Blood Coagulation, Fibrinolysis and Cellular Haemostasis
Schattauer GmbH
Plasminogen binds to plasmin-modulated factor Xa by Ca2+- and C-terminal lysine-dependent and -independent interactions
Jean E. Grundy
1
Canadian Blood Services, R&D Department, University of British Columbia/Centre for Blood Research, Department of Pathology and Laboratory Medicine, Vancouver, British Columbia, Canada
,
Mark A. Hancock
2
Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada
,
Scott C. Meixner
1
Canadian Blood Services, R&D Department, University of British Columbia/Centre for Blood Research, Department of Pathology and Laboratory Medicine, Vancouver, British Columbia, Canada
,
Roger C. MacKenzie
3
Institute of Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada
,
Marlys L. Koschinsky
2
Department of Biochemistry, Queen’s University, Kingston, Ontario, Canada
,
Edward L. G. Pryzdial
1
Canadian Blood Services, R&D Department, University of British Columbia/Centre for Blood Research, Department of Pathology and Laboratory Medicine, Vancouver, British Columbia, Canada
› Author AffiliationsFinancial support: *Supported by a Heart and Stroke Foundation of British Columbia and Yukon Operating Grant (E.P.) and a Heart and Stroke Foundation of Ontario Operating Grant (M.K.; T5653). J.G. is the recipient of a Career Development Fellowship Award from Canadian Blood Services. M.K. is a Career Investigator of the Heart and Stroke Foundation of Ontario.
Plasminogen binding to receptors involves both C-terminal lysine-dependent and -independent interactions. The latter are poorly understood. Our earlier work demonstrated a novel Ca2+-enhanced bivalent interaction between plasmin-cleaved FXa (FXa33/13) and plasminogen truncated at Lys78 (Lys-Pg). Here we hypothesized that the effects of Ca2+may enable dissection of the C-terminal lysine-dependent and -independent interactions. To evaluate the role of the Glu-plasminogen (Glu-Pg) amino acids 1–77, binding of FXa33/13 to immobilized Glu-Pg was compared to Lys-Pg by surface plasmon resonance. Under identical conditions, approximately half the amount of FXa33/13 bound to Glu-Pg. The simplest fit of data suggested a 2:1 plasminogen:FXa33/13 stoichiometry for both, which were proportionately enhanced by Ca2+. Only Lys-Pg demonstrated significant Ca2+-independent binding to FXa33/13. In the presence of Ca2+, weak C-terminal lysine-independent binding could be detected, but only for Glu-Pg. The elastase-generated plasminogen fragment encompassing the angiostatin-like kringle domains 1 to 3 (K1–3) inhibited binding of FXa33/13 to Lys-Pg, whereas fragments corresponding to kringle 4- and kringle 5-protease domain had no effect. Immobilized K1–3 binding to FXa33/13 had both Ca2+-dependent and -independent components. The principal KMd for the interaction was 10-fold higher than Lys-Pg. In the presence of Ca2+, eACA inhibited FXa33/13 binding to K1–3 by 30%, but eliminated binding in the absence of Ca2+. These studies suggest that Ca2+-dependent and -independent binding of Lys-Pg to FXa33/13 are C-terminal lysine-dependent. The N-terminal 1–77 amino acids of Glu-Pg confer significant C-terminal lysine-independent binding, which may play a role during the initiating stages of plasminogen activation.
Keywords
Coagulation factors -
protein function / activity -
plasminogen (-fragments) -
Gla-domain proteins
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