VWF-targeted thrombolysis to overcome rh-tPA resistance in experimental murine ischemic stroke models.

Recombinant human tissue plasminogen activator (rh-tPA) is an important thrombolytic agent for treatment of acute ischemic stroke. It requires fibrin binding for plasminogen activation. In contrast, Microlyse, a novel thrombolytic agent, requires von Willebrand factor (VWF) binding for plasminogen activation. We compared rh-tPA with Microlyse, administered 20 minutes after inducing thrombosis, in 2 randomized blinded acute ischemic stroke mouse models. Thrombosis was induced in the middle cerebral artery with different experimental triggers. Where thrombin infusion generates fibrin-rich thrombi, topical FeCl3 application generates platelet-rich thrombi. In the fibrin-rich model, both rh-tPA and Microlyse increased cortical reperfusion (determined by laser speckle imaging) 10 minutes after therapy administration (35.8 ± 17.1%; P = .001 39.3 ± 13.1%; P < .0001; 15.6 ± 7.5%, respectively, vs vehicle). In addition, both thrombolytic agents reduced cerebral lesion volume (determined by magnetic resonance imaging) after 24 hours (18.9 ± 11.2 mm3; P = .033; 16.1 ± 13.9 mm3; P = .018; 26.6 ± 5.6 mm3, respectively, vs vehicle). In the platelet-rich model, neither rh-tPA nor Microlyse increased cortical reperfusion 10 minutes after therapy (7.6 ± 8.8%; P = .216; 16.3 ± 13.9%; P = .151; 10.1 ± 7.9%, respectively, vs vehicle). However, Microlyse, but not rh-tPA, decreased cerebral lesion volumes (13.9 ± 11.4 mm3; P < .001; 23.6 ± 11.1 mm3; P = .188; 30.3 ± 10.9 mm3, respectively, vs vehicle). These findings support broad applicability of Microlyse in ischemic stroke, irrespective of the thrombus composition.

Microlyse: a thrombolytic agent that targets VWF for clearance of microvascular thrombosis.

Thrombotic microangiopathies are hallmarked by attacks of disseminated microvascular thrombosis. In thrombotic thrombocytopenic purpura (TTP), this is caused by a rise in thrombogenic ultra-large von Willebrand factor (VWF) multimers because of ADAMTS13 deficiency. We previously reported that systemic plasminogen activation is therapeutic in a TTP mouse model. In contrast to its natural activators (ie, tissue plasminogen activator and urokinase plasminogen activator [uPA]), plasminogen can directly bind to VWF. For optimal efficacy and safety, we aimed to focus and accelerate plasminogen activation at sites of microvascular occlusion. We here describe the development and characterization of Microlyse, a fusion protein consisting of a high-affinity VHH targeting the CT/CK domain of VWF and the protease domain of uPA, for localized plasminogen activation on microthrombi. Microlyse triggers targeted destruction of platelet-VWF complexes by plasmin on activated endothelial cells and in agglutination studies. At equal molar concentrations, Microlyse degrades microthrombi sevenfold more rapidly than blockade of platelet-VWF interactions with a bivalent humanized VHH (caplacizumab*). Finally, Microlyse attenuates thrombocytopenia and tissue damage (reflected by increased plasma lactate dehydrogenase activity, as well as PAI-1 and fibrinogen levels) more efficiently than caplacizumab* in an ADAMTS13-/- mouse model of TTP, without affecting hemostasis in a tail-clip bleeding model. These findings show that targeted thrombolysis of VWF by Microlyse is an effective strategy for the treatment of TTP and might hold value for other forms of VWF-driven thrombotic disease.

A reactive center loop-based prediction platform to enhance the design of therapeutic SERPINs.

