HK1116438A - Prevention of thrombus formation and/or stabilization - Google Patents

Description

Prevention of thrombus formation and/or stabilization

In its most general aspect, the subject of the present invention is the prevention of the formation and/or stabilization of a three-dimensional arterial or venous thrombus.

In particular, the present invention relates to the use of at least one antibody and/or one inhibitor for inhibiting the activity of factor XII and preventing the formation and/or stabilization of thrombi and thrombus growth. The invention also relates to pharmaceutical preparations and to the use of factor XII as an antithrombotic target.

Injury to the vessel wall initiates a sudden adhesion and aggregation of platelets, followed by activation of the plasma coagulation system and formation of fibrin-containing thrombi, which occlude the injury site. These events are critical to limiting post-traumatic blood loss, but can also occlude diseased vessels, leading to ischemia and infarction of vital organs. In a cascade or cascade, blood coagulation proceeds through a series of reactions involving activation of zymogens by limited proteolysis, ultimately resulting in the explosive generation of thrombin, which converts plasma fibrinogen to fibrin and effectively activates platelets. Platelets that adhere to collagen or fibrin, in turn, promote thrombin generation to an extent of several orders of magnitude by exposing procoagulant Phosphatidylserine (PS) on their outer surface, which amplifies the assembly and activation of the thrombin protease complex, and by direct interaction between platelet receptors and coagulation factors.

There are two convergent pathways of coagulation that are initiated by either the extrinsic (vascular wall) or intrinsic (blood-borne) components of the vascular system. The "extrinsic" pathway is initiated by a complex of plasma coagulation factor vii (fvii) and integral membrane Tissue Factor (TF), an essential coagulation cofactor that is not present at the luminal surface of blood vessels but is strongly expressed in the subendothelium. TF expressed in circulating microvesicles can also promote thrombus enlargement by maintaining thrombin generation on the surface of activated platelets.

When factor XII (FXII, Hageman factor) contacts a negatively charged surface in a reaction involving high molecular weight kininogen and plasma kallikrein, the "intrinsic" or contact activation pathway is initiated. FXII can be activated by macromolecular components of the subendothelial matrix such as glycosaminoglycans and collagen, sulfatides, nucleotides and other soluble polyanions or non-physiological materials such as glass or polymers. One of the most effective contact activators is kaolin, and this reactant serves as the basis for the mechanism of the major clinical coagulation test ((activated) partial thromboplastin time (PTT, aPTT)). In a response amplified by platelets, activated FXII then activates FXI, which in turn activates coagulation factor IX. Despite its high efficiency in inducing blood coagulation in vitro, the (patho-) physiological importance of the FXII-initiated intrinsic coagulation pathway is questioned by the fact that: genetic defects in FXII as well as high molecular weight kininogen and plasma kallikrein are not associated with bleeding complications. The observation that humans and mice lack exogenous pathway components such as TF, FVII or coagulation factor IX suffer from severe bleeding, together with this observation leads to the current hypothesis: fibrin formation is initiated in vivo only by the exogenous cascade (Mackman, N. (2004). Role of tissue factor in haemostasis, and vaculardevelopment. Arterioscler. Thromb. Vasc. biol.24, 1015. 1022).

Like all physiological mechanisms, the coagulation cascade can become inappropriately activated and lead to the formation of a tampon within the blood vessel. Thus, the blood vessel may become occluded and the supply of blood to the distal organs is limited. This process is known as thromboembolism and is associated with high mortality rates. Furthermore, the use of prosthetic devices in contact with blood is severely limited due to the activation of the coagulation cascade and the coating of the prosthetic surface, which often impairs its function. Examples of such prosthetic devices are hemodialyzers, cardiopulmonary bypass circuits, vascular stents and indwelling catheters. For the case of using such devices, an anticoagulant such as heparin is used to prevent fibrin deposition on the surface. However, some patients are intolerant to heparin, which can lead to heparin-induced thrombocytopenia (HIT), leading to platelet aggregation and life-threatening thrombosis. Furthermore, the inherent risk of all anticoagulants used in the clinic is also associated with an increased risk of severe bleeding. Therefore, there is a need for new anticoagulants that are not associated with such complications and that can be used in affected patients or as an excellent therapeutic concept for preventing thrombosis without increased bleeding tendency.

It is therefore apparent that there remains a need for improved drug therapies for the treatment or prevention of thrombosis and similar disorders. It is therefore an object of the present invention to meet this need. For more than 50 years, it has been known that deficiencies in coagulation factor XII are not associated with increased spontaneous or injury-related bleeding complications (Ratnoff, o.d. & coloury, j.e. (1955) a family hemorrhagitta associated with a deficiency of a clot-promoting fraction of plasma. j.in Invest 34, 602-13). Indeed, despite the presence of pathological aPTT (clinical coagulation test focused on the intrinsic pathway of coagulation), FXII deficient persons do not show abnormal bleeding even in major surgical procedures (Colman, r.w. hemostatis and thrombosis.basic principles & clinical practice (eds. Colman r.w., hirsch.j., Mader v.j., Clowes a.w., & George J.)103 + 122 Lippincott Williams & Wilkins, philidelphia, 2001). In contrast, FXII deficiency is associated with an increased risk of venous thrombosis (Kuhli, C., Scharrer, I., Koch, F., Ohrloff, C. & Hattenbach, L.O. (2004) Factor XII specificity: a thombophagic risk Factor for regenerative infection.A.J.Ophthalmol.137, 459-464; Halbmayer, W.M., nhalter, C., Feichtinger, C., Rubi, K. & Fischer, M. (1993) Factor XII (Hageman Factor) specificity. a Factor for optimizing of thrombus. Studies and case reports supporting this view relate to the indicated cases of FXII deficiency: mr. John Hageman, who died from pulmonary embolism. The hypothesis that FXII Deficiency and increased Risk of thrombogenicity are challenged by recent reevaluations of several case reports linking FXII Deficiency to thrombosis (gilolamin, a., Randi, m.l., Gavasso, s., Lombardi, A.M. & Spiezia, F. (2004) The objective Venous thrombosis Seen linkage with Severe (Homozygous) FXII Deficiency area foundation: a Study of prevalen 21 patents and Review of The same life. In most cases, the authors identified a concomitant congenital or acquired prothrombotic risk factor in combination with a deficiency in the coagulation factor FXII, which could even cause thrombotic events independent of FXII. The greatest epidemiological studies carried out using well-characterized patients (Koster, T., Rosendal, F.R., Briet, E. & Vandenbroucke, J.P. (1994) John Hageman's factor and deep-vessel thrombosis: Leidenthrombophilia study. Br. J. Haematol.87, 422, 424) and FXII deficient families (Zeerleder, S. et al (1999) evaluation of the inhibition of thrombosis in genetic factor XII deficiency. Thromb. Haemost.82, 1240-1246) showed no risk of thrombosis or anti-thrombosis related between thrombosis II deficiency and any thrombogenic or anti-thrombosis related factor.

Surprisingly and contrary to what is generally believed by those skilled in the art, the applicants have found that the intrinsic coagulation pathway driven by factor XII is essential for arterial thrombosis in vivo, but not for normal tissue-specific hemostasis. Unexpectedly, these results alter the concept that blood coagulation in vivo is mediated solely by the extrinsic pathway, a long-standing concept and place factor XII centrally in the process of pathological thrombosis.

