Matrix Metalloproteinases Collagenase-2, Macrophage Elastase, Collagenase-3, and Membrane Type 1-Matrix Metalloproteinase Impair Clotting by Degradation of Fibrinogen and Factor XII

The effects of plasma proteins on controlling the activity of matrix metalloproteinases (MMPs, matrixins) have been the focus of numerous studies, although only a few have examined the influence of matrixins on plasma proteins. Recently, it has been shown that MMPs may play a role in the degradation of fibrin. We have now investigated the role of collagenase-2 (MMP-8), macrophage elastase (MMP-12), collagenase-3 (MMP-13), and membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) in the degradation of fibrinogen and Factor XII of the plasma clotting system. Our data demonstrate that the catalytic domains of MMP-8, MMP-12, MMP-13, and MMP-14 can proteolytically process fibrinogen and, with the exception of MMP-8, also inactivate Factor XII (Hageman factor). We have identified the amino termini of the major protein fragments. Cleavage of fibrinogen occurred in all chains and resulted in significantly impaired clotting. Moreover, rapid proteolytic inactivation of Factor XII (Hageman factor) by MMP-12, MMP-13, and MMP-14 was noted. These results support the hypothesis of an impaired thrombolytic potential of MMP-degraded Factor XII in vivo. MMP-induced degradation of fibrinogen supports a plasmin-independent fibrinolysis mechanism. Consequently, degradation of these proteins may be important in inflammation, atherosclerosis, and angiogenesis, all of which are known to be influenced by MMP activity. The effects of plasma proteins on controlling the activity of matrix metalloproteinases (MMPs, matrixins) have been the focus of numerous studies, although only a few have examined the influence of matrixins on plasma proteins. Recently, it has been shown that MMPs may play a role in the degradation of fibrin. We have now investigated the role of collagenase-2 (MMP-8), macrophage elastase (MMP-12), collagenase-3 (MMP-13), and membrane type 1-matrix metalloproteinase (MT1-MMP, MMP-14) in the degradation of fibrinogen and Factor XII of the plasma clotting system. Our data demonstrate that the catalytic domains of MMP-8, MMP-12, MMP-13, and MMP-14 can proteolytically process fibrinogen and, with the exception of MMP-8, also inactivate Factor XII (Hageman factor). We have identified the amino termini of the major protein fragments. Cleavage of fibrinogen occurred in all chains and resulted in significantly impaired clotting. Moreover, rapid proteolytic inactivation of Factor XII (Hageman factor) by MMP-12, MMP-13, and MMP-14 was noted. These results support the hypothesis of an impaired thrombolytic potential of MMP-degraded Factor XII in vivo. MMP-induced degradation of fibrinogen supports a plasmin-independent fibrinolysis mechanism. Consequently, degradation of these proteins may be important in inflammation, atherosclerosis, and angiogenesis, all of which are known to be influenced by MMP activity. matrix metalloproteinase batimastat catalytic domain fibrinopeptide A high performance liquid chromatography membrane-type MMP polyacrylamide gel electrophoresis polyvinylidene difluoride The matrix metalloproteinases, MMPs1 and matrixins, form a family of structurally and functionally related zinc-containing endopeptidases. Together they are able to degrade most of the constituents of the extracellular matrix such as basement membrane, collagens, proteoglycans, fibronectin, and laminin (1Woessner J.F. FASEB J. 1991; 5: 2145-2155Crossref PubMed Scopus (3091) Google Scholar). Thus, they are implicated in connective tissue remodeling processes associated with embryonic development, pregnancy, growth, and wound repair (2Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (704) Google Scholar). The deleterious potential of the MMPs is normally controlled by the endogenous and specific tissue inhibitors of metalloproteinases or the more general nonspecific α2-macroglobulin (3Nagase H. Woessner Jr., J.F. J. Biol. Chem. 1999; 274: 