Serine proteases are essential for many physiological processes and require tight regulation by serine protease inhibitors (SERPINs). A disturbed SERPIN-protease balance may result in disease. The reactive center loop (RCL) contains an enzymatic cleavage site between the P1 through P1' residues that controls SERPIN specificity. This RCL can be modified to improve SERPIN function; however, a lack of insight into sequence-function relationships limits SERPIN development. This is complicated by more than 25 billion mutants needed to screen the entire P4 to P4' region. Here, we developed a platform to predict the effects of RCL mutagenesis by using α1-antitrypsin as a model SERPIN. We generated variants for each of the residues in P4 to P4' region, mutating them into each of the 20 naturally occurring amino acids. Subsequently, we profiled the reactivity of the resulting 160 variants against seven proteases involved in coagulation. These profiles formed the basis of an in silico prediction platform for SERPIN inhibitory behavior with combined P4 to P4' RCL mutations, which were validated experimentally. This prediction platform accurately predicted SERPIN behavior against five out of the seven screened proteases, one of which was activated protein C (APC). Using these findings, a next-generation APC-inhibiting α1-antitrypsin variant was designed (KMPR/RIRA; / indicates the cleavage site). This variant attenuates blood loss in an in vivo hemophilia A model at a lower dosage than the previously developed variant AIKR/KIPP because of improved potency and specificity. We propose that this SERPIN-based RCL mutagenesis approach improves our understanding of SERPIN behavior and will facilitate the design of therapeutic SERPINs.

Restriction of C1-inhibitor activity in hereditary angioedema by dominant-negative effects of disease-associated SERPING1 gene variants.

BACKGROUND: Patients with hereditary angioedema experience recurrent, sometimes life-threatening, attacks of edema. It is a rare genetic disorder characterized by genetic and clinical heterogenicity. Most cases are caused by genetic variants in the SERPING1 gene leading to plasma deficiency of the encoded protein C1 inhibitor (C1INH). More than 500 different hereditary angioedema-causing variants have been identified in the SERPING1 gene, but the disease mechanisms by which they result in pathologically low C1INH plasma levels remain largely unknown. OBJECTIVES: The aim was to describe trans-inhibitory effects of full-length or near full-length C1INH encoded by 28 disease-associated SERPING1 variants. METHODS: HeLa cells were transfected with expression constructs encoding the studied SERPING1 variants. Extensive and comparative studies of C1INH expression, secretion, functionality, and intracellular localization were carried out. RESULTS: Our findings characterized functional properties of a subset of SERPING1 variants allowing the examined variants to be subdivided into 5 different clusters, each containing variants sharing specific molecular characteristics. For all variants except 2, we found that coexpression of mutant and normal C1INH negatively affected the overall capacity to target proteases. Strikingly, for a subset of variants, intracellular formation of C1INH foci was detectable only in heterozygous configurations enabling simultaneous expression of normal and mutant C1INH. CONCLUSIONS: We provide a functional classification of SERPING1 gene variants suggesting that different SERPING1 variants drive the pathogenicity through different and in some cases overlapping molecular disease mechanisms. For a subset of gene variants, our data define some types of hereditary angioedema with C1INH deficiency as serpinopathies driven by dominant-negative disease mechanisms.

Xenotropic and polytropic retrovirus receptor 1 regulates procoagulant platelet polyphosphate.

Polyphosphate is a procoagulant inorganic polymer of linear-linked orthophosphate residues. Multiple investigations have established the importance of platelet polyphosphate in blood coagulation; however, the mechanistic details of polyphosphate homeostasis in mammalian species remain largely undefined. In this study, xenotropic and polytropic retrovirus receptor 1 (XPR1) regulated polyphosphate in platelets and was implicated in thrombosis in vivo. We used bioinformatic analyses of omics data to identify XPR1 as a major phosphate transporter in platelets. XPR1 messenger RNA and protein expression inversely correlated with intracellular polyphosphate content and release. Pharmacological interference with XPR1 activity increased polyphosphate stores, led to enhanced platelet-driven coagulation, and amplified thrombus formation under flow via the polyphosphate/factor XII pathway. Conditional gene deletion of Xpr1 in platelets resulted in polyphosphate accumulation, accelerated arterial thrombosis, and augmented activated platelet-driven pulmonary embolism without increasing bleeding in mice. These data identify platelet XPR1 as an integral regulator of platelet polyphosphate metabolism and reveal a fundamental role for phosphate homeostasis in thrombosis.

