Steven de Maat

Defective glycosylation of coagulation factor XII underlies hereditary angioedema type III

Hereditary angioedema type III (HAEIII) is a rare inherited swelling disorder that is associated with point mutations in the gene encoding the plasma protease factor XII (FXII). Here, we demonstrate that HAEIII-associated mutant FXII, derived either from HAEIII patients or recombinantly produced, is defective in mucin-type Thr309-linked glycosylation. Loss of glycosylation led to increased contact-mediated autoactivation of zymogen FXII, resulting in excessive activation of the bradykinin-forming kallikrein-kinin pathway. In contrast, both FXII-driven coagulation and the ability of C1-esterase inhibitor to bind and inhibit activated FXII were not affected by the mutation. Intravital laser-scanning microscopy revealed that, compared with control animals, both F12-/- mice reconstituted with recombinant mutant forms of FXII and humanized HAEIII mouse models with inducible liver-specific expression of Thr309Lys-mutated FXII exhibited increased contact-driven microvascular leakage. An FXII-neutralizing antibody abolished bradykinin generation in HAEIII patient plasma and blunted edema in HAEIII mice. Together, the results of this study characterize the mechanism of HAEIII and establish FXII inhibition as a potential therapeutic strategy to interfere with excessive vascular leakage in HAEIII and potentially alleviate edema due to other causes.

Plasmin Cleavage of von Willebrand Factor as an Emergency Bypass for ADAMTS13 Deficiency in Thrombotic Microangiopathy

Background— Von Willebrand factor (VWF) multimer size is controlled through continuous proteolysis by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type I motif, member 13). This prevents spontaneous platelet agglutination and microvascular obstructions. ADAMTS13 deficiency is associated with thrombotic thrombocytopenic purpura, in which life-threatening episodes of microangiopathy damage kidneys, heart, and brain. Enigmatically, a complete ADAMTS13 deficiency does not lead to continuous microangiopathy. We hypothesized that plasmin, the key enzyme of the fibrinolytic system, serves as a physiological backup enzyme for ADAMTS13 in the degradation of pathological platelet–VWF complexes. Methods and Results— Using real-time microscopy, we determined that plasmin rapidly degrades platelet–VWF complexes on endothelial cells in absence of ADAMTS13, after activation by urokinase-type plasminogen activator or the thrombolytic agent streptokinase. Similarly, plasmin degrades platelet–VWF complexes in platelet agglutination studies. Plasminogen directly binds to VWF and its A1 domain in a lysine-dependent manner, as determined by enzyme-linked immunosorbent assay. Plasma levels of plasmin–&agr;2-antiplasmin complexes increase with the extent of thrombocytopenia in patients with acute episodes of thrombotic thrombocytopenic purpura, independent of ADAMTS13 activity. This indicates that plasminogen activation takes place during microangiopathy. Finally, we show that the thrombolytic agent streptokinase has therapeutic value for Adamts13−/− mice in a model of thrombotic thrombocytopenic purpura. Conclusions— We propose that plasminogen activation on endothelial cells acts as a natural backup for ADAMTS13 to degrade obstructive platelet–VWF complexes. Our findings indicate that thrombolytic agents may have therapeutic value in the treatment of microangiopathies and may be useful to bypass inhibitory antibodies against ADAMTS13 that cause thrombotic thrombocytopenic purpura.

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.

Bradykinin: Inflammatory Product of the Coagulation System

Episodic and recurrent local cutaneous or mucosal swelling are key features of angioedema. The vasoactive agents histamine and bradykinin are highly implicated as mediators of these swelling attacks. It is challenging to assess the contribution of bradykinin to the clinical expression of angioedema, as accurate biomarkers for the generation of this vasoactive peptide are still lacking. In this review, we will describe the mechanisms that are responsible for bradykinin production in hereditary angioedema (HAE) and the central role that the coagulation factor XII (FXII) plays in it. Evidently, several plasma parameters of coagulation change during attacks of HAE and may prove valuable biomarkers for disease activity. We propose that these changes are secondary to vascular leakage, rather than a direct consequence of FXII activation. Furthermore, biomarkers for fibrinolytic system activation (i.e. plasminogen activation) also change during attacks of HAE. These changes may reflect triggering of the bradykinin-forming mechanisms by plasmin. Finally, multiple lines of evidence suggest that neutrophil activation and mast-cell activation are functionally linked to bradykinin production. We put forward the paradigm that FXII functions as a ‘sensor molecule’ to detect conditions that require bradykinin release via crosstalk with cell-derived enzymes. Understanding the mechanisms that drive bradykinin generation may help to identify angioedema patients that have bradykinin-mediated disease and could benefit from a targeted treatment.

