Factor XII in coagulation, inflammation and beyond.

Factor XII (FXII) is a protease that is mainly produced in the liver and circulates in plasma as a single chain zymogen. Following contact with negatively charged surfaces, FXII is converted into the two-chain active form, FXIIa. FXIIa initiates the intrinsic blood coagulation pathway via activation of factor XI. Furthermore, it converts plasma prekallikrein to kallikrein (PK), which reciprocally activates FXII and liberates bradykinin from high molecular weight kininogen. In addition, FXIIa initiates fibrinolysis via PK-mediated urokinase activation and activates the classical complement pathway. Even though the main function of FXII seems to relate to the activation of the intrinsic coagulation pathway and the kallikrein-kinin system, a growing body of evidence suggests that FXII may also directly regulate cellular responses. In this regard, it has been found that FXII/FXIIa induces the expression of inflammatory mediators, promotes cell proliferation, and enhances the migration of neutrophils and lung fibroblasts. In addition, it has been reported that genetic ablation of FXII protects against neuroinflammation, reduces the formation of atherosclerotic lesions in Apoe-/- mice, improves wound healing, and inhibits postnatal angiogenesis. Although the aforementioned effects can be partially explained by the downstream products of FXII activation, the ability of FXII/FXIIa to directly regulate cellular responses has recently emerged as an alternative hypothesis. These direct cellular reactions to FXII/FXIIa will be discussed in the review.

In vivo activation and functions of the protease factor XII.

Combinations of proinflammatory and procoagulant reactions are the unifying principle for a variety of disorders affecting the cardiovascular system. Factor XII (FXII, Hageman factor) is a plasma protease that initiates the contact system. The biochemistry of the contact system in vitro is well understood; however, its in vivo functions are just beginning to emerge. The current review concentrates on activators and functions of the FXII-driven contact system in vivo. Elucidating its physiologic activities offers the exciting opportunity to develop strategies for the safe interference with both thrombotic and inflammatory diseases.

In vivo roles of factor XII.

Coagulation factor XII (FXII, Hageman factor, EC = 3.4.21.38) is the zymogen of the serine protease, factor XIIa (FXIIa). FXII is converted to FXIIa through autoactivation induced by "contact" to charged surfaces. FXIIa is of crucial importance for fibrin formation in vitro, but deficiency in the protease is not associated with excessive bleeding. For decades, FXII was considered to have no function for coagulation in vivo. Our laboratory developed the first murine knockout model of FXII. Consistent with their human counterparts, FXII(-/-) mice have a normal hemostatic capacity. However, thrombus formation in FXII(-/-) mice is largely defective, and the animals are protected from experimental cerebral ischemia and pulmonary embolism. This murine model has created new interest in FXII because it raises the possibility for safe anticoagulation, which targets thrombosis without influence on hemostasis. We recently have identified platelet polyphosphate (an inorganic polymer) and mast cell heparin as in vivo FXII activators with implications on the initiation of thrombosis and edema during hypersensitivity reactions. Independent of its protease activity, FXII exerts mitogenic activity with implications for angiogenesis. The goal of this review is to summarize the in vivo functions of FXII, with special focus to its functions in thrombosis and vascular biology.

The plasma contact system 2.0.

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.

Intrinsic coagulation activation and the risk of arterial thrombosis in young women: results from the Risk of Arterial Thrombosis in relation to Oral contraceptives (RATIO) case-control study.