Small-Molecule Cyclophilin Inhibitors Potently Reduce Platelet Procoagulant Activity.

Procoagulant platelets are associated with an increased risk for thrombosis. Procoagulant platelet formation is mediated via Cyclophilin D (CypD) mediated opening of the mitochondrial permeability transition pore. Inhibiting CypD activity could therefore be an interesting approach to limiting thrombosis. In this study, we investigated the potential of two novel, non-immunosuppressive, non-peptidic small-molecule cyclophilin inhibitors (SMCypIs) to limit thrombosis in vitro, in comparison with the cyclophilin inhibitor and immunosuppressant Cyclosporin A (CsA). Both cyclophilin inhibitors significantly decreased procoagulant platelet formation upon dual-agonist stimulation, shown by a decreased phosphatidylserine (PS) exposure, as well as a reduction in the loss of mitochondrial membrane potential. Furthermore, the SMCypIs potently reduced procoagulant platelet-dependent clotting time, as well as fibrin formation under flow, comparable to CsA. No effect was observed on agonist-induced platelet activation measured by P-selectin expression, as well as CypA-mediated integrin αIIbß3 activation. Importantly, whereas CsA increased Adenosine 5'-diphosphate (ADP)-induced platelet aggregation, this was unaffected in the presence of the SMCypIs. We here demonstrate specific cyclophilin inhibition does not affect normal platelet function, while a clear reduction in procoagulant platelets is observed. Reducing platelet procoagulant activity by inhibiting cyclophilins with SMCypIs forms a promising strategy to limit thrombosis.

Dysregulated Innate and Adaptive Immune Responses Discriminate Disease Severity in COVID-19.

The clinical spectrum of COVID-19 varies and the differences in host response characterizing this variation have not been fully elucidated. COVID-19 disease severity correlates with an excessive proinflammatory immune response and profound lymphopenia. Inflammatory responses according to disease severity were explored by plasma cytokine measurements and proteomics analysis in 147 COVID-19 patients. Furthermore, peripheral blood mononuclear cell cytokine production assays and whole blood flow cytometry were performed. Results confirm a hyperinflammatory innate immune state, while highlighting hepatocyte growth factor and stem cell factor as potential biomarkers for disease severity. Clustering analysis revealed no specific inflammatory endotypes in COVID-19 patients. Functional assays revealed abrogated adaptive cytokine production (interferon-γ, interleukin-17, and interleukin-22) and prominent T-cell exhaustion in critically ill patients, whereas innate immune responses were intact or hyperresponsive. Collectively, this extensive analysis provides a comprehensive insight into the pathobiology of severe to critical COVID-19 and highlights potential biomarkers of disease severity.

A mutation in the kringle domain of human factor XII that causes autoinflammation, disturbs zymogen quiescence, and accelerates activation.