Contact system activation on endothelial cells.

When the contact system assembles and activates on negatively charged surface materials, plasma coagulation rapidly follows. This mechanism is redundant for hemostasis but mediates pathological thrombus formation, as was reported in a multitude of in vivo studies. The epidemiological data are presently scarce to firmly support a role for the contact system in human thrombotic disease, while its physiological function and mode of activation remains mysterious. Besides its role in blood coagulation in vitro, the contact system is responsible for the production of bradykinin. This inflammatory peptide is involved in episodes of pathological tissue swelling in (hereditary) angioedema, but potentially also in the physiological regulation of vascular permeability. A body of evidence indicates that contact system factors are recruited to the surface of activated endothelial cells, where proteins that are locally released can activate them. Furthermore, clinical and biochemical studies indicate that plasmin, the effector enzyme of the fibrinolytic system, can evoke contact system activation. This auxiliary role for plasmin may so far not have been fully appreciated in pathophysiology. To conclude this review, we propose a complementary model for contact system activation on the endothelial cell surface that is initiated by plasmin activity.

Antihistone Properties of C1 Esterase Inhibitor Protect against Lung Injury

&NA; 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.

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.

Processing of Factor XII during Inflammatory Reactions

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.

Invasive surgery reduces infarct size and preserves cardiac function in a porcine model of myocardial infarction

Reperfusion injury following myocardial infarction (MI) increases infarct size (IS) and deteriorates cardiac function. Cardioprotective strategies in large animal MI models often failed in clinical trials, suggesting translational failure. Experimentally, MI is induced artificially and the effect of the experimental procedures may influence outcome and thus clinical applicability. The aim of this study was to investigate if invasive surgery, as in the common open chest MI model affects IS and cardiac function. Twenty female landrace pigs were subjected to MI by transluminal balloon occlusion. In 10 of 20 pigs, balloon occlusion was preceded by invasive surgery (medial sternotomy). After 72 hrs, pigs were subjected to echocardiography and Evans blue/triphenyl tetrazoliumchloride double staining to determine IS and area at risk. Quantification of IS showed a significant IS reduction in the open chest group compared to the closed chest group (IS versus area at risk: 50.9 ± 5.4% versus 69.9 ± 3.4%, P = 0.007). End systolic LV volume and LV ejection fraction measured by echocardiography at follow‐up differed significantly between both groups (51 ± 5 ml versus 65 ± 3 ml, P = 0.033; 47.5 ± 2.6% versus 38.8 ± 1.2%, P = 0.005). The inflammatory response in the damaged myocardium did not differ between groups. This study indicates that invasive surgery reduces IS and preserves cardiac function in a porcine MI model. Future studies need to elucidate the effect of infarct induction technique on the efficacy of pharmacological therapies in large animal cardioprotection studies.

Truncation of ADAMTS13 by Plasmin Enhances Its Activity in Plasma

ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) cleaves von Willebrand Factor (VWF) multimers to control their thrombogenicity. The fibrinolytic enzyme plasmin can cleave VWF in a similar manner. However, plasmin can also cleave ADAMTS13, which ultimately inactivates it. This leaves the overall role of plasmin in primary haemostasis uncertain.We investigated the combined molecular effects of plasmin on VWF and ADAMTS13. We first identified that plasmin destroys FRETS-VWF73 substrate by cleaving the ADAMTS13 binding region in a buffered system. We next investigated how plasmin affects both VWF and ADAMTS13 under static conditions in plasma by western blotting. We found that globular VWF is largely protected from plasmin cleavage. However, ADAMTS13 is rapidly cleaved under these conditions, suggesting inactivation. Surprisingly, we observed that plasmin enhances ADAMTS13 activity in a modified two-stage FRETS-VWF73 assay that protects FRETS-VWF73 substrate from degradation. In direct binding studies under the same conditions, we found that plasmin generates multiple C-terminally truncated forms of ADAMTS13 with VWF-binding capacity. In an effort to seek evidence for this mechanism in vivo, we analysed plasma from patients with systemic amyloidosis, which is hallmarked by a hyperfibrinolytic state. We found that their plasma contained increased levels of C-terminally truncated forms of ADAMTS13, which correlated with their hyperfibrinolytic state.We propose that truncation of ADAMTS13 by plasmin abolishes intramolecular self-association, which improves interaction with unfolded VWF.