Search

Background Inorganic polyphosphate modulates the contact pathway of blood clotting, which is implicated in thrombosis and inflammation. Polyphosphate polymer lengths are highly variable, with shorter polymers (approximately 60-100 phosphates) secreted from human platelets, and longer polymers (up to thousands of phosphates) in microbes. We previously reported that optimal triggering of clotting via the contact pathway requires very long polyphosphates, although the impact of shorter polyphosphate polymers on individual proteolytic reactions of the contact pathway was not interrogated. Objectives and methods We conducted in vitro measurements of enzyme kinetics to investigate the ability of varying polyphosphate sizes, together with high molecular weight kininogen and Zn2+ , to mediate four individual proteolytic reactions of the contact pathway: factor XII autoactivation, factor XII activation by kallikrein, prekallikrein activation by factor XIIa, and prekallikrein autoactivation. Results The individual contact pathway reactions were differentially dependent on polyphosphate length. Very long-chain polyphosphate was required to support factor XII autoactivation, whereas platelet-size polyphosphate significantly accelerated the activation of factor XII by kallikrein, and the activation of prekallikrein by factor XIIa. Intriguingly, polyphosphate did not support prekallikrein autoactivation. We also report that high molecular weight kininogen was required only when kallikrein was the enzyme (ie, FXII activation by kallikrein), whereas Zn2+ was required only when FXII was the substrate (ie, FXII activation by either kallikrein or FXIIa). Activation of prekallikrein by FXIIa required neither Zn2+ nor high molecular weight kininogen. Conclusions Platelet polyphosphate and Zn2+ can promote subsets of the reactions of the contact pathway, with implications for a variety of disease states.

International audience; Hypozincemia is prevalent in severe acute respiratory syndrome coronavirus-2 (SARS-COV-2)-infected patients and has been considered as a risk factor in severe coronavirus disease-2019 (COVID-19). Whereas zinc might affect SARS-COV-2 replication and cell entry, the link between zinc deficiency and COVID-19 severity could also be attributed to the effects of COVID-19 on the body metabolism and immune response. Zinc deficiency is more prevalent in the elderly and patients with underlying chronic diseases, with established deleterious consequences such as the increased risk of respiratory infection. We reviewed the expected effects of zinc deficiency on COVID-19-related pathophysiological mechanisms focusing on both the renin-angiotensin and kinin-kallikrein systems. Mechanisms and effects were extrapolated from the available scientific literature. Zinc deficiency alters angiotensin-converting enzyme-2 (ACE2) function, leading to the accumulation of angiotensin II, des-Arg9-bradykinin and Lys-des-Arg9-bradykinin, which results in an exaggerated pro-inflammatory response, vasoconstriction and pro-thrombotic effects. Additionally, zinc deficiency blocks the activation of the plasma contact system, a protease cascade initiated by factor VII activation. Suggested mechanisms include the inhibition of Factor XII activation and limitation of high-molecular-weight kininogen, prekallikrein and Factor XII to bind to endothelial cells. The subsequent accumulation of Factor XII and deficiency in bradykinin are responsible for increased production of inflammatory mediators and marked hypercoagulability, as typically observed in COVID-19 patients. To conclude, zinc deficiency may affect both the renin-angiotensin and kinin-kallikrein systems, leading to the exaggerated inflammatory manifestations characteristic of severe COVID-19.

Hereditary autoinflammatory diseases are caused by gene mutations of the innate immune pathway, e.g. nucleotide receptor protein 3 (NLRP3). Here, we report a four-generation family with cold-induced urticarial rash, arthralgia, chills, headache and malaise associated with an autosomal-dominant inheritance. Genetic studies identify a substitution mutation in gene F12 (T859A, resulting in p.W268R) which encodes coagulation factor XII (FXII). Functional analysis reveals enhanced autocatalytic cleavage of the mutated protein and spontaneous FXII activation in patient plasma and in supernatant of transfected HEK293 cells expressing recombinant W268R-mutated proteins. Furthermore, we observe reduced plasma prekallikrein, cleaved high molecular weight kininogen and elevated plasma bradykinin. Neutrophils are identified as a local source of FXII. Interleukin-1β (IL-1β) is upregulated in lesional skin and mononuclear donor cells exposed to recombinant mutant proteins. Treatment with icatibant (bradykinin-B2-antagonist) or anakinra (interleukin-1-antagonist) reduces disease activity in patients. In conclusion, our findings provide a link between contact system activation and cytokine-mediated inflammation., Systemic autoinflammatory syndromes such as cryopyrin-associated periodic syndrome (CAPS) are rare and often involve genes related to the inflammasome. Here, the authors report a syndrome characterised by systemic inflammation and cold-induced urticarial rash associated with a Factor XII-activating mutation.

Recent studies have shown that the contact activation of blood coagulation can be initiated on the surface of circulating microparticles–particles formed as a result of the activation or apoptosis of blood cells or endothelial cells. In the present work, by means of a mathematical model, we investigated the mechanism of the activation of contact pathway of blood plasma coagulation. The model describes membrane-dependent reactions of the activation of factors XII and XI with account of the presence of blood plasma inhibitors. All reactions were described by ordinary differential equations integrated by an implicit multistep method. The current mathematical model is based on our previous model of factor XII activation on the platelet surface. The initial model is modified by the addition of factor XI, kallikrein, and blood plasma inhibitors. We show that the amidolytic activity of the contact pathway factors associated with the microparticles is proportional to the concentration of microparticles. In previous studies, an increase in the overall solution amidolytic activity after the dilution of plasma was observed. Computational analysis of the contact pathway activation in the diluted plasma shows that the increase in the activation appears from the dilution of blood plasma inhibitors. Thus, a well-known experimental phenomenon of the hypercoagulability of plasma after dilution can be explained by an increased activation of the blood plasma coagulation through the contact pathway on the circulating microparticles. In addition, the computational analysis reveals that a rapid stop of the contact pathway activation on the microparticles observed in the experiments could be explained by the rapid depletion of the free activation surface.

SummaryBacterial pneumonia, the most common cause of sepsis, is associated with activation of coagulation. Factor XI (FXI), the key component of the intrinsic pathway, can be activated via factor XII (FXII), part of the contact system, or via thrombin. To determine whether intrinsic coagulation is involved in host defence during pneumonia and whether this is dependent on FXII activation, we infected in parallel wild-type (WT), FXI knockout (KO) and FXII KO mice with two different clinically relevant pathogens, the Gram-positive bacterium Streptococcus pneumoniae and the Gram-negative bacterium Klebsiella pneumoniae, via the airways. FXI deficiency worsened survival and enhanced bacterial outgrowth in both pneumonia models. This was accompanied with enhanced inflammatory responses in FXI KO mice. FXII KO mice were comparable with WT mice in Streptococcus pneumoniae pneumonia. On the contrary, FXII deficiency improved survival and reduced bacterial outgrowth following infection with Klebsiella pneumoniae. In both pneumonia models, local coagulation was not impaired in either FXI KO or FXII KO mice. The capacity to phagocytose bacteria was impaired in FXI KO neutrophils and in human neutrophils where activation of FXI was inhibited. Deficiency for FXII or blocking activation of FXI via FXIIa had no effect on phagocytosis. Taken together, these data suggest that FXI protects against sepsis derived from Streptococcus pneumoniae or Klebsiella pneumoniae pneumonia at least in part by enhancing the phagocytic capacity of neutrophils by a mechanism that is independent of activation via FXIIa.Supplementary Material to this article is available online at www.thrombosis-online.com.

