©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Coagulation Factor V Is Activated to the Functional Cofactor by Elastase and Cathepsin G Expressed at the Monocyte Surface (*)

(Received for publication, October 3, 1994)

Debra H. Allen Paula B. Tracy (§)

From the Department of Biochemistry, University of Vermont College of Medicine, Burlington, Vermont 05405

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 times 10^6/ml) resulted in the time-dependent cleavage of the 330-kDa protein. Activation occurred via several high molecular mass intermediates (geq200 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 (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.


INTRODUCTION

Monocytes and macrophages play key roles in the physiological and pathophysiological processes of wound repair, acute/chronic inflammation, and atherosclerosis. These processes, which in several instances proceed at extravascular tissue sites, are characterized by extensive fibrin deposition and an active fibroproliferative response (1, 2, 3, 4) . 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. Thus, we and others have begun to explore the hypothesis that the ability of these cells to participate in the molecular events leading to thrombin formation is an important mechanism by which they function.

Monocytes/macrophages can provide the appropriate membrane surface for the assembly and function of virtually all the coagulation complexes involved in thrombin production. Monocytes can be stimulated to express tissue factor at their membrane surface, which binds factor VIIa, and catalyzes the activation of factor X to factor Xa, thereby initiating the extrinsic pathway of coagulation (for review, see (7) ). The tissue factor-factor VIIa complex also activates factor IX to factor IXabeta(8) , which in complex with the cofactor factor VIIIa bound to the monocyte/macrophage membrane can provide additional factor Xa(9) . 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 IXabeta 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) . For example, factor Va is responsible for mediating the majority of the protein-protein and protein-membrane interactions required for prothrombinase assembly, such that the deletion of factor Va from the complex reduces the rate of thrombin generation by 4 orders of magnitude(13) . The central role that factor Va assumes in complex assembly, coupled with its profound influence on the rate of thrombin formation, provides strong evidence that the activation of factor V to factor Va is a key regulatory event. This notion is underscored by the inability of the procofactor factor V to participate, to any significant degree, in thrombin generation(13, 14) .

Several proteases have been identified that cleave human factor V to yield different levels of cofactor activity. Thrombin (12) and factor Xa (15) are believed to be the most potent physiological activators of human factor V and by far the most widely recognized with respect to coagulant and hemostatic responses. In contrast to thrombin activation of factor V, the factor Xa-catalyzed activation of factor V is absolutely dependent on the presence of a membrane surface(14, 15) . Whether the membrane surface is required for substrate or enzyme binding, or both, has not yet been determined. Factor V activators that can be released from platelets(16, 17) , endothelial cells(18) , and neutrophils (19) have also been described. In contrast to the other cell-derived activators, leukocyte elastase released from neutrophils first activates factor V and then rapidly inactivates the formed factor Va(19) . The extent to which these cell-associated proteases contribute to factor V activation/inactivation in normal or pathophysiological events has not been elucidated.

In attempts to determine if factor Xa bound to the monocyte membrane (20, 21) could activate factor V, the observation was made that freshly isolated peripheral blood monocytes alone effected the rapid cleavage and activation of plasma concentrations of factor V. Cofactor activity was expressed at the monocyte membrane surface, which supported the factor Xa-catalyzed activation of prothrombin. 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.


EXPERIMENTAL PROCEDURES

Materials

Purified human neutrophil elastase and alpha(1)-proteinase inhibitor were obtained from Calbiochem. Purified human neutrophil cathepsin G, alpha(2)-macroglobulin, and alpha(1)-antichymotrypsin were obtained from Athens Research and Technology, Inc. (Athens, GA). L-Ala-L-Ala-L-Pro-L-Val chloromethyl ketone was obtained from BACHEM, Inc. (Torrance, CA). Heparin was obtained from ESI Pharmaceuticals (Cherry Hill, NJ). Phenylmethanesulfonyl fluoride, soybean trypsin inhibitor, N-p-tosyl-L-lysine chloromethyl ketone, and diisopropyl fluorophosphate were obtained from Sigma. Sheep anti-human neutrophil elastase was obtained from Biodesign International (Kennebunkport, ME), and sheep non-immune IgG was obtained from ICN Biomedicals, Inc. (Costa Mesa, CA).

