U.S. patent application number 13/339793 was filed with the patent office on 2012-06-14 for prevention of thrombus formation and/or stabilization.
This patent application is currently assigned to CSL Behring GMBH. Invention is credited to Bernhard Nieswandt, Thomas Renne.
Application Number | 20120148688 13/339793 |
Document ID | / |
Family ID | 34927953 |
Filed Date | 2012-06-14 |
United States Patent
Application |
20120148688 |
Kind Code |
A1 |
Nieswandt; Bernhard ; et
al. |
June 14, 2012 |
PREVENTION OF THROMBUS FORMATION AND/OR STABILIZATION
Abstract
The present invention relates to the use of at least one
antibody and/or one inhibitor for inhibiting factor XII and
preventing the formation and/or the stabilization of three
dimensional thrombi. It also relates to a pharmaceutical
formulation and the use of factor XII as an anti-thrombotic
target.
Inventors: |
Nieswandt; Bernhard;
(Wurzburg, DE) ; Renne; Thomas; (Wurzburg,
DE) |
Assignee: |
CSL Behring GMBH
|
Family ID: |
34927953 |
Appl. No.: |
13/339793 |
Filed: |
December 29, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11793820 |
Nov 26, 2007 |
8119137 |
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PCT/EP2005/013714 |
Dec 20, 2005 |
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13339793 |
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Current U.S.
Class: |
424/750 ;
424/758; 514/14.2 |
Current CPC
Class: |
A61P 7/10 20180101; Y02A
50/414 20180101; C07K 16/36 20130101; A61K 38/556 20130101; A61K
2039/505 20130101; A61K 38/55 20130101; A61K 39/3955 20130101; A61P
31/04 20180101; A61P 19/06 20180101; C07K 2317/76 20130101; A61P
7/02 20180101; A61P 37/00 20180101; A61P 9/10 20180101; A61P 1/18
20180101; A61P 43/00 20180101; A61K 38/56 20130101; A61P 33/00
20180101; A61P 29/00 20180101; C07K 16/40 20130101; Y02A 50/30
20180101; A61P 9/00 20180101; A61P 31/00 20180101; A61K 38/57
20130101 |
Class at
Publication: |
424/750 ;
514/14.2; 424/758 |
International
Class: |
A61K 38/57 20060101
A61K038/57; A61P 7/02 20060101 A61P007/02; A61K 36/899 20060101
A61K036/899; A61K 38/05 20060101 A61K038/05; A61K 36/42 20060101
A61K036/42 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 23, 2004 |
EP |
04030593.0 |
Claims
1.-16. (canceled)
17. A method of treating a patient at risk for the formation and/or
the stabilization of thrombi comprising administering to said
patient at least one inhibitor of Factor XII or activated Factor
XII (collectively FXII), wherein the patient has, has had, or is at
risk for: venous thrombosis, arterial thrombosis, stroke,
myocardial infarction, or use of a prosthetic device, and wherein
the at least one inhibitor of FXII is not ATIII inhibitor,
angiotensin converting enzyme inhibitor, C1 inhibitor, aprotinin,
alpha-1 protease inhibitor, leupeptin, ecotin, bovine pancreatic
trypsin inhibitor mutants, antipain, or DX88.
18. The method of claim 17, wherein the at least one inhibitor is
at least one protease inhibitor.
19. The method of claim 18, wherein the at least one protease
inhibitor is a serine protease inhibitor.
20. The method of claim 18, wherein the at least one protease
inhibitor is Z-Pro-Pro-aldehyde-dimethyl acetate.
21. The method of claim 18, wherein the at least one protease
inhibitor is chosen from one or more of: Fmoc-Ala-Pyr-CN,
corn-trypsin inhibitor, yellowfin sole anticoagulant protein,
Curcurbita maxima trypsin inhibitor-V, and Curcurbita maxima
isoinhibitor.
22. The method of claim 17, wherein the prosthetic device is a
haemodialyser, cardiopulmonary by-pass circuit, vascular stent, or
in-dwelling catheter.
23. The method of claim 17, wherein the patient has, has had, or is
at risk for venous thrombosis.
24. The method of claim 17, wherein the patient has, has had, or is
at risk for arterial thrombosis.
25. The method of claim 17, wherein the patient has, has had, or is
at risk for stroke.
26. The method of claim 17, wherein the patient has, has had, or is
at risk for myocardial infarction.
Description
[0001] The subject of the present invention is, in the most general
aspect, the prevention of the formation and/or stabilization of
three-dimensional arterial or venous thrombi.
[0002] In particular the present invention relates to the use of at
least one antibody and/or one inhibitor for inhibiting factor XII
activity and preventing the formation and/or the stabilization of
thrombi and thrombus growth. It also relates to a pharmaceutical
formulation and the use of factor XII as an anti-thrombotic
target.
[0003] Vessel wall injury triggers sudden adhesion and aggregation
of blood platelets, followed by the activation of the plasma
coagulation system and the formation of fibrin-containing thrombi,
which occlude the site of injury. These events are crucial to limit
posttraumatic blood loss but may also occlude diseased vessels
leading to ischemia and infarction of vital organs. In the
waterfall or cascade model, blood coagulation proceeds by a series
of reactions involving the activation of zymogens by limited
proteolysis culminating in the fulminant generation of thrombin,
which converts plasma fibrinogen to fibrin and potently activates
platelets. In turn, collagen- or fibrin-adherent platelets
facilitate thrombin generation by several orders of magnitude by
exposing procoagulant phosphatidyl serine (PS) on their outer
surface which propagates assembly and activation of coagulation
protease complexes and by direct interaction between platelet
receptors and coagulation factors.
[0004] Two converging pathways for coagulation exist that are
triggered by either extrinsic (vessel wall) or intrinsic
(blood-borne) components of the vascular system. The "extrinsic"
pathway is initiated by the complex of the plasma factor VII (FVII)
with the integral membrane protein tissue factor (TF), an essential
coagulation cofactor that is absent on the luminal surface but
strongly expressed in subendothelial layers of the vessel. TF
expressed in circulating microvesicles might also contribute to
thrombus propagation by sustaining thrombin generation on the
surface of activated platelets.
[0005] The "intrinsic" or contact activation pathway is initiated
when factor XII (FXII, Hageman factor) comes into contact to
negatively charged surfaces in a reaction involving high molecular
weight kininogen and plasma kallikrein. FXII can be activated by
macromolecular constituents of the subendothelial matrix such as
glycosaminoglycans and collagens, sulfatides, nucleotides and other
soluble polyanions or non-physiological material such as glass or
polymers. One of the most potent contact activators is kaolin and
this reaction serves as the mechanistic basis for the major
clinical clotting test, the (activated) partial thromboplastin time
(PTT, aPTT). In reactions propagated by platelets, activated FXII
then activates FXI and FXIa in turn activates factor IX. Despite
its high potency to induce blood clotting in vitro, the
(patho)physiological significance of the FXII-triggered intrinsic
coagulation pathway is questioned by the fact that hereditary
deficiency of FXII as well as of high molecular weight kininogen
and plasma kallikrein is not associated with bleeding
complications. Together with the observation that humans and mice
lacking extrinsic pathway constituents, such as TF, FVII or factor
IX, suffer from severe bleeding this has lead to the current
hypothesis that fibrin formation is in vivo exclusively initiated
by the extrinsic cascade (Mackman, N. (2004). Role of tissue factor
in hemostasis, thrombosis, and vascular development. Arterioscler.
Thromb. Vasc. Biol. 24, 1015-1022).