21491-21494Abstract Full Text Full Text PDF PubMed Scopus (3903) Google Scholar). Disturbance of the well balanced equilibrium of MMPs and tissue inhibitors of metalloproteinases results in pathological situations such as rheumatoid and osteoarthritis, atherosclerosis, tumor growth, metastasis, and fibrosis (4Nagase H. Das S.K. Dey S.K. Fowlkes J.L. Huang W. Brew K. Hawkes S.P. Edwards D.R. Khokha R. Inhibitors of Metalloproteinases in Development and Disease. Harwood, Lausanne1997Google Scholar, 5Johnson L.L. Dyer R. Hupe D.J. Curr. Opin. Chem. Biol. 1998; 2: 466-471Crossref PubMed Scopus (257) Google Scholar, 6Yong V.W. Krekoski C.A. Forsyth P.A. Bell R. Edwards D.R. Trends Neurosci. 1998; 21: 75-80Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar, 7Coussens L.M. Werb Z. Chem. Biol. 1996; 3: 895-904Abstract Full Text PDF PubMed Scopus (506) Google Scholar, 8Chambers A.F. Matrisian L.M. J. Natl. Cancer Inst. 1997; 89: 1260-1270Crossref PubMed Scopus (1436) Google Scholar). In addition to degradation of extracellular matrix constituents, plasma proteins such as serpins (9Knäuper V. Reinke H. Tschesche H. FEBS Lett. 1990; 263: 355-357Crossref PubMed Scopus (53) Google Scholar) or fibrinogen and cross-linked fibrin (10Bini A. Itoh Y. Kudryk B.J. Nagase H. Biochemistry. 1996; 35: 13056-13063Crossref PubMed Scopus (90) Google Scholar, 11Bini A. Wu D. Schnuer J. Kudryk B.J. Biochemistry. 1999; 38: 13928-13936Crossref PubMed Scopus (68) Google Scholar, 12Hiraoka N. Allen E. Apel I.J. Gyetko M.R. Weiss S.J. Cell. 1998; 95: 365-377Abstract Full Text Full Text PDF PubMed Scopus (646) Google Scholar) are also cleaved. Fibrinogen is a 340-kDa dimeric glycoprotein consisting of a pair of three polypeptide chains Aα, Bβ, and γ that are interconnected by 29 disulfide bonds. The amino termini of these chains are joined together in a central domain that can be isolated as a single fragment from a plasmin digestion of fibrinogen (13Doolittle R.F. Annu. Rev. Biochem. 1984; 53: 193-229Crossref Scopus (528) Google Scholar). During blood coagulation, fibrinogen participates in both the cellular phase and the fluid phase of blood clot formation (14Herrick S. Blanc-Brude O. Gray A. Laurent G. Int. J. Biochem. Cell Biol. 1999; 31: 741-746Crossref PubMed Scopus (238) Google Scholar, 15Henschen-Edman A.H. Haemost. 1999; 29: 179-186PubMed Google Scholar). Fibrinogen can be converted into an insoluble fibrin clot as a consequence of thrombin-catalyzed removal of fibrinopeptides A (FpA, Aα-(20–35)) 2Numbering of amino acids includes signal peptide sequences. and B (FpB, Bβ-(31–44)) from the Aα and Bβ chains (16Gorkun O.V. Veklich Y.I. Weisel J.W. Lord S.T. Blood. 1997; 89: 4407-4414Crossref PubMed Google Scholar). In addition to the ordinary route of thrombin generation (17Butenas S. Van't Veer C. Mann K.G. Blood. 1999; 94: 2169-2178Crossref PubMed Google Scholar) via the tissue factor pathway, an alternative route exists that is initiated by the activation of Factor XII (Hageman factor) (18Mann K.G. Thromb. Haemostasis. 1999; 82: 165-174Crossref PubMed Scopus (416) Google Scholar). The activation of Hageman factor to yield active α-Factor XIIa takes place by a single cleavage at 372R↓V373 (numbering includes signal peptide) (19McMullen B.A. Fujikawa K. J. Biol. Chem. 1985; 260: 5328-5338Abstract Full Text PDF PubMed Google Scholar). Eventually, cleavage at353R↓N354 and362R↓L363 leads to the β-Factor XII (20Fujikawa K. McMullen B.A. J. Biol. Chem. 1983; 258: 10924-10933Abstract Full Text PDF PubMed Google Scholar), which still exhibits full catalytic activity. After several more steps of zymogen activation, this alternative route leads into the ordinary pathway of blood coagulation, terminating in the proteolytic conversion of fibrinogen into fibrin. The purpose of this work is to examine the role of MMPs in the degradation of fibrinogen and Factor XII. We therefore examined the clotting of MMP-digested fibrinogen to support the idea that some of the biological functions of fibrinogen might be hampered (11Bini A. Wu D. Schnuer J. Kudryk B.J. Biochemistry. 