A first subject of the present invention is therefore the use of at least one antibody and/or at least one inhibitor for inhibiting factor XII and for preventing the formation and/or stabilization of three-dimensional arterial or venous thrombi. Thus, anti-FXII antibodies or inhibitors may function to inhibit activation of FXII and/or interfere with other portions of the FXII molecule that are critically involved in FXII activation.

Together with the fact that the intrinsic pathway is not required for hemostasis, this establishes factor XII as a promising new target for effective antithrombotic therapy. Furthermore, these results are also important for the development of anti-FXII agents to control other (pathological) mechanisms associated with the contact system, such as inflammation, complement activation, fibrinolysis, angiogenesis and kinin formation.

Accordingly, the invention also provides the use of such antibodies and/or inhibitors in the treatment or prevention of conditions or disorders associated with thrombosis of arteries, i.e. stroke or myocardial infarction, inflammation, complement activation, fibrinolysis, angiogenesis and/or diseases associated with pathological kinin formation such as hypotonic shock, oedema (including hereditary angioedema), bacterial infection, arthritis, pancreatitis or joint gout.

In particular, according to the present invention there is provided the use of at least one anti-FXII antibody (such as, for example, the F1 antibody (MoAb F1, Ravon et al, blood.1995 Dec 1; 86 (11): 4134-43)) and/or at least one protease inhibitor for inhibiting FXII-driven thrombosis.

Particularly preferred are protease inhibitors, for example selected from the group consisting of: AT III inhibitors, angiotensin converting enzyme inhibitors, C1 inhibitors, aprotinin, alpha-1 protease inhibitors, antipain ([ (S) -1-carboxy-2-phenylethyl ] -carbamoyl-L-Arg-L-Val-Arginal), Z-Pro-Pro-aldehyde-dimethylacetate (Z-Pro-Pro-aldehyde-dimethylacetate), DX88 (Dyax., 300Technology Square, Cambridge, MA 02139, USA; cited in Williams A.andBacard, Transfus Apheresis Sci.2003 Dec: 29 (3): 255-8), leupeptin, inhibitors of prolyl oligopeptidase such as Fmoc-Ala-Pyr-CN, maize trypsin inhibitors, mutants of bovine trypsin inhibitor, colicin, E-II, E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E, YAP (yellowtail sole (yellowfin sole) anticoagulant protein) and Marigold (Cucurbita maxima) trypsin inhibitor-V (including Marigold equivalent inhibitor).

Accordingly, the present invention provides the use of such antibodies and/or inhibitors described herein in medicine; and the use of such antibodies and/or inhibitors in the manufacture of a medicament.

Thus, according to a further aspect of the present invention, a pharmaceutical formulation comprising at least one antibody and/or one inhibitor is provided, which is suitable for inhibiting factor XII and for preventing the formation and/or stabilization of a three-dimensional arterial or venous thrombus.

In particular, the antibody used in the pharmaceutical formulation is an anti-FXII antibody (e.g., like the F1 antibody (MoAb F1, Rayon et al, blood.1995 Dec 1; 86 (11): 4134-43)) and the inhibitor is a protease inhibitor, such as, but not limited to, AT III inhibitors, angiotensin converting enzyme inhibitors, C1 inhibitors, aprotinin, alpha-1 protease inhibitors, antiprotease ([ (S) -1-carboxy-2-phenylethyl ] -carbamoyl-L-Arg-L-Val-Arginal), Z-Pro-Pro-aldehyde-dimethylacetate, DX88(DyaxInc., 300Technology Square, Cambridge, MA 02139, USA; in Williams A.and Baird. LG, Transfus Apheresis Sci.2003 Dec: 29 (3): 255-8), leupeptin Leulin aprotinin, Inhibitors of prolyl oligopeptidase such as Fmoc-Ala-Pyr-CN, maize trypsin inhibitor, mutants of bovine pancreatic trypsin inhibitor, colicin, YAP (yellowtail flounder anticoagulant protein) and cucurbita pepstatin-V (including cucurbita moschata synergic inhibitors).

The antibody may also be an antibody fragment or mimetic that retains inhibitory activity, for example, an analogue of the Kunitz protease inhibitor domain of amyloid precursor protein disclosed in us patent 6,613,890 (especially columns 4 to 8). Other suitable inhibitors may be Hamadarin as disclosed in The Journal of Biological Chemistry, Vol.277, No.31(August 2, pp.27651-27658, 2002) by Harahiko Isawa et al. Suitable corn trypsin inhibitors and methods for their preparation are disclosed in Zhi-Yuan Chen et al, Applied and Environmental Microbiology, March 1999, p.1320-1324 and reference 19 cited therein. All cited references are incorporated by reference in their entirety into this application. Last but not least, small molecules isolated e.g. by assays using FXII or FXIIa inhibition as the basis for selection and their respective uses described above or below are part of the present invention. These small molecule FXIIa inhibitors can be designed based on the crystal structure of FXII. Thus, some FXII domains or light chains can be recombinantly expressed in expression systems such as e.coli (e.coli), yeast or mammalian cells. The protein was then purified and crystallized using standard methods described for FXII substrate FXI (Jin L, et al (2005) Crystal structures, FfxIa catalytic domain in complex with organic molecules, J Biol chem.280 (6): 4704-12). Alternatively, small molecule serine protease inhibitors may be included to stabilize the FXII structure. Such formulations comprising small molecule inhibitors of protein targets (which may be designed, for example, under the guidance of crystals of these target proteins) are well known in the art and include pharmaceutical formulations that may be administered to a patient, for example, systemically, such as parenterally or orally, or topically.

As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraarterial, and intratracheal injections, instillations, spray administrations, and infusion techniques. The parenteral formulation is preferably administered intravenously, in the form of a bolus injection or a constant volume infusion, or subcutaneously, according to known procedures. Preferred liquid carriers well known for parenteral use include sterile water, saline, aqueous dextrose, sugar solutions, ethanol, glycols and oils.

Tablets and capsules for oral administration may contain conventional excipients such as binding agents, fillers, lubricants, and wetting agents and the like. Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups, elixirs and the like, and may be presented as a dry product for reconstitution with water or other suitable vehicle at the time of use. Such liquid formulations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous carriers and preservatives.

Formulations suitable for topical application may be in the form of aqueous or oily suspensions, solutions, emulsions, gels or, preferably, emulsion ointments. Formulations for spray application may be in the form of sprayable liquids or dry powders.

According to a third aspect of the present invention, there is provided the use of factor XII as an antithrombotic target by inhibiting factor XII by at least one antibody and/or one inhibitor and thus preventing the formation and/or stabilization of a three-dimensional thrombus in a blood vessel.

The nature, benefits and other features of the present invention will become apparent from the following detailed description of the experiments performed and their results when read in conjunction with the following drawings.

Factor XII deficient mice were used to analyze the function of the intrinsic coagulation cascade in hemostasis and thrombosis. In vivo fluorescence microscopy and ultrasound flow measurements reveal serious defects in the formation and stabilization of three-dimensional thrombi in the different arterial branches of the vascular system. Reconstitution of mutant mice with human coagulation factor XII restored the intrinsic coagulation pathway in vitro and arterial thrombosis in vivo. Mechanistically, the procoagulant activity of the endogenous pathway is critically promoted by activated platelets. These results place the FXII-induced endogenous blood coagulation cascade in the central position during arterial thrombosis, which links plasma coagulation to platelet aggregation.