[0006] Like all physiological mechanisms, the coagulation cascade
can become activated inappropriately and result in the formation of
haemostatic plugs inside the blood vessels. Thereby, vessels can
become blocked and the blood supply to distal organs limited. This
process is known as thromboembolism and is associated with high
mortality. In addition, the use of prosthetic devices that are in
contact with blood is severely limited because of activation of the
coagulation cascade and coating of the prosthetic surface, often
compromising its function. Examples of such prosthetic devices are
haemodialysers, cardiopulmonary by-pass circuits, vascular stents
and in-dwelling catheters. In cases where such devices are used,
anticoagulants, such as heparin, are used to prevent fibrin from
depositing on the surface. However, some patients are intolerant of
heparin, which can cause heparin induced thrombocytopenia (HIT)
resulting in platelet aggregation and life threatening thrombosis.
Furthermore, an intrinsic risk of all anticoagulants used in
clinics is an associated increased risk of serious bleeding.
Therefore, a need for new types of anticoagulant exists that is not
associated with such complications and that can be used in affected
patients or as superior therapy concept preventing thrombosis
without increased bleeding tendencies.
[0007] Hence, it is apparent that there still exists a need for an
improved medication for the treatment or prophylaxis of thrombosis
and similar disorders. Therefore, it is an object of the present
invention to satisfy such a need. For more than five decades it has
been known that deficiency of coagulation factor XII is not
associated with increases spontaneous or injury related bleeding
complications (Ratnoff, O. D. & Colopy, J. E. (1955) A familial
hemorrhagic trait associated with a deficiency of a clot-promoting
fraction of plasma. J Clin Invest 34, 602-13). Indeed, although
presenting a pathological aPTT (a clinical clotting test that
addresses the intrinsic pathway of coagulation) humans that are
deficient in FXII do not suffer from abnormal bleeding even during
major surgical procedures (Colman, R. W. Hemostasis 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, Philadelphia, 2001). In
contrast, deficiency of FXII had been associated with increased
risk of venous thrombosis (Kuhli, C., Scharrer, I., Koch, F.,
Ohrloff, C. & Hattenbach, L. O. (2004) Factor XII deficiency: a
thrombophilic risk factor for retinal vein occlusion. Am. J.
Ophthalmol. 137, 459-464., Halbmayer, W. M., Mannhalter, C.,
Feichtinger, C., Rubi, K. & Fischer, M. (1993) Factor XII
(Hageman factor) deficiency: a risk factor for development of
thromboembolism. Incidence of factor XII deficiency in patients
after recurrent venous or arterial thromboembolism and myocardial
infarction. Wien. Med. Wochenschr. 143, 43-50.). Studies and case
reports supporting this idea refer to the index case for FXII
deficiency, Mr. John Hageman, who died of pulmonary embolism. The
hypothesis that FXII deficiency is associated with an increased
prothrombotic risk is challenged by a recent reevaluation of
several case reports linking FXII deficiency with thrombosis
(Girolami, A., Randi, Gavasso, S., Lombardi, A. M. & Spiezia,
F. (2004) The Occasional Venous Thromboses Seen in Patients with
Severe (Homozygous) FXII Deficiency are Probably Due to Associated
Risk Factors: A Study of Prevalence in 21 Patients and Review of
the Literature. J. Thromb. Thrombolysis 17, 139-143). In most cases
the authors identified concomitant congenital or acquired
prothrombotic risk factors in combination with factor FXII
deficiency that could be responsible for the thrombotic event
independently of FXII. The largest epidemiological studies using
well characterized patients (Koster, T., Rosendaal, F. R., Briet,
E. & Vandenbroucke, J. P. (1994) John Hageman's factor and
deep-vein thrombosis: Leiden thrombophilia Study. Br. J. Haematol.
87, 422-424) and FXII-deficient families (Zeerleder, S. et al.
(1999) Reevaluation of the incidence of thromboembolic
complications in congenital factor XII deficiency-a study on 73
subjects from 14 Swiss families. Thromb. Haemost. 82, 1240-1246)
indicated that there is no correlation of FXII deficiency and any
pro- or anti-thrombotic risk.
[0008] Surprisingly and in contrast to common believe of those
skilled in the art the applicant has discovered that the factor
XII-driven intrinsic coagulation pathway is essential for arterial
thrombus formation in vivo but not necessary for normal
tissue-specific hemostasis. Unexpectedly, these results change the
long-standing concept that blood clotting in vivo is exclusively
mediated by the extrinsic pathway and place factor XII in a central
position in the process of pathological thrombus formation.
[0009] Accordingly, the first subject of the invention is the use
of at least one antibody and/or at least one inhibitor for
inhibiting factor XII and preventing the formation and/or the
stabilization of three-dimensional arterial or venous thrombi. The
anti-FXII antibody respective inhibitor may hereby function so as
inhibiting the activation of FXII and/or interfere with other
portions of the FXII molecule that are critically involved in FXII
activation.
[0010] Together with the fact that the intrinsic pathway is not
required for hemostasis, this establishes factor XII as a promising
new target for powerful antithrombotic therapy. In addition these
results are important for the development of anti-FXII agents to
control other contact system-linked (patho)mechanisms such as
inflammation, complement activation, fibrinolysis, angiogenesis and
kinin formation.
[0011] Therefore, the present invention further provides the use of
such an antibody and/or inhibitor in the treatment or prophylaxis
of a condition or disorder related to arterial thrombus formation,
i. e. stroke or myocardial infarction, inflammation, complement
activation, fibrinolysis, angiogenesis and/or diseases linked to
pathological kinin formation such as hypotonic shock, edema
including hereditary angioedema, bacterial infections, arthritis,
pancreatitis, or articular gout.
[0012] In particular, the use of at least one anti-FXII antibody
(e.g. like F1 antibody (MoAb F1, Rayon et al., Blood. 1995 Dec. 1;
86(11):4134-43)) and/or the use of at least one protease inhibitor
to inhibit FXII-driven thrombus formation is according to the
present invention.
[0013] Especially preferred is the protease inhibitor selected from
for example AT III inhibitor, angiotensin converting enzyme
inhibitor, Cl inhibitor, aprotinin, alpha-1 protease inhibitor,
antipain
([(5)-1-Carboxy-2-Phenylethyl]-Carbamoyl-L-Arg-L-Val-Arginal),
Z-Pro-Pro-aldehyde-dimethyl acetate, DX88 (Dyax Inc., 300
Technology Square, Cambridge, Mass. 02139, USA; cited in: Williams
A. and Baird LG., Transfus Apheresis Sci. 2003 Dec. 29 (3):255-8),
leupeptin, inhibitors of prolyl oligopeptidase such as
Fmoc-Ala-Pyr-CN, corn-trypsin inhibitor, mutants of the bovine
pancreatic trypsin inhibitor, ecotin, YAP (yellowfin sole
anticoagulant protein) and Cucurbita maxima trypsin inhibitor-V
including Curcurbita maxima isoinhibitors.
[0014] Accordingly, the present invention provides the use of such
an antibody and/or inhibitor described herein in medicine; and also
the use of such an antibody and/or inhibitor in the manufacture of
a medicament.
[0015] Therefore, according to another aspect of the present
invention, a pharmaceutical formulation is provided comprising at
least one antibody and/or one inhibitor, which is suitable for
inhibiting factor XII and which prevents the formation and/or the
stabilization of three-dimensional arterial or venous thrombi.
[0016] In particular, the antibody used for the pharmaceutical
formulation is an anti-FXII antibody (e.g. like F1 antibody (MoAb
F1, Rayon et al., Blood. 1995 Dec. 1; 86(11):4134-43)), and the
inhibitor is a protease inhibitor, for example but not limited to
AT III inhibitor, angiotensin converting enzyme inhibitor, C1
inhibitor, aprotinin, alpha-1 protease inhibitor, antipain
([(S)-1-Carboxy-2-Phenylethyl]-Carbamoyl-L-Arg-L-Val-Arginal),
Z-Pro-Pro-aldehyde-dimethyl acetate, DX88 (Dyax Inc., 300
Technology Square, Cambridge, Mass. 02139, USA; cited in: Williams
A. and Baird LG., Transfus Apheresis Sci. 2003 Dec. 29 (3):255-8),
leupeptin, inhibitors of prolyl oligopeptidase such as
Fmoc-Ala-Pyr-CN, corn-trypsin inhibitor, mutants of the bovine
pancreatic trypsin inhibitor, ecotin, YAP (yellowfin sole
anticoagulant protein) and Cucurbita maxima trypsin inhibitor-V
including Curcurbita maxima isoinhibitors.