Cell Isolation

Peripheral blood mononuclear cells, devoid of platelets, were prepared daily from 1 unit of citrate phosphate dextrose-adenine anticoagulated blood using a modification (10) of the method of Boyum (22) as described(21) . Monocytes were purified from mononuclear cell suspensions using two protocols that employ density gradient centrifugation as previously described(21) . Purified suspensions of T-lymphocytes were isolated from mononuclear cell suspensions by rosette formation with sheep red blood cells treated with 2-aminoethylisothiouronium bromide (23) (Sigma) as previously described(10) . Neutrophils were purified from whole blood by discontinuous gradient centrifugation(11) . Purified cell populations were suspended in 20 mM HEPES, 0.15 M NaCl, pH 7.4. Cell identity was confirmed by observing typical morphology after staining with a modified Wright's stain. Typically, the purity of the cell suspensions was as follows: monocytes, geq80% (with the main contaminant being lymphocytes); lymphocytes, geq95%; and neutrophils, geq99%. Cell viability determined by monitoring either nigrosin or trypan blue uptake was geq98% for all purified cell populations.

Isolation of Coagulant Proteins

Coagulation proteins were isolated from human, fresh-frozen plasma obtained from the American Red Cross (Burlington, VT). Prothrombin and factor X were isolated as described by Bajaj et al.(24) Factor X was applied to an alpha-human protein C immunoaffinity column to remove trace protein C contamination undetectable by gel electrophoresis. Factor Xa was prepared as described by Jesty and Nemerson(25) , using the factor X activator isolated from Russell's viper venom(26) . Taipan snake venom (Sigma) was used to activate prothrombin to alpha-thrombin as previously described(27) . Factor V was isolated using immunoaffinity chromatography as described (28) and activated with 3 NIH units/ml of thrombin for 10 min at 37 °C. Antithrombin III was purified as described(29) . Protein purity was assessed by sodium dodecyl SDS-polyacrylamide gel electrophoresis (PAGE) (^1)prior to and following reduction with 5% 2-mercaptoethanol (v/v) in either 5-15% gradient or 10% slab gels according to methods described by Laemmli(30) . Gels were stained with Coomassie Brilliant Blue R-250 to visualize proteins. Molecular weights and extinction coefficients, , of the various proteins were taken as follows: factor V, 330,000, 9.6(31) ; factor Xa, 50,000, 11.6(24) ; prothrombin, 72,000, 14.2(24) ; thrombin, 37,000, 17.4(32) ; and antithrombin III, 58,000, 6.5(33) .

Factor V was radioiodinated using the IODO-GEN (Pierce) transfer technique purified and characterized as previously detailed(15) . I-Factor V was >95% precipitable with 10% trichloroacetic acid, expressed specific radioactivities of 1000-5000 cpm/ng (0.1-0.5 mol of iodine/mol of protein), and was stored at -20 °C in 50% glycerol. I-Factor V retained full clotting activity.

Cleavage ofI-Factor V by Cell-associated Proteases

All assays were performed in 20 mM HEPES, 0.15 M NaCl, 5 mM CaCl(2), pH 7.4. Suspensions of monocyte, T-lymphocyte, or neutrophil populations (0.1-5 times 10^6 cells/ml) were incubated with factor V (20-67 nM containing trace I-factor V) at 37 °C. At various time points, an aliquot (100 µl) was removed from the reaction mixture and centrifuged at 12,000 times g for 10 s. 30 µl of the supernatant fraction was assayed immediately to assess factor Va cofactor activity as detailed below. 2 volumes of SDS-PAGE sample preparation buffer were added to the remaining supernatant fraction, which was used to monitor cleavage of I-factor V by SDS-PAGE using 5-15% polyacrylamide gels as described. An equal amount of isotope was applied to each lane. Following electrophoresis, dried gels were subjected to autoradiography at -70 °C using Kodak XR-1 film and Dupont ``Lightning Plus'' intensifying screens. The extent of factor V cleavage and formation of the various peptides was determined by densitometric analyses of the autoradiographs using a Microscan 1000 scanning densitometer (Technology Resources Inc., Nashville, TN) as previously described(15) . Data were expressed as integrated volumes for each protein band using the arbitrary density units of the scanning system.