Figure 1 depicts the coagulation assay of FXII deficient mice: (A) tail bleeding time in wild-type (n ═ 12) and FXII-/- (n ═ 11) mice. Each symbol represents an individual. (B) Peripheral blood cell count (thousand/. mu.l) and overall coagulation parameters of FXII-/-and wt mice. Abbreviated as white blood cell count (WBC), activated partial thromboplastin time (aPTT), and Prothrombin Time (PT). Values are given as mean ± SD of 10 mice for each genotype. (C) Contact system proteins FXII, Plasma Kallikrein (PK) and high molecular weight kininogen (HK) detected in 0.3. mu.l of wt and FXII-/-plasma by Western blotting using specific antibodies. Molecular weight standards are given on the left. (D) Recalcification clotting times were determined in platelet-free (upper panel) and platelet-rich (lower panel) plasma from C57BL/6 and 129sv wt, FXII-/-, FcR γ -/-and integrin α 2 deficient mice after activation with either kaolin (dark bars) or collagen (light bars). The effect of JON/A was analyzed in C57BL/6 plasma supplemented with 50. mu.g/ml antibody. Mean ± STD from 6 experiments are given.

Fig. 2. (A) Thromboembolic death was observed following intravenous injection of collagen (0.8mg/kg) and epinephrine (60 μ g/kg). All wild type mice died within 5 minutes. Animals that remained alive 30 minutes after challenge were considered survivors. (B) Platelets were counted in control (n ═ 19), FXII-/- (n ═ 14) and FcR γ -/- (n ═ 5) mice 2 minutes after collagen/epinephrine infusion. (C) Heparinized platelet rich plasma from wild type and FXII-/-mice was stimulated with collagen (10. mu.g/ml) or ADP (5. mu.M) and light transmission was recorded using a standard aggregometer. Results shown are representative of 6 mice per group. (D) Hematoxylin/eosin stained sections from the lungs of the indicated mice 2 min after collagen/epinephrine injection. The thrombi were counted at 20-fold magnification for each field. Bars represent mean ± SDT from 100 fields.

Figure 3 depicts defective thrombosis in mice lacking factor XII. Using 20% FeCl₃In the case of induced injury, in vivo thrombosis on mesenteric arterioles was monitored. (A) Single platelet adhesion was detected 5 min post injury in all mouse strains, with the first thrombus observed 7 to 8 min post injury in wild type mice, the first thrombus occurring 14 to 35 min post injury in FXII-/-and the first thrombus occurring 5 to 35 min post injury in FXI-/-. (B) Thrombosis was observed in 100% of mesenteric arteries in wild-type mice, but only in 50% and 44.4% of FXII-/-mice. (C) Thrombosis in wild-type miceOn average, 25 minutes after injury blocked blood vessels, whereas thrombi formed in FXII and FXI deficient mice did not result in occlusion. Each symbol represents a single monitored arteriole. (D) Representative photograph of one experiment.

Fig. 4. Wild-type (n ═ 10), FXII-/- (n ═ 10) and FXI-/- (n ═ 11) were analyzed in the arterial occlusion model. Thrombosis was induced in the aorta by a single forceful compression with forceps. Blood flow was monitored with a perivascular ultrasound flow probe until total occlusion. The experiment was stopped after 40 minutes. Each symbol represents an individual. (B) Mechanical injury in the carotid artery was induced by ligation. After removal of the filamentous thrombus, in μm²The areas in wild type (n ═ 10) and FXII-/- (n ═ 10) were measured. (C) The micrographs show representative images 2 minutes after injury.

FIG. 5 depicts a defect in thrombosis in FXII deficient animals, which is restored by human FXII. (A) In wild-type mice as well as FXII-/-mice injected with human FXII, it was observed that when FeCl was carried out in 100% of mesenteric arteries₃Thrombosis upon induced injury. (B) In wild-type mice, the resulting thrombus occludes the blood vessels within an average of 25 minutes after injury, and in FXII-/-mice injected with human FXII, the resulting thrombus occludes the blood vessels within 22.7 minutes after injury. Each symbol represents an individual. (C) A representative photograph is shown. (D) FXII-/-mice received 2mg/kg hFXII-/-and induced thrombosis in the aorta by a single forceful compression with forceps. Blood flow was monitored with a perivascular ultrasound flow probe until total occlusion. The experiment was stopped after 40 minutes. Each symbol represents an individual.

FIG. 6 depicts the inhibition of thrombosis by anti-FXII antibodies in mice. Wild type mice received 2mg/kg of anti-FXII antibody or non-immune IgG intravenously. After 15 minutes, with 20% FeCl₃In the case of induced injury, in vivo thrombosis on mesenteric arterioles was monitored. (A) Single platelet adhesion was detected 5 minutes after injury in both groups. After 7 to 8 minutes, treated with control IgGThe first thrombus was observed in mice, whereas in anti-FXII treated mice the first thrombus appeared 12 to 32 minutes after injury. (B) Thrombosis was observed in 100% of the mesenteric arteries in control mice, but only in 60% of the anti-FXII treated mice. (C) The time to complete occlusion is shown. Each symbol represents an individual.

FIG. 7 depicts a modified model of arterial thrombosis. Initially, at the site of vascular injury, thrombosis was primarily due to exposure of Tissue Factor (TF) in the endothelial stroma. TF complexed with FVII initiates the extrinsic coagulation pathway. At the site of injury, FXII, which drives the intrinsic pathway via FXI, contributes little to thrombin (FII) production and is negligible for normal hemostasis. Thus, individuals with FXII defects do not suffer from bleeding. The thrombin generated initiates clot formation by forming fibrin and activating platelets. Expansion of thrombus growth: on the exposed surface of the growing thrombus, the intrinsic pathway induced by FXII critically promotes thrombin generation. Activated FXII produces additional fibrin through FXI. Thus, FXII as well as FXI defects severely impair thrombosis.

In the present invention, the potential contribution of the intrinsic pathway of coagulation to pathological thrombosis in vivo was evaluated by an arterial thrombosis model based on in vivo microscopy and flow measurements using mice lacking coagulation factor XII. Although the initial adhesion of platelets at the site of injury was unchanged in mutant animals, the subsequent formation and stabilization of three-dimensional thrombi was severely deficient. This defect is observed in different branches of the vasculature and can be fully restored by exogenous human coagulation factor XII. These findings establish the intrinsic coagulation pathway driven by coagulation factor XII as the primary link between primary and secondary hemostasis in a modified model of thrombosis.

