title: Differential effect of calcium phosphate and calcium pyrophosphate on binding of matrix metalloproteinases to fibrin: comparison to a fibrin-binding protease from inflammatory joint fluids.
abstract: The ability of calcium phosphate (CaP) and calcium pyrophosphate (CaPPi) to mediate matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9) binding to fibrin was evaluated. Substrate gel electrophoresis (gelatin zymography) revealed that CaP bound MMP-2 and MMP-9, forming a high molecular weight aggregate with lowered electrophoretic mobility. Formation of the CaP : MMP aggregate was necessary for fibrin binding. In contrast, CaPPi did not aggregate MMPs and did not promote uptake of MMPs into fibrin. Scatchard analysis (Ca/P ratio) revealed that CaPPi (1.96) was chemically similar to calcium pyrophosphate dihydrate (2.00) compared to amorphous CaP (1.50) or crystalline CaP, hydroxyapatite (1.66). MMP : CaP interaction appeared to be electrostatic in nature as high salt concentration (NaCl > 150 mm) reduced binding. In contrast, two non-ionic detergents (Brij-35 and Tween-20) did not prevent MMP : CaP binding. MMP : CaP interaction did not involve the C-terminal MMP region because the specific tissue inhibitor of metalloproteinases (TIMPs) also did not block MMP : CaP interaction and fibrin binding. Although MMP : CaP binding could be decreased with albumin, this effect appeared non-specific due to the high albumin concentration required. High albumin concentration could also partially dissociate preformed MMP : CaP complexes. Interestingly, type I and type IV collagen substantially increased MMP : fibrin-binding activity, whereas denatured collagen, gelatin, did not. Inflammatory joint fluid from five patients also demonstrated similar MMP fibrin-binding activity consistent with CaP mediation. The relevance of these findings to CaP and CaPPi in the pathogenesis of crystal arthropathies such as basic calcium phosphate (BCP) and calcium pyrophosphate dihydrate crystal disease (CPPD) is discussed.
authors.affiliation: Department of Laboratory Medicine, University of Connecticut Health Center, Farmington, CT 06030-2235, USA. makowski@nso1.uchc.edu
authors.forename: G S
authors.initials: GS
authors.lastname: Makowski
authors.forename: M L
authors.initials: ML
authors.lastname: Ramsby
mesh_terms: D000328:Adult; D000368:Aged; D001168:Arthritis / Q000378:metabolism*; D002130:Calcium Phosphates / Q000737:chemistry / Q000378:metabolism*; D002131:Calcium Pyrophosphate / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D005337:Fibrin / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D013582:Synovial Fluid / Q000378:metabolism
chemical_list: D002130:Calcium Phosphates; D005337:Fibrin; C020243:calcium phosphate; D020782:Matrix Metalloproteinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D002131:Calcium Pyrophosphate