Abstract
The ability of intact peripheral blood monocytes to modulate factor V procoagulant activity was studied using electrophoretic and autoradiographic techniques coupled to functional assessment of cofactor activity. Incubation of plasma concentrations of factor V with monocytes (5 x 10(6)/ml) resulted in the time-dependent cleavage of the 330-kDa protein. Activation occurred via several high molecular mass intermediates (> or = 200 kDa) to yield peptides of 150, 140, 120, 94, 91, 82, and 80 kDa, which paralleled the expression of cofactor activity. The cleavage pattern observed differed from that obtained with either thrombin or factor Xa as an activator. The incubation time required to achieve full cofactor activity was dependent on the monocyte donor and ranged from 10 min to 1 h and was consistently slightly lower than that obtained with thrombin-activated factor Va. Cofactor activity was not diminished by additional incubation. The cofactor activity generated bound to the monocyte such that a competent prothrombinase complex was formed at the monocyte membrane surface. Furthermore, within 5 min of factor V addition to monocytes, near maximal cofactor activity (approximately 70%) was bound and expressed on the monocyte membrane. The proteolytic activity toward factor V was associated primarily with the monocyte membrane, as little proteolytic activity was released into the cell-free supernatant. Proteolytic activity was inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride. However, the inhibitor profile obtained with alpha 1-antiproteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin suggested membrane-bound forms of elastase and cathepsin G were mediating, in large part, the proteolysis observed. These data were confirmed using purified preparations of both proteases and a specific anti-human leukocyte elastase antibody. Thus, expression of these proteases at the monocyte surface may contribute to thrombin generation at extravascular tissue sites by catalyzing the activation of the essential cofactor, factor Va, which binds to the monocyte surface and supports the factor Xa-catalyzed activation of prothrombin.
Highlights
Since thrombin serves as a potential effector of fibrin deposition, leukocyte chemoattraction [5], and mesenchymal cell growth [6], it seems likely that the production of thrombin at the monocyte/macrophage membrane surface provides an important bioregulatory effector molecule at these extravascular sites
Propagation of the coagulant response is accomplished by the assembly and function of prothrombinase, a stoichiometric complex of the nonenzymatic cofactor factor Va and the enzyme factor Xa bound to the monocyte surface in the presence of calcium ions and which effects the proteolytic conversion of prothrombin to thrombin [10, 11]
While it is well established that both factor IXa/3 and factor Xa formation are accomplished at the monocyte/macrophage membrane surface, little is known concerning how these cells may participate in the events resulting in the proteolytic activation of the plasma procofactors, factors V and VIII
Summary
Vol 270, No., Issue of January 20, pp. 1408-1415, 1995 Printed in U.S.A. Human Coagulation Factor V Is Activated to the Functional Cofactor by Elastase and Cathepsin G Expressed at the Monocyte Surface*. Propagation of the coagulant response is accomplished by the assembly and function of prothrombinase, a stoichiometric complex of the nonenzymatic cofactor factor Va and the enzyme factor Xa bound to the monocyte surface in the presence of calcium ions and which effects the proteolytic conversion of prothrombin to thrombin [10, 11]. While it is well established that both factor IXa/3 and factor Xa formation are accomplished at the monocyte/macrophage membrane surface, little is known concerning how these cells may participate in the events resulting in the proteolytic activation of the plasma procofactors, factors V and VIII The provision of these cofactors is essential to the proper assembly and function of the coagulant enzyme complexes in which they participate [12]. As detailed in this report, membrane-bound forms of elastase and cathepsin G appear to be responsible, at least in part, for the monocyte-mediated activation of factor V
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