To analyze the function of FXII for in vivo coagulation, FXII deficient mice were generated. FXII-/-mice are healthy, phenotypically indistinguishable from their wild-type littermates, and fertile. Detailed histological and hemostatic (hemostasiological) analyses did not show a link to increased thrombosis or bleeding in FXII-/-mice, despite prolonged aPTT (68 ± 17 seconds) and recalcification times (412 ± 78 seconds) (wt: 23 ± 4 and 210 ± 31 seconds) in plasma collected postorbitally (Pauer, h.u. et al (2004). Targeted deletion of muscle coagulation factor XII-a model for contact phase activation in video, blood, haem.92, 503-. Similar to FXII-deficient humans, FXII-/-mice had no increased bleeding tendency, as indicated by the similarity of tail bleeding times to those found in wild-type animals (369.5 ± 201.7 and 355.9 ± 176.1 seconds, respectively, with each group n ═ 12, fig. 1A). The peripheral blood cell count of the mutant mice was not different from the wild-type control. Notably, the Prothrombin Time (PT) of FXII-/-mice was similar to wild type (8.9 ± 1.3 vs. 9.1 ± 1.3 seconds), indicating that FXII deficiency did not affect fibrin formation by the extrinsic coagulation system (fig. 1B). To assess possible differences in FXII procoagulant activity between human and mouse, FXII deficient humans (FXII < 1%) or vice versa were reconstituted with murine wild-type plasma and PTT of the mixture was determined. In either case, clot formation became normal, supporting the notion that FXII is functionally equivalent for coagulation in humans and mice.

In humans, similar to FXII deficiency, deficiency in the contact system protein Plasma Kallikrein (PK) and high molecular weight kininogen (HK) does not lead to increased risk of bleeding despite prolonged aPTT. To confirm that the aPTT prolongation in FXII-/-mice was not due to an additional defect in the contact phase proteins, we analyzed PK and HK in FXII-/-and wt plasma. Western blots showed that HK and PK levels were equal in mutant and wild type mice (fig. 1C). Functionally, HK processing and thrombin formation are severely impaired compared to the wild type in FXII-/-plasma exposed to collagen or kaolin.

Blood coagulation and platelet activation are complementary and interdependent processes. Platelets interact with and promote activation of several coagulation factors, the central coagulation product thrombin being a potent platelet activator. Thus, the contribution of platelets and FXII to clot formation was examined in more detail next. To this end, we induced coagulation using kaolin, which normally activates FXII but has no direct effect on platelets, or collagen, which activates FXII and platelets, where it interacts with many receptors, most importantly α 2 β 1 integrin and GPVI. Collagen outperformed kaolin for clot formation in wild-type plasma in the presence but not absence of platelets (fig. 1D). In contrast, the relative potency of kaolin and collagen in plasma containing activation-deficient FcR γ -/-platelets is similar to PFP, and similar effects are seen with PRP from integrin α 2-/-mice. Platelet procoagulant activity is also efficiently triggered in clotting plasma, and the fibrin (ogen) receptor α IIb β 3 has been shown to play a key role in this process, although the underlying mechanisms are not fully understood. Consistent with these reports, blocking α IIb β 3 function of antibody JON/a greatly inhibited platelet-dependent shortening of clotting time (fig. 1D). Together, these results demonstrate that platelets in a procoagulant state can promote FXI I-induced clot formation.

To test whether collagen-induced FXII activation has functional consequences in vivo, wild-type and FXII-/-mice were tested in a lethal pulmonary thromboembolic model induced by infusion of a mixture of collagen (0.8mg/kg body weight) and epinephrine (60 μ g/kg body weight). All control mice (19/19) died within 5 minutes from extensive pulmonary thrombosis and cardiac arrest, with a > 95% reduction in circulating platelet counts at 2 minutes post challenge (fig. 2A, B). Under these experimental conditions, 35.7% (5/14) of FXII-/-mice survived, although their peripheral platelet counts decreased similarly to that in the wild-type control, suggesting that the observed protection was not based on a defect in platelet activation. This hypothesis was confirmed by in vitro studies that showed that FXII-/-platelets express normal levels of major surface glycoproteins, including collagen receptors, and that collagen-related peptides can normally activate cells (as measured by activation of integrin α IIb β 3 and P-selectin expression) by typical agonists such as thrombin, Adenosine Diphosphate (ADP), or GPVI-specific agonists. Consistent with this, FXII-/-platelets showed an unaltered aggregation response to collagen, ADP (fig. 2C), PMA or thrombin.

In a parallel set of experiments, FcR γ -/-mice were challenged with collagen/epinephrine. These mice were completely protected from death and platelet counts decreased only moderately 2 minutes after challenge, confirming that platelet activation is strictly required for lethality in this model. These data are further supported by histological analysis of lung sections from different groups of mice. Although most of the blood vessels were occluded in wild-type mice, this was significantly reduced in FXII-/-mice (survivors and non-survivors). Consistent with previous reports, little thrombus was found in the lungs from FcR γ -/-mice (fig. 2D). These results suggest that in vivo collagen initiates platelet activation and FXII dependent intrinsic coagulation pathways that act synergistically in this model to form an occlusive lung thrombus.

Pathological thrombosis is usually initiated by a crack or sudden rupture of an atherosclerotic plaque in the arterial branches of the vasculature, which leads to non-physiologically strong platelet activation and procoagulant activity on the surface of the subendothelial layer. To test the role of FXII in these processes, thrombosis was studied in wild-type and FXII-/-mice using different models of arterial injury. In the first model, oxidative damage was induced in mesenteric arterioles (60-100 μm in diameter) and thrombosis was examined by in vivo fluorescence microscopy. Wild-type and FXII-/-mice received fluorescently labeled platelets of the same genotype (1X 10)⁸) And applied topically with 20% ferric chloride (FeCl)₃) The saturated filter paper was used for 1 minute to induce injury, and ferric chloride caused the formation of free radicals, resulting in the destruction of endothelium. The interaction of platelets with the damaged vessel wall started rapidly and was firmly adherent in both groups of mice 5 minutes after injuryThe number of platelets was similar (fig. 3A). However, although in wild-type mice adherent platelets continually recruit additional platelets from the circulatory system, resulting in the formation of aggregates, this process is severely deficient in mutant mice. In 100% of the control vessels (17/17), a stable thrombus with a diameter > 20 μm had formed 10 minutes after the injury, which grew continuously over time and finally resulted in total occlusion in 94.1% (16/17) of the vessels within an observation period of 40 minutes (mean occlusion time: 25.6. + -. 8.9 minutes) (FIG. 3). In sharp contrast, in the mutant mice, the appearance of microaggregates or thrombus formation was completely absent in 50% (7/14) of the blood vessels. In the remaining 50% (7/14) of the vessels, a thrombus formed, however it was always unstable and rapidly detached from the vessel wall. No thrombus > 20 μm in diameter in any blood vessel remained attached at the site of injury for more than 1 minute. Thus, there was no vascular occlusion in FXII-/-mice during the observation period (40 min). This unexpected result demonstrates that FXII is FeCl₃The production and stabilization of platelet rich thrombi in the injured arterioles, and suggesting that the FXII-induced coagulation pathway contributes substantially to the observed thrombotic response. This hypothesis was confirmed when FXI deficient mice were analyzed in the same model. Since FXI is the major substrate for FXII in the "endogenous" cascade, similar defects in thrombosis must be expected in those mice. In fact, very similar to FXII-/-mice, almost normal platelet adhesion at the site of injury could be detected within the first 3 minutes after injury, while thrombus formation was completely inhibited in 55.6% (5/9) of the blood vessels. In the remaining vessels, the formed microaggregates and thrombi are unstable and are continuously embolized (fig. 3). As a result, no blood vessels were occluded during the observation period (40 minutes). This data shows that in FeCl₃FXI deficient mice are protected in an induced carotid occlusion model.

