The Journal of Immunology, Vol 137, Issue 9 2864-2870, Copyright © 1986 by American Association of Immunologists

ARTICLES

Characterization and purification of mouse macrophage procoagulant- inducing factor

J Ryan and CL Geczy

Macrophage procoagulant-inducing factor (MPIF) is a product of mouse Lyt-1+2- cells that induces macrophage procoagulant activity (MPCA) on mouse peritoneal exudate cells or on the macrophage-like tumor cell line WEHI-265. Supernatants from Sepharose-bound concanavalin A- stimulated cells were fractionated by using DEAE-Sephacel, heparin- Sepharose, and isoelectric focusing. This procedure resolved three different MPIF: MPIF alpha (pI 8.5), MPIF beta (pI 8.8 to 9.2), and MPIF gamma (pI 5 to 5.5). MPIF alpha and beta were small molecules (approximately 14 kD and 20 to 25 kD) as determined by gel filtration on Sephadex G200 and Biogel P100. MPIF beta was sharply resolved as a peak eluting after lysozyme by gel filtration on HPLC columns I-150 and I-125, although SDS-PAGE of the HPLC-enriched material resolved two well-defined bands of 70 and 120 kD and some poorly defined material of 14 kD. Silver staining failed to detect components of MPIF alpha after SDS-PAGE. MPIF gamma activity was associated with material that separated over a broad range (20 to 60 kD and 60 to 200 kD), possibly due to aggregation with other components of the supernatants. Crude supernatants were stable to heating at 56 degrees C for 30 min and pH 2 treatment, although more highly enriched fractions were unstable to these treatments. Heating at 90 degrees C for 5 min totally destroyed MPIF activity. The properties of the two basic MPIF differ from other lymphokines known to affect macrophage function, e.g., colony- stimulating factor, migration-inhibition factor, interferon-gamma, and interleukin 1.