.85] and 1.84 [1.30, 2.59], respectively). Participants undergoing cashew, peanut, pecan, sesame, and walnut challenges had more severe reactions as ED increased. In participants who underwent repeat challenges, the ED did not change (p = 0.66), but reactions were more severe (p = 0.02). Conclusions: Participants with a history of asthma, high sIgEr, and/or high values of sIgE were found to be at higher risk for severe reactions during food challenges. These findings may help to optimize food challenge dosing schemes in multi-food allergic, atopic patients, specifically at lower doses where the majority of reactions occur. Trials Registration Number: ClinicalTrials. gov number NCT03539692; https://clinicaltrials.gov/ct2/show/NCT03539692.