BACKGROUND: Classically, intrinsic coagulation proteins are thought to have a minor role in hemostasis. Recently, these proteins, especially FXII, were implicated as possible key players in the pathogenesis of arterial thrombosis. This study aims to determine the risks of arterial thrombosis conferred by increased activation of intrinsic coagulation proteins in young women and the effect of oral contraceptive use on this association. METHODS AND RESULTS: The Risk of Arterial Thrombosis In relation to Oral contraceptives (RATIO) study is a population-based case-control study including young women (age 18 to 50 years) with myocardial infarction (n=205) and ischemic stroke (n=175) and 638 healthy controls. Intrinsic coagulation protein activation was determined by measuring activated protein-inhibitor complexes. This complex is with C1 esterase inhibitor (FXIIa-C1-INH, FXIa-C1-INH, Kallikrein-C1-INH) or antitrypsin inhibitor (FXIa-AT-INH). Odds ratios (ORs) and corresponding confidence intervals (95% CIs) were calculated with logistic regression. High levels of protein activation (>90th percentile of controls) showed an increased risk of ischemic stroke: FXIIa-C1-INH (OR, 2.1; 95% CI, 1.3 to 3.5), FXIa-C1-INH (OR, 2.8; 95% CI, 1.6 to 4.7), FXIa-AT-INH (OR, 2.3; 95% CI, 1.4 to 4.0), and Kallikrein-C1 (OR, 4.3; 95% CI, 2.6 to 7.2). If anything, myocardial infarction risk was only increased by Kallikrein-C1-INH (OR, 1.5; 95% CI, 0.9 to 2.5). Oral contraceptive use further increased the risks. CONCLUSIONS: High levels of activated proteins of the intrinsic coagulation system are associated with arterial thrombosis, whereas the strength of these associations differs for myocardial infarction and ischemic stroke. This contradicts similar analyses among men in the Northwick Park Heart Study. Together with the finding that oral contraceptive use further increases the risks, the question of whether the role of intrinsic coagulation proteins in the pathogenesis of arterial thrombosis is sex-specific is raised.

Factor XII: what does it contribute to our understanding of the physiology and pathophysiology of hemostasis & thrombosis.

Factor XII (FXII) is a coagulation protein that is essential for surface-activated blood coagulation tests but whose deficiency is not associated with bleeding. For over forty years, investigators in hemostasis have not considered FXII important because its deficiency is not associated with bleeding. It is because there is a dichotomy between abnormal laboratory assay findings due to FXII deficiency and clinical hemostasis that investigators sought explanations for physiologic hemostasis independent of FXII. FXII is a multidomain protein that contains two fibronectin binding consensual sequences, two epidermal growth factor regions, a kringle region, a proline-rich domain, and a catalytic domain that when proteolyzed turns into a plasma serine protease. Recent investigations with FXII deleted mice that are protected from thrombosis indicate that it contributes to the extent of developing thrombus in the intravascular compartment. These findings suggest that it has a role in thrombus formation without influencing hemostasis. Last, FXII has been newly appreciated to be a growth factor that may influence tissue injury repair and angiogenesis. These combined studies suggest that FXII may become a pharmacologic target to reduce arterial thrombosis risk and promote cell repair after injury, without influencing hemostasis.

The elusive physiologic role of Factor XII.

Physiologic hemostasis upon injury involves many plasma proteins in a well-regulated cascade of proteolytic reactions to form a clot. Deficiency of blood coagulation Factors VIII, IX, or XI is associated with hemophilia. Factor XII (FXII) autoactivates by contact with a variety of artificial or biologic negatively charged surfaces (contact activation), resulting in blood coagulation and activation of the inflammatory kallikrein-kinin and complement systems. However, surprisingly, individuals deficient in FXII rarely suffer from bleeding disorders. Most biologic surfaces that activate FXII become expressed in disease states. Investigators have long searched for physiologic activators of FXII and its role in vivo. In this issue of the JCI, Maas et al. show that misfolded protein aggregates produced during systemic amyloidosis allow for plasma FXIIa and prekallikrein activation and increased formation of kallikrein-C1 inhibitor complexes, without Factor XIa activation and coagulation (see the related article beginning on page 3208). This study describes a novel biologic surface for FXII activation and activity, which initiates inflammatory events independent of hemostasis.

Coagulation factor XII (FXII) activity, activated FXII, distribution of FXII C46T gene polymorphism and coronary risk.