FeCl is known₃Induced arterial thrombosis is dependent on platelet and thrombin generation, but it is not clear how this type of injury resembles in diseased vessels (e.g. when arterial congee)Like the rupture of a sclerotic plaque) in the blood vessel. Therefore, to exclude a large amount of FeCl₃The possibility that induced oxidative damage creates non-physiological conditions that may artificially favor FXII-dependent contact phase activation, FXII function was assessed in another mature arterial thrombosis model in which injury was mechanically induced in the aorta and blood flow monitored with an ultrasound flow probe. Blood flow decreased continuously over a few minutes in all mice tested, after a temporary increase in blood flow immediately after injury. In all tested wild type mice (10/10), this reduction resulted in complete and irreversible occlusion of the vessels within 1.6 to 11.1 minutes post injury (mean occlusion time 5.3 ± 3.0 minutes, fig. 4A). Different images were found in FXII-/-mice, where stable thrombosis was severely defective. Although in all animals a gradual decrease in blood flow was observed within the first few minutes after injury, occlusion occurred only in 4 out of 10 mice. Furthermore, occlusive thrombi in those mice were unstable and rapidly embolized in all cases, reestablishing blood flow within 10 to 115 seconds post occlusion. The reopened vessel is not reoccluded. Thus, all FXII-/-mice showed a substantially normal flow rate through the damaged vessels at the end of the observation period (40 min). Very similar results were obtained with FXI-/-mice, of which 9 out of 11 failed to establish occlusive thrombosis within the observation period (30 min) (fig. 5A).

A serious defect in arterial thrombosis in FXII-/-mice was demonstrated in a third independent model in which platelet recruitment in the damaged carotid artery was studied by in vivo fluorescence microscopy. Platelets were purified from donor mice, fluorescently labeled, and injected into recipient mice of the same genotype. Vascular injury is caused by a forceful ligation of the carotid artery, which invariably results in the disruption of the endothelial layer and the frequent rupture of the internal elastic lamina, followed by rapid collagen-induced platelet adhesion and thrombosis at the site of injury (Gruner et al, Blood 102: 7/4/20072003). Despite the wild type actionThe material quickly forms large and stable thrombus (thrombus area: 102.821 +/-39.344 mu m)²(ii) a t ═ 5 minutes), it did not embolize, but only small and medium size aggregates were formed in mutant mice, which often shed from the site of injury (fig. 4B, C). Therefore, thrombus area was significantly reduced in mutant mice (8.120 ± 13.900 μm)²(ii) a t 5 min), although initial platelet adhesion on the vessel wall appears to be defect free.

To test whether the severe defect in thrombosis in FXII-/-mice is due to a lack of plasma FXII or platelet FXII, or possibly to a secondary unidentified effect of FXII deficiency, such as a change in the vasculature, arterial thrombosis was investigated in FXII-/-mice after administration of human FXII (2. mu.g/g body weight). This treatment normalized PTT (27 ± 6 seconds) and completely restored arterial thrombosis. At 100% FeCl₃In the injured mesenteric arterioles, a thrombus of > 20 μm had formed within 10 minutes after injury and all vessels were completely occluded during the observation period (FIGS. 5A-C). Even a tendency to faster occlusion was detectable in the reconstituted FXII-/-mice compared to untreated wild-type control mice (mean occlusion time: 22.7. + -. 8.2 min vs. 25.6. + -. 8.9 min). Similar results were obtained when the injury was mechanically induced in the aorta. In all tested vessels, complete and irreversible occlusion occurred within 10 minutes after injury (fig. 5D), confirming that the lack of plasma FXII caused the thrombotic defect observed in FXII-/-mice.

The above studies demonstrate that FXII is critical for arterial thrombosis and therefore can be used as an antithrombotic target.

To assess this directly, mice were treated with 2mg/kg body weight of polyclonal rabbit anti-mouse FXII antibody or non-immune rabbit antibody and assessed in FeCl₃Platelet recruitment and thrombosis in mesenteric arteries after induced injury. As shown in fig. 6A, platelet adhesion at the site of injury was comparable in both groups of mice. However, although in 100% of the control vessels, it had already formed within 10 minutes after injuryA thrombus of > 20 μm had formed and all vessels were completely occluded during the observation period (fig. 6B, C), but in animals treated with anti-FXII antibody, a thrombus of > 20 μm was observed in only 67% of the vessels and occlusion occurred in only 50% of the vessels.

Alternatively, to test the effect of small molecule FXII inhibitors, FeCl in carotid arteries₃5 min before induced injury, wild type mice were infused with the FXII inhibitor maize trypsin inhibitor (CTI, 50. mu.g/g body weight) (Wang et al (2005) J.Thromb.Heamost.3: 695-quick 702). Inhibitor treatment extended aPTT (62 ± 11 seconds, n ═ 4), but did not affect bleeding during the surgical procedure. Of the animals tested, none (0/4) was FeCl applied₃Within 30 minutes after the start, a thrombus was generated which occludes the blood vessel.

These results demonstrate that anti-FXII therapeutic agents, like anti-FXII antibodies or small molecule FXII inhibitors, provide significant protection against arterial thrombosis.

Although contact activation of FXII has been considered the start of the intrinsic coagulation cascade for over 50 years, this pathway is considered to be unrelated to blood coagulation. In the present invention, platelet recruitment and thrombus formation at the site of arterial injury in FXI I-deficient mice were analyzed by in situ video microscopy and ultrasound flux measurements using three different in vivo models and demonstrated a significant inability to form stable three-dimensional thrombi. This defect is based on FXII deficiency in plasma but not in other compartments, as it is completely reversed by intravenous injection of exogenous human FXII (figure 6), thereby also precluding the possibility that the secondary effects of FXII deficiency contribute to the observed phenotype.

These results were unexpected because FXII was considered an antithrombotic rather than prothrombotic enzyme based on reports indicating that FXII deficiency was associated with increased incidence of venous thrombosis (Kuhli, C., Scharrer, I., Koch, F., Ohrloff, C., and Hattenbach, L.O. (2004) Factor XII discovery: atheromic risk Factor for therapeutic blood circulation. am.J.Ophthalmol.137, 459. 464; Halbmayer, W.M., Mannhalter, C., Feichingler, C., Rubi, K. and Fischer, M. XII. [ Factor (Hagememor) discovery: a. Factor for blood circulation. 33. see blood circulation. III. binder. 43. see.

FXII deficient mice exhibited normal bleeding times (fig. 1A) and showed no signs of spontaneous or increased post-traumatic (intra-operative) bleeding, confirming that FXII is dispensable for normal hemostasis. Initially, these results seem to contradict the central laws of hemostasis that relate only to those factors whose defects are associated with bleeding or thrombosis, with blood coagulation. However, carefully considered, these data do not challenge this law but give rise to interesting possibilities: hemostasis and arterial thrombosis may occur by different mechanisms.

