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.

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.

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.

Excessive enzyme activity often has pathological consequences. This for example is the case in thrombosis and hereditary angioedema, where serine proteases of the coagulation system and kallikrein-kinin system are excessively active. Serine proteases are controlled by SERPINs ( se rine p rotease in hibitors). We here describe the basic biochemical mechanisms behind SERPIN activity and identify key determinants that influence their function. We explore the clinical phenotypes of several SERPIN deficiencies and review studies where SERPINs are being used beyond replacement therapy. Excitingly, rare human SERPIN mutations have led us and others to believe that it is possible to refine SERPINs toward desired behavior for the treatment of enzyme-driven pathology.

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.

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–α 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.

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.

Summary. Factor XII is a mysterious plasma protein without a clear physiologic function. It was identified as a clotting factor, but has no clear role in hemostasis. However, FXII also contributes to the production of bradykinin, a short-lived inflammatory peptide. A growing body of mechanistic research from animal models indicates that FXII contributes to thrombotic disease by triggering excessive coagulation. FXII is evolutionarily conserved, suggesting that this molecule does have a physiologic function. This leads to intriguing questions: What does FXII really do? Is it even a real clotting factor at all? Before the groundbreaking discovery of a role for FXII in thrombotic disease, many studies investigated the biochemical properties of FXII and its activators. In this review, we highlight several biochemical studies that reveal much about the natural behavior of FXII. On the basis of these findings, it is possible to draft a conceptual model to explain how FXII reacts to surface materials. We then discuss how this model applies to the activities of FXII in its natural environment. There are two tentative physiologic functions of FXII that can operate exclusively: (i) maintenance of thrombus stability; (ii) local regulation of vascular permeability. Either, or both, of these natural functions may explain the evolutionary development and maintenance of FXII.