[0017] The antibody may also be a fragment of same or mimetic
retaining the inhibitory activity, for example analogues of Kunitz
Protease Inhibitor domain of amyloid precursor protein as disclosed
in U.S. Pat. No. 6,613,890 especially in columns 4 through 8. Other
suitable inhibitors may be Hamadarin as disclosed by Harahiko Isawa
et al. in The Journal of Biological Chemistry, Vol. 277, No. 31
(August 2, pp. 27651-27658, 2002). A suitable Corn Trypsin
Inhibitor and methods of its production are disclosed in Zhi-Yuan
Chen et al., Applied and Environmental Microbiology, March 1999, p.
1320-1324 and reference 19 cited ibidem. All references cited are
incorporated for reference including their entire content in this
application. Last not least, small molecules isolated for example
via use of FXII respective FXIIa inhibition as the assay on which
selection is based are part of the invention, as well as their
respective use described above or below. These small molecule FXIIa
inhibitors could be designed on the bases of a crystal structure of
FXII. Therefore several FXII domains or the light chain could be
expressed recombinantly in expression systems such as E. coli,
yeast or mammalian cells. Then the protein is purified and
crystallized using standard procedures as described for the FXII
substrate FXI (Jin L, et al. (2005) Crystal structures of the FXIa
catalytic domain in complex with ecotin mutants reveal
substrate-like interactions. J Biol Chem. 280(6):4704-12.)
Alternatively, small molecule serine protease inhibitors could be
included to stabilize the FXII structure. Such formulations
comprising small molecule inhibitors of protein targets, which can
be for example designed guided by crystals of these target
proteins, are well known in the art and include pharmaceutical
formulations that may be, for example, administered to a patient
systemically, such as parenterally, or orally or topically.
[0018] The term "parenteral" as used here includes subcutaneous,
intravenous, intramuscular, intra-arterial and intra-tracheal
injection, instillation, spray application and infusion techniques.
Parenteral formulations are preferably administered intravenously,
either in bolus form or as a constant infusion, or subcutaneously,
according to known procedures. Preferred liquid carriers, which are
well known for parenteral use, include sterile water, saline,
aqueous dextrose, sugar solutions, ethanol, glycols and oils.
[0019] Tablets and capsules for oral administration may contain
conventional excipients such as binding agents, fillers, lubricants
and wetting agents, etc. Oral liquid preparations may be in the
form of aqueous or oily suspensions, solutions, emulsions, syrups,
elixirs or the like, or may be presented as a dry product for
reconstitution with water or other suitable vehicle for use. Such
liquid preparations may contain conventional additives, such as
suspending agents, emulsifying agents, non-aqueous vehicles and
preservatives.
[0020] Formulations suitable for topical application may be in the
form of aqueous or oily suspensions, solutions, emulsions, gels or,
preferably, emulsion ointments. Formulations useful for spray
application may be in the form of a sprayable liquid or a dry
powder.
[0021] According to a third aspect of the present invention, the
use of factor XII as an anti-thrombotic target by inhibiting factor
XII by at least one antibody and/or one inhibitor and preventing
therefore the formation and/or the stabilization of
three-dimensional thrombi in the vessel is provided.
[0022] The nature, benefit, and further features of the present
invention become apparent from the following detailed description
of the performed experiments and their results when read in
conjunction with the accompanying figures described below.
[0023] Factor XII-deficient mice were used to analyze the function
of the intrinsic coagulation cascade in hemostasis and thrombosis.
Intravital fluorescence microscopy and ultrasonic flow measurements
revealed a severe defect in the formation and stabilization of
three-dimensional thrombi in different arterial branches of the
vascular system. Reconstitution of the mutant mice with human
factor XII restored the intrinsic coagulation pathway in vitro and
arterial thrombus formation in vivo. Mechanistically, the
procoagulant activity of the intrinsic pathway was critically
promoted by activated platelets. These results place the
FXII-induced intrinsic blood coagulation cascade in a central
position in the process of arterial thrombus formation linking
plasmatic coagulation with platelet aggregation.
[0024] FIGS. 1A, 1B, 1C, and 1D describe the coagulation analysis
of FXII deficient mice: (A) Tail bleeding times of wild-type (n=12)
and FXII-/- (n=11) mice. Each symbol represents one individual. (B)
Peripheral blood counts in thousands/.mu.l and global coagulation
parameters of FXII-/- and wt mice. The abbreviations are white
blood counts (WBC), activated partial thromboplastin time (aPTT)
and prothrombin time (PT). Values give mean values.+-.SD of 10 mice
of each genotype. (C) Contact system proteins FXII, plasma
kallikrein (PK) and high molecular weight kininogen (HK) probed in
0.3 .mu.l wt and FXII-/- plasma by Western blotting using specific
antibodies. A molecular weight standard is 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.gamma.-/- and integrin
.alpha.2-deficient mice following activation with kaolin (dark
columns) or collagen (light columns). The effect of JON/A was
analyzed in C57BL/6 plasma supplemented with 50 .mu.g/ml antibody.
Means.+-.STD from 6 experiments are given.
[0025] FIG. 2(A) Thromboembolic mortality was observed following
the intravenous injection of collagen (0.8 mg/kg) and epinephrine
(60 .mu.g/kg). All wild-type mice died within 5 min. Animals that
were alive 30 min after challenge were considered survivors. FIG.
2(B) Platelet counts in control (n=19), FXII-/- (n=14) and
FcR.gamma.-/- (n=5) mice 2 min after infusion of
collagen/epinephrine. FIG. 2(C) Heparinized platelet rich plasma
from wild-type and FXII-/- mice was stimulated with collagen (10
.mu.g/m1) or ADP (5 .mu.M) and light transmission was recorded in a
standard aggregometer. The results shown are representative of six
mice per group. FIG. 2(D)
[0026] Hematoxilin/Eosin-stained sections from lungs of the
indicated mice 2 min after collagen/epinephrine injection. Thrombi
per eyefield ware counted in 20.times. magnification. The bars
represent means .+-.SDT from 100 eyefields.
[0027] FIGS. 3A, 3B, 3C, and 3D describe the defective thrombus
formation in mice lacking factor XII in vivo. Thrombus formation in
vivo was monitored on mesenteric arterioles upon injury induced
with 20% FeCl3. (A) Single platelet adhesion is detected 5 min
after injury in all mouse strains, 7 to 8 minutes after injury the
first thrombi in wt mice were observed, whereas in FXII-/- the
first thrombi occurred 14 to 35 minutes after injury and in FXI-/-
5 to 35 minutes after injury. (B) Thrombus formation was observed
in 100% of mesenteric arteries in wild type mice, but only in 50%
of FXII-/- mice and in 44.4% of FXI-/- mice. (C) Thrombi formed in
wt mice occluded the vessel in average 25 minutes after injury
whereas thrombi formed in FXII and FXI deficient mice did not lead
to occlusion. Each symbol represents one single monitored
arteriole. (D) Representative pictures of one experiment.
[0028] FIG. 4(A) Wild-type (n=10), FXII-/- (n=10) and FXI-/- (n=11)
were analyzed in an arterial occlusion model. Thrombosis was
induced in the aorta by one firm compression with a forceps. Blood
flow was monitored with an perivascular ultrasonic flow probe until
complete occlusion. The experiment was stopped after 40 min. Each
symbol represents one individual. FIG. 4(B) Mechanical injury in
the carotid artery was induced by a ligation. After removal of the
filament thrombus area in wild-type (n=10) and FXII-/- (n=10) was
measured in .mu.m2. FIG. 4(C) The photomicrographs show
representative images 2 min after injury.