Background In diabetic retinopathy (DR), neutrophil extracellular traps (NET) and kallikrein-kinin system are considered as contributing factors. However, the detail activation mechanisms has not been fully understood. Since the NET could provide negative-charged surface for factor XII activation and the activated factor XII (XIIa) can initiate kallikrein-kinin system, this study investigated whether patients with DR show activation of NET, factor XII and kallikrein-kinin system. Methods The markers related to NET (DNA-histone complex) and kallikrein-kinin system (high-molecular-weight kininogen, prekallikrein, bradykinin) and factor XIIa were measured in 253 patients with diabetes. To access ex vivo effect of glucose, DNA-histone complex and factor XIIa were measured in whole blood stimulated by glucose. Results The circulating level of DNA-histone complex and factor XIIa were significantly higher in patients with DR than those without DR. In logistic regression analysis, DNA-histone complex, factor XIIa, and high-molecular-weight kininogen were the risk factors of DR. In recursive partitioning analysis, among patients with diabetes duration less than 10 years, patients with high level of DNA-histone complex (>426 AU) showed high risk of DR. In ex vivo experiment, glucose significantly elevated both DNA-histone complex and factor XIIa. Conclusion Our findings suggest that activation of factor XII and kallikrein-kinin system combined with NET formation actively occur in patients with DR and circulating levels of DNA-histone complex, factor XIIa and HMWK can be potential biomarkers to estimate the risk of DR. Strategies against factor XII activation may be beneficial to inhibit DR.

The contact system initiates the intrinsic pathway of coagulation and is started by Factor XII activation, which then activates prekallicrein to kallicrein and Factor XI to Factor XIa and, in the presence of high molecular weight kininogen, forms a “contact phase activation loop”, that amplifies Factor XII activation. FXII deficiency is not associated with bleeding tendencies, but when the blood clots, the thrombus is less dense, thus favoring antithrombotic protection. Activated Factor XII inhibition emerges as an efficient target for preventing thrombo-embolic diseases without inducing a hemorrhagic risk. Activated Factor XII exhibits other activities, in that it can activate complement and provoke inflammation, contributing to innate immunity. It also stimulates fibrinolysis through uPA activation from scu-PA. Among the other components of the contact phase, Factor XI has a more important role in coagulation pathways and can directly activate FX, FVIII and FV, in a FIX independent pathway. Its deficiency is associated with a mild bleeding diathesis (“pseudo-hemophilia” or hemophilia C), with a variable incidence among kindreds. Recently, the occurrence of thrombotic events the same day following infusion of immunoglobulin concentrates has been demonstrated to be caused by the presence of trace amounts of activated Factor XI, pointing out the key role of this factor for thrombogenicity. Prekallicrein can be activated at the endothelial surface in the presence of high molecular weight kininogen, whose cleavage generates bradykinins and contributes to vessel tonicity and inflammation. The contact phase, through its activation loop, is then an important physiological system, which can initiate and regulate various biological functions and is at the crossroads of various biological activities. Many of the body’s physiological functions are intimately linked between them, making the global approach of special usefulness for understanding the interactions which can result from any abnormality of one of them. New pharmaceutical drugs targeting a defined activity need to be investigated for all the possible interferences or side effects. In this article we aim to present and summarize the present understanding of contact phase system activation and regulation, its involvement in various physiological functions, and the laboratory tools for its exploration.

The need to improve blood biocompatibility of medical devices is urgent. As soon as blood encounters a biomaterial implant, proteins adsorb on its surfaces, often leading to several complications such as thrombosis and failure of the device. Therefore, controlling protein adsorption plays a major role in developing hemocompatible materials. In this study, the interaction of key blood plasma proteins with superhemophobic titania nanotube substrates and the blood clotting responses was investigated. The substrate stability was evaluated and fibrinogen adsorption and thrombin formation from plasma were assessed using ELISA. Whole blood clotting kinetics was also investigated, and Factor XII activation on the substrates was characterized by an in vitro plasma coagulation time assay. The results show that superhemophobic titania nanotubes are stable and considerably decrease surface protein adsorption/Factor XII activation as well as delay the whole blood clotting, and thus can be a promising approach for designing blood contacting medical devices.

SummaryBlood coagulation is essential for hemostasis, however excessive coagulation can lead to thrombosis. Factor XII starts the intrinsic coagulation pathway and contact-induced factor XII activation provides the mechanistic basis for the diagnostic aPTT clotting assay. Despite its function for fibrin formation in test tubes, patients and animals lacking factor XII have a completely normal hemostasis. The lack of a bleeding tendency observed in factor XII deficiency states is in sharp contrast to deficiencies of other components of the coagulation cascade and factor XII has been considered to have no function for coagulation in vivo. Recently, experimental animal models showed that factor XII is activated by an inorganic polymer, polyphosphate, which is released from procoagulant platelets and that polyphosphate-driven factor XII activation has an essential role in pathologic thrombus formation. Cumulatively, the data suggest to target polyphosphate, factor XII, or its activated form factor XIIa for anticoagulation. As the factor XII pathway specifically contributes to thrombosis but not to hemostasis, interference with this pathway provides a unique opportunity for safe anticoagulation that is not associated with excess bleeding.The review summarizes current knowledge on factor XII functions, activators and inhibitors.

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, respectively. Heparin and polyphosphate supply a negative surface for factor XII activation, a serine protease that drives contact system-mediated coagulation and inflammation. Activation of the factor XII substrate plasma kallikrein leads to further activation of zymogen factor XII and triggers the pro-inflammatory kallikrein-kinin system that results in the release of the mediator bradykinin. The severity of anaphylaxis is correlated with the intensity of contact system activation, the magnitude of mast cell activation, and bradykinin formation. The main inhibitor of the complement system, C1 esterase inhibitor, potently interferes with factor XII activity, indicating a meaningful crosslink between complement and kallikrein-kinin systems. Deficiency in a functional C1 esterase inhibitor leads to a severe swelling disorder called hereditary angioedema. The significance of factor XII 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 factor XII integrates with inflammation and the complement system to cause anaphylaxis and hereditary angioedema as well as highlight current diagnosis and treatments of bradykinin-related diseases.

The role of factor XII (FXII) as the main trigger of the coagulation cascade during haemodialysis has been recently challenged. Polyvinylchloride (PVC) is the standard polymer for haemodialysis circuit tubings, but its interaction with FXII has not been extensively characterized. In a modified Chandler tubing loop model using heparinized fresh human whole blood we selectively inhibited coagulation factors VII, X or XII with monospecific antibodies. Contact of whole blood with PVC induced a strong thrombin generation [thrombin–antithrombin complexes (TAT) 64 ± 24 μg/l, before

Pollen Typhae is the traditional Chinese herbal medicine widely used to treat the hemorrhagic diseases both by external and oral application. The present study examines the hemostatic properties and its components of Pollen Typhae. Pollen extract significantly reduced prothrombin time (PT), activated partial prothrombin time (APTT) and recalcification time. Pollen extract directly activated factor XII in the coagulation cascade. Acidic polysaccharide in the pollen that adsorbed to the diethylaminoethyl (DEAE) column was the causative agent of factor XII activation. These results suggested that an electronegative charge attributed to an acidic polysaccharide in the pollen extract contributed to the hemostatic activity. We then examined the hemostatic activity of administered pollen extract in the mouse tail bleeding model. Tail bleeding was significantly decreased after oral administration of the pollen extract, whereas the acidic polysaccharide fraction did not affect the duration of tail bleeding. These results suggest that the oral anticoagulant effect of Pollen Typhae is attributed to compounds other than acidic polysaccharides. We concluded that the activation of the intrinsic coagulation pathway by the acidic polysaccharide contributes to the external hemostatic property of Pollen Typhae, and the action of components such as flavonoids that possess anticoagulant activity are causative agent when orally administered.

For decades it has been known that activated platelets promote plasma clotting and that the fibrin forming activity of activated platelets is dependent on blood coagulation factor XII. However, because factor XII deficiency is not associated with any bleeding disorder, platelet-driven factor XII activation was believed to be an in vitro artefact with no importance for coagulation in vivo. Using arterial and venous thrombosis models in factor XII deficient mice we recently demonstrated that factor XII is essential for thrombosis in vivo. However it was not known how factor XII is activated by procoagulant platelets within the growing thrombus. Here, we review our studies of the last 5 years that led to the identification of the endogenous factor XII activator. We found that platelets release polyphosphates, linear inorganic polymers of 60-100 phosphate residues that directly bound to and activated factor XII. Platelet polyphosphates potently initiate fibrin formation via the factor XII-driven intrinsic pathway. Inhibition of factor XII or polyphosphates protected mice from lethal thrombotic disease and strongly reduced clot stability in patients. Our data identify polyphosphates as the endogenous factor XII activator in vivo linking platelet activation (primary hemostasis) and fibrin production (secondary hemostasis). Targeting polyphosphate-mediated factor XII activation offers the exciting possibility for a safe anticoagulation with minimal therapy-associated bleeding.