Coagulation factor XII (FXII) drives production of the inflammatory peptide bradykinin. Pathological mutations in the F12 gene, which encodes FXII, provoke acute tissue swelling in hereditary angioedema (HAE). Interestingly, a recently identified F12 mutation, causing a W268R substitution, is not associated with HAE. Instead, FXII-W268R carriers experience cold-inducible urticarial rash, arthralgia, fever, and fatigue. Here, we aimed to investigate the molecular characteristics of the FXII-W268R variant. We expressed wild type FXII (FXII-WT), FXII-W268R, and FXII-T309R (which causes HAE), as well as other FXII variants in HEK293 freestyle cells. Using chromogenic substrate assays, immunoblotting, and ELISA, we analyzed expression media, cell lysates, and purified proteins for FXII activation. Recombinant FXII-W268R forms increased amounts of intracellular cleavage products that are also present in expression medium and display enzymatic activity. The active site-incapacitated variant FXII-W268R/S544A reveals that intracellular fragmentation is largely dependent on autoactivation. Purified FXII-W268R is highly sensitive to activation by plasma kallikrein and plasmin, compared with FXII-WT or FXII-T309R. Furthermore, binding studies indicated that the FXII-W268R variant leads to the exposure of a plasminogen-binding site that is cryptic in FXII-WT. In plasma, recombinant FXII-W268R spontaneously triggers high-molecular-weight kininogen cleavage. Our findings suggest that the W268R substitution influences FXII protein conformation and exposure of the activation loop, which is concealed in FXII-WT. This results in intracellular autoactivation and constitutive low-grade secretion of activated FXII. These findings help to explain the chronically increased contact activation in carriers of the FXII-W268R variant.

Design and characterization of α1-antitrypsin variants for treatment of contact system-driven thromboinflammation.

The contact system produces the inflammatory peptide bradykinin and contributes to experimental thrombosis. C1 esterase-inhibitor (C1INH) deficiency or gain-of-function mutations in factor XII (FXII) cause hereditary angioedema, a life-threatening tissue swelling disease. C1INH is a relatively weak contact system enzyme inhibitor. Although α1-antitrypsin (α1AT) does not naturally inhibit contact system enzymes, a human mutation (M358R; α1AT-Pittsburgh) changes it into a powerful broad-spectrum enzyme inhibitor. It blocks the contact system, but also thrombin and activated protein C (APC), making it an unattractive candidate for therapeutic contact system blockade. We adapted the reactive center loop of α1AT-Pittsburgh (AIPR/S) to overcome these obstacles. Two α1AT variants (SMTR/S and SLLR/S) strongly inhibit plasma kallikrein, activated FXII, and plasmin. α1AT-SMTR/S no longer inhibits thrombin, but residually inhibits APC. In contrast, α1AT-SLLR/S residually inhibits thrombin, but no longer APC. Additional modification at the P1' position (S→V) eliminates residual inhibition of thrombin and APC for both variants, while retaining their properties as contact system inhibitors. Both α1AT-SMTR/V and -SLLR/V are superior to C1INH in reducing bradykinin production in plasma. Owing to their capacity to selectively block contact system-driven coagulation, both variants block vascular occlusion in an in vivo model for arterial thrombosis. Furthermore, both variants block acute carrageenan-induced tissue edema in mice. Finally, α1AT-SLLR/V, our most powerful candidate, suppresses epithelial leakage of the gut in a mouse model of colitis. Our findings confirm that redesign of α1AT strongly alters its inhibitory behavior and can be used for the treatment of contact system-mediated thrombosis and inflammation.

Evidence for bradykinin release in chronic spontaneous urticaria.