Although the sustained thrombin generation mechanism discussed above may be sufficient to produce a tampon, the data suggest that, at least in mice, additional activation of the intrinsic coagulation pathway is required for the formation of a stable arterial thrombus. There is no evidence for the possibility of species-specific differences in function of FXII or in the substrate of the enzyme. All coagulation parameters and hemostatic phenotypes of mutant mice were consistent with human FXII deficiency, and all changes observed in animals were normalized by reconstitution with human FXII (figure 5). Furthermore, it is excluded that the thrombotic defect is limited to a specific experimental model, since thrombotic defects are found in different arterial branches of the vascular system and are independent of the type of injury. It may be difficult to determine which type of lesion best reflects the vascular damage resulting from the rupture of atherosclerotic plaques, which are considered to be the major trigger of acute cardiovascular syndrome. Atherosclerotic lesions are rich in thrombogenic components, most importantly TF and fibrillar collagen. During atherogenesis, enhanced collagen synthesis by intimal smooth muscle cells and fibroblasts has been shown to contribute significantly to luminal narrowing. Rupture or cracking of the plaque results in exposure of collagen fibrils to flowing blood, which initiates platelet adhesion and aggregation. Furthermore, they induce FXII activation, as shown herein for type I fibrocollagen, which is the predominant type of collagen found in the vessel wall. Collagen may not be the only (patho-) physiological activator of FXII at the site of injury. Other candidates may be substances released from lysed cells or exposed to the ECM, including HSP90 or soluble and insoluble polyanions, such as nucleosomes or glycosaminoglycans.

Among these FXII activators, collagen is the most thrombogenic, since it also activates platelets efficiently. At the site of injury, platelets are bound to the ECM by reversible interaction of platelets GPIb-V-IX with collagen-bound vWf (which reduces the velocity of the cell, allowing binding of other receptors). Among these, the collagen receptor GPVI is most important because it activates integrins α 2 β 1 and α IIb β 3, which then mediate stable adhesion and promote cellular activation. In addition, platelet activation by the GPVI/FcR γ -chain complex induces a procoagulant state in cells characterized by Phosphatidylserine (PS) exposure and the production of (PS-exposed) membrane vesicles and microvesicles. Integrin α 2 β 1 promotes this process directly through "outside-in" signaling and indirectly through enhancement of GPVI-collagen interactions. It has been determined that membranes containing PS strongly accelerate two central reactions of the coagulation process: tenase and prothrombinase. The present invention shows that procoagulant platelets promote FXII-dependent coagulation in vitro by a mechanism involving the GPVI/FcR γ -chain complex as well as α 2 β 1 (figure 2). This may explain, at least in part, why α 2 β 1 deficient mice show a partial defect in occlusive thrombosis, although platelet adhesion is unchanged at the site of arterial injury. In addition to collagen, clotting plasma also effectively stimulates platelet procoagulant activity through integrin α IIb β 3-dependent mechanisms. In this experiment, blockade of α IIb β 3 almost completely inhibited platelet precipitation in FXII-dependent coagulation, suggesting that the well-known anticoagulant activity of α IIb β 3 antagonists may be based in part on inhibition of the intrinsic coagulation pathway driven by FXII. In summary, the present invention suggests that FXII driven contact system and platelet activation may be interdependent processes that play a role in pathological thrombosis.

Based on the experimental results, a model of pathological thrombosis schematically depicted in fig. 7 was proposed. At the site of vascular injury, the first layer of platelets is contacted with collagen in an environment that is additionally rich in TF and fibrin. Thus, it is not surprising that the adhesion of platelets to damaged vessel walls is not impaired in FXII-/-mice, and that these cells are very likely to be fully activated and in a procoagulant state. However, in growing thrombi, collagen is not present and the concentration of TF provided by the microvesicles may be lower compared to the vessel wall and their activity is reduced by TFPI released in large quantities from activated platelets. Under these conditions, an additional mechanism is required to maintain spatio-temporal (spatio-temporal) thrombin generation to activate newly recruited platelets and to induce their clotting activity by forming fibrin. The severe inability of FXII-/-mice to establish stable thrombi clearly indicates that the intrinsic coagulation pathway driven by FXII is an essential participant in this process. Together with the observation that low TF mice also exhibit compromised arterial thrombosis, these results suggest that both exogenous and endogenous pathways must be effectively operating and cooperate to promote the formation of three-dimensional and ultimately occlusive thrombi. Conversely, the lack of bleeding in FXII-/-mice suggests that thrombus growth in the third dimension may not be necessary to seal the pores in the vessel wall. This may explain why the extrinsic pathway of generating a first thin layer of fibrin and activated platelets is sufficient to mediate normal hemostasis. Our results create the following interesting possibilities: the formation of three-dimensional thrombi performs a function different from hemostasis. These functions may include stopping blood flow in certain areas of the tissue wound to prevent distribution of invading pathogens or toxins with the blood stream.

Experimental procedures

Animal(s) production

All experiments and Care were approved by the local Animal Care and Use Committee (Animal Care & Use Committee). Typical mouse mutants (Gailani, D., Lasky, N.M. and Broze, G.J., Jr. (1997) A.mut of factor XI deficiency. blood Cooagul. 8, 134. 144; Pauer, H.U., Renne, T., Hemmerlein, B.Legler, T.Fritzlars, S.A., Adham, I., Muller-Esterl, W.Emonens, G.Sanken, U.Engel, W.and Burfee, P.gered (2004) Tageted, S.E.E., U.S., Schgelet, W.S., and P.E.E.S., Ocular, W.E.E., and Haemarke.S., Haemarke, J.S., Biocement, K.S.S., N.S., J., P.S., Biocement, K.S.S., Haemarke, K.S.S., Haemark, N.S.S., Haemark, Haemarke, Haemark, J.S.S., Haemark, Haemarke, Haemark, J., Haemark, 10789-10794). C57B/6J mice (FXI-/-) or Sv129(FXII-/-) were used as controls. Mice lacking the FcR gamma chain (Takai, t., Li, m., Sylvestre, d., Clynes, r. and ravech, J.V. (1994). FcR gamma chain deletion reactions in bioinformatic effector cell devices. cell 76, 519-529) were from (Taconics, Germantown).

Production of anti-FXII antibodies

Total cellular RNA was isolated from the liver of 129sv wt mice and FXII-cDNA synthesis was performed using the "one-step RT-PCR kit" from Qiagen according to the manufacturer's instructions. The coagulation factor FXII heavy chain (positions 61-1062, corresponding to residues 21-354) was amplified using 25pmol each of 5-and 3-primers (ttggatccccaccatggaaagactccaag and ttgaattcgcgcatgaacgaggacag) introducing BamH I and EcoR I restriction sites, respectively, according to the following protocol: 30 cycles of 95 ℃ for 30 seconds, 58 ℃ for 60 seconds and 72 ℃ for 1 minute on a thermocycler (Biometra, G ö ttingen, Germany). The PCR product was cloned into the BamH I and EcoR I sites of pGEX-2T expression vector (Pharmacia). Subsequently, the protein to be sequenced was expressed in E.coli strain BL 21. Exponentially growing bacteria were stimulated with 0.5mM isopropyl-. beta. -D-thiogalactopyranoside for 1 hour, harvested, resuspended in 10mM Tris-HCl (pH7.4) containing 1mM EDTA, 200mM NaCl, 10. mu.g/ml benzamidine hydrochloride, 10. mu.g/ml phenylmethylsulfonyl fluoride and sonicated for 3 minutes in 15 second pulses each. After centrifugation at 15,000 Xg for 20 minutes at 4 ℃, the supernatant was removed and transferred to a GST-sepharose column (Pharmacia) for purification. The eluted protein was > 95% pure as inferred from Coomassie-stained SDS-PAGE. Polyclonal antibodies against GST-heavy chain FXII were generated in rabbits according to standard procedures. Antibodies were selected from hyperimmune serum using a column with FXII heavy chain fused to Maltose Binding Protein (MBP). These fusion proteins were expressed and purified using the pMAL-c2 expression system and an amylose resin column, similar to that described for the GST-fusion construct.