The continued search for the ideal antithrombotic agent that would prevent or reduce thrombus growth inside blood vessels without an effect on the essential hemostatic functions of blood, including extraluminal thrombin generation and platelet activation, has been going in new directions in the past decade. These directions include studies suggesting that activation of the intrinsic coagulation cascade through contact activation of factor XII, and the resultant thrombin generation is a pathologic event that leads to undesirable consequences. Recent animal studies of contact pathway inhibitors in experimental thrombogenesis suggest that the contact activation pathway of blood coagulation may play a pathogenic role in thrombosis, and pharmacologic inhibition of contact activation may have antithrombotic effects. Development of reasonably potent selective inhibitors of contact activation pathway components or activities now allow for the conduct of studies in various animal species, including primates. These studies have generated interesting data that now support the hypothesis that thrombogenesis in humans may also involve the pathological activation of the intrinsic coagulation cascade. Moreover, baboons, mice, and rabbits, pretreated with antibodies that selectively inhibit factor XII activation, procoagulant factor XIIa activity, or all enzymatic activities of factor XIIa show significantly reduced propensity for occlusive thrombus propagation in various models of acute thrombogenesis on vascular grafts, membrane oxygenators, and even on injured arteries. Since contact activation of blood does not play a demonstrable role in normal hemostasis, and there has been no evidence generated to date suggesting that activation of factor XII has a physiologic function, current research now supports the original concept, developed over 2 decades ago, that temporal pharmacologic inhibition of thrombin generation through the contact pathway may have therapeutic potential and could produce beneficial antithrombotic activities without hemostasis impairment.

Platelet activation and blood coagulation are essential for hemostasis and contribute to a variety of other biological processes such as inflammation, complement activation and tissue repair. Factor XII, originally called Hageman factor, plays an important role in the kallikrein-kinin system by activating prekallikrein. In the 1960s, a platelet activity that promoted factor XII activation was identified but its biochemical nature remained unknown. Inorganic polyphosphates (poly P) are polymers that consist of many phosphate residues linked by phosphoanhydride bonds. These polymers exist in all living organisms. In bacteria, poly P is important for growth and survival. Recently, poly P has been identified in human platelet dense granules. Studied have shown that upon platelet activation and secretion, poly P activates factor XII, indicating that it is most likely the elusive platelet factor XII activator. Poly P also regulates coagulation and fibrinolysis. In this review, we focus on early studies of factor XII and the identification of platelet factor XII activation activity, and discuss recent findings of poly P in factor XII activation and coagulation.

SummaryPlatelets play a central role in thrombosis, hemostasis, and inflammation. We show that activated platelets release inorganic polyphosphate (polyP), a polymer of 60–100 phosphate residues that directly bound to and activated the plasma protease factor XII. PolyP-driven factor XII activation triggered release of the inflammatory mediator bradykinin by plasma kallikrein-mediated kininogen processing. PolyP increased vascular permeability and induced fluid extravasation in skin microvessels of mice. Mice deficient in factor XII or bradykinin receptors were resistant to polyP-induced leakage. PolyP initiated clotting of plasma via the contact pathway. Ablation of intrinsic coagulation pathway proteases factor XII and factor XI protected mice from polyP-triggered lethal pulmonary embolism. Targeting polyP with phosphatases interfered with procoagulant activity of activated platelets and blocked platelet-induced thrombosis in mice. Addition of polyP restored defective plasma clotting of Hermansky-Pudlak Syndrome patients, who lack platelet polyP. The data identify polyP as a new class of mediator having fundamental roles in platelet-driven proinflammatory and procoagulant disorders.

In vivo mouse models have indicated that the intrinsic coagulation pathway, initiated by factor XII, contributes to thrombus formation in response to major vascular damage. Here, we show that fibrillar type I collagen provoked a dose-dependent shortening of the clotting time of human plasma via activation of factor XII. This activation was mediated by factor XII binding to collagen. Factor XII activation also contributed to the stimulating effect of collagen on thrombin generation in plasma, and increased the effect of platelets via glycoprotein VI activation. Furthermore, in flow-dependent thrombus formation under coagulant conditions, collagen promoted the appearance of phosphatidylserine-exposing platelets and the formation of fibrin. Defective glycoprotein VI signaling (with platelets deficient in LAT or phospholipase Cγ2) delayed and suppressed phosphatidylserine exposure and thrombus formation. Markedly, these processes were also suppressed by absence of factor XII or XI, whereas blocking of tissue factor/factor VIIa was of little effect. Together, these results point to a dual role of collagen in thrombus formation: stimulation of glycoprotein VI signaling via LAT and PLCγ2 to form procoagulant platelets; and activation of factor XII to stimulate thrombin generation and potentiate the formation of platelet-fibrin thrombi.

SummarySulfated galactan from the red alga Botryocladia occidentalis has a potent anticoagulant activity,due to its ability to enhance thrombin and factor Xa inhibition by antithrombin and/or heparin cofactor II. It is less active than unfractionated heparin in arterial thrombosis,but in a venous thrombosis presents a dual effect,inhibiting thrombosis in low but not in high doses.This dual effect on venous thrombosis is a consequence of two actions, one that inhibits thrombin and factor Xa and one that induces factor XII activation. In order to dissociate these effects, we prepared derivatives of the sulfated galactan with low molecular weights. Two fractions that were similar in size to unfractionated heparin and low-molecular-weight heparin were obtained. As the molec- ular weight decreased, the ability to activate factor XII and to promote inhibition of coagulation proteases in the presence of antithrombin and heparin cofactor II diminished.At ∼5 kDa, the sulfated galactan fragment had no effect on factor XII activation, and showed the same effect as unfractionated heparin in a venous thrombosis model.The ∼5-kDa fragment is an antithrombotic with several advantages: i) It is as active as unfractionated heparin in venous thrombosis, but it has little activity in arterial thrombosis; ii) It inhibits venous thrombosis with very little anticoagulant effect; iii) It does not cause bleeding; and iv) It is not obtained from mammals.

The polymer poly-N-acetylglucosamine (pGlcNAc) containing fiber material is becoming increasingly important as a topical agent for hemostasis at wound sites. The pGlcNAc polymeric fiber provides hemostasis through redundant mechanisms that include platelet activation for fibrin network formation. The research presented here better defines the mechanism for the effect of pGlcNAc containing fibers on platelet-mediated processes. Adsorption experiments demonstrated that pGlcNAc fibers tightly bind most major plasma proteins and a specific sub-set of platelet surface proteins, including the integrin beta(3) subunit (CD61) and the von Willebrand receptor GP1b (CD42b). The result of this interaction is a platelet-dependent acceleration of fibrin gel formation. Accelerated fibrin polymerization is sensitive to factor XII inhibition by corn trypsin inhibitor and integrin inactivation with integrilin. Confocal microscopy studies show that when platelet integrins contact plasma protein-saturated pGlcNAc fibers, an increase in intracellular free calcium for platelet activation occurs to drive surface expression of phosphatidyl serine (PS). Thus, a catalytic surface for thrombin generation and accelerated fibrin clot formation results from the interaction of platelets with pGlcNAc. These findings, when considered with the observation that pGlcNAc fibers also induce red blood cell agglutination and vasoconstriction, provides an explanation for the ability of the pGlcNAc material to provide hemostasis in a wide variety of clinical applications.