Certain experimental protocols required the analysis of I-factor V either free in solution or bound to the cell surface. Accordingly, free and bound I-factor V/Va were obtained by centrifugation (12,000 times g, 30 s) of an aliquot (100 µl) of the reaction mixtures over an oil mixture containing 1 part Apiezon A oil (Apiezon Products, Ltd., London) and 9 parts n-butyl phthalate. The supernatant fractions were made 10% in acetic acid, and the cell pellets were lysed by addition of 10% acetic acid. Both fractions were subsequently frozen and lyophilized and then processed for SDS-PAGE and autoradiography as outlined above.

In experimental protocols where the effect of a protease inhibitor or a specific anti-protease antibody on factor V proteolysis was being assessed, the inhibitor or antibody was incubated at 37 °C with the monocytes for 2 or 15 min, respectively, prior to the addition of factor V to the reaction mixture.

Functional Assessment of Factor Va Cofactor Activity

The ability of the cell-associated proteases to cleave factor V to factor Va was assessed by monitoring the generation of factor Va functional activity. The assay measures the effect of factor Va on prothrombin activation through the assembly and function of the prothrombinase complex. Thrombin concentrations are measured in one of two ways. The formation of thrombin in the assay can be continuously monitored by the change in fluorescence intensity of dansylarginine-N-(3-ethyl-1,5-pentanediyl)amine as it interacts with thrombin(13) . Assay mixtures contained 1.39 µM prothrombin, 3 µM dansylarginine-N-(3-ethyl-1,5-pentanediyl)amine, 20 µM phospholipid vesicles (containing 75% phosphotidylcholine, 25% phosphotidylserine), 5 mM CaCl(2) in 20 mM HEPES, 0.15 M NaCl, pH 7.4. Factor V (Va) samples removed from activation mixtures described above were assayed at concentrations leq 1 nM. Reactions were initiated by addition of 10 nM factor Xa. Under these conditions, factor Va is the limiting component; thus, initial rates of thrombin formation are proportional to the concentration of factor Va in the assay. Fluorescence measurements were obtained routinely on a Perkin-Elmer LS-3b fluorescence spectrometer with the excitation and emission wavelengths set at 335 and 565 nm, respectively. Alternatively, aliquots of prothrombin activation reaction mixtures were removed at various timed intervals (0, 1, 2, 4, 7, 10 min) and added to an equal volume of 20 mM TRIS, 0.15 M NaCl, 50 mM EDTA, 0.1% polyethylene glycol 8000, pH 7.4, to quench the reaction. The thrombin concentration in each sample was determined using the chromogenic substrate Spectrozyme TH (0.4 mM, American Diagnostica, Inc., Greenwich, CT). The change in sample absorbance at 405 nM was monitored using a Molecular Devices V(max) spectrophotometer and compared with a thrombin standard curve (0-200 nM) prepared daily using purified thrombin. The initial rate of thrombin generated in the various assay mixtures was calculated by linear regression analysis of the data obtained from the subsamples removed over time.


