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Fibrinolytic activity of normal human blood monocytes
1Harry Webster Thorp Laboratories, Division of Clinical Immunology, McGill University Clinic, Royal Victoria Hospital, Montreal, Quebec, Canada.
Abstract:
Neutrophils (PMN) are important in the cellular phase of blood fibrino lytic activity (FA). The contribution of monocytes (MC), which have FA, is unclear. To determine the relative roles of these cells to activity in normal blood, we examined, by solid phase radiofibrin assay, FA of normal blood and plasma, and of purified PMN and MC, with and without plasminogen (PLG), mini-plasminogen (mPLG), the other major elastase fragment of PLG, or autologous plasma. PMN alone (0.5 x 10(6)/ml) had striking activity (292 +/- 25 SEM ng fibrin lysed/h; n = 10 normal subjects) while MC alone (0.5 x 10(6)/ml) had mean FA of 32 +/- 4 ng/h, which could be accounted for by contaminating PMN (36 +/- 8 ng/h). Thus, in a 1 h assay (when cellular FA accounts for 70-80% of FA in whole blood), normal numbers of MC (0.5 x 10(6)/ml) had no detectable FA when assayed with PLG or normal plasma. With longer assay times (2-6 h), PLG-dependent (plasminogen activator, PA) activity was demonstrated with mixtures of MC and PLG or plasma. This PA activity was released into the medium and required prior contact of MC and an intact, soluble PLG molecule for PA activity to be detected in medium (suggesting a PLG-MC triggering mechanism), since activity was reduced or absent when MC were exposed to mPLG, the other major elastase fragment of PLG, or solid phase PLG. Exposure of MC to solid phase fibrin did not result in PA release. MC PA activity was little affected by cycloheximide pretreatment, indicating preformed rather than newly synthesized PA. By SDS-PAGE and fibrin zymography, MC extracts revealed a single PA band with features of pro-urokinase (single chain urinary-type PA): Mr 55,000, inhibition by antiurokinase antibody (but not by anti-tPA), and resistance to inhibition by DFP. By ELISA assay, approximate normal monocyte content of this PA (as Mr 55,000 urokinase) was 0.03 fg (3.3 x 10(8) molecules) per cell.
Insights
Monocytes (MC) exhibit plasminogen activator (PA) activity, primarily urokinase, after prolonged incubation with plasminogen (PLG). This indicates a delayed but significant role for monocytes in blood fibrinolytic activity.
Area of Science:
- Hematology
- Biochemistry
- Cell Biology
Background:
- Neutrophils (PMN) are key contributors to blood fibrinolytic activity (FA).
- The specific role of monocytes (MC) in FA, despite possessing FA, remains unclear.
- Understanding cellular contributions to FA is crucial for hemostasis and thrombosis research.
Purpose of the Study:
- To elucidate the relative contributions of neutrophils and monocytes to blood fibrinolytic activity.
- To investigate the plasminogen activator (PA) activity of monocytes under various conditions.
- To characterize the nature and regulation of monocyte-derived PA.
Main Methods:
- Solid phase radiofibrin assay to measure FA of purified PMN and MC.
- Assays performed with varying concentrations of plasminogen (PLG), mini-plasminogen (mPLG), and autologous plasma.
- SDS-PAGE, fibrin zymography, ELISA, and cycloheximide treatment to characterize PA.
- Investigated PA release mechanisms and requirements.
Main Results:
- Purified neutrophils exhibited significant fibrinolytic activity.
- Monocytes alone showed minimal FA in short-term assays, attributable to contaminating neutrophils.
- Monocytes demonstrated PLG-dependent PA activity upon prolonged incubation (2-6 hours) with soluble PLG or plasma.
- Monocyte PA activity was identified as pro-urokinase (Mr 55,000) and appeared to be preformed.
Conclusions:
- Monocytes contribute to blood fibrinolytic activity through a delayed, PLG-dependent release of pro-urokinase.
- This suggests a distinct, albeit slower, mechanism of fibrinolysis involving monocytes compared to neutrophils.
- The findings highlight the importance of considering monocyte-derived PA in understanding blood clot lysis.