Blood coagulation is thought to be initiated by plasma protease factor VIIa in complex with the membrane protein tissue factor. In contrast, coagulation factor XII (FXII)–mediated fibrin formation is not believed to play an important role for coagulation in vivo. We used FXII-deficient mice to study the contributions of FXII to thrombus formation in vivo. Intravital fluorescence microscopy and blood flow measurements in three distinct arterial beds revealed a severe defect in the formation and stabilization of platelet-rich occlusive thrombi. Although FXII-deficient mice do not experience spontaneous or excessive injury-related bleeding, they are protected against collagen- and epinephrine-induced thromboembolism. Infusion of human FXII into FXII-null mice restored injury-induced thrombus formation. These unexpected findings change the long-standing concept that the FXII-induced intrinsic coagulation pathway is not important for clotting in vivo. The results establish FXII as essential for thrombus formation, and identify FXII as a novel target for antithrombotic therapy.

Summary. β2-glycoprotein I (β2GPI) bears the epitope(s) for autoimmune anticardiolipin antibodies (aCL) frequently present in patients with antiphospholipid syndrome (APS). β2GPI is involved in coagulation and fibrinolytic systems, including inhibition of contact activation. Coagulation factor XII is an initiator of intrinsic coagulation and also of intrinsic fibrinolysis. We investigated the effect of aCL (= anti-β2GPI antibodies), regarding intrinsic fibrinolysis using autoimmune monoclonal anti-β2GPI antibodies derived from a patient with APS or from an NZW/BXSB-F1 mouse. We developed a chromogenic assay system to determine intrinsic fibrinolytic activity. The reaction was activated by kaolin in the euglobulin fraction. Exogenous β2GPI slightly suppressed intrinsic fibrinolytic activity of the euglobulin fraction from normal plasma. Human monoclonal anti-β2GPI antibody (EY2C9) and mouse monoclonal anti-β2GPI antibody (WBCAL-1) in the presence of β2GPI decreased the activity. In this system, the suppression remained significant in the presence of an excess of exogenous activated factor XII. Euglobulin fractions from APS patients' plasma paralleled low activities of intrinsic fibrinolysis compared with those from healthy subjects. Our results suggest that β2GPI and anti-β2GPI antibodies suppress intrinsic fibrinolytic activities. This suppression was not only due to inhibition of factor XII activation but was also related to function of activated factor XII (XIIa). These phenomena partly explain the mechanisms of thrombosis in APS.

Hereditary angioedema (HAE) is a term coined for a familial form of potentially life-threatening angioedema first described by William Osler in 1888. It is inherited as an autosomal dominant and the older literature often employs the term angioneurotic edema because it was believed that swelling could be a consequence of an emotional disorder. This type of angioedema, in particular, often included family histories of death by asphyxiation so that family members appeared “neurotic,” that is, overly fearful, anxious, depressed, and so on. It also turns out that episodes of angioedema may well be triggered by emotional events in addition to other known triggers such as trauma, infection, and estrogen-containing hormones, but the biochemical explanation for this is not yet known. However, the conception of the disorder changed in 1953 when Donaldson and Evans discovered that it is due to a mutation in a plasma enzyme inhibitor known as C1 inhibitor (C1 INH).1 It was then assumed that some vasoactive mediator from the complement system was the proximate cause of the angioedema. The next era dealt with attempts to identify this vasoactive factor. It then became clear that his inhibitor could inactivate enzymes that are not part of the complement cascade. The first discovery was that it inhibited plasma kallikrein,2 an enzyme that cleaves a plasma kininogen to produce bradykinin. At that point, no one seriously considered bradykinin as a possible cause of the swelling. The focus was on a kinin thought to be derived from the complement cascade termed C2 kinin. The key experiment, published in a nonrefereed journal,3 was that activated C1 will cleave C2 (in the presence of C4 as a cofactor) to yield C2a C2b, and that plasmin digestion of C2b would produce a kinin that could be distinguished from bradykinin by its inactivation by tryptic digestion. It could be assayed by contraction of an estrous rat uterus or with lesser sensitivity (but greater reliability), employing a guinea pig ileum. This observation was never confirmed by other workers but additional observations seemed to substantiate the idea. Studies of vascular permeability in human and rabbit skin indicated that injection of activated C1 led to increased vascular permeability that required C2; the swelling was augmented when skin testing of patients with hereditary angioedema was compared with normal controls.4 There was even one publication by Donaldson5 suggesting a linkage between activated Factor XII (activated Hageman factor) and activation of the complement cascade so that simultaneous production of bradykinin (inhibited by Factor XII activation) and the C2-kinin seemed possible. During the years 1972–1990 I was studying the mechanism of activation of the plasma bradykinin cascade. Two forms of activated Factor XII were described, that is, Factor XIIa at about 80 kd and Factor XII fragment (XIIf) at 28.5–30 kd6,7; soon thereafter, the Factor XII native protein was purified.8 Factor XIIa or Factor XIIf was shown to be required for kallikrein activity to be produced in plasma; prekallikrein was purified,9 and its mechanism of conversion to kallikrein described. A controversy regarding the number of kininogens in plasma was resolved to indicate that there is a high molecular weight kininogen (HK) that is rather selectively cleaved by plasma kallikrein10,11 to yield bradykinin, whereas low molecular weight kininogen is preferentially cleaved by tissue kallikrein to produce lysylbradykinin (kallidin). About 80% of plasma kallikrein was found to circulate as a complex with HK12 and the distal light chain segment of HK was shown to be the feature that distinguishes it from low molecular weight kininogen (LK). This HK domain has the site of prekallikrein binding13,14 and the site responsible for the cofactor activity of HK, which accelerates the conversion of prekallikrein to kallikrein and also conversion of coagulation factor XI to factor XIa.14 Activation of the bradykininforming cascade was known to occur upon binding to negatively charged surfaces (eg, a glass test tube to clot blood) and this acceleration by surface binding was studied in some detail.15,16 Factor XII was shown to autoactivate upon binding17 so a molecular mechanism for initiation of the cascade seemed to be in place. From the onset of these studies, in the late 1970s, I was of the opinion that bradykinin is the mediator of the swelling seen in hereditary angioedema and that C2 kinin might be an artifact. My bias was based on a conversation with Frank Austen that occurred when the C2 kinin data were presented at a plenary session of The Association of American Physicians and I was seated in the audience. A rat uterus–based bioassay was used to demonstrate the new kinin and I had been trying to use such an assay to measure bradykinin for months. Its baseline was poor, it contracted when agonist (any agonist, at times including the buffer control) was added to the bath solution, and I could not be sure what was real and what was not. I switched to using a guinea pig ileum. I looked at the data being presented, I turned to Frank Austen, who was sitting next to me, and exclaimed, “That’s an artifact!” I could not, however, explain the C2–dependent increase in vascular permeability reported by skin testing and am not sure I could do so even now. One issue is that anything injected into hereditary angioedema Medical University of South Carolina, Charleston, South Carolina. Correspondence to Allen P. Kaplan, MD, FAAAAI, 17 Logan Street, Charleston, SC 29401. Telephone: (843) 573-9373. E-mail: kaplana@musc.edu. Copyright © 2011 by World Allergy Organization

Fucosylated chondroitin sulfate (FCS), a structurally unusual glycosaminoglycan, has distinct anticoagulant properties, and is an especially strong inhibitor of the intrinsic factor Xase (anti-Xase). To obtain a highly selective inhibitor of human Xase, we purified six native FCSs with various sulfation patterns, prepared a series of FCS derivatives, and then elucidated the relationship between the structures and the anticoagulant activities of FCSs. FCSs 1-3 containing higher Fuc2S4S exhibit stronger AT-dependent anti-IIa activities, whereas 4-6 containing more Fuc3S4S produce potent HCII-dependent anti-IIa activities. Saccharides containing a minimum of 6-8 trisaccharide units, free carboxyl groups, and full fucosylation of GlcA may be required for potent anti-Xase activity, and approximately six trisaccharide units and partial fucosylation of GlcA may contribute to potent HCII-dependent activity. Decreasing of the molecular weights markedly reduces their AT-dependent anti-IIa activities, and even eliminates human platelet and factor XII activation. Furthermore, in vitro and in vivo studies suggested that fractions of 6-12 kDa may be very promising compounds as putative selective intrinsic Xase inhibitors with antithrombotic action, but without the consequences of major bleeding and factor XII activation. (C) 2014 Elsevier Masson SAS. All rights reserved.