BACKGROUND: Whether factor XII (FXII) activity, its 46C>T polymorphism and activated FXII (FXIIa) are associated with coronary heart disease (CHD) remains to be determined. METHODS: FXII, FXIIa and the FXII 46C>T polymorphism were determined in a hospital-based cohort of 2615 patients undergoing coronary angiography. RESULTS: Fifty-seven per cent of the participants were identified as wild-type (46CC), 38% as heterozygous (46CT) and 5% as homozygous (46TT) for FXII 46C>T. FXII and FXIIa levels were significantly lower in carriers of the T-allele: 132 (97-151) U dL(-1) FXII in 46CC, 87 (77-99) U dL(-1) FXII in 46CT and 53 (42-67) U dL(-1) FXII in 46TT carriers (P < 0.001), and 2.8 (2.3-3.5) microg L(-1) FXIIa in CC, 2.1 (1.6-2.6) microg L(-1) FXIIa in CT and 1.2 (0.9-1.5) microg L(-1) FXIIa in TT carriers (P < 0.001; medians, lower and upper quartiles). Patients with stable CHD (n = 935), a history of myocardial infarction (n = 785) or who were suffering from acute coronary syndromes (ACS; n = 323) had significantly lower FXII levels than controls (n = 572). The differences remained statistically significant after adjustments for age, sex, diabetes mellitus, smoking, hypercholesterolemia and hypertension. Significantly reduced FXIIa levels in ACS patients lost significance once adjusted for covariates. FXII genotype was not associated with any clinical phenotype. CONCLUSION: Lower FXII activity represents an independent risk for CHD and ACS. This is not the case for FXIIa levels or the FXII 46C>T variation.

Platelets promote coagulation factor XII-mediated proteolytic cascade systems in plasma.

Blood coagulation factor XII (FXII, Hageman factor) is a plasma serine protease which is autoactivated following contact with negatively charged surfaces in a reaction involving plasma kallikrein and high-molecular-weight kininogen (contact phase activation). Active FXII has the ability to initiate blood clotting via the intrinsic pathway of coagulation and inflammatory reactions via the kallikrein-kinin system. Here we have determined FXII-mediated bradykinin formation and clotting in plasma. Western blotting analysis with specific antibodies against various parts of the contact factors revealed that limited activation of FXII is sufficient to promote plasma kallikrein activation, resulting in the conversion of high-molecular-weight kininogen and bradykinin generation. The presence of platelets significantly promoted FXII-initiated bradykinin formation. Similarly, in vitro clotting assays revealed that platelets critically promoted FXII-driven thrombin and fibrin formation. In summary, our data suggest that FXII-initiated protease cascades may proceed on platelet surfaces, with implications for inflammation and clotting.

Factor XII, kininogen and plasma prekallikrein in abnormal pregnancies.

Factor XII, plasma prekallikrein and high molecular weight kininogen were first identified as coagulation proteins in the intrinsic pathway because patients deficient in these proteins had marked prolongation of in vitro surface-activated coagulation time. However, deficiencies of these proteins are not associated with clinical bleeding. Paradoxically, studies suggest that these proteins have anticoagulant and profibrinolytic activities. In fact, association between deficiencies of these proteins and thrombosis has been reported. Also, deficiencies of these proteins, auto-antibodies to these proteins and anti-phospholipid antibodies are frequent hemostatis-related abnormalities found in unexplained recurrent aborters. Recently, evidence has accumulated for the presence of the kallikrein-kininogen-kinin system in the fetoplacental unit. Since contact proteins or kallikrein-kininogen-kinin system may play an important role in pregnancy especially in the fetoplacental unit, deficiencies of these proteins and/or auto-antibodies to these proteins may be associated with pregnancy losses. These possibilities will be reviewed, the functions of the individual components will be summarized, and their role in blood coagulation and pregnancy discussed.