RESULTS

Activation of Human Factor V by Monocyte-associated Proteases

The ability of intact peripheral blood monocytes to modulate factor V and factor Va procoagulant activity was studied using electrophoretic and autoradiographic techniques, coupled to functional assessment of factor Va cofactor activity. Incubation of plasma concentrations of human factor V (20-67 nM containing trace radiolabeled protein) with isolated peripheral blood monocytes (5 times 10^6 cells/ml) resulted in the time-dependent cleavage of the 330-kDa single chain protein (Fig. 1A). Cleavage was demonstrated by the appearance of several high molecular mass peptides (geq200 kDa) as well as peptides of approximately 150, 140, 120, 94, 91, 82, and 80 kDa, over a course of 60 min. Significantly more proteolysis was observed than with any of the other well characterized factor V activators including thrombin, factor Xa, and plasmin. The observed cleavage pattern and end products formed were different as well. The 220- and 150-kDa monocyte-derived peptides were the only two that migrated similarly to peptides that appear during the factor Xa-(15) , thrombin- (12, 15) , or plasmin-catalyzed (34) activation of factor V. Addition of thrombin (3 NIH units/ml) to the monocyte-derived peptides (rightlane) at the end of a 60-min incubation resulted in further cleavage of all the high molecular mass (geq200 kDa) peptides to generate the 150-kDa activation peptide and the 105- and 74-kDa subunits of thrombin-activated factor Va (factor Va). Interestingly, the 82- and 80-kDa monocyte-derived peptides were further cleaved to yield the 74-kDa factor Va light chain.


Figure 1: Cleavage and activation of human factor V by a monocyte-associated protease(s). Monocytes (5 times 10^6/ml) were incubated with human factor V (67 nM) plus trace I-factor V (1000 cpm/µl) in 20 mM HEPES, 0.15 M NaCl, 5 mM CaCl(2), pH 7.4, at 37 °C. At the time points indicated, aliquots were removed and centrifuged at 12,000 times g for 10 s. The supernatant was removed, and the aliquots were assayed. A depicts SDS-PAGE (5-10% gradient) followed by autoradiography. Equal amounts of isotope were applied to each lane. The symbols at the bottom of each lane correspond to the factor Va cofactor activity assays made during the course of the incubation as shown in B. In B, factor Va cofactor activity (leq1 nM) was assessed by its ability to support the factor Xa-catalyzed (5 nM) activation of prothrombin (1.39 µM) in the presence of defined phospholipid vesicles (20 µM). --, activity observed with factor Va. Inset depicts initial rates of thrombin generation for each assay.



The time-dependent cleavages observed paralleled the expression of factor Va cofactor activity (Fig. 1B), as indicated by the shortening of the lag period preceding steady state thrombin formation in the assay and an increase in the steady state rate of thrombin generation. Both observations were dependent on the length of time monocytes were incubated with the factor V. Increases in cofactor activity were apparent with loss of the 330-kDa parent molecule but could not be ascribed with any certainty to a particular combination of the peptides observed.

Based on the five replicate experiments performed, the length of time required to achieve full cofactor activity was donor-dependent and in some cases required less than a 15-min incubation of monocytes with factor V, in marked contrast to the 1-h incubation shown in Fig. 1A. As can be seen clearly in Fig. 1B, total cofactor activity generated was consistently slightly lower than that observed with thrombin as activator (--), since addition of thrombin following a 60-min incubation of factor V with monocytes (-circle-) did not result in rates of prothrombin activation achieved with thrombin-activated factor V, factor Va (-up triangle-). Even though lymphocytes represented as much as 20% of the cells present in the reaction mixtures, purified populations of T-lymphocytes (geq98%) neither activated nor cleaved factor V when identical experimental protocols to those described in Fig. 1were used (data not shown). These combined observations indicate that intact, freshly isolated peripheral blood monocytes express a protease(s) capable of activating factor V, that the factor Va generated is a slightly less effective cofactor than factor Va, and that the cofactor is comprised of novel peptides.