Platelet preparation

Mice were blood collected from the retroorbital plexus under ether anesthesia. Blood was collected in tubes containing 20U/mL heparin and platelet rich plasma (prp) was obtained by centrifugation at 300g for 10 min at Room Temperature (RT). To obtain washed platelets, prp was centrifuged at 1000g for 8 min, and the pellet was washed in the presence of prostacyclin (0.1. mu.g/mL) and apyrase (0.02U/mL) in modified Tyrodes-Hepes buffer (134mM NaCl, 0.34mM Na₂HPO₄，2.9mM KCl，12mM NaHCO₃20mM Hepes, 5mM glucose, 0.35% bovine serum albumin, pH6.6) were resuspended twice. The platelets were then resuspended in the same buffer (pH7.0, 0.02U/mL apyrase) and analyzed after incubation at 37 ℃ for at least 30 minutes.

Flow cytometry

Heparinized whole blood was treated with a composition containing 5mM glucose, 0.35% Bovine Serum Albumin (BSA) and 1mM CaCl₂Modified Tyrode-HEPES buffer (13)4mM NaCl，0.34mM Na₂HPO₄，2.9mM KCl，12mM NaHCO₃20mM HEPES [ N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid]pH7.0) was diluted 1: 20. The sample and fluorophore-labeled antibody were incubated at room temperature for 15 minutes and analyzed directly on FACScalibur (Becton Dickinson, Heidelberg, Germany) (Nieswandt, B., Schulte, V., and Bergmaier, W. (2004). Flow-cytometry analysis of mobilatelet function. methods mol. biol.272, 255-268).

Aggregation determination (aggregation)

For the determination of platelet aggregation, prp (200. mu.L, with 0.5X 10) was used⁶Individual platelets/. mu.L) was measured for light transmission. The transmission was recorded over 10 minutes using a fiberscope 4 channel aggregometer (apatlorberger und analysisystem, Hamburg, Germany) and expressed in arbitrary units, 100% transmission was adjusted with plasma. Platelet aggregation was induced by the addition of collagen (10. mu.g/mL) and ADP (5. mu.M).

Bleeding time test

Mice were anesthetized by intraperitoneal injection of tribromoethanol (Aldrich) (0.15ml/10g body weight) and 3mm segments of the tail tip were dissected with a scalpel. Tail bleeding was monitored by gently absorbing the blood beads with filter paper without touching the wound site. Bleeding was determined to stop when no blood was observed on the paper after a 15 second interval. Bleeding was stopped manually after 20 minutes, if necessary. Mice were treated with 100 μ g/mouse of hFXII as indicated.

Preparation of platelets for in vivo microscopy

Mouse blood (1 volume) was collected in 0.5 volume Hepes buffer containing 20U/mL heparin. The blood was centrifuged at 250g for 10 minutes and the platelet rich plasma was carefully transferred to a new tube. Platelets were labeled with 5-carboxyfluorescein diacetate succinimidyl ester (DCF) and adjusted to 200X 10⁶Platelets per 250 μ l final concentration (Massberg, s., Sausbier, m., Klatt,p., Bauer, M., Pfeifer, A., Siess, W., Fassler, R., Ruth, P., Krombach, F. and Hofmann, F. (1999). Increased addition and aggregation of plattens lacking cycloguanosine 3 ', 5' -monophosphosphates kinase I.J Exp Med 189, 1255-1264).

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With FeCl₃In vivo thrombosis model of induced lesions

Male and female mice 4-5 weeks old were anesthetized by intraperitoneal injection of 2, 2, 2-tribromoethanol and 2-methyl-2-butanol (Sigma) (2.5% solution, 0.15ml/10g body weight). Fluorescently labeled platelets were injected intravenously. Mesentery was carefully removed through a midline abdominal incision. Arterioles (35-50 μ M diameter) were visualized with a Zeiss Axiovert 200 inverted microscope (x10) equipped with a 100-W HBO fluorescent lamp source and a CCD camera (CV-M300) connected to a S-VHS video recorder (AG-7355, Panasonic, Matsushita Electric, Japan). Local application of FeCl to induce vascular injury and endothelial denudation₃After (20%) the arterioles were monitored for 40 minutes until complete occlusion occurred (> 1 minute duration of blood flow cessation). Firm platelet adhesion was determined as the number of fluorescently labeled platelets deposited on the vessel wall up to 5 minutes post injury, thrombi were characterized as platelet aggregates larger than 20 μm in diameter, and occlusion time of the vessel was characterized as the time required to reach a cessation of blood flow for at least 1 minute. In all experiments, a maximum of two arterioles from each mouse were selected based on the nature of the exposure. A total of 17 wt, 14 FXII-/-and 9 FXI-/-arterioles were studied.

Living body microscopic examination-carotid artery

Biomicroscopy of damaged carotid arteries was performed essentially as described (Massberg, s., Gawaz, m., Gruner, s., Schulte, v., Konrad, i., Zohlnhofer, d., Heinzmann, u., and Nieswandt, B. (2003). Briefly, mice were anesthetized by intraperitoneal injection of ketamine/xylazine (ketamine 100mg/kg, Parke-Davis, Karlsruhe, Gemany; xylazine 5mg/kg, Bayer AG, Leverkusen, Germany). Polyethylene catheters (Portex, Hyhe, England) were implanted in the right jugular vein and intravenously infused with fluorescent platelets (200X 106/250. mu.l). Carotid injury for endothelial denudation was induced by vigorous ligation. Before and after vascular injury, fluorescent platelets were visualized in situ by in vivo video microscopy of the right common carotid artery using a Zeiss Axiotech microscope (20x water immersion objective, W20x/0.5, Zeiss, G ö ttingen, Germany) with a 100W HBO mercury lamp for epi-illumination. Platelet adhesion and thrombus formation were recorded 5 minutes after induction of injury and videotape images were evaluated using a computer-aided image analysis program (Visitron, Munich, Germany).

Pulmonary thromboembolism

Mice were anesthetized by intraperitoneal injection of 2, 2, 2-tribromoethanol and 2-methyl-2-butanol (Aldrich) (2.5% solution, 0.15ml/10g body weight). The anesthetized mice received a mixture of collagen (0.8mg/kg) and epinephrine (60 μ g/kg) injected into the jugular vein. Incisions in surviving mice were sutured and allowed to recover. Anatomical and histological studies were performed on lungs fixed in 4% formaldehyde and paraffin sections were stained with hematoxylin/eosin.

Platelet count

Platelet counts were determined by flow cytometry on a FACScalibur (Becton Dickinson, Heidelberg, Germany). Results are expressed as mean ± s.d, or as a percentage of the control (wt, n ═ 19; FXII-/-, n ═ 14; and FcR γ -/-, n ═ 5).

Time of occlusion

The abdominal cavity of the anesthetized mice was opened longitudinally and the abdominal aorta was prepared. An ultrasonic flow probe was placed around the aorta and thrombus formation was induced by a single forced compression with forceps. Blood flow was monitored until total occlusion occurred. The experiment was manually stopped after 45 minutes. As indicated, human factor XII was replaced intravenously immediately prior to the experiment.