BACKGROUND: Chronic spontaneous urticaria (CSU) is characterized by recurrent itchy weals and/or angioedema and is believed to be driven by mast cell activation. It was shown that excessive mast cell activation during anaphylaxis initiates contact activation, resulting in bradykinin release. Evidence for bradykinin release was never demonstrated in CSU. OBJECTIVE: To study biomarkers of bradykinin release in CSU. METHODS: Plasma samples of CSU patients were collected during routine visits at the outpatient clinic. Cleaved high molecular weight kininogen (cHK) was used as a biomarker for bradykinin release. cHK, factor XIIa-C1-inhibitor (FXIIa-C1-INH), kallikrein-C1-INH, plasmin-antiplasmin (PAP) complexes and soluble urokinase-type plasminogen activator receptor (suPAR) levels were determined by ELISA. Clinical data and data on tryptase levels were collected from medical records. cHK levels were compared to previously determined levels in hereditary angioedema (HAE). RESULTS: One hundred seventeen samples from 88 CSU patients and 28 samples from healthy controls were analysed. Median cHK level in CSU was 9.1% (range: 1.4%-21.5%), significantly increased compared to healthy controls (median 6.0% range: 0%-19.9%; P = .0005) and comparable to HAE (n = 46, median 10.3%, range 0%-44.3%, P > .9999). cHK levels normalized in patients during disease remission (median 6.5% range 1.5%-20.8%) but were not dependent on the presence of angioedema, acute angioedema attacks or response to antihistamines. Surprisingly, cHK levels were inversely correlated to serum tryptase (r = -0.65 P = .0137). C1-INH complexes and suPAR levels were not elevated in patients compared to healthy controls. PAP-complex levels in patients were elevated compared to healthy controls but there was no correlation between PAP-complex and cHK levels. CONCLUSIONS: cHK levels are elevated in symptomatic CSU patients compared to healthy controls, indicating increased bradykinin production. Increased cHK levels are not limited to patients with angioedema. CLINICAL RELEVANCE: If elevated bradykinin generation has clinical implications in the pathology of CSU is open to debate.

Coagulation factor XII in thrombosis and inflammation.

Combinations of proinflammatory and procoagulant reactions are the unifying principle for a variety of disorders affecting the cardiovascular system. The factor XII-driven contact system starts coagulation and inflammatory mechanisms via the intrinsic pathway of coagulation and the bradykinin-producing kallikrein-kinin system, respectively. The biochemistry of the contact system in vitro is well understood; however, its in vivo functions are just beginning to emerge. Challenging the concept of the coagulation balance, targeting factor XII or its activator polyphosphate, provides protection from thromboembolic diseases without interfering with hemostasis. This suggests that the polyphosphate/factor XII axis contributes to thrombus formation while being dispensable for hemostatic processes. In contrast to deficiency in factor XII providing safe thromboprotection, excessive FXII activity is associated with the life-threatening inflammatory disorder hereditary angioedema. The current review summarizes recent findings of the polyphosphate/factor XII-driven contact system at the intersection of procoagulant and proinflammatory disease states. Elucidating the contact system offers the exciting opportunity to develop strategies for safe interference with both thrombotic and inflammatory disorders.

Mast cells increase vascular permeability by heparin-initiated bradykinin formation in vivo.

Activated mast cells trigger edema in allergic and inflammatory disease. We report a paracrine mechanism by which mast cell-released heparin increases vascular permeability in vivo. Heparin activated the protease factor XII, which initiates bradykinin formation in plasma. Targeting factor XII or kinin B2 receptors abolished heparin-triggered leukocyte-endothelium adhesion and interfered with a mast cell-driven drop in blood pressure in rodents. Intravital laser scanning microscopy and tracer measurements showed heparin-driven fluid extravasation in mouse skin microvessels. Ablation of factor XII or kinin B2 receptors abolished heparin-induced skin edema and protected mice from allergen-activated mast cell-driven leakage. In contrast, heparin and activated mast cells induced excessive edema in mice deficient in the major inhibitor of factor XII, C1 esterase inhibitor. Allergen exposure triggered edema attacks in hereditary angioedema patients, lacking C1 esterase inhibitor. The data indicate that heparin-initiated bradykinin formation plays a fundamental role in mast cell-mediated diseases.

Polyphosphate nanoparticles on the platelet surface trigger contact system activation.