Proteolytic Activity Is Associated with the Monocyte Membrane Surface

To determine if the monocyte protease was membrane associated, experiments were done to determine if factor V cleavage required the continued presence of cells. In five separate experiments using different monocyte donors, reaction mixtures containing monocytes (5 times 10^6/ml) and factor V (20 nM plus trace labeled protein) were incubated as described above in Fig. 1. After 5 min, the reaction mixture was divided, and each sample was centrifuged at 200 times g for 10 min. One sample was merely resuspended (Fig. 2A, panelI) and was used to represent factor V cleavages resulting from the continued presence of cells as well as to control for any proteolytic activity released due to centrifugation of the cells in the presence of factor V. The supernatant fraction from the second sample was removed and transferred to a separate tube (Fig. 2A, panelII) to determine if any proteolytic activity was expressed in the cell supernatant. Both reaction mixtures were returned to 37 °C, and aliquots were removed at 30, 45, and 60 min and processed for autoradiography. In a second reaction mixture, monocytes were incubated in buffer alone for 5 min at 37 °C and centrifuged as above. The supernatant was removed, factor V (20 nM plus trace I-factor V) was added and incubated at 37 °C, and aliquots were removed at 45 and 60 min and processed for autoradiography (Fig. 2A, panelIII). Examination of the autoradiographs indicated that significantly more proteolytic activity was expressed by the pelleted, resuspended cells (panelI) than that which was observed in the cell-free supernatant (panelII). However, comparison of panels II and III suggested that the release of proteolytic activity into the supernatant might be triggered by the addition of factor V to the cells and not by buffer alone, since panel III served as the control for protease release into the supernatant, which may have resulted from centrifugation of cells. These observations were confirmed by densitometric scanning analyses (Fig. 2B) in which the appearance of the 94/91-kDa doublet was plotted as a function of time for each experimental protocol. This peptide pair was chosen for quantitation since it appeared to be a bona fide cleavage end product. The rate of appearance of these peptides was three times faster in the presence of cells (-bullet-) than that expressed by the cell-free supernatant (--). However, of the activity present in the cell-free supernatant, only 40-50% could be attributed to protease release during cell centrifugation. Since this observation was a consistent finding in four additional experiments, it would suggest that factor V may trigger some small extent of protease release from the cells. Identical conclusions could be drawn by densitometric analysis of the disappearance of the 330-kDa factor V molecule (data not shown). These combined data strongly suggest that the monocyte-associated proteolytic activity toward factor V was primarily located at the cell membrane surface.


Figure 2: Monocyte-mediated proteolysis of factor V requires the continued presence of cells. Monocytes (5 times 10^6/ml) were incubated with factor V (20 nM plus trace label) as described in Fig. 1. After 5 min, the reaction mixture was divided, and both aliquots were centrifuged at 200 times g for 10 min. One aliquot was resuspended and assayed for proteolytic activity toward factor V (panelI). The cell-free supernatant was removed from the second and assayed similarly (panelII). In a third reaction mixture (panelIII), factor V was added to a cell-free supernatant obtained subsequent to the centrifugation step. A, SDS-PAGE and autoradiographic visualization of the factor V peptides. B, densitometric analysis of the data shown in A monitoring the appearance of the 94/91 peptide doublet (indicated by the arrow).



Factor Va Cofactor Activity Was Expressed at the Monocyte Membrane Surface

To establish the potential physiological relevance of this reaction, four experiments using different monocyte donors were done to determine which cleavage products associated with the monocyte membrane and if the monocytes and the monocyte-bound factor Va peptides could provide both the membrane surface and factor V(a) cofactor activity required to support the factor Xa-catalyzed activation of prothrombin. Monocytes (5 times 10^6/ml) were incubated at 37 °C with factor V (20 nM) containing trace labeled protein. At the time points indicated in Fig. 3A (a single representative experiment), two aliquots were removed. One aliquot was prepared for autoradiographic visualization of both the free (supernatant) and bound (pellet) peptides (panelsI and II, respectively) as detailed under ``Experimental Procedures.'' The second aliquot was centrifuged at 200 times g for 10 min. After removal of the supernatant, the cell pellet was resuspended in fresh buffer to which factor Xa (10 nM) and prothrombin (1.39 µM) were added to monitor prothrombin activation. Initial rates of prothrombin activation were determined and are indicated above each lane in panelII. Control reactions were prepared by incubating 20 nM factor Va (containing trace label) with purified monocytes in analogous protocols (Fig. 3B). We reasoned that incubation of the fully active cofactor with monocytes would reflect the maximum amount of cofactor that could bind to the monocytes throughout the incubation period.