Histopathological analysis

The mice were sacrificed, lungs were rapidly removed, and fixed in buffered 4% formalin (pH 7.4; Kebo) for 24 hours at 4 ℃. Tissues were dehydrated and embedded in paraffin (Histolab ProductsAB), cut into 4 μm sections, and mounted on glass slides. After paraffin removal, tissues were stained with Mayers hematoxylin (Histolab Products AB) and eosin (Surgipath medical industries, Inc.).

SDS-polyacrylamide gel electrophoresis, Western blotting, and immunoblotting (immunoblotting)

Plasma was separated by 12.5% (w/v) polyacrylamide gel electrophoresis in the presence of 1% (w/v) SDS (Laemmli, 1970) (0, 3. mu.l/lane). Proteins were transferred to nitrocellulose membranes for 30 min at 100 mA. The membranes were blocked with PBS (pH7.4) containing 4% (w/v) milk powder and 0.05% (w/v) Tween-20. Membranes were probed with 0.5. mu.g/ml monoclonal antibody against MBK3 (Haaseman J. immunology 1988). Bound antibody was detected with a peroxidase-conjugated secondary antibody against mouse IgG (dilution 1: 5000) followed by a chemiluminescent detection method.

Blood coagulation assay

To determine the recalcification clotting time, 100. mu.l of mouse plasma treated with citrate as an anticoagulant (0.38% sodium citrate) was incubated with 100. mu.l each of Horm type collagen (Nycomed, Munich, Germany), ellagic acid, chondroitin sulfate (all from Sigma), kaolin or buffer (final concentration 30. mu.g/ml) in KC10 "Kugelkoagulometer" (Amelung, Lemgo, Germany) at 37 ℃ for 120 seconds. To test the effect of platelet activation on FXII-dependent coagulation, washed platelets were resuspended in a suspension containing 4mM Ca²⁺And 5. mu.M Ca²⁺Ion carrier A23187(Sigma) in Tyrode buffer for 10 min before addition to platelet-free plasma. By using 100. mu.l of 25mM CaCl₂Recalcification of the solution to initiate blood clot formation,and the time until coagulation occurred was recorded using coagulation timer KC4 (Amelung).

Coagulation assay

The automated blood coagulation system (BCS, Dade Behring) was used to determine both total and single coagulation parameters using Dade Behring's reagent according to the protocol described in detail by the manufacturer for human samples. The principles of the BCS assay protocol are available from the Dade Behring Package insert, which is found on the Dade Behring's website (http:// www.dadebehring.com). D-dimer was measured by ELISA from Asserachrom (Roche). Peripheral blood counts were determined on Sysmex XE 2100 according to standard protocols.

Thrombin measurement

Thrombin production was measured according to the method of Aronson et al (Circulation, 1985) with minor modifications. An aliquot (0.5ml) of platelet rich or platelet free plasma was placed in a round-bottomed polypropylene tube coated with Horms collagen (100. mu.g/ml, 24 hours, 4 ℃) and 20. mu.l 1M Ca was added²⁺To initiate coagulation. Samples (10. mu.l) were added to the wells of a microtiter plate containing 90. mu.l of 3.8% sodium citrate at intervals of 2.5-10 minutes over 60 minutes. By adding 50L of 2mmol/LS-2238 (H-D-Phe-Arg-NH-NO) in 1mol/L Tris (pH8.1)₂-HCl, a thrombin specific substrate; chromogenix, M ö lndal, Sweden) for 2 minutes. The absorbance of the released colored product was measured spectrophotometrically AT a wavelength of 405nm using a Vmax microtiter plate reader (EasyReader, EAR 340AT, SLT Lab Instruments GmbH, Vienna, Austria). Measurements were obtained in triplicate at each time point.

Statistical evaluation

Statistical analysis was performed using the unpaired Student's t test.

Although only the preferred embodiments have been specifically illustrated and described herein, it will be appreciated that many modifications and variations of the present invention are possible in light of the above teachings and are within the purview of the appended claims without departing from the spirit and intended scope of the invention.

Claims (16)

1. Use of at least one antibody and/or one inhibitor for inhibiting coagulation factor XII and for preventing the formation and/or stabilization of a thrombus and thereby preventing the growth of a three-dimensional endoluminal thrombus.

2. Use according to claim 1, wherein the antibody is an anti-FXII antibody.

3. Use according to claim 1, wherein the antibody inhibits FXII activation.

4. Use according to claim 1, wherein the inhibitor is a protease inhibitor.

5. Use according to claim 1, wherein the inhibitor is a serine protease inhibitor.

6. Use according to claim 3, wherein the protease inhibitor is selected from the group consisting of AT III inhibitors, angiotensin converting enzyme inhibitors, C1 inhibitors, aprotinin, alpha-1 protease inhibitors, antiproteinase ([ (S) -1-carboxy-2-phenylethyl ] -carbamoyl-L-Arg-L-Val-Arginal), Z-Pro-Pro-aldehyde-dimethylacetate, DX88, leupeptin, inhibitors of prolyl oligopeptidase such as Fmoc-Ala-Pyr-CN, maize trypsin inhibitor, mutants of bovine trypsin inhibitor, colicin, YAP (yellowtail flounder anticoagulant protein) and melon trypsin inhibitor-V and melon isoinhibitors.

7. Use of an antibody and/or inhibitor according to any one of claims 1 to 4 in medicine.

8. Use of an antibody and/or inhibitor according to any one of claims 1 to 4 in the manufacture of a medicament.

9. Use of an antibody and/or inhibitor according to any one of claims 1 to 4 for the treatment or prevention of a condition or disorder associated with the formation of venous or arterial thrombosis, in particular stroke or myocardial infarction, inflammation, complement activation, fibrinolysis, angiogenesis and/or diseases associated with FXII-induced kinin formation such as hereditary angioedema, bacterial infection of the lung, trypanosoma infection, hypotensive shock, pancreatitis, chagas disease or joint gout.

10. Pharmaceutical formulation comprising at least one antibody and/or one inhibitor, which is suitable for inhibiting factor XII and for preventing the formation and/or stabilization of pathological thrombi.

11. The formulation according to claim 8, wherein said antibody is an anti-FXII antibody.

12. A formulation according to claim 8 wherein the antibody inhibits FXII activation.

13. The formulation according to claim 8, wherein the inhibitor is a protease inhibitor.

14. The formulation according to claim 8, wherein the inhibitor is a serine protease inhibitor.

15. The formulation according to claim 10, wherein the protease inhibitor is selected from the group consisting of AT III inhibitors, angiotensin converting enzyme inhibitors, C1 inhibitors, aprotinin, alpha-1 protease inhibitors, antiproteinase ([ (S) -1-carboxy-2-phenylethyl ] -carbamoyl-L-Arg-L-Val-Arginal), Z-Pro-aldehyde-dimethylacetate, DX88, leupeptin, inhibitors of prolyl oligopeptidase such as Fmoc-Ala-Pyr-CN, maize trypsin inhibitor, mutants of bovine trypsin inhibitor, colicin, YAP (yellowtail flounder anticoagulant protein), and squash trypsin inhibitor-V and squash isoinhibitors.

16. Use of factor XII as an anti-thrombotic target by inhibiting factor XII by at least one antibody and/or one inhibitor and thus preventing the formation and/or stabilization of a thrombus or thrombus growth.