Polyphosphate is an inorganic polymer that can potentiate several interactions in the blood coagulation system. Blood platelets contain polyphosphate, and the secretion of platelet-derived polyphosphate has been associated with increased thrombus formation and activation of coagulation factor XII. However, the small polymer size of secreted platelet polyphosphate limits its capacity to activate factor XII in vitro. Thus, the mechanism by which platelet polyphosphate contributes to thrombus formation remains unclear. Using live-cell imaging, confocal and electron microscopy, we show that activated platelets retain polyphosphate on their cell surface. The apparent polymer size of membrane-associated polyphosphate largely exceeds that of secreted polyphosphate. Ultracentrifugation fractionation experiments revealed that membrane-associated platelet polyphosphate is condensed into insoluble spherical nanoparticles with divalent metal ions. In contrast to soluble polyphosphate, membrane-associated polyphosphate nanoparticles potently activate factor XII. Our findings identify membrane-associated polyphosphate in a nanoparticle state on the surface of activated platelets. We propose that these polyphosphate nanoparticles mechanistically link the procoagulant activity of platelets with the activation of coagulation factor XII.

Factor XII Activation Promotes Platelet Consumption in the Presence of Bacterial-Type Long-Chain Polyphosphate In Vitro and In Vivo.

Objective- Terminal complications of bacterial sepsis include development of disseminated intravascular consumptive coagulopathy. Bacterial constituents, including long-chain polyphosphates (polyP), have been shown to activate the contact pathway of coagulation in plasma. Recent work shows that activation of the contact pathway in flowing whole blood promotes thrombin generation and platelet activation and consumption distal to thrombus formation ex vivo and in vivo. Here, we sought to determine whether presence of long-chain polyP or bacteria in the bloodstream promotes platelet activation and consumption in a coagulation factor (F)XII-dependent manner. Approach and Results- Long-chain polyP promoted platelet P-selectin expression, microaggregate formation, and platelet consumption in flowing whole blood in a contact activation pathway-dependent manner. Moreover, long-chain polyP promoted local fibrin formation on collagen under shear flow in a FXI-dependent manner. Distal to the site of thrombus formation, platelet consumption was dramatically enhanced in the presence of long-chain polyP in the blood flow in a FXI- and FXII-dependent manner. In a murine model, long-chain polyP promoted platelet deposition and fibrin generation in lungs in a FXII-dependent manner. In a nonhuman primate model of bacterial sepsis, pre-treatment of animals with an antibody blocking FXI activation by FXIIa reduced lethal dose100 Staphylococcus aureus-induced platelet and fibrinogen consumption. Conclusions- This study demonstrates that bacterial-type long-chain polyP promotes platelet activation in a FXII-dependent manner in flowing blood, which may contribute to sepsis-associated thrombotic processes, consumptive coagulopathy, and thrombocytopenia.

Antihistone Properties of C1 Esterase Inhibitor Protect against Lung Injury.

RATIONALE: Acute respiratory distress syndrome is characterized by alveolar epithelial cell injury, edema formation, and intraalveolar contact phase activation. OBJECTIVES: To explore whether C1 esterase inhibitor (C1INH), an endogenous inhibitor of the contact phase, may protect from lung injury in vivo and to decipher the possible underlying mechanisms mediating protection. METHODS: The ability of C1INH to control the inflammatory processes was studied in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: Here, we demonstrate that application of C1INH alleviates bleomycin-induced lung injury via direct interaction with extracellular histones. In vitro, C1INH was found to bind all histone types. Interaction with histones was independent of its protease inhibitory activity, as demonstrated by the use of reactive-center-cleaved C1INH, but dependent on its glycosylation status. C1INH sialylated-N- and -O-glycans were not only essential for its interaction with histones but also to protect against histone-induced cell death. In vivo, histone-C1INH complexes were detected in bronchoalveolar lavage fluid from patients with acute respiratory distress syndrome and multiple models of lung injury. Furthermore, reactive-center-cleaved C1INH attenuated pulmonary damage evoked by intravenous histone instillation. CONCLUSIONS: Collectively, C1INH administration provides a new therapeutic option for disorders associated with histone release.

Coagulation Factor XI Promotes Distal Platelet Activation and Single Platelet Consumption in the Bloodstream Under Shear Flow.