The formation of bradykinin in plasma requires interaction of three proteins, namely coagulation factor XII (Hageman factor), prekallikrein and high-molecular-weight kininogen (HK). Prekallikrein and HK circulate as a bimolecular complex. Initiation of the cascade upon binding to negatively charged surfaces (or macromolecules) is dependent on factor XII autoactivation, conversion of prekallikrein to kallikrein, and a feedback activation of factor XII by kallikrein. The latter reaction is extremely rapid relative to factor XII autoactivation. The kallikrein then digests HK to liberate bradykinin. The natural surface appears to be vascular endothelial cells which express binding proteins for factor XII and HK, and activation can proceed along the cell surface. Recent findings demonstrate that prekallikrein has enzymatic activity separate from that of kallikrein such that it can stoichiometrically bind and cleave HK to liberate bradykinin. It is normally prevented from doing so by the plasma C1 inhibitor. Release of heat shock protein 90 (HSP-90) from endothelial cells can convert prekallikrein to kallikrein (stoichiometrically) within the prekallikrein-HK complex, even in the absence of factor XII, and the prekallikrein-HK complex can autoactivate to generate kallikrein if phosphate is the buffering ion. The effects of phosphate ion and HSP-90 are additive. Thus, an active site appears to be induced in prekallikrein by binding to HK and any of the aforementioned reactions can generate kallikrein prior to factor XII activation by autoactivation of the HK-PK complex. This brief review highlights the major discoveries made over the past 50 years which have led to our current concepts regarding the constituents and mechanisms of activation of the plasma bradykinin-forming cascade.

Background We have previously reported that prekallikrein expresses an active site when it is bound to high-molecular-weight kininogen (HK) and can digest HK to produce bradykinin. The reaction is stoichiometric and inhibited by C1 inhibitor (C1-INH) or corn trypsin inhibitor. Addition of heat shock protein 90 leads to conversion of prekallikrein to kallikrein in a zinc-dependent reaction. Objective Our goal was to determine whether these reactions are demonstrable in plasma and distinguish them from activation through factor XII. Methods Plasma was incubated in polystyrene plates and assayed for kallikrein formation. C1-INH was removed from factor XII–deficient plasma by means of immunoadsorption. Results We demonstrate that prekallikrein-HK will activate to kallikrein in phosphate-containing buffers and that the rate is further accelerated on addition of heat shock protein 90. Prolonged incubation of plasma deficient in both factor XII and C1-INH led to conversion of prekallikrein to kallikrein and cleavage of HK, as was seen in plasma from patients with hereditary angioedema but not plasma from healthy subjects. Conclusions These results indicate that C1-INH stabilizes the prekallikrein-HK complex to prevent HK cleavage either by prekallikrein or by prekallikrein-HK autoactivation to generate kallikrein. In patients with hereditary angioedema, kallikrein and bradykinin formation can occur without invoking factor XII activation, although the kallikrein formed can rapidly activate factor XII if it is surface bound.

SummaryIt is now well recognised that antiphospholipid antibodies are associated with thrombosis and recurrent fetal loss. Some antiphospholipid antibodies (aPAs) have been shown to require a cofactor, β2 glyco-protein-I (β2GPI), for binding to phospholipids, and recently β2GPI has been identified as the antigenic target for some aPAs. β2GPI possesses in vitro anticoagulant properties and modulation of β2GPI function may therefore result in altered haemostatic regulation. In the present study, the influence of plasma derived aPAs and β2GPI on factor XII activation on the surface of very low density lipoprotein (VLDL) was investigated. Factor XIIa generation was dependent on lipoprotein lipase treatment of VLDL and β2GPI inhibited the factor XIIa generation in a concentration dependent manner. No consistent effects on factor XIIa generation were demonstrated with the IgG fractions from patients with aPAs. Inhibition of the β2GPI activity was demonstrated by some antibodies, and study with cardiolipin affinity purified antibody indicated that antibody concentration is critical. These results suggest that perturbation of β2GPI function may contribute to the pathogenic mechanism for thrombosis in some patients with aPAs. .

SummaryThe physiological role of the plasma protein factor XII (FXII), as well as its involvement in human pathology, is poorly understood. While FXII is implicated in thrombotic pathology as a coagulation factor, it can contribute to inflammatory conditions without triggering coagulation. We recently generated nanobodies against the catalytic domain of activated FXII (FXIIa). Here, we describe two of these nanobodies, A10 and B7, both of which do not recognise FXII. Nanobody A10 recognises the catalytic domain of purified β-FXIIa (80 kDa), but not that of purified α-FXIIa (28 kDa), whereas nanobody B7 recognises both. This suggests minute differences in the catalytic domain between these isoforms of FXIIa. The detection of FXIIa by these nanobodies in plasma can become compromised through inactivation by serine protease inhibitors. This effect can be efficiently countered through the addition of the small-molecular protease inhibitor PPACK. Finally, we show that our nanobody-based assays in vitro distinguish various activation products of FXII that differ with the type of activator present: whereas procoagulant activators solely trigger the formation of a species that is captured by B7, proinflammatory activators first generate a species that is recognised by B7, which is later converted into a species that is recognised by A10. These findings suggest that a progressive proteolysis of FXIIa results in the generation a non-procoagulant form of FXIIa, whereas retention of intermediate forms triggers coagulation. Moreover, our findings indicate the development of nanobodies against activated enzymes offers improved opportunities to investigate their contribution to health and disease.

The contact system is a protease cascade that is initiated by factor XII activation on cardiovascular cells. The system starts procoagulant and proinflammatory reactions, via the intrinsic pathway of coagulation and the kallikrein-kinin system, respectively. The biochemistry of the contact system in vitro is well understood. However, activators of the system in vivo and their contributions to disease states have remained enigmatic. Recent experimental and clinical data have identified misfolded proteins, collagens, and polyphosphates as the long-sought activators of the contact system in vivo. Here we present an overview about contact system activators and their contributions to health and pathology.

Background: Particulate matter (PM) is a key component of ambient air pollution and has been associated with an increased risk of thrombotic events and mortality. The underlying mechanisms remain unclear. Objectives: To study the mechanisms of PM-driven procoagulant activity in human plasma and to investigate mainly, the coagulation driven by ultrafine particles (UFPs

The effect of zinc ions (Zn(II)) on the activation of factor XII in the absence of a procoagulant surface was investigated by initial velocity kinetic studies at I = 0.15, pH 7.4, and 25 degrees C. Zinc ions at concentrations greater than 160 microM potentiated 99-fold the kcat/KM for the activation of factor XII by kallikrein and, at an optimum concentration of 110 microM, accelerated 140-fold the apparent kcat/KM for factor XII autoactivation. High molecular weight kininogen had no effect on either metal-potentiated reaction. Analysis of the factor XII concentration dependence of initial activation rates revealed that Zn(II), at levels that saturate the effect, accelerates kallikrein activation of factor XII by lowering KM (from 52 to 7.3 microM) and raising kcat (from 2.6 to 31 min-1). For the autocatalytic activation reaction of factor XII in the presence of optimal Zn(II), apparent KM and kcat values of 2.4 microM and 0.041 min-1, respectively, were determined, but these parameters were not resolvable in the absence of the metal ion. Zinc ions minimally affected kallikrein enzymatic activity and inhibited factor XIIa enzymatic activity with KI values of 20-40 microM, suggesting that the rate-enhancing effects of the metal ion are due to interactions with the substrate (factor XII) rather than with the enzyme. The Zn(II) inhibition of factor XIIa enzymatic activity accounted for a decreased Zn(II) enhancement of factor XII autoactivation at high metal ion concentrations (> 110 microM). The Zn(II) concentration dependence of the acceleration of factor XII activation reactions were sigmoid and characterized by Hill coefficients of 3.3-4.3, suggesting that cooperative binding of at least four zinc ions to factor XII was responsible for the Zn(II) potentiating effect. The Zn(II) enhancement of the rates of factor XII activation decreased both above and below pH 7.4 with midpoint pH values of 6.5-7.0 and 8.0, consistent with histidine and possibly water ligands mediating Zn(II) binding to the protein. Despite an apparent weaker binding of Zn(II) to factor XII at pH 6.5, indistinguishable maximum accelerating effects of the metal ion were observed at saturation at this pH, indicating that the increased positive charge of factor XII resulting from protonation at the lower pH did not mimic the effect of Zn(II) binding. These results imply that zinc ions induce a conformational change in factor XII that makes it a better substrate for its enzyme activators.

