Abstract
Previously, we demonstrated that amorphous calcium phosphate (ACP), chemical precursor to apatite, strongly interacted with fibrin and facilitated binding of matrix metalloproteinase (MMP)-9, a type IV collagenase. Plasmin-dependent fibrinolysis resulted in coordinate MMP-9 activation. Here we report on the effect(s) of ACP on fibrin degradation and binding of endogenous plasma proteases. Electrophoresis (8.5% SDS-PAGE) revealed that fibrin formed in the presence of ACP demonstrated characteristic γ-γ dimers (90-kDa) and β-monomers (55-kDa), but resisted spontaneous fibrinolysis (72 h, 37°C) or degradation by plasminogen activators (uPA, tPA). Casein zymography revealed an ACP-dependent decrease in fibrin binding of a low molecular weight (Mw) protease triplet (47-, 43-, 42-kDa) and increased fibrin binding of two high Mw proteases (94- and 84-kDa). The low Mw triplet also possessed gelatinolytic activity, but was not an MMP since 1,10-phenanthroline was ineffective as an inhibitor. Fibrin-binding proteases were inhibited to some degree by the serine protease inhibitor aprotinin. Competition/dissociation experiments with ∈-aminocaproic acid revealed that the low Mw triplet lacked kringle regions whereas the 94- and 84-kDa proteases were tentatively identified and glu-/lys-plasmin(ogen)s. The triplet may, however, represent one or more kringle deficient mini-plasminogen(s), since electrophoretic mobility and substrate specificity was similar to elastase-generated mini-plasminogen. To explore these findings in a clinically relevant setting, a series of plasma samples was collected from a patient with unstable angina prior to, during, and post coronary artery bypass graft (CABG) surgery. Fibrin formed from plasma collected during and immediately post CABG was associated with increased fibrinolytic capacity and enhanced binding of a) MMP-9, b) the low Mw protease triplet (described above), and c) PA (as putative 110-kDa tPA:PAI-1 complex). The relevance of these findings to pathologic calcification of atherosclerotic plaques is discussed.
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References
Rakoczi, I., B. Wiman, and D. Collen. 1978. On the biological significance of the specific interaction between fibrin, plasminogen and antiplasmin. Biochim. Biophys. Acta. 540:295–300.
Mosesson, M. W. 1990. Fibrin polymerization and its regulatory role in hemostasis. J. Clin. Lab. Med. 116:8–17.
Dvorak, H. F. 1986. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. New Engl. J. Med. 315:1650–1659.
Richardson, D. L., D. S. Pepper, and A. B. Kay. 1976. Chemotaxis for human monocytes by fibrinogen-derived peptides. Brit. J. Haematol. 32:507–513.
Makowski, G. S., and M. L. Ramsby. 1998. Binding of latent matrix metalloproteinase 9 to fibrin: activation via a plasmindependent pathway. Inflammation 22:287–305.
Makowski, G. S., and M. L. Ramsby. 1998. Binding of latent matrix metalloproteinase 9 to fibrin is mediated by amorphous calcium-phosphate. Inflammation 22:599–617.
Boskey, A. L. 1997. Amorphous calcium phosphate: The contention of bone. J Dental Res. 76:1433–1436.
Fleisch, H., and W. F. Neuman. 1961. Mechanisms of calcification: role of collagen, polyphosphates, and phosphatase. Am. J. Physiol. 200:1296–1300.
Makowski, G. S., and M. L. Ramsby. 1999. Amorphous calcium phosphate-mediated binding of matrix metalloproteinase-9 to fibrin is inhibited by pyrophosphate and bisphosphonate. Inflammation 23:333–360.
McGann, T. C. A., R. D. Kearney, W. Buchheim, A. S. Posner, F. Betts, and N. C. Blumenthal. 1983. Amorphous calcium phosphate in casein micelles of bovine milk. Calcif. Tiss. Int'l. 35:821–823.