[0029] FIGS. 5A, 5B, 5C, and 5D describe the defect in thrombus
formation in FXII deficient animals which is restored by human
FXII. (A) Thrombus formation upon FeCl.sub.3 induced injury was
observed in 100% of mesenteric arteries in wild-type mice as well
as in FXII-/- mice injected with human FXII. (B) Formed thrombi
occluded the vessel in average 25 minutes after injury in wild-type
mice and in 22.7 minutes after injury in FXII-/- mice injected with
human FXII. Each symbol represents one individual. (C)
Representative pictures are shown. (D) FXII-/- mice received 2
mg/kg hFXII-/- and thrombosis was induced in the aorta by one firm
compression with a forceps. Blood flow was monitored with an
perivascular ultrasonic flow probe until complete occlusion. The
experiment was stopped after 40 min. Each symbol represents one
individual.
[0030] FIGS. 6A, 6B, and 6C describe the anti-FXII antibodies
inhibiting thrombus formation in mice in vivo. Wild-type mice
received 2 mg/kg anti-FXII antibodies or non-immune IgG i.v. After
15 min, thrombus formation in vivo was monitored on mesenteric
arterioles upon injury induced with 20% FeCl.sub.3. (A) Single
platelet adhesion is detected 5 min after injury in both groups.
After 7 to 8 minutes the first thrombi mice in control IgG-treated
mice were observed, whereas in anti-FXII-treated mice the first
thrombi occurred 12 to 32 minutes after injury. (B) Thrombus
formation was observed in 100% of mesenteric arteries in control
mice, but only in 60% of anti-FXII-treated mice. (C) Time to
complete occlusion is shown. Each symbol represents one
individual.
[0031] FIGS. 7A and 7B describe a revised model of arterial
thrombus formation. (A) Initially, at sites of vascular lesions
thrombin formation is predominantly due to tissue factor (TF)
exposure in the subendothelial matrix. TF in complex with FVII
initiates the extrinsic pathway of coagulation. At the site of
injury the contribution of the FXII driving the intrinsic pathway
via FXI for thrombin (FII) generation is minor and negligible for
normal hemostasis. Accordingly individuals with FXII-deficiency do
not suffer from bleeding. Generated thrombin initiates clot
formation by forming fibrin and activating platelets. (B)
Propagation of thrombus growth: On surfaces exposed in the growing
thrombus the FXII-induced intrinsic pathway critically contributes
to thrombin generation. Activated FXII generates additional fibrin
through FXI. Accordingly, FXII- as well as FXI-deficiency severely
impairs thrombus formation.
[0032] In the present invention a potential contribution of the
intrinsic pathway of coagulation for pathological thrombus
formation in vivo was assessed by intravital microscopy- and flow
measurement-based models of arterial thrombosis using mice lacking
factor XII. While initial adhesion of platelets at sites of injury
is not altered in the mutant animals, the subsequent formation and
stabilization of three-dimensional thrombi is severely defective.
This defect was seen in different branches of the vasculature and
could be completely restored by exogenous human factor XII. These
findings establish the factor XII-driven intrinsic coagulation
pathway as a major link between primary and secondary hemostasis in
a revised model of thrombus formation.
[0033] To analyze the function of FXII for clotting in vivo,
FXII-deficient mice were generated. FXII-/- mice are healthy,
phenotypically indistinguishable from their wild-type littermates,
and fertile. Detailed histological and hemostasiological analyses
showed no correlates for increased thrombosis or bleeding in
FXII-/- mice despite a prolonged aPTT of 68.+-.17 sec and
recalcification time of 412.+-.78 sec in retroorbitally collected
plasma (wt: 23.+-.4 and 210.+-.31 sec) (Pauer, H. U., et al.
(2004). Targeted deletion of murine coagulation factor XII gene-a
model for contact phase activation in vivo. Thromb. Haemost. 92,
503-508). Similarly to FXII-deficient humans, FXII-/- mice present
with no increased bleeding tendency as indicated by tail bleeding
times similar to those found in wild-type animals (369.5.+-.201.7
and 355.9.+-.176.1 sec, respectively, n=12 per group, FIG. 1A).
Peripheral blood cell counts of mutant mice did not differ from
wild-type controls. Notably, the prothrombin time (PT) of FXII-/-
mice was similar to the wild-type (8.9.+-.1.3 vs. 9.1.+-.1.3 sec)
indicating that FXII deficiency does not affect fibrin formation by
the extrinsic coagulation system (FIG. 1B). To assess potential
differences in FXII procoagulant activity between humans and mice,
FXII-deficient human (FXII<1%) with murine wild-type plasma or
vice versa were reconstituted and the PTT of the mixtures was
determined. In either case, clot formation was normalized
supporting the notion that FXII function for clotting is comparable
in humans and mice.
[0034] In humans similarly to FXII deficiency the deficiency of
contact system proteins plasma kallikrein (PK) and high molecular
weight kininogen (HK) does not result in an increased bleeding risk
despite a prolonged aPTT. To confirm that the aPTT prolongation in
FXII-/- mice is not due to additional defects of contact phase
proteins, we analyzed PK and HK in FXII-/- and wt plasma. The
Western blot indicated that HK and PK levels are equivalent in
mutant and wild-type mice (FIG. 1C). Functionally, in FXII-/-
plasma exposed to collagen or kaolin HK procession and thrombin
formation was severely impaired compared to wild-type.
[0035] Blood coagulation and platelet activation are complementary
and mutually dependent processes. Platelets interact with and
contribute to the activation of several coagulation factors and the
central coagulation product thrombin is a potent platelet
activator. Therefore, next the contribution of platelets and FXII
was examined to clot formation in more detail. For this, we induced
clotting using either kaolin that classically activates FXII but
has no direct effect on platelets or collagen, which activates both
FXII and platelets where it interacts with numerous receptors, most
importantly .alpha.2.beta.1 integrin and GPVI. In the presence, but
not in the absence of platelets, collagen was superior to kaolin
for clot formation in wild-type plasma (FIG. 1D). In contrast, in
plasma containing activation-defective FcR.gamma.-/- platelets, the
relative potency of kaolin and collagen was similar to PFP and a
similar effect was seen with PRP from integrin .alpha.2-/- mice.
Platelet procoagulant activity is also efficiently triggered in
coagulating plasma and the fibrin(ogen) receptor .alpha.IIb.beta.3
has been shown to play a crucial role in this process although the
underlying mechanisms are not fully understood. In agreement with
these reports, the .alpha.IIb.beta.3-function blocking antibody
JON/A largely inhibited the platelet-dependent decrease in the
clotting time (FIG. 1D). Together, these results demonstrated that
platelets in a procoagulant state can promote FXII-induced clot
formation.
[0036] To test whether collagen-induced FXII activation has
functional consequences in vivo, wild-type and FXII-/- mice were
subjected to a model of lethal pulmonary thromboembolism induced by
the infusion of a mixture of collagen (0.8 mg/kg body weight) and
epinephrine (60 .mu.g/kg body weight). All of the control mice
(19/19) died within 5 min from widespread pulmonary thrombosis and
cardiac arrest which was accompanied by a >95% reduction in
circulating platelet counts as soon as 2 min after challenge (FIG.
2A, B). Under these experimental conditions, 35.7% (5/14) of the
FXII-/- mice survived although their peripheral platelet counts
were similarly reduced as in the wild-type control, suggesting that
the observed protection was not based on a platelet activation
defect. This assumption was confirmed by in vitro studies showing
that FXII-/- platelets express normal levels of the major surface
glycoproteins, including collagen receptors, and that the cells are
normally activatable by classical agonists such as thrombin,
adenosine diphosphate (ADP), or the GPVI-specific agonist,
collagen-related peptide (as measured by activation of integrin
.alpha.IIb.beta.3 and P-selectin expression). In agreement with
this, FXII-/- platelets exhibited an unaltered aggregation response
to collagen, ADP (FIG. 2C), PMA, or thrombin.