The activation of factor XI initiates the intrinsic coagulation pathway. Until recently it was believed that the main activator of factor XI is factor XIIa in conjunction with the cofactor high molecular weight kininogen on a negatively charged surface. Two recent reports have presented evidence that in a purified system factor XI is activatable by thrombin together with the soluble polyanion dextran sulfate. To assess the physiological relevance of these findings we studied the activation of factor XI in normal and factor XII-deficient plasma. We used either kaolin/cephalin or dextran sulfate as a surface for the intrinsic coagulation pathway, tissue factor to generate thrombin via the extrinsic pathway, or the addition of alpha-thrombin directly. 125I-factor XI, added to factor XI-deficient plasma at physiologic concentrations (35 nmol/L), is rapidly cleaved on incubation with kaolin. The kinetics appear to be exponential with half the maximum cleavage at 5 minutes. Similar kinetics of factor XI cleavage are seen when 40 nmol/L factor XIIa (equal to 10% of factor XII activation) is added to factor XII-deficient plasma if an activating surface is provided. Tissue factor (1:500) added to plasma did not induce cleavage of factor XI during a 90-minute incubation, although fibrin formation within 30 seconds indicated that thrombin was generated via the extrinsic pathway. Adding 1 mumol/L alpha-thrombin (equivalent to 50% prothrombin activation) directly to factor XII deficient or normal plasma (with or without kaolin/cephalin/Ca2+ or dextran sulfate) led to instantaneous fibrinogen cleavage, but again no cleavage of factor XI was observable. We conclude that in plasma surroundings factor XI is not activated by thrombin, and that proposals of thrombin initiation of the intrinsic coagulation cascade are not supportable.

Platelets play a central role in thrombosis, hemostasis, and inflammation. We show that activated platelets release inorganic polyphosphate (polyP), a polymer of 60-100 phosphate residues that directly bound to and activated the plasma protease factor XII. PolyP-driven factor XII-activation triggered release of the inflammatory mediator bradykinin by plasma kallikrein-mediated kininogen processing. PolyP increased vascular permeability and induced fluid extravasation in skin microvessels of mice. Mice deficient in factor XII or bradykinin receptors were resistant to polyP-induced leakage. PolyP initiated clotting of plasma via the contact pathway. Ablation of intrinsic coagulation pathway proteases factor XII and factor XI protected mice from polyP-triggered lethal pulmonary embolism. Targeting polyP with phosphatases interfered with procoagulant activity of activated platelets and blocked platelet-induced thrombosis in mice. Infusion of polyP restored defective plasma clotting of Hermansky-Pudlak Syndrome patients, which lack platelet polyP. The data identify polyP as a new class of mediator having fundamental roles in platelet-driven proinflammatory and procoagulant disorders.

New interest in factor XII has been found due to the fact that its murine knockout animal is protected from arterial thrombosis in several models (1,2). The mechanism(s) for these observations have not been precisely described. However, several investigators have shown that factor XII converting to factor XIIa presumably by autoactivation on surfaces such as platelet polysomes, expressed RNA, exposed vascular collagen, and/or aggregated protein contributes to developing thrombus (3–6). Since factor XII participates in the size of an induced-clot formation it has a role in thrombosis. This activity describes for the first time an in vivo pathophysiologic event contributed to by factor XII/XIIa. It is known from the observations of Ratnoff and others that factor XII is essential for surface activated blood coagulation reactions (7). Recognition that factor XII initiates test-tube blood coagulations was the basis for the waterfall hypothesis for the blood coagulation system (8). However, factor XII deficient patients do not have a bleeding defect even though their surface-activated blood coagulation tests are prolonged. Thus, factor XII is not a hemostatic protein. The dichotomy of factor XII being needed for normal blood coagulation tests, but not hemostasis has confused this field for decades. Now that it is generally agreed that tissue factor is the physiologic initiator of blood coagulation leading to hemostasis, the contact activation system needs to be redefined (9,10). Contact activation mechanisms explain how common blood coagulations tests like the activated partial thromboplastin time (APTT) or activated coagulation time (ACT) which are performed on millions of patients annually work. The molecular mechanism for factor XII autoactivation still is not known. Factor XII autoactivation at the molecular level occurs by imposing specific orientation and ordering on the adsorbed protein molecules on sum frequency generation vibrational spectroscopy (11). Alternatively, factor XII in vivo may be activated by contact as described above with various biologic substances in developing thrombus or secondary to formed plasma kallikrein, its only known enzyme activator. The kinetics of factor XII activation by plasma kallikrein (Km=11 µM) is similar to factor XIIa activation of prekallikrein (Km=2.4 µM). However, the Km of prekallikrein activation in vivo by its endothelial cell membrane expressed serine protease, prolylcarboxypeptidase, is 9 nM (12). These latter kinetic data suggest that physiologic factor XII activation is secondary to plasma kallikrein formation. However, it has yet to be shown if prolylcarboxypeptidase is important for in vivo prekallikrein activation for factor XII activation. Although C1 inhibitor deficient mice have angioedema, indicating higher constitutive bradykinin formation from either unregulated plasma kallikrein or factor XIIa, factor XII deficient mice have lower levels of basal plasma bradykinin, indicating factor XII’s importance in the peptide’s formation (13,14). The observation that the presence of factor XII contributes to the size of induced thrombus formation indicates that its inhibition may be a pharmacologic target to decrease the extent of thrombosis. Although factor XII deficiency was initially thought to be a risk factor for venous thrombosis, more recent studies indicate that it is not. However, human in vivo studies on the role of factor XII in arterial thrombosis also are not clear. Kanaji et al. reported that a polymorphism 46C to T substitution in the 5’-untranslated region of factor XII gene is associated with low translational efficiency and decreased plasma levels (15). This polymorphism may account for the commonly recognized fact that factor XII levels are lower in Asians when compared to Caucasians. An initial report suggested that the homozygous 46TT allele protects from acute coronary syndrome and that lower XII levels may be associated with lower thrombosis risk (16,17). Two other reports suggested that opposite, i.e., the TT genotype is associated with a higher risk of coronary artery disease (18,19). The degree of lowering of factor XII levels by the TT allele is probably insufficient to significantly reduce the amplifying effect of factor XII’s autoactivation on developing clot to really influence thrombosis risk (20). Not much is known how factor XII levels in plasma are regulated. Estrogen therapy increases plasma factor XII levels which are mainly produced in the liver as zymogens. The factor XII gene contains a sequence resembling the consensus estrogen-responsive element (20). The hepatocyte nuclear factor-4 (HNF-4) transcription factor inhibits estrogen induction of factor XII promoter in fibroblasts but not in HepG2 cells where its binding contributes to estrogen induction of the gene (21). HNF-4 null mice exhibit reduced expression of factor XII (22). In the present report of Thrombosis Research, Sabater-Lleal et al. contribute to our understanding on what regulates factor XII plasma levels (23). These authors describe two new mutations, a C/G substitution at position -8 and a C/T substitution at position -13 of the factor XII gene that result in decreased expression levels. These mutations interfere with nuclear proteins interacting with the factor XII promoter. This region is in the putative HNF-4 binding site of the factor XII gene promoter. These polymorphisms summated with the 46C/T polymorphism to lower plasma factor XII levels. What is the physiologic role of factor XII? The answer to this question also is not completely known but new interpretations are coming to this field. In addition to its possible influence on arterial thrombosis, factor XII also may be a growth factor. In the intravascular compartment, factor XII has a multiprotein receptor that consists of the urokinase plasminogen activator receptor, gC1qR, and cytokeratin 1 (24). Factor XII has been recognized as a growth factor leading to ERK1/2 activation (25). Factor XII’s artificial and cell binding site has been localized to its fibronectin type II domain which is adjacent to its epidermal growth factor domain, suggesting its growth promoting activities may be mediated by its cell binding site. Since collagen-activated platelets provide sufficient zinc ion to promote factor XII binding to cells and developing clot and collagen exposure is sufficient for its activation in flowing blood, factor XII in the developing thrombosis may also stimulate repair for this injury (6,24–26). An appreciation for the role of factor XII as a growth factor is emerging. This activity may be important for repair after thrombosis or inflammation. Better understanding of factor XII expression and activities should lead to better characterization of thrombus formation and its repair.