1999; 38: 13928-13936Crossref PubMed Scopus (68) Google Scholar). In addition to this, the previously unreported degradation (and inactivation) of Factor XII is shown here. All digestions were subjected to SDS-PAGE followed by automated sequencing to characterize the generated fragments and identify the cleavage sites. Lyophilized human fibrinogen (F4883, >95% clottable and essentially plasminogen-free according to the manufacturer) was purchased from Sigma and dissolved to a final concentration of 2 mg/ml. Thrombin and Owren's Veronal buffer (28.4 mm sodium barbital in 125 mm NaCl, pH 7.35) were supplied by DADE (Aguada). EDTA and the synthetic serine proteinase inhibitor Pefabloc® SC (4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride) were delivered from Merck. The chromogenic thrombin substrate S-2302 was supplied by Chromogenix (Moelndal, Sweden). Active recombinant human cdMT1-MMP-(Ile114–Ile318) and cdMMP-8-(Met80–Gly242) were prepared as described previously (21Lichte A. Kolkenbrock H. Tschesche H. FEBS Lett. 1996; 397: 277-282Crossref PubMed Scopus (62) Google Scholar, 22Kleine T. Bartsch S. Bläser J. Schnierer S. Triebel S. Valentin M. Gote T. Tschesche H. Biochemistry. 1993; 32: 14125-14131Crossref PubMed Scopus (29) Google Scholar). Active human neutrophil gelatinase B (MMP-9) was purified as described previously (23Triebel S. Bläser J. Reinke H. Tschesche H. FEBS Lett. 1992; 314: 386-388Crossref PubMed Scopus (214) Google Scholar). Active human recombinant cdMT2-MMP was a generous gift from Dr. Horst Will of INVITEK, Berlin-Buch, Germany (24Will H. Hinzmann B. Eur. J. Biochem. 1995; 231: 602-608Crossref PubMed Scopus (317) Google Scholar). The cdMMP-12 cDNA (base pairs 310–801) was obtained by reverse transcription-polymerase chain reaction from total RNA isolation of placental tissue. The fragment was ligated into the vector pET-11a. pET-11a/cdMMP-12 was transformed intoEscherichia coli strain BL-21(DE3). Cells were grown to mid-log growth phase (A 560, 0.6) and induced with isopropyl-β-d-thiogalactopyranoside for 3 h. Harvested cells were lysed, and the overexpressed protein was isolated as inclusion bodies. The inclusion bodies were dissolved in a urea buffer and dialysed three times against Tris buffer. The protein was purified by affinity chromatography (25Moore W.M. Spilburg C.A. Biochemistry. 1986; 25: 5189-5195Crossref PubMed Scopus (102) Google Scholar). After elution with urea buffer, cdMMP-12 was refolded by dialyzing three times against Tris buffer, pH 7.5. The proΔMMP-13 cDNA (base pairs 71–805) was amplified by polymerase chain reaction from the plasmid pEMBL-19 containing the complete cDNA for procollagenase-3 derived by reverse transcription-polymerase chain reaction from total RNA isolated from breast cancer cells (26Freije J.M.P. Dietz-Itza I. Balbin M. Sanchez L.M. Blasco R. Tolivia J. Lopez-Otin C. J. Biol. Chem. 1994; 269: 16766-16773Abstract Full Text PDF PubMed Google Scholar). The resulting 734-base pair fragment was cloned into the expression vector pET-12b and E. coli strain BL21(DE3). The expression and purification of the inclusion bodies were performed as described for cdMMP-12. The protein was purified by Q-Sepharose chromatography and gel filtration with Sephacryl S-100. The isolated proΔMMP-13 was activated before use by incubation with 5 mm HgCl2 for 2 h at 37 °C. MMP activity was determined by gelatin zymography (27Kleiner D.E. Stetler-Stevenson W.G. Anal. Biochem. 1994; 218: 325-329Crossref PubMed Scopus (817) Google Scholar) and by a continuous assay using MCA peptide ((7-methoxy-coumarin-4-yl)acetyl-Pro-Leu-Gly-Leu-(3-[2, 4-dinitrophenyl]-1–2-3-diaminopropionyl)-Ala-Arg-NH2) as a synthetic substrate (28Knight C.G. Willenbrock F. Murphy G. FEBS Lett. 1992; 296: 263-266Crossref PubMed Scopus (679) Google Scholar). Fibrinogen was incubated with cdMMP-8 in a 1:50 enzyme/substrate molar ratio at 37 °C for different time intervals. In the case of cdMMP-12, MMP-13, and MT1-MMP, a ratio of 1:10 was employed. All reactions were performed in 20 mm Tris-HCl buffer, pH 7.3, containing 100 mmNaCl, 5 mm CaCl2, 0.3 mg/ml Pefabloc® SC, and 100 μm ZnCl2. Digestions were terminated by addition of 0.1 μl of EDTA (0.1 μmol) and a third part of denaturing buffer (50 mm Tris-HCl pH 6.8, containing 1% SDS, 8 m urea, 30 mm NaCl, 1% 2-mercaptoethanol, and 0.05% bromphenol blue). All reaction products were subjected to SDS-PAGE. All reaction products were subjected to SDS-PAGE on 10% gels under reducing conditions followed by silver staining (29Heukeshoven J. Dernick R. Electrophoresis. 