[0037] In a parallel set of experiments, FcR.gamma.-/- mice were
challenged with collagen/epinephrine. These mice were completely
protected from lethality and platelet counts were only moderately
reduced 2 min after challenge confirming the strict requirement for
platelet activation for lethality in this model. These data were
further supported by histological analysis of lung sections derived
from mice of the different groups. While the large majority of the
vessels was obstructed in wild-type mice, this was significantly
reduced in FXII-/- mice (survivors and non survivors). In agreement
with previous reports, virtually no thrombi were found in lungs
from FcR.gamma.-/- mice (FIG. 2D). These results suggested that in
vivo collagen triggers both platelet activation and the
FXII-dependent intrinsic coagulation pathway which in this model
synergize to form occlusive pulmonary thrombi.
[0038] Pathological thrombus formation is frequently initiated by
fissuring or abrupt disruption of the atherosclerotic plaque in the
arterial branch of the vasculature leading to unphysiologically
strong platelet activation and procoagulant activity on the surface
on the subendothelial layers. To test the role of FXII in these
processes, thrombus formation was studied in wild-type and FXII-/-
mice, employing different models of arterial injury. In the first
model, oxidative injury was induced in mesenteric arterioles
(60-100 .mu.m in diameter) and thrombus formation was examined by
in vivo fluorescence microscopy. Wild-type and FXII-/- mice
received fluorescently labeled platelets (1.times.108) of the same
genotype and injury was induced by topical application of a filter
paper saturated with 20% ferric chloride (FeCl.sub.3) for 1 min
which provokes the formation of free radicals leading to the
disruption of the endothelium. Platelet interactions with the
injured vessel wall started rapidly and five minutes after injury
the number of firmly adherent platelets was similar in both groups
of mice (FIG. 3A). However, while in wild-type mice the adherent
platelets consistently recruited additional platelets from the
circulation, resulting in the formation of aggregates, this process
was severely defective in the mutant mice. In 100% of the control
vessels (17/17), stable thrombi >20 .mu.m in diameter had formed
with 10 min after injury which continuously grew over time and
finally lead to complete occlusion in 94.1% (16/17) of the vessels
within the observation period of 40 min (mean occlusion time:
25.6.+-.8.9 min)(FIG. 3). In sharp contrast, in mutant mice the
formation of microaggregates or thrombi occurred was completely
absent in 50% (7/14) of the vessels. In the remaining 50% (7/14) of
the vessels, thrombi were formed which were, however, consistently
unstable and rapidly detached from the vessel wall. In none of the
vessels, a thrombus >20 .mu.m in diameter remained attached at
the site of injury for more than 1 min. Consequently, no vessel
occluded in FXII-/- mice within the observation period (40 min).
This unexpected result demonstrated that FXII is required for the
generation and stabilization of plateletrich thrombi in
FeCl.sub.3-injured arterioles and suggested that FXII-induced the
coagulation pathway essentially contributes to the observed
thrombotic response. This assumption was confirmed when mice
deficient in FXI were analysed in the same model. Since FXI is the
principal substrate of FXII in the "intrinsic" cascade, a similar
defect in thrombus formation would have to be expected in those
mice. Indeed, very similar to FXII-/- mice, virtually normal
platelet adhesion at the site of injury was detectable during the
first three minutes after injury, whereas the formation of thrombi
was completely inhibited in 55.6% (5/9) of the vessels. In the
remaining vessels, the formed microaggregates and thrombi were
unstable and continuously embolized (FIG. 3). As a result, none of
the vessels occluded within the observation period (40 min). This
data shows that FXI-deficient mice are protected in a model of
FeCl.sub.3-induced occlusion of the carotid artery.
[0039] FeCl.sub.3-induced arterial thrombus formation is known to
depend on platelets and thrombin generation but it is unclear how
well this type of injury resembles the thrombogenic milieu produced
in diseased vessels, e.g. upon rupture of the atherosclerotic
plaque. Therefore, to exclude the possibility that the massive
FeCl.sub.3-induced oxidative damage produces unphysiological
conditions which may artificially favor FXII-dependent contact
phase activation, the function of FXII was assessed in a second
well-established arterial thrombosis model where injury is induced
mechanically in the aorta and blood flow is monitored with an
ultrasonic flow probe. After a transient increase directly after
injury, blood flow progressively decreased for several minutes in
all mice tested. In all tested wild-type mice (10/10), this
decrease resulted in complete and irreversible occlusion of the
vessel within 1.6 to 11.1 min after injury (mean occlusion time
5.3.+-.3.0 min, FIG. 4A). A different picture was found in FXII-/-
mice where stable thrombus formation was severely defective. While
in all animals a progressive reduction in blood flow was observed
during the first minutes after injury, occlusion occurred only in 4
of 10 mice. Moreover, the occlusive thrombi in those mice were in
all cases unstable and rapidly embolized so that blood flow was
re-established between 10 s and 115 s after occlusion. None of the
re-opened vessels occluded a second time. Consequently, all FXII-/-
mice displayed essentially normal flow rates through the injured
vessel at the end of the observation period (40 min). Very similar
results were obtained with FXI-/- mice, where 9 of 11 were unable
to establish an occlusive thrombus within the observation period
(30 min)(FIG. 5A).
[0040] The severe defect in arterial thrombus formation in FXII-/-
mice was confirmed in a third independent model where platelet
recruitment in the injured 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 was induced by vigorous ligation of the
carotid artery which consistently causes disruption of the
endothelial layer and frequently breaching of the internal elastic
lamina followed by rapid collagen-triggered platelet adhesion and
thrombus formation at the site of injury (Gruner et al., Blood 102:
4021-27, 2003). While wild-type animals rapidly formed large stable
thrombi (thrombus area: 102.821.+-.39.344 .mu.m2; t=5 min), which
did not embolize, only small and medium-sized aggregates formed in
the mutant mice, which were frequently detached from the site of
injury (FIG. 4B, C). Consequently, the thrombus area was
dramatically reduced in the mutant mice (8.120.+-.13.900 .mu.m2;
t=5 min) although primary platelet adhesion on the vessel wall
appeared not to be defective. To test whether the severe defect in
thrombus formation in FXII-/- mice results from the lack of plasma
FXII or platelet FXII, or possibly from secondary, unidentified
effects of FXII deficiency such as alterations in the vasculature,
arterial thrombus formation was studied in FXII-/- mice following
administration human of FXII (2 .mu.g/g body weight). This
treatment normalized the PTT (27.+-.6 sec) and fully restored
arterial thrombus formation. In 100% of the FeCl3-injured
mesenterial arterioles, thrombi >20 .mu.m had formed within 10
min after injury and all of the vessels completely occluded within
the observation period (FIG. 5A-C). There was even a tendency
towards faster occlusion detectable in the reconstituted FXII-/-
mice as compared to untreated wild-type control mice (mean
occlusion time: 22.7.+-.8.2 min vs. 25.6.+-.8.9 min). A similar
result was obtained when injury was induced mechanically in the
aorta. In all tested vessels, complete and irreversible occlusion
occurred within 10 min after injury (FIG. 5D), confirming that the
lack of plasma FXII accounts for the thrombotic defect observed in
FXII-/- mice.
[0041] The above-described studies demonstrated that FXII is
crucial for arterial thrombus formation and may, therefore, serve
as an antithrombotic target.