FXII was identified 50 years ago as a coagulation protein in the intrinsic pathway of blood coagulation as FXII deficient patients had marked prolongation of the in vitro surface-activated coagulation time. However, series of investigations have convincingly shown that FXII has no role in normal hemostasis. Recently, experimentally induced thrombosis in factor XII-knockout mice has provided evidence that factor XII (FXII) deficient mice are protected against ischemic brain injury after obstructive clot formation. Based on these experiments it has, therefore, been suggested, that blocking of FXII could be a unique target to prevent obstructive clot formation in arterial thrombosis without side effect of increased bleeding. FXII deficiency has, however, not convincingly been shown to protect against arterial thrombosis in humans. The target mentioned above may either be an inhibition of FXII activation or an inhibition of its proteolytic activity. FXII is a zymogen of the proteolytic enzyme, FXIIa, the substrates of which are factor XI and prekallikrein. Thus, FXIIa is not only involved in the activation of the coagulation system, but is also associated with the kallikrein/kinin system. The activation of the latter is deeply involved in inflammation and pain sensation. Furthermore, FXIIa binds to endothelial cells and to the extracellular matrix, indicating a role in vascular repair. Therefore, a complete evaluation of all these properties of FXII and FXIIa has to be considered when formulating a strategy for blocking FXII activation.

Beta2-glycoprotein-I (beta2GPI) is a phospholipid-binding plasma protein that consists of five homologous domains. Domain V is distinguished from others by bearing a positively charged lysine cluster and hydrophobic extra C-terminal loop. Beta2GPI has been known as a natural anticoagulant regulator. Beta2GPI exerts anticoagulant activity by inhibition of phospholipid-dependent coagulation reactions such as prothrombinase, tenase, and factor XII activation. It also binds factor XI and inhibits its activation. On the other hand, beta2GPI inhibits anticoagulant activity of activated protein C. According to the data from knockout mice, beta2GPI may contribute to thrombin generation in vivo. Phospholipid-bound beta2GPI is one of the major target antigens for antiphospholipid antibodies present in patients with antiphospholipid syndrome (APS). Binding of pathogenic anti-beta2GPI antibodies increases the affinity of beta2GPI to the cell surface and disrupts the coagulation/fibrinolysis balance on the cell surface. These pathogenic antibodies activate endothelial cells via signal transduction events in the presence of beta2GPI. Impaired fibrinolysis has been reported in patients with APS. Using a newly developed chromogenic assay, we demonstrated lower activity of intrinsic fibrinolysis in euglobulin fractions from APS patients. Addition of monoclonal anti-beta2GPI antibodies with beta2GPI also decreased fibrinolytic activity in this assay system. beta2GPI is proteolytically cleaved by plasmin in domain V (nicked beta2GPI) and becomes unable to bind to phospholipids, reducing antigenicity against antiphospholipid antibodies. This cleavage occurs in patients with increased fibrinolysis turnover. Nicked beta2GPI binds to plasminogen and suppresses plasmin generation in the presence of fibrin, plasminogen, and tissue plasminogen activator (tPA). Thus, nicked beta2GPI plays a role in the extrinsic fibrinolysis via a negative feedback pathway loop.

Thrombolytic therapy activates the contact system, and factor XII activation may activate the coagulation cascade and inflammation. It is not known whether an early inflammatory response is induced by thrombolytic therapy in patients with acute myocardial infarction (AMI). We prospectively measured the plasma levels of activated factor XII, cleaved kininogen, prothrombin fragment 1 + 2 (as indexes of the contact phase and coagulation activation), and interleukin-6 and C-reactive protein (CRP) (as indexes of inflammation) in 39 patients hospitalized for AMI within 12 hours of symptom onset: 26 receiving thrombolytic therapy and 13 heparin alone. Blood samples were collected at baseline and after 90 minutes and 24 hours. Patients undergoing thrombolysis had a significant early increase in activated factor XII (from 2.2 ng/ml at baseline to 4.7 ng/ml after 90 minutes; p = 0.0001), cleaved kininogen (from 26% to 37%; p = 0.001), and fragment 1 + 2 (from 1.4 to 2.1 nmol/L; p = 0.0001), whereas the 24-hour levels were similar to baseline levels. The levels of interleukin-6 significantly increased during the first 90 minutes (from 3.9 to 6.3 microg/ml; p = 0.001), and were even higher after 24 hours (11.9 ng/ml, p = 0.0001). CRP levels increased only after 24 hours (p = 0.0001). There were no changes in these parameters in patients receiving heparin alone, except for a 24-hour increase in interleukin-6 and CRP levels. Thus, in patients with AMI receiving thrombolytic therapy, early activation of inflammation parallels the activation of the contact system and the coagulation cascade, which might contribute to microvascular obstruction and reperfusion injury.

The rate of plasma clotting was measured in order to investigate two different processes. In both cases normal, pooled platelet-poor plasma was used as a substrate for measurement of clotting. The intrinsic coagulation pathway was studied by bringing a variety of biomaterials into contact with a plasma aliquot and observing the rate of clotting diminish by virtue of factor XII activation. The efficacy of heparinization was investigated by measuring the increase in clotting time of a plasma aliquot during biomaterial contact. In both cases, clotting time was measured turbidometrically. Marked differences in intrinsic pathway activation were observed between a variety of materials. There were clear differences between the materials and the negative and positive controls. The assay showed that heparinized materials could be distinguished from non-heparinized materials and a non-activated plasma control.

It is estimated from circulating levels of coagulation and fibrinolysis activators (and inhibitors) and breakdown products that many, but not all, patients with sepsis and allied conditions show evidence of diffuse intravascular coagulation (DIC) that may contribute, by thrombin generation, widespread microvascular fibrin deposits and platelet aggregates, to development of multiple organ damage and ultimate demise [1, 2]. The DIC may involve activation of coagulation and activation and subsequent inhibition of fibrinolysis, which are, in part, regulated differently. Activation of inflammatory cascades, including release of cytokines such as tumor necrosis factor (TNF-α), may lead to harmful activation of the coagulation cascade via tissue factor synthesis, while the contact system and factor XII activation may contribute to hypotension and inhibition of activated fibrinolysis.

The contribution of various enzymes in the activation of factor VII, determined from the increase in factor VII coagulant activity (VIIc), was investigated following the exposure of citrated plasma to low temperature. The contact system of coagulation was initiated either by the contact surface present in certain plasmas (i.e. plasma from women in late pregnancy) or by micellar stearate added to plasma diluted with an equal volume of buffer (plasma from normal healthy subjects or from women in late pregnancy). With either of the contact surfaces, increase of VIIc and the concentration of enzymes derived from factor XII (XIIa) depended on the potency of the contact surface. The stearate-induced VIIc in diluted plasmas from women in late pregnancy or from normal subjects was inhibited by 60-70% in the presence of anti-factor IX monoclonal antibody. VIIc was not increased in XII-deficient plasma following the addition of stearate. The addition of purified human factor XII to this plasma restored the increase in VIIc and the activation of factor XII. In factor IX-deficient plasma, the stearate-induced increase in VIIc was only 38% of that seen in normal plasma and was restored by the addition of purified factor IX. Similarly in factor XI-deficient plasma, the stearate-induced increase in VIIc and the factor XII activation were 48% and 69% of that found in normal plasma. The addition of EDTA (2 mM) did not alter the extent of factor XII activation induced by contact surface, but it did inhibit the rise in VIIc. It is concluded that in the presence of contact surface the activation of factor XII and the sequential activation of factor XI and of factor IX results in the activation of factor VII. Activated factor IX is responsible for the major part of the factor VII activation whereas the rest may be through the direct activation by XIIa.

