Activation of Mac-1 (CD11b/CD18)-bound factor X by released cathepsin G defines an alternative pathway of leucocyte initiation of coagulation.
Leucocyte initiation of coagulation preserves the haemostatic balance and may aberrantly contribute to vascular injury. In addition to the extrinsic activation mediated by tissue factor: factor VIIa, monocytes express an alternative procoagulant response after binding of the zymogen factor X to the integrin Mac-1 (CD11b/CD18). Here, factor X-activating activity was found in purified monocyte granules, and coincided with size-chromatographed fractions containing cathepsin G. In contrast, elastase-containing granule fractions did not activate factor X. In the presence of Ca2+ ions, purified cathepsin G, but not elastase, cleaved factor X to a approximately 54 kDa catalytically active derivative, structurally indistinguishable from the procoagulant product generated on monocytes after binding to Mac-1. Factor X activation by purified cathepsin G involved limited proteolysis of a novel Leu177-Leu178 peptide bond in the zymogen's activation peptide. Cathepsin G activation of factor X was completely inhibited by alpha 1 antichymotrypsin, or soybean trypsin inhibitor, or by a neutralizing antiserum to cathepsin G, while eglin, or an anti-elastase antibody, were ineffective. Affinity chromatography on active-site-dependent inhibitors Glu-Gly-Arg-chloromethyl ketone or benzamidine completely abolished factor Xa activity generated by cathepsin G. Cathepsin G was not constitutively detected on the monocyte surface by flow cytometry. However, inflammatory stimuli, including formyl peptide or phorbol ester, or Mac-1 engagement with its ligands fibrinogen, factor X or serum-opsonized zymosan, triggered monocyte degranulation and cathepsin G activation of factor X. These findings demonstrate that monocytes can alternatively initiate coagulation in a sequential three-step cascade, including (i) binding of factor X to Mac-1, (ii) discharge of azurophil granules, and (iii) limited proteolytic activation of membranebound factor X by cathepsin G. By rapidly forming thrombin and factor Xa in a protected membrane microenvironment, this pathway may contribute a "priming' signal for clotting, anticoagulation and vascular cell signal transduction, in vivo.
Plescia
J
J
Boyer Center for Molecular Medicine, Department of Pathology, Yale University School of Medicine, New Haven, CT 06536, USA.
Altieri
D C
DC
D001777:Blood Coagulation; D056649:Cathepsin G; D002403:Cathepsins / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007962:Leukocytes / Q000187:drug effects / Q000502:physiology*; D016177:Macrophage-1 Antigen / Q000502:physiology*; D009000:Monocytes; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010196:Pancreatic Elastase / Q000378:metabolism; D012697:Serine Endopeptidases; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D016177:Macrophage-1 Antigen; D009240:N-Formylmethionine Leucyl-Phenylalanine; D005170:Factor X; D002403:Cathepsins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D015951:Factor Xa; D013755:Tetradecanoylphorbol Acetate