[0042] To assess this directly, mice were treated with 2 mg/kg body
weight polyclonal rabbit anti-mouse FXII antibodies or non-immune
rabbit antibodies and assessed platelet recruitment and thrombus
formation in mesenterial arteries following FeCl.sub.3-induced
injury. As shown in FIG. 6A, platelet adhesion at sites of injury
was comparable in both groups of mice. However, while in 100% of
the control vessels, thrombi >20 .mu.m had formed within 10 min
after injury and all of the vessels completely occluded within the
observation period (FIG. 6B, C), thrombi >20 .mu.m were only
observed in 67% of the vessels and occlusion occurred only in 50%
of the vessels of the animals treated with anti-FXII antibody.
[0043] Alternatively, to test the impact of small molecule FXII
inhibitors, wildtype mice were infused with the FXII inhibitor corn
trypsin inhibitor (CTI, 50 .mu.g/g body weight) 5 min prior to
FeCl.sub.3-induced injury in the carotic artery (Wang et al. (2005)
J. Thromb. Heamost. 3: 695-702). Inhibitor treatment prolonged the
aPTT (62.+-.11 sec, n=4) but did not affect bleeding during the
surgical procedure. In none of the animals tested (0/4) vessel
occluding thrombi developed within 30 min following application of
FeCl.sub.3.
[0044] These results demonstrated that anti-FXII therapeutics like
anti-FXII antibodies or small molecule FXII inhibitors provide
significant protection against arterial thrombus formation.
[0045] Although contact activation of FXII has been recognized as
the starting point of the intrinsic blood coagulation cascade for
more than 50 years this pathway was considered to be irrelevant for
blood clotting. In the present invention, three different in vivo
models were used to analyze platelet recruitment and thrombus
formation at sites of arterial injury in FXII-deficient mice by in
situ video microscopy and ultra-sonic flow measurements and showed
a severe inability to form stable three-dimensional thrombi. This
defect was based on the lack of FXII in plasma, but not other
compartments, as it was completely reversed by intravenous
injection of exogenous human FXII (FIG. 6) thereby also excluding
that secondary effects of FXII deficiency contribute to the
observed phenotype.
[0046] These results are unexpected as FXII has been regarded as an
antithrombotic rather than a prothrombotic enzyme based on a few
reports showing an association of FXII-deficiency with an increased
incidence of venous thrombosis (Kuhli, C., Scharrer, I., Koch, F.,
Ohrloff, C., and Hattenbach, L. O. (2004). Factor XII deficiency: a
thrombophilic risk factor for retinal vein occlusion. Am. J.
Ophthalmol. 137, 459-464; Halbmayer, W. M., Mannhalter, C.,
Feichtinger, C., Rubi, K., and Fischer, M. (1993). [Factor XII
(Hageman factor) deficiency: a risk factor for development of
thromboembolism. Incidence of factor XII deficiency in patients
after recurrent venous or arterial thromboembolism and myocardial
infarction]. Wien. Med. Wochenschr. 143, 43-50). FXII-deficient
mice display normal bleeding times (FIG. 1A) and do not show signs
of spontaneous or increased posttraumatic (intraoperative) bleeding
confirming that FXII is dispensable for normal hemostasis. At the
first sight, these results seem to contradict with a central dogma
of hemostasis that only those factors whose deficiency is
associated with bleeding or thrombosis are relevant to blood
clotting. On a closer look, however, the data do not challenge this
theorem but rather raise the interesting possibility that
hemostasis and arterial thrombosis may occur through different
mechanism.
[0047] Although the above discussed mechanisms of sustained
thrombin generation may be sufficient to generate a hemostatic
plug, the data show that the formation of stable arterial thrombi
requires the additional activation of the intrinsic coagulation
pathway, at least in mice. There is no evidence for the possibility
that species-specific differences exist in the function of FXII or
a substrate of the enzyme. All coagulation parameters and the
hemostatic phenotype of the mutant mice are in line with human
FXII-deficiency and all alterations observed in animals were
normalized by reconstitution with human FXII (FIG. 5). Furthermore,
it is excluded that the thrombotic defect is restricted to a
particular experimental model as it was found in different arterial
branches of the vasculature and independent of the type of injury.
It may be difficult to determine what type of damage best reflects
the vascular lesion produced by rupture of an atherosclerotic
plaque, which is considered the major trigger of acute
cardiovascular syndromes. Atherosclerotic lesions are rich in
thrombogenic constituents, most importantly TF and fibrillar
collagens. In the process of atherogenesis, enhanced collagen
synthesis by intimal smooth muscle cells and fibroblasts has been
shown to significantly contribute luminal narrowing. Plaque rupture
or fissuring results in exposure of collagen fibrils to the flowing
blood which triggers platelet adhesion and aggregation. In
addition, they induce FXII activation as shown here for fibrillar
collagen type I, which is the major collagen type found in the
vessel wall. But the collagens are likely not the only
(patho)physiological activator of FXII at sites of injury. Other
candidates could be substances liberated from disintegrating cells
or exposed in the ECM including HSP90 or soluble and insoluble
polyanions, e.g. nucleosomes or glycosaminogly-cans.
[0048] Among these FXII activators, collagens are by far most
thrombogenic because they also potently activate platelets. At
sites of injury, platelets tether to the ECM by the reversible
interaction of platelet GPIb-V-IX with collagen-bound vWf which
reduces the velocity of the cells and thereby allows binding of
other receptors. Among these, the collagen receptor GPVI is of
central importance as it activates integrins .alpha.2.beta.1 and
.alpha.IIb.beta.3 which then mediate stable adhesion and contribute
to cellular activation. In addition, platelet activation through
the GPVI/FcR.gamma.-chain complex induces a procoagulant state of
the cells which is characterized by the exposure of
phosphati-dylserine (PS) and the production of (PS exposing)
membrane blebs and microve-sicles. Integrin .alpha.2.beta.1
facilitates this process directly by "outside-in" signals and
indirectly by reinforcing GPVI-collagen interactions. It is
established that PS-containing membranes strongly accelerate two
central reactions of the coagulation process, the tenase and
prothrombinase reactions. The present invention shows that
procoagulant platelets facilitate FXII-dependent clotting in vitro
by a mechanism involving both the GPVI/FcR.gamma.-chain complex as
well as .alpha.2.beta.1 (FIG. 2). This could at least partly
explain why .alpha.2.beta.1-deficient mice, despite unaltered
platelet adhesion at sites of arterial injury, show partial defects
in the formation of occlusive thrombi. Besides collagens,
coagulating plasma potently stimulates platelet procoagulant
activity by an integrin .alpha.IIb.beta.3-dependent mechanism. In
the present experiments, .alpha.IIb.beta.3 blockade almost
completely inhibited platelet participation in FXII-dependent
clotting, indicating that the well-known anticoagulant activity of
.alpha.IIb.beta.3 antagonists may partly be based on the inhibition
of the FXII-driven intrinsic coagulation pathway. Together, the
present invention indicates that the FXII-driven contact system and
platelet activation may be mutually dependent processes that
cooperate in pathological thrombus formation.
[0049] Based on the experimental results, a model of pathological
thrombus formation depicted schematically in FIG. 7 was proposed.
At sites of vascular injury, the first layer of platelets comes in
contact with collagens in an environment that is additionally
enriched in TF and fibrin. It is therefore not surprising that
platelet adhesion to the damaged vessel wall is not impaired in
FXII-/- mice and it is very likely that these cells were fully
activated and in a procoagulant state. In a growing thrombus,
however, collagens are absent and TF concentrations provided by
microvesicles may be lower as compared to the vessel wall and
reduced in their activity by TFPI released in large amounts from
activated platelets. Under these conditions, additional mechanisms
are required to maintain spatio-temporal thrombin generation to
activate newly recruited platelets and, via the formation of fibrin
provoke their coagulant activity. The severe inability of FXII-/-
mice to establish stable thrombi unambiguously demonstrates that
the FXII-driven intrinsic coagulation pathway is an essential
player in this process. Together with the observation that low
TF-mice also display impaired arterial thrombosis, these results
suggest that both extrinsic and intrinsic pathway must be operative
and synergize to promote the formation of a three-dimensional and
eventually occluding thrombus. In contrast, the lack of bleeding in
FXII-/- mice indicates that thrombus growth in the third dimension
may not be necessary to seal a hole in the vessel wall. This could
explain why the extrinsic pathway, which produces the first thin
layer of fibrin and activated platelets, is sufficient to mediate
normal hemostasis. Our results raise the interesting possibility
the formation of a three-dimensional thrombus serves functions
other than hemostasis. These could include the arrest of blood flow
in certain areas of tissue trauma in order to prevent the
distribution of invading pathogens or toxins with the blood
stream.