Background Factor XII–dependent bradykinin formation is thought to be responsible for the swelling associated with the various forms of C1 inhibitor deficiency, and complement activation is augmented during attacks of swelling. Objectives To further elucidate the interactions of the kinin-forming cascade that lead to complement activation during attacks of swelling and to determine whether fibrinolysis is augmented as well. Methods We compared spontaneous and kaolin-induced activation of normal plasma with the plasma of patients with hereditary angioedema. Results Hereditary angioedema plasma demonstrated augmented factor XII activation, production of factor XIIf, prekallikrein activation, and high-molecular-weight kininogen cleavage, and, as a result, bradykinin formation was markedly increased. Baseline levels of C4a and plasmin-α 2 antiplasmin complexes increased, and, on activation with kaolin, levels increased further. Conclusions All parameters indicative of activation of the bradykinin-forming cascade are activated in hereditary angioedema plasma vs normal plasma. Production of factor XIIf, demonstrated for the first time in whole plasma, may be responsible for C1 activation based on C4a production. The factor XII–dependent fibrinolytic cascade is also activated.

In this article we have reviewed the current knowledge regarding the involvement of Factor XII in contact activation. Clearly in the past decade an overwhelming amount of data and hypotheses have been published regarding the central role of this zymogen in the initiation and further propagation of contact activation reactions. Therefore we feel that it will be helpful to conclude this article with a figure that summarizes those interactions and reactions that are generally believed to reflect the major molecular events occurring during surface-dependent contact activation. The contact factors are capable of very efficient interation with each other, provided a suitable negatively charged surface is present. Such surfaces are thought to stimulate the interactions between the contact factors through binding of the proteins and thus bringing the proteins together. Factor XII readily binds to the negatively charged surface, but for the binding of prekallikrein and Factor XI, the cofactor HMW kininogen is likely to be necessary. Bound at the surface, the zymogens Factor XII and prekallikrein are thought to be involved in a so-called reciprocal activation mechanism in which Factor XIIa activates prekallikrein to kallikrein, which in turn converts Factor XII to Factor XIIa. The formation of Factor XIIa is further promoted by the fact that surface-bound Factor XII is likely more susceptible to proteolytic cleavage and by the fact that the activated Factor XIIa is capable of auto-activating its own zymogen Factor XII. However, the latter effect, although undoubtedly contributing to the formation of Factor XIIa at the surface, seems to be of less importance than the reciprocal activation mechanism. This is underscored by the fact that Factor XII activation is rather slow in prekallikrein-deficient plasma. Surface-bound Factor XIIa is then responsible for the activation of Factor XI to Factor XIa, thereby propagating the initial trigger. Presumably, Factor XIa must leave the surface in order to be able to become involved in the activation of blood coagulation Factor IX.

Para-amino-methyl benzoic acid (PAMBA) in a dose of 50 mg/kg body weight was unable to alter the ellagic acid induced hypercoagulable state. No change in the hypercoagulability pattern was observed regardless of the time of administration of the compound (before or after the ellagic infusion).

The effect of divalent metal ions on the rate of dextran sulfate dependent autocatalytic activation of human blood coagulation factor XII was studied at pH 7.4 and 25 degrees C. Zn2+ and Cu2+, but not Co2+, increased the rate of factor XII activation induced by dextran sulfate with optimum effects at approximately 5 and 1 microM, respectively, while Ca2+ acceleration required much higher concentrations (millimolar). Further investigation of the effect of Zn2+ on factor XII activation demonstrated a complete dependence on the presence of dextran sulfate, lack of inhibition by soybean trypsin inhibitor, the appearance of alpha-XIIa as the primary reaction product, and reaction kinetics characteristic of an autocatalytic process. These results were consistent with Zn2+ affecting only the rate of surface-mediated factor XII autoactivation. The initial turnover velocity of dextran sulfate induced factor XII autoactivation increased linearly with factor XII concentration in the absence of Zn2+ up to 0.9 microM factor XII but showed saturation behavior over this same concentration range in the presence of 5 microM Zn2+, indicating that Zn2+ increased the reaction rate primarily by lowering the apparent Km. Comparison of the kinetics of autoactivation at mu = 0.15 and 0.24 revealed that the enhancement in the apparent kcat/Km brought about by Zn2+ increased from 19-fold to 520-fold, respectively, due to a differential dependence of the Zn2+-stimulated and unstimulated reactions on ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

The mechanism by which negatively charged substances such as celite, kaolin, or ellagic acid contribute to the surface-dependent activation of Hageman factor (Factor XII) was studied. Kinetic studies of the proteolytic activation of 125 I-labeled human Hageman factor by human plasma kallikrein, plasma, activated Factor XI, and trypsin were performed in the presence and absence of high molecular weight kininogen and surface materials such as celite, kaolin, or ellagic acid. The results showed that surface-bound Hageman factor was 500 times more susceptible than soluble Hageman factor to proteolytic activation by kallikrein in the presence of high molecular weight kininogen. Surface binding of Hageman factor enhanced its cleavage by plasmin, activated Factor XI, and trypsin by 100-fold, 30-fold, and 5-fold, respectively. On a molar basis, trypsin was twice as potent as kallikrein in the cleavage of the surface-bound Hageman factor, while plasmin and activated Factor XI were an order of magnitude less potent than kallikrein. Kallikrein even at concentrations as low as 0.5 nM (i.e., 1/1000th of the concentration of prekallikrein in plasma) was very potent in the limited proteolysis of the surface-bound Hageman factor. These results suggest that substances classically known as “activating surfaces” promote the activation of Hageman factor indirectly by altering its structure such that it is much more susceptible to proteolytic activation by other plasma or cellular proteases.

Bradykinin release, an end product of contact system activation, is thought to play a significant role in the pathophysiology of asthma. We have found an increased level of native α2-macroglobulin-kallikrein (α2M-KK) in asthma plasmas, and have demonstrated increased levels of contact system activity in these plasmas under certain laboratory conditions. We investigated the possible role of α2M-KK as a modulator of the contact system activity. α2M-KK potentiated the factor XII activation on kaolin and the kallikrein production in a dextran-sulfate-mediated assay. This potentiation presumably involves a proteolytic effect of α2M-KK on high molecular weight kininogen.

Bradykinin, the end product of activation of the plasma contact system, may play a significant role in reactions to contrast material and in other forms of anaphylactoid and anaphylactic responses to drugs or antigens. Activation of factor XII initiates activity in the plasma contact system, and we have identified factors (negatively charged surfaces) present in elevated concentrations in the plasma of patients who are asthmatic or allergic that can "prime" their plasma for the initiation and/or potentiation of factor XII activation. In other studies, we have shown that persons who react to contrast material and persons who are asthmatic or allergic share both a potential for accelerated contact system activity and evidence of an increased mean concentration of low-level contact system products. Recently, we have found that contrast media can inhibit angiotensin-converting enzyme, the substance that hydrolyzes bradykinin and limits its systemic effects. Thus, a number of factors suggest that it is the potential for increased production of bradykinin in persons who are allergic or asthmatic that may account for the greatly increased susceptibility of these patients to contrast material. This susceptibility may be critically triggered by the contrast media-induced inhibition of angiotensin converting enzyme. In view of these findings, the possibility exists that most, if not all, significant reactions to contrast material require an underlying allergic diathesis that may, or may not, be apparent by history and conventional diagnostic testing.