1988; 9: PubMed Scopus Google Scholar). The of were K. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The clotting time of fibrinogen was determined with a by the of A. Scopus Google Scholar). of fibrinogen in Owren's Veronal buffer was for different time at 37 °C with the of MMP-8, MMP-12, MMP-13, and MT1-MMP, cleavage was by a molar of was initiated by μl of thrombin to μl of the fibrinogen A was with addition of the thrombin and at the of clotting by a the conditions clotting time on the concentration of The concentration of the clottable fibrinogen was from the clotting time according to a that was with known of After digestion Factor XII fragments were by SDS-PAGE on 10% gels and to a membrane J. Biol. Chem. Full Text PDF PubMed Google Scholar). Fibrinogen digestion products were by and on 10% gels under reducing and denaturing After the protein fragments to a membrane in buffer, pH containing the membrane was with were of the and subjected to automated sequencing on a with amino H. S. F. Tschesche H. H. in Google Scholar). identify the MMP cleavage in a of the protein was subjected to by using a of fibrinogen fragments was performed at using a obtained with and in was were in denaturing buffer, subjected to and to a μl of Factor XII was incubated with 5 μl of active MMP in pH 7.3, containing 100 mm NaCl, 5 mm CaCl2, and 100 for and at 37 °C. After the incubation time was μl of batimastat was and incubated for 5 to MMP activity. μl of this was with 100 μl of S-2302 and the at was with an of 2 for of the buffer the batimastat the active MMP and the Factor XII active MMP were as We examined the of several MMPs on of digestion were at different time in denaturing buffer, and subjected to The time of fibrinogen degradation incubation with is shown in A. reducing conditions in fibrinogen was into The Aα, Bβ, and γ chains be The catalytic domain of fibrinogen and an enzyme/substrate molar ratio of 1:10 was employed. of fibrinogen resulted in the complete of fibrinogen a In several fragments with of and and be During fibrinogen of the and the were to fragments at and These fragments degradation to yield digestion Thus, 30 of incubation be and 2 h the was also cleaved. MMP-12, of the fibrinogen also be was a B that the and were to a with with the of fibrinogen the generation of products was with time to a of be identified by of amino degradation of the fibrinogen by MMP-8, MMP-12, MMP-13, and incubation degradation of and were also noted. The identified amino termini of the generated fibrinogen fragments are in of the identified of of amino acids of all proteins includes signal peptide was subjected to and the resulting fragments were The amino were determined by automated The of amino acids of all proteins includes signal peptide in a Fibrinogen was subjected to and the resulting fragments were The amino were determined by automated identify the fibrinogen fragments generated by and by was a for We the of the by a of to SDS-PAGE under reducing containing more fragment were by SDS-PAGE and a characterize the of fibrinogen degradation by the clotting of fibrinogen was by the of A. Scopus Google Scholar). Fibrinogen was incubated with cdMMP-12, and for to 2 and the fibrinogen degradation by a molar of the clotting of fibrinogen was 3 the of clottable fibrinogen at different of The fibrinogen were from of fibrinogen After an incubation with fibrinogen clotting activity was to Moreover, the clotting was to an fibrinogen was incubated with MMP-8, MMP-12, or