Perioperative use of the Thrombelastograph in patients with inherited bleeding disorders.

There are few reports of the use of Thrombelastgraph (TEG) in patients with isolated or inherited bleeding disorders. Three surgical patients are presented with von Willebrand's disease, factor XII deficiency, and hemophilia A with dysfibrinogenemia in which the abnormalities detected by the TEG were similar to the expected coagulation abnormalities based on preoperative laboratory studies. This initial report suggests that this potential application of the TEG be further investigated.

Glycosaminoglycans affect the interaction of human plasma kallikrein with plasminogen, factor XII and inhibitors.

Human plasma kallikrein, a serine proteinase, plays a key role in intrinsic blood clotting, in the kallikrein-kinin system, and in fibrinolysis. The proteolytic enzymes involved in these processes are usually controlled by specific inhibitors and may be influenced by several factors including glycosaminoglycans, as recently demonstrated by our group. The aim of the present study was to investigate the effect of glycosaminoglycans (30 to 250 micro/ml) on kallikrein activity on plasminogen and factor XII and on the inhibition of kallikrein by the plasma proteins C1-inhibitor and antithrombin. Almost all available glycosaminoglycans (heparin, heparan sulfate, bovine and tuna dermatan sulfate, chondroitin 4- and 6-sulfates) reduced (1.2 to 3.0 times) the catalytic efficiency of kallikrein (in a nanomolar range) on the hydrolysis of plasminogen (0.3 to 1.8 microM) and increased (1.9 to 7.7 times) the enzyme efficiency in factor XII (0.1 to 10 microM) activation. On the other hand, heparin, heparan sulfate, and bovine and tuna dermatan sulfate improved (1.2 to 3.4 times) kallikrein inhibition by antithrombin (1.4 microM), while chondroitin 4- and 6-sulfates reduced it (1.3 times). Heparin and heparan sulfate increased (1.4 times) the enzyme inhibition by the C1-inhibitor (150 nM).

Platelet adhesion receptors and (patho)physiological thrombus formation.

In thrombus formation associated with hemostasis or thrombotic disease, blood platelets first undergo a rapid transition from a circulating state to an adherent state, followed by activation and aggregation. Under flow conditions in the bloodstream, this process potentially involves platelet-platelet, platelet-endothelium, platelet-subendothelial matrix, and platelet-leukocyte interactions. Specific adhesion receptors on platelets mediate these interactions, by engaging counter-receptors on other cells, or noncellular ligands in the plasma or matrix. The glycoprotein (GP) Ib-IX-V complex on platelets initiates adhesion at high shear stress by binding the adhesive ligand, von Willebrand Factor (vWF). GP Ib-IX-V may also mediate platelet-endothelium or platelet-leukocyte adhesion, by recognition of P-selectin or Mac-1, respectively. Other membrane glycoproteins, such as the collagen receptor GP VI, may trigger platelet activation at low shear rates. Engagement of GP Ib-IX-V or GP VI leads ultimately to platelet aggregation mediated by the integrin, alphaIIbbeta3 (GP IIb-IIIa). This review will focus on recent advances in understanding structure-activity relationships of GP Ib-IX-V, its role in initiating thrombus formation, and its emerging relationships with other vascular cell adhesion receptors.

Acquired inhibitors to the coagulation factor XII associated with liver disease.

Three patients with liver disease and prolonged activated partial thromboplastine time (APTT) on routine tests are presented. One woman had metastatic liver disease from gastric carcinoma, a second woman had autoimmune hepatitis, and one man had severe chronic hepatitis B. APTT was not corrected after mixing experiments with 25%, 50%, and 75% of normal pool plasma, indicating the presence of an acquired inhibitor. In all three cases, factor XII coagulant activity was reduced: <1%, <1%, and 3%, respectively, while all of the other coagulation factors were normal. In all three cases no other auto-antibody was detected. In the first patient, APTT was normalized after a left liver lobectomy, whereas the primary lesion remained unresected. In the second patient, the FXII activity was improved after corticosteroid therapy but never returned to normal values. In the third patient, the APTT was improved after hydroxychloroquine therapy. None of the patients had hemorrhagic or thrombotic phenomena.