Figure 3: Assessment of factor Va cofactor activity associated with peptides bound to the monocyte membrane as visualized by autoradiography. Monocytes (5 times 10^6/ml) were incubated with 20 nM factor V (A) or factor Va (B) each containing trace label as previously described. At the specified time points, aliquots were removed and centrifuged at 12,000 times g (10 s). The supernatant (I) or pellet (II) fractions were then processed for SDS-PAGE and autoradiography. In parallel reaction mixtures, the pelleted cells with bound factor Va peptides were resuspended in 20 mM HEPES, 0.15 M NaCl, 5 mM CaCl 0.35% bovine serum albumin, pH 7.4, and assayed for their ability to support the factor Xa-catalyzed (5 nM) activation of prothrombin (1.4 µM) in the absence of additional factor Va or lipid. Initial rates are indicated above the corresponding lanes in panelII.



Comparison of the prothrombin activation rate data listed in panelsA and B indicated that at any time during the course of the reaction, the factor V(a) peptides associated with the monocyte membrane due to cell-mediated proteolysis (panelA) expressed 60% of the cofactor activity provided by bound factor Va (panelB). The time-dependent increase in expression of cofactor activity observed for both species of monocyte-bound peptides most likely reflects their increased binding over time. It is interesting to note that within 5 min of factor V addition, 60-70% of the total cofactor activity, which can associate with the monocyte membrane surface, is expressed. Thus, at the earliest time point measured (5 min subsequent to factor V addition to a monocyte suspension), the factor Va cofactor activity expressed at the monocyte membrane surface was capable of promoting the generation of 50 nmol/liter/min (5 NIH units/ml/min).

Comparison of the peptides bound to the monocyte surface versus those remaining in the supernatant in panelA allows for several observations: 1) only one peptide resulting from factor V cleavage by a monocyte-associated protease(s) did not bind to the monocyte membrane (arrow); 2) factor V bound poorly or alternatively was rapidly cleaved upon binding; and 3) peptides of leq 94 kDa selectively bound, suggesting that they comprise the active cofactor. In contrast, the data shown in panelB indicated that only the heavy (105 kDa) and light chain (74 kDa) subunits comprising factor Va(12, 15) bound to the monocyte to any appreciable degree. The 150-kDa activation peptide did not bind nor did a peptide migrating similarly to the 280-kDa factor V activation intermediate as indicated by the arrows. Bound to a very small degree were the 220- and 150-kDa activation intermediates, which contain the 74- and 105-kDa factor Va subunits, respectively. It is interesting to note that the bound factor Va was further cleaved to products not observed in the supernatant fraction, suggesting that factor Va is also a substrate for the monocyte-associated protease(s). However, this additional proteolysis did not appear to affect cofactor activity. Although it is not yet clear which peptides are expressing cofactor activity, these data clearly demonstrate that incubation of plasma concentrations of factor V with monocytes results in the production of a cofactor bound to the monocyte surface that expresses significant cofactor activity and facilitates the assembly of a functional prothrombinase complex at the monocyte surface.

Identification of the Monocyte Proteases Responsible for Factor V Activation

A variety of protease inhibitors were used to begin to identify the protease(s) involved in factor V cleavage and activation. The ability of diisopropyl fluorophosphate (data not shown) and phenylmethanesulfonyl fluoride to nearly abolish proteolytic activity suggested the activity could be attributed to a serine protease(s) (Fig. 4). Several serine proteases have been reported to be expressed at the monocyte membrane surface; one of which has been reported to exhibit elastase-like characteristics(35) . However, when more specific inhibitors were used, the inhibitor profile obtained was somewhat inconsistent with the activity being totally attributable to a single enzyme. For example, even though in this particular experiment densitometric analyses indicated that alpha(1)-proteinase inhibitor could abolish 90% of the protease activity (lane3versus10), the specific elastase inhibitor L-Ala-L-Ala-L-Pro-L-Val chloromethyl ketone was only partially inhibitory (lane5) and inhibited the proteolytic activity to the same extent as alpha(2)-macroglobulin (lane4). Thus, we would hypothesize that in this particular instance, 60% of the proteolytic activity observed was due to elastase or an elastase-like enzyme. However, the substantial inhibition observed with plasma concentrations of alpha(1)-antichymotrypsin (lane8) suggested that a membrane-bound form of cathepsin G, which may be susceptible to inhibition by alpha(1)-proteinase inhibitor, may be playing a role as well. The lack of inhibition effected by the antithrombin III-heparin complex (lane7) and the peptide products formed indicated that membrane-bound forms of factor Xa or thrombin were not responsible.


