Contact activation triggers stimulation of the monocyte 5-lipoxygenase pathway via plasmin

Abstract

The purpose of this study was to characterize the stimulus that activates the 5-lipoxygenase pathway in human peripheral monocytes (PM) during the process of contact activation. Incubation of PM, but not of polymorphonuclear leukocytes (PMN), in contact-activated, recalcified plasma induced a time-dependent release of leukotrienes (LT). The presence of platelets was required for the generation of cysteinyl-LT, but LTB4 formation also proceeded in their absence, although to a lesser extent. Plasmin, presumably generated via the intrinsic fibrinolytic pathway, was liable for the 5-lipoxygenase stimulation during contact activation inasmuch as (1) the 5-lipoxygenase pathway in PM was stimulated by contact-activated, recalcified, autologous or homologous plasma, but not by factor XII-deficient or prekallikrein- deficient plasma; (2) lysine analogs such as N alpha-acetyl-L-lysine, 6- aminohexanoic acid (6-AHA), or trans-4- (aminomethyl)cyclohexane-1- carboxylic acid (t-AMCA), which inhibit plasmin(ogen) binding to PM plasmin(ogen) binding sites, concentration-dependently reduced the cysteinyl-LT release; (3) plasminogen activators such as urokinase or streptokinase concentration-dependently enhanced the cysteinyl-LT release up to 10 and 1,000 IU/mL, respectively, while higher concentrations were less effective leading to bell-shaped concentration- response curves; (4) plasmin inhibitors such as aprotinin or alpha 2- antiplasmin concentration-dependently inhibited the cysteinyl-LT release; and (5) preincubation of plasma with monoclonal antibodies directed against plasminogen and capable of preventing plasminogen activation blocked the contact-mediated 5-lipoxygenase stimulation. Moreover, incubation of PM with plasmin, but not with plasma kallikrein, in Hanks' balanced salt solution (HBSS)-bovine serum albumin (BSA) 0.4% triggered a concentration-dependent release of LTB4 up to 0.1 caseinolytic units (CU)/mL, with higher concentrations being less effective. By contrast, release of cyclooxygenase metabolites such as thromboxane (TX) B2 and prostaglandin (PG) E2 was not stimulated by plasmin, indicating specificity for the 5-lipoxygenase pathway. With plasmin as a hitherto unknown stimulus of the 5-lipoxygenase pathway in PM, a novel link between contact activation and inflammation has been established.

Plasmin as a proinflammatory cell activator

“…Approximately 10 g/ml tranexamic acid is sufficient to inhibit fibrinolysis [99]. In our studies, up to 80 g/ml tranexamic acid was not sufficient to completely inhibit the plasmin-induced cysteinyl-leukotriene synthesis [44] and chemotaxis [43] of human monocytes. In addition, plasma concentrations of tranexamic acid decrease rapidly after infusion (t ½ ϳ1 h) [100], whereas the plasmin generation on the monocyte surface may be delayed.…”

“…At variance to monocytes, macrophages, and DCs, plasmin does not directly activate neutrophils [44,48]. However, neutrophils can be indirectly activated by plasmin, for example, through the plasmin-mediated activation of perivascular mast cells and the secondary generation of lipid mediators.…”

“…Plasmin generated as a result of contact activation of the intrinsic coagulation cascade or when added to cells kept in culture triggers biosynthesis of cysteinyl-leukotrienes and leukotriene B 4 by monocytes via the 5-lipoxygenase pathway. The effect is specific for plasmin, as a number of other proteases, including coagulation factors and other serine proteases, are unable to trigger a selective activation of the 5-lipoxygenase pathway in monocytes [44,46,48]. Plasmin binding to the monocyte surface occurs via kringle structures and lysine-binding sites, and the cell activation is dependent on the proteolytic activity of plasmin [48].…”

Plasmin: A Modulator of Immune Function

“…33 Plasmin stimulates NF-kB and AP-1, resulting in the production of TNF-α, IL-1α, IL-1β, and TF. 34 Plasmin also activates the 5-lipoxygenase pathway in monocytes 35,36 and macrophages, 37 resulting in the synthesis of proinflammatory leukotrienes. 38 In human monocytes, plasmin stimulates JAK/STAT signaling resulting in MCP-1 release, 39 thereby promoting further monocyte recruitment.…”

Plasminogen Activators in Inflammation and Sepsis

“…Plasmin stimulates inflammatory mediators and receptors, platelets, and monocytes (118, 152, 154, 195), is a specific chemoattractant for human monocytes (175), and induces expression of cytokines and tissue factor (174).…”