MMP-13, These results a impaired activity of fibrinogen with results were obtained by clotting of MMP-8, MMP-12, or fibrinogen the cleavage of Factor XII by MMP-13, were incubated at 37 °C for to 3 h The proteolytic resulted in of Factor XII into several fragments of at and 30 incubation to degradation and of these and of and of these fragments that cleavage occurred at the of the type and the growth Moreover, a cleavage the catalytic was identified of the cleavage that leads to the activation of Factor XII. is that the fragment generated by MMP-12, MMP-13, and catalytic activity against the chromogenic substrate In addition to this, have that Factor XII be activated by MMP-induced was shown by a in which Factor XII was subjected to by MMPs and for activation with the synthetic substrate After addition of the of Factor XII the cleavage of the chromogenic substrate MMP-digested Hageman factor activity. Cleavage of Hageman factor by cdMMP-12 is an as of this matrix The time of this degradation is shown in B. The amino termini were also identified as and cleavage by The amino at the of the type domain was identified as a cleavage generated by cdMMP-12. Moreover, the for the cleavage of human at at the of the catalytic was also Cleavage of Hageman factor by MMP-14 fragments with of and The identified amino was the as that obtained with and The was also generated by MMP-14 of Hageman of amino acids of all proteins includes signal peptide XII was by MMPs and the generated termini were isolated and identified by The of amino acids of all proteins includes signal peptide in a Factor XII was by MMPs and the generated termini were isolated and identified by The data in this work demonstrate the of the catalytic domains of MMP-8, MMP-12, MMP-13, and All degrade the of fibrinogen and at incubation times the and as The in fibrinogen by different of the matrix metalloproteinases were by electrophoresis of fibrinogen in polyacrylamide gels under denaturing and reducing We that the fibrinogen was to the time the were the and were the incubation time was to 3 the staining of the and were also of the generated fibrinogen fragments were identified by sequencing data that these fragments resulted from in the and of fibrinogen were able to the amino termini of all generated fragments. of digestion products is MMP of fibrinogen thrombin-catalyzed fibrin the clotting time of fibrinogen digestion with cdMMP-12, and The clotting time the of fibrinogen proteolytic by the We have that the degradation of fibrinogen by MMPs resulted in a of fibrinogen the fibrinogen impaired clotting In this to that were able to clotting times of fibrinogen for times 5 of a of fibrinogen degradation by addition of Thus, the determined of fibrinogen in the of digestion to be the obtained data that of fibrinogen with matrixins the of and The clotting of fibrinogen was the catalytic domain of was active in human plasma to the of or The of to the of may influence a of cellular tumor growth, wound and Fibrinogen cellular of a of different and tumor Fibrinogen both at the amino and termini of the These as well as the of the to the glycoprotein J. G. J. PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar). During incubation of fibrinogen with both were as well as the MMP-8, MMP-12, and MMP-13, at of the is These fibrinogen fragments may still be by and to glycoprotein may be the are The data in this for the time the degradation of Factor XII of the blood clotting by matrix MMP-12, MMP-13, and MMP-14 at cleavage of the cleavage of Hageman factor into active Factor takes place with cleavage of the peptide (19McMullen B.A. Fujikawa K. J. Biol. Chem. 1985; 260: 5328-5338Abstract Full Text PDF PubMed Google Scholar, D.E. J. Biol. Chem. 1985; 260: Full Text PDF PubMed Google Scholar). activity of Factor XII can be MMP-induced Moreover, Hageman factor be activated by is therefore that the activation of Factor XII the of to a as known from W. R. J. Biol. PubMed Scopus Google Scholar, R. W. Chem. 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