Figure 4: Effect of protease inhibitors on the cleavage of factor V by a monocyte-associated protease(s). Monocytes (5 times 10^6/ml) were incubated with the protease inhibitors indicated for 2 min at 37 °C prior to the addition of factor V (20 nM) plus trace label. Following a 30-min incubation, the reaction mixtures were processed for SDS-PAGE and autoradiography as previously described. Densitometric analysis was used to calculate the amount of single chain factor V remaining, as its loss represented the presence of proteolytic activity. PMSF, phenylmethanesulfonyl fluoride; TLCK, N-p-tosyl-L-lysine chloromethyl ketone.



In five similar experiments using different donors for monocyte isolation, similar inhibition trends were observed. Whereas alpha(1)-proteinase inhibitor consistently elicited the most pronounced inhibition (geq90%), the inhibition effected by alpha(2)-macroglobulin, the elastase-specific peptide, and alpha(1)-antichymotrypsin was substantially more variable. These data were interpreted to indicate that monocytes exhibit significant heterogeneity with respect to the levels of the proteases expressed.

Based on these observations, experiments with purified human leukocyte elastase (HLE) and cathepsin G were performed to determine their ability to cleave and activate factor V. Preliminary experiments were done to determine concentrations of both proteases, which produced factor V cleavages over a similar time frame as 5 times 10^6 monocytes/ml to accurately compare the cleavage patterns obtained. Low concentrations of both proteases were required as detailed in Fig. 5. Both proteases cleaved factor V (Fig. 5), concomitant with an increase in cofactor activity (data not shown). The cleavage patterns were remarkably similar to each other and to that obtained with monocytes alone. The co-migration studies shown allowed for the unequivocal identification of two peptides unique to elastase-mediated cleavage as indicated by the arrows. These observations were supported by data shown in Fig. 6in which an alpha-HLE antibody, at a concentration that substantially inhibited purified HLE, was only partially inhibitory toward total monocyte proteolytic activity yet completely inhibited the formation of peptides unique to elastase cleavage, as indicated by the arrows. The inhibitory activity could not be increased by higher concentrations of antibody in subsequent experiments (data not shown). These combined data strongly suggest that both elastase and cathepsin G may be active on the monocyte surface to effect the cleavage and activation of factor V.


Figure 5: Monocyte-, cathepsin G-, and HLE-mediated proteolysis of factor V. Factor V (20 nM) plus trace label was incubated with monocytes (5 times 10^6/ml), cathepsin G (0.1 nM), or HLE (0.3 nM) for 5 min at 37 °C and then prepared for autoradiography (middle, left, and rightlanes, respectively). The lanes to the left and right of middle represent equal mixtures of the monocyte and cathepsin G or monocyte and HLE gel samples, respectively. Right arrows represent cleavages specific to HLE.




Figure 6: Partial inhibition of monocyte-associated proteolytic activity with an alpha-HLE antibody. Monocytes (5 times 10^6/ml) or purified HLE (0.3 nM) were incubated ± sheep alpha-HLE antibody (10 µM) for 15 min at 37 °C prior to the addition of factor V (20 nM) plus trace label. At the times indicated, aliquots were removed and processed for autoradiography. A, -alpha-HLE; B, +alpha-HLE; C, control sheep IgG plus monocytes or HLE.



Since a membrane-bound form of HLE appeared to be responsible for the proteolytic activity observed, experiments were done to ensure that neutrophils present at extremely low levels (geq0.05%) in our monocyte preparations were not responsible for the activity observed. The factor V cleavage pattern obtained with neutrophils was identical to that obtained with