Blumenthal, N. C., F. Betts, and A. S. Posner. 1977. Stabilization of amorphous calcium phosphate by Mg and ATP. Calcif. Tiss. Res. 23:245–250.
Hidaka, S., K. Abe, and S. Y. Liu. 1991. A new method for the study of the formation and transformation of calcium phosphate precipitates: effects of several chemical agents and chinese folk medicines. Arch. Oral Biol. 36:49–54.
Koolwijk, P. A., M. M. Miltenburg, M. G. M. van Erck, M. Oudshoorn, M. J. Niedbala, F. C. Breedveld, and V. W. M. van Hinsbergh. 1995. Activated gelatinase-B (MMP-9) and urokinase-type plasminogen activator in synovial fluids of patients with arthritis. Correlation with clinical and experimental variables of inflammation. J. Rheumatol. 22:385–393.
Weinberg, J. B., A. M. M. Pippen, and C. S. Greenberg. 1991. Extravascular fibrin formation and dissolution in synovial tissue of patients with osteoarthritis and rheumatoid arthritis. Arth. Rheum. 34:996–1005.
Bini, A., K. G. Mann, B. J. Kudryk, and F. J. Schoen. 1999. Noncollagenous bone matrix proteins, calcification, and thrombosis in carotid artery atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 19:1852–1861.
Hermann S. M., C. Whatling, E. Brand, V. Nicaud, J. Gariepy, A. Simon, A. Evans, J. B. Ruidavets, D. Arveiler, G. Luc, L. Tiret, A. Henney, and F. Cambien. 2000. Polymorphisms of the human matrix gla protein (MCP) gene, vascular calcification, and myocardial infarction. Arterioscler. Thromb. Vasc. Biol. 20:2386–2393.
Makowski G. S., and M. L. Ramsby. 1996. Calibrating gelatin zymograms with human gelatinase standards. Anal. Biochem. 236:353–356.
Clauss, A. 1957. Rapid physiological coagulation method for the determination of fibrinogen. Acta. Haematol. 17:237–246.
Regoeczi, E. 1968. Occlusion of plasma proteins by human fibrin: studies using trace-labelled proteins. Br. J. Haematol. 14:279–290.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685.
Heussen, C., and E. B. Dowdle. 1980. Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates. Anal. Biochem. 102:196–202.
Makowski, G. S., and M. L. Ramsby. 1993. pH modification to enhance the molecular seiving properties of sodium dodecyl sulfate-10% polyacrylamide gels. Anal. Biochem. 212:283–285.
Francis, C. W. and V. J. Marder. 1982. A molecular model of plasmic degradation of crosslinked fibrin. Semin. Thromb. Hemost. 8:25–35.
Ramsby, M. L. and G. S. Makowski. 1999. Overlay assay: enzyme zymography of plasminogen activators and inhibitors. In: The Encyclopedia of Molecular Biology, T. E. Creighton and L. Hood, eds. New York: Wiley & Sons, pp. 1734–1736.
Ramsby, M. L., and D. L. Kreutzer. 1993. Fibrin induction of tissue plasminogen activator in corneal endothelial cells in vitro. Invest. Ophthalmol. Vis. Sci. 34:3207–3219.
Chandler, W. L., G. Schimer, and J. R. Stratton. 1989. Optimum conditions for the stabilization of tissue plasminogen activator activity in human plasma. J. Lab. Clin. Med. 113:362–371.
Thorsen, S., I. Clemmensen, L. Sottrup-Jensen, and S. Magnusson. 1981. Adsorption to fibrin of native fragments of known primary structure from human plasminogen. Biochim. Biophys. Acta 668:377–387.
Lijnen, H. R., B. Van Hoef, and D. Collen. 1981. On the role of carbohydrate side chains of human plasminogen and its interaction with a2-antiplasmin and fibrin. Eur. J. Biochem. 120:149–154.
Machovich, R. and W. G. Owen. 1989. An elastase-dependent pathway of plasminogen activation. Biochemistry 28:4517–4522.