Experimental Procedures
Animals
[0050] All experiments and care were approved by the local Animal
Care & Use Committee. Classical mouse mutants lacking factor XI
(FXI-/-), factor XII (FXII-/-), .alpha.2 integrin (.alpha.2-/-)
were produced as described (Gailani, D., Lasky, N. M., and Broze,
G. J., Jr. (1997). A murine model of factor XI deficiency. Blood
Coagul. Fibrinolysis 8, 134-144; Pauer, H. U., Renne, T.,
Hemmerlein, B., Legler, T., Fritzlar, S., Adham, I., Muller-Esterl,
W., Emons, G., Sancken, U., Engel, W., and Burfeind, P. (2004).
Targeted deletion of murine coagulation factor XII gene-a model for
contact phase activation in vivo. Thromb. Haemost. 92, 503-508;
Holtkotter, O., Nieswandt, B., Smyth, N., Muller, W., Hafner, M.,
Schulte, V., Krieg, T., and Eckes, B. (2002). Integrin alpha
2-deficient mice develop normally, are fertile, but display
partially defective platelet interaction with collagen. J Biol Chem
JID--2985121R 277, 10789-10794). As a control C57B/6J mice (FXI-/-)
or Sv129 (FXII-/-) were used. Mice deficient in the
FcR.gamma.-chain (Takai, T., Li, M., Sylvestre, D., Clynes, R., and
Ravetch, J. V. (1994). FcR gamma chain deletion results in
pleiotrophic effector cell defects. Cell 76, 519-529) were from
(Taconics, Germantown).
Generation of Anti-FXII Antibodies
[0051] Total cellular RNA was isolated from a liver of a 129sv wt
mouse and the FXII-cDNA synthesis was performed with the "one-step
RT-PCR Kit" from Qiagen according to the manufacturers
instructions. The factor FXII heavy chain (positions 61-1062,
corresponding to residues 21-354) was amplified using 25 pmol each
of the 5- and 3-primers (ttggatccccaccatggaaagactccaag and
ttgaattcgcgcatgaacgaggaca g) introducing a BamH I and EcoR I
restriction site, respectively with the following protocol: 30 s at
95.degree. C., 60 s at 58.degree. C., and 1 min at 72.degree. C.
for 30 cycles on a thermal cycler (Biometra, Gottingen, Germany).
The PCR product was cloned into the BamH I and EcoR I site of the
pGEX-2T expression vector (Pharmacia). Following sequencing protein
was expressed in E.coli strain BL21. Exponentially growing bacteria
were stimulated with 0.5 mM
isopropyl-.beta.-D-thiogalactopyranoside for 1 h, harvested,
resuspended in 10 mM Tris-HCl, pH 7.4, containing 1 mM EDTA, 200 mM
NaCl, 10 .mu.g/ml benzamidine hydrochloride, 10 .mu.g/ml
phenylmethylsulfonyl fluoride and sonicated for 3 min in pulses of
15 s each. After centrifugation at 15,000.times.g for 20 min at
4.degree. C., the supernatant was removed and transferred to a
GST-sepharose column (Pharmacia) for purification. Eluted protein
was >95% pure as deduced from Coomassie stained SDS-PAGE.
Polyclonal antibodies against GST-heavy chain FXII were raised in
rabbits following standard procedures. Antibodies were selected
from the hyperimmunserum using columns with FXII-heavy chain fused
to the maltose binding protein (MBP). These fusion proteins were
expressed and purified using the pMAL-c2 expression system and
amylose resin columns similarly as described for the GST-fusion
construct.
Platelet Preparation
[0052] Mice were bled under ether anesthesia from the retroorbital
plexus. Blood was collected in a tube containing 20 U/mL heparin,
and platelet rich plasma (prp) was obtained by centrifugation at
300 g for 10 min at room temperature (RT). For washed platelets,
prp was centrifuged at 1000 g for 8 min and the pellet was
resuspended twice in modified Tyrodes-Hepes buffer (134 mM NaCl,
0.34 mM Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM Hepes, 5 mM
glucose, 0.35% bovine serum albumin, pH 6.6) in the presence of
prostacyclin (0.1 .mu.g/ml) and apyrase (0.02 U/mL). Platelets were
then resuspended in the same buffer (pH 7.0, 0.02 U/mL of apyrase)
and incubated at 37.degree. C. for at least 30 min before
analysis.
Flow Cytometry
[0053] Heparinized whole blood was diluted 1:20 with modified
Tyrode-HEPES buffer (134 mM NaCl, 0.34 mM Na2HPO4, 2.9 mM KCl, 12
mM NaHCO3, 20 mM HEPES
[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid], pH 7.0)
containing 5 mM glucose, 0.35% bovine serum albumin (BSA), and 1 mM
CaCl2. The samples were incubated with fluorophore-labeled
antibodies for 15 minutes at room temperature and directly analyzed
on a FACScalibur (Becton Dickinson, Heidelberg, Germany)
(Nieswandt, B., Schulte, V., and Bergmeier, W. (2004).
Flow-cytometric analysis of mouse platelet function. Methods Mol.
Biol. 272, 255-268).
Aggregometry
[0054] To determine platelet aggregation, light transmission was
measured using prp (200 .mu.L with 0.5.times.106 platelets/.mu.L).
Transmission was recorded in a Fibrintimer 4 channel aggregometer
(APACT Laborgerate and Analysensysteme, Hamburg, Germany) over 10
min and was expressed as arbitrary units with 100% transmission
adjusted with plasma. Platelet aggregation was induced by addition
of collagen (10 .mu.g/mL) and ADP (5 .mu.M).
Bleeding Time Experiments
[0055] Mice were anesthetized by intraperitoneal injection of
tribromoethanol (Aldrich) (0.15 m1/10 g of body weight) and 3 mm
segment of the tail tip was cut off with a scalpel. Tail bleeding
was monitored by gently absorbing the bead of blood with a filter
paper without contacting the wound site. When no blood was observed
on the paper after 15 second intervals, bleeding was determined to
have ceased. When necessary, bleeding was stopped manually after 20
minutes. Where indicated, mice were treated with 100 .mu.g/mouse of
hFXII.
Preparation of Platelets for Intravital Microscopy
[0056] Mouse blood (1 vol) was collected into 0.5 vol of Hepes
buffer containing 20 U/mL heparin. The blood was centrifuged at 250
g for 10 minutes and platelet-rich plasma was gently transferred to
a fresh tube. Platelets were labelled with 5-carboxyfluorescein
diacetate succinimidyl ester (DCF) and adjusted to a final
concentration of 200.times.106 platelets/250 .mu.l (Massberg, S.,
Sausbier, M., Klatt, P., Bauer, M., Pfeifer, A., Siess, W.,
Fassler, R., Ruth, P., Krombach, F., and Hofmann, F. (1999).
Increased adhesion and aggregation of platelets lacking cyclic
guanosine 3',5'-monophosphate kinase I. J Exp Med 189,
1255-1264).
In Vivo Thrombosis Model with FeCl3-Induced Injury.