OBJECTIVE: Coagulation factor XI (FXI) has been shown to contribute to thrombus formation on collagen or tissue factor-coated surfaces in vitro and in vivo by enhancing thrombin generation. Whether the role of the intrinsic pathway of coagulation is restricted to the local site of thrombus formation is unknown. This study was aimed to determine whether FXI could promote both proximal and distal platelet activation and aggregate formation in the bloodstream. APPROACH AND RESULTS: Pharmacological blockade of FXI activation or thrombin activity in blood did not affect local platelet adhesion, yet reduced local platelet aggregation, thrombin localization, and fibrin formation on immobilized collagen and tissue factor under shear flow, ex vivo. Downstream of the thrombus formed on immobilized collagen or collagen and 10 pmol/L tissue factor, platelet CD62P expression, microaggregate formation, and progressive platelet consumption were significantly reduced in the presence of FXI function-blocking antibodies or a thrombin inhibitor in a shear rate- and time-dependent manner. In a non-human primate model of thrombus formation, we found that inhibition of FXI reduced single platelet consumption in the bloodstream distal to a site of thrombus formation. CONCLUSIONS: This study demonstrates that the FXI-thrombin axis contributes to distal platelet activation and procoagulant microaggregate formation in the blood flow downstream of the site of thrombus formation. Our data highlight FXI as a novel therapeutic target for inhibiting distal platelet consumption without affecting proximal platelet adhesion.

Plasmin is a natural trigger for bradykinin production in patients with hereditary angioedema with factor XII mutations.

BACKGROUND: Patients with angioedema experience unpredictable attacks of tissue swelling in which bradykinin is implicated. Several distinct mutations in Factor XII (FXII) are associated with hereditary angioedema (HAE) in the presence of normal C1 esterase inhibitor activity (FXII-HAE). The underlying disease mechanisms are unclear, which complicates diagnosis and treatment. OBJECTIVE: We sought to identify the natural trigger for FXII activation, which causes uncontrolled bradykinin production in patients with FXII-HAE. METHODS: We generated recombinant variants of FXII, representing health and disease, and studied their behavior in functional studies. We investigated bradykinin-forming pathways in blood plasma with newly developed nanobody-based analytic methods. RESULTS: We here report that FXII-HAE mutations collectively introduce new sites that are sensitive to enzymatic cleavage by plasmin. These FXII mutants rapidly activate after cleavage by plasmin, escape from inhibition through C1 esterase inhibitor, and elicit excessive bradykinin formation. Furthermore, our findings indicate that plasmin modulates disease activity in patients with FXII-HAE. Finally, we show that soluble lysine analogs attenuate this mechanism, explaining their therapeutic value in patients with HAE. CONCLUSION: Our findings indicate a new pathway for bradykinin formation in patients with HAE, in which FXII is cleaved and activated by plasmin. This should lead to the identification of new markers for diagnosis and targets for treatment.

Keeping von Willebrand Factor under Control: Alternatives for ADAMTS13.

Von Willebrand factor (VWF) is one of the most important proteins of the hemostatic system. Its multimeric state is essential for its natural function to guide platelets to sites of injury. ADAMTS13 is the key protease that regulates the multimeric state of VWF. Without ADAMTS13, VWF multimers can grow to pathologically large sizes. This is a risk factor for the life-threatening condition thrombotic thrombocytopenic purpura (TTP). In this condition, VWF-rich thrombi occlude the microvasculature of various tissues. Intriguingly, a complete ADAMTS13 deficiency does not cause continuous TTP, either in patients or genetically targeted mice. Instead, TTP occurs in episodes of disease, separated by extended periods of remission. This indicates that regulating factors beyond ADAMTS13 are likely involved in this pathologic cascade of events. This raises the question of what really happens when ADAMTS13 is (temporarily) unavailable. In this review, we explore the possible role of complementary mechanisms that are capable of modifying the thrombogenic potential of VWF.