Thrombin activates factor XI on activated platelets in the absence of factor XII.

Thrombin can activate factor XI in the presence of dextran sulfate or sulfatides. However, a physiological cofactor for thrombin activation of factor XI has not been identified. We examined this question in a cell-based, tissue factor-initiated model system. In the absence of factor XII, factor XI enhanced thrombin generation in this model. The effect on thrombin generation was reproduced by 2 to 5 pmol/L factor XIa. A specific inhibitor of factor XIIa did not diminish the effect of factor XI. Thus, factor XI can be activated in a model system that does not contain factor XIIa or nonphysiological cofactors. Preincubation of factor XI with activated platelets and thrombin or factor Xa enhanced subsequent thrombin generation in the model system. Preincubation of factor XI with thrombin or factor Xa, but without platelets, did not enhance thrombin generation, suggesting that these proteases might activate factor XI on platelet surfaces. Thrombin and factor Xa were then directly tested for their ability to activate factor XI. In the presence of dextran sulfate, thrombin or factor Xa activated factor XI. Thrombin, but not factor Xa, also cleaved detectable amounts of factor XI in the presence of activated platelets. Thus, thrombin activates enough factor XI to enhance subsequent thrombin generation in a model system. Platelet surfaces might provide the site for thrombin activation of functionally significant amounts of factor XI in vivo.

Activation of the contact system in cerebrospinal fluid of patients with Alzheimer disease.

Several converging lines of evidence suggest that beta-amyloid and inflammation may be linked in the pathogenesis of Alzheimer disease (AD), but the mechanism of beta-amyloid neurotoxicity is unclear. In this study, by demonstrating that high molecular weight kininogen may be massively cleaved in the cerebrospinal fluid (CSF) of patients with AD, we provide evidence of the potential involvement of the contact system in the inflammatory processes taking place in this disease. In the CSF of patients with neuroimmune inflammatory disease (multiple sclerosis, chronic inflammatory demyelinating polyneuropathy), there was no evidence of increased cleavage of high molecular weight kininogen, suggesting that this finding may be characteristic of the Alzheimer brain. The data obtained from in vitro experiments seem to indicate that the cleavage of high molecular weight kininogen in vivo may be the result of the interaction of beta-amyloid with factor XII and of kallikrein generation. The actual relevance of such a phenomenon remains to be established in vivo. However, the demonstration that the contact system may be activated in the brains of Alzheimer patients points to the potential involvement of the kallikrein-kinin system in the inflammatory process of this disease.

Contact activation: a revision.

In conclusion, a revised view of the contact system has been presented. This system has little to do with the initiation of hemostasis. Like lupus anticoagulants, deficiencies of contact proteins give prolonged APTTs but may be risk factors for thrombosis. BK from kininogens is a potent modulator of vascular biology inducing vasodilation, tissue plasminogen activator release, and prostacyclin liberation. Kininogens, themselves, are selective inhibitors of alpha-thrombin-induced platelet activation preventing alpha-thrombin from cleaving the cloned thrombin receptor after arginine41. Kininogens' alpha-thrombin inhibitory activity exists in intact kininogens, BK, and all of BK's breakdown products. HK also is the pivotal protein for contact protein assembly on endothelium. It is the receptor for prekallikrein which when bound to HK becomes activated to kallikrein by an endothelial cell enzyme system independent of activated forms of plasma factor XII. Prekallikrein activation on endothelial cells results in kinetically favorable single chain urokinase and plasminogen activation. Thus the "physiologic, negatively charged surface" for contact system activation is really the assembly of these proteins on cell membranes and activation by membrane-associated enzymes.