[0057] Male and female mice in the age of 4-5 weeks were
anesthetized by intraperitoneal injection of 2,2,2-tribromoethanol
and 2-methyl-2-butanol (Sigma) (0.15 m1/10 g of body weight from
2.5% solution). Fluorescently labeled platelets were injected
intravenously. Mesentery was exteriorized gently through a midline
abdominal incision. Arterioles (35-50 .mu.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 an S-VHS video recorder (AG-7355, Panasonic,
Matsushita Electric, Japan). After topical application of FeCl3
(20%) which induced vessel injury and denudation of the
endothelium, were arterioles monitored for 40 min or until complete
occlusion occurred (blood flow stopped for >1 min). Firm
platelet adhesion is determined as number of fluorescently labeled
platelets that deposited on the vessel wall until 5 minutes after
injury, thrombus is characterized as a platelet aggregate in a
diameter larger than 20 .mu.m, occlusion time of vessel is
characterized as time required for blood to stop flowing for at
least one minute. In all experiments maximum of two arterioles were
chosen from each mouse based on quality of exposure. A total of 17
wt, 14 FXII-/- and 9 FXI-/- arterioles were studied.
Intravital Microscopy--Carotid Artery
[0058] Intravital microscopy of the injured carotid artery was
performed essentially as described (Massberg, S., Gawaz, M.,
Gruner, S., Schulte, V., Konrad, I., Zohlnhofer, D., Heinzmann, U.,
and Nieswandt, B. (2003). A crucial role of glycoprotein VI for
platelet recruitment to the injured arterial wall in vivo. J Exp
Med JID--2985109R 197, 41-49). Briefly, mice were anesthetized by
intraperitoneal injection of keta-mine/xylazine (ketamine 100
mg/kg, Parke-Davis, Karlsruhe, Germany; xylazine 5 mg/kg, Bayer AG,
Leverkusen, Germany). Polyethylene catheters (Portex, Hythe,
England) were implanted into the right jugular vein and fluorescent
platelets (200.times.106/250 .mu.l) were infused intravenously.
Carotid injury for endothelial denudation was induced by vigorous
ligation. Prior to and following vascular injury, the fluorescent
platelets were visualized in situ by in vivo video microscopy of
the right common carotid artery using a Zeiss Axiotech microscope
(20.times.water immersion objective, W 20.times./0.5, Zeiss,
Gottingen, Germany) with a 100 W HBO mercury lamp for
epi-illumination. Platelet adhesion and thrombus formation was
recorded for 5 min after the induction of injury and the
video-taped images were evaluated using a computer-assisted image
analysis program (Visitron, Munich, Germany).
Pulmonary Thromboembolism
[0059] Mice were anesthetized by intraperitoneal injection of
2,2,2-tribromoethanol and 2-methyl-2-butanol (Aldrich) (0.15 ml/10
g of body weight from 2.5% solution). Anesthetized mice received a
mixture of collagen (0.8 mg/kg) and epinephrine (60 .mu.g/kg)
injected into the jugular vein. The incisions of surviving mice
were stitched, and they were allowed to recover. Necroscopy and
histological studies were performed on lungs fixed in 4%
formaldehyde and paraffin sections were stained with
hematoxylin/eosin.
Platelet Count
[0060] Platelet count was determined by flow cytometry on a
FACScalibur (Becton Dickinson, Heidelberg, Germany). Results are
expressed as mean.+-.S.D or as percent of control (wt, n=19;
FXII-/-, n=14 and FcR.gamma.-/-, n=5).
Occlusion Time
[0061] The abdominal cavity of anesthetized mice was longitudinally
opened and the abdominal aorta was prepared. An ultrasonic flow
probe was placed around the aorta and thrombosis was induced by one
firm compression with a forceps. Blood flow was monitored until
complete occlusion occurred. The experiment was stopped manually
after 45 minutes. Where indicated, human Factor XII was substituted
intravenously directly before the experiment.
Histopathologic Analyses
[0062] Mice were sacrificed, lungs rapidly removed and fixed at 4 C
for 24 hr in buffered 4% formalin (pH 7.4; Kebo). Tissues were
dehydrated and imbedded in paraffin (Histolab Products AB), cut
into 4 .mu.m sections, and mounted. After removal of the paraffin,
tissues were stained with Mayers hematoxylin (Histolab Products AB)
and eosin (Surgipath Medical Industries, Inc.).
SDS-Polyacrylamide Gel Electrophoresis, Western Blotting, and
Immunoprinting
[0063] Plasma (0.3 .mu.l/lane) was separated by 12.5% (w/v)
polyacrylamide gel electrophoresis in the presence of 1% (w/v) SDS
(Laemmli, 1970). Proteins were transferred onto nitrocellulose
membranes for 30 min at 100 mA. The membranes were blocked with PBS
containing 4% (w/v) dry milk powder and 0.05% (w/v) Tween-20, pH
7.4. Membranes were probed with 0.5 .mu.g/ml of the monoclonal
antibody against MBK3 (Haaseman J. Immunology 1988). Bound
antibodies were detected using peroxidase-conjugated secondary
antibodies against mouse IgG (dilution 1:5000) followed by a
chemiluminescence detection method.
Coagulation Assays.
[0064] For the determination of the recalcification clotting time,
100 .mu.l citrate anticoagulated mouse plasma (0.38% sodium
citrate), was incubated with 100 .mu.l each of Horm type collagen
(Nycomed, Munchen, Germany), ellagic acid, chondroitin sulfate
(both from Sigma), kaolin or buffer (final concentrations 30
.mu.g/ml) for 120 sec at 37.degree. C. in a KC10
"Kugelkoagulometer" (Amelung, Lemgo, Germany). To test the effect
of platelets activation on FXII-dependent clotting washed platelets
were resuspended in Tyrode buffer including 4 mM Ca2+ and 5 .mu.M
Ca2+-ionophor A23187 (Sigma) for 10 min prior to addition to
platelet free plasma. Clot formation was initiated by
recalcification with 100 .mu.l 25 mM CaCl2-solution, and the time
until clotting occurred was recorded using the coagulation timer
KC4 (Amelung).
Coagulation Analysis
[0065] Global and single coagulation parameters were determined
with an automated blood coagulation system (BCS, Dade Behring) with
Dade Behring reagents according to the protocols for human samples
detailed by the manufacturer. Principles of BCS assay protocols are
available from Dade Behring package inserts, which can be found on
Dade Behring's web site (http://www.dadebehring.com). D-dimers
werde measured with the ELISA from Asserachrom (Roche). Peripheral
blood counts were determines on the Sysmex XE 2100 according to
standard protocols.
Thrombin Measurements
[0066] Thrombin generation was measured according to the method of
Aronson et al. (Circulation, 1985), with slight modifications.
Platelet-rich or platelet free plasma aliquots (0.5 ml) were placed
into round-bottomed polypropylene tubes that were coated with Norms
type collagen (100 .mu.g/ml, 24 h, 4.degree. C.), and 20 .mu.l of 1
M Ca2+ was added to initiate clotting. Samples (10 .mu.l) were
added to the wells of a microtiter plate containing 90 .mu.l of
3.8% sodium citrate at 2.5-10 min intervals for 60 min. Color was
developed for 2 min by the addition of 50 l of 2 mmol/liter S-2238
(H-D-Phe-Arg-NH-NO2-HCl, a thrombin-specific substrate;
Chromogenix, Molndal, Sweden) in 1 mol/liter Tris (pH 8.1). The
absorbance of the released color product was measured
spectrophotometrically at a wavelength of 405 nm using a Vmax
microtiter plate reader (Easy Reader, EAR 340AT, SLT Lab
Instruments GmbH, Vienna, Austria). Measurements were obtained in
triplicate at each time point.
Statistical Evaluation
[0067] Statistical analysis was performed using the unpaired
Student's t test.
[0068] Although only preferred embodiments are 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 within the purview of the
appended claims without departing from the spirit and intended
scope of the invention.
* * * * *
References