Fearnley, G. R. and J. M. Tweed. 1953. Evidence of an active fibrinolytic enzyme in the plasma of normal people with observation on inhibition associated with the presence of calcium. Clin. Sci. 12:81–89.
Dvorak, H. F., V. S. Harvey, P. Estrella, L. F. Brown, J. McDonagh, and A. M. Dvorak. 1987. Fibrin containing gels induce angiogenesis: implication for tumor stroma generation and wound healing. Lab. Invet. 57:673–683.
Ramsby, M. L., P. C. Donshik, and G. S. Makowski. 2000. Ligneous conjunctivitis: biochemical evidence for hypofibrinolysis. Inflammation 24:45–71.
Cheras, P. A., A. N. Whitaker, E. A. Blackwell, T. J. Sinton, M. D. Chapman, and K. A. Peacock. 1997. Hypercoagulability and hypofibrinolysis in primary osteoarthritis. Clin. Orthopaed. Rel. Res. 334:57–67.
Kawashima, Y., S. Saika, O. Yamanaka, Y. Okede, K. Ohkawa, and Y. Ohnishi. 1998. Immunolocalization of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human subconjunctival tissues. Curr. Eye Res. 17:445–451.
Bini, A., J. J. Fenoglio, Jr, R. Mesa-Tejada, B. Kudryk, and K. L. Kaplan. 1989. Identification and distribution of fibrinogen, fibrin, and fibrin(ogen) degradation products in atherosclerosis. Arteriosclerosis 9:109–121.
Stary, H. C., A. B. Chandler, R. E. Dinsore, V. Fuster, S. Glagov, W. Insull, Jr., M. E. Rosenfeld, C. J. Schwartz, W. D. Wagner, and R. W. Wissler. 1995. A definition of advanced types of athersclerotic lesions and a histological classification of atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 15:1512–1531.
Anderson, H. C. 1988. Mechanisms of pathologic calcification. Rheum. Dis. Clin. No. Amer. 14:302–315.
Butler, J., R. Pillai, G. M. Rocker, S. Westaby, D. Parker, and D. J. Shale. 1993. Effect of cardiopulmonary bypass on systemic release of neutrophil elastase and tumor necrosis factor. J. Thor. Cardiovasc. Surg. 102:309–317.
Carney, D. E., C. J. Lutz, A. L. Picone, L. A. Gatto, N. S. Ramamurthy, L. M. Golub, S. R. Simon, B. Searles, A. Paskanik, K. Snyder, C. Finck, H. J. Schiller, and G. F. Nieman. 1999. Matrix metalloproteinase inhibitor prevents acute lung injury after cardiopulmonary bypass. Circulation 100:400–406.
Chandler, W. L., J. C. K. Fitch, M. H. Wall, E. D. Verrier, R. P. Cochran, L. O. Soltow, and B. D. Spiess. 1995. Individual variations in the fibrinolytic response during and after cardiopulmonary bypass. Thromb. Haemost. 74:1293–1297.
Paramo, J. A., J. Rifon, R. Llornes, J. Casares, M. J. Paloma, and E. Rocha. 1991. Intra-and postoperative fibrinolysis in patients undergoing cardiopulmonary bypass surgery. Haemostasis 21:58–64.
Stibbe, J., C. Kluft, E. Brommer, M. Gomes, D. de Jong, and J. Nauta. 1984. Enhanced fibrinolytic activity during cardiopulmonary bypass in open-heart surgery in man is caused by extrinsic (tissue-type) plasminogen activator. Eur. J. Clin. Invest. 14:375–382.
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Makowski, G.S., Ramsby, M.L. Interaction of Amorphous Calcium Phosphate with Fibrin In Vitro Causes Decreased Fibrinolysis and Altered Protease Profiles: Implications for Atherosclerotic Disease. Inflammation 25, 319–329 (2001). https://doi.org/10.1023/A:1012831900153
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DOI: https://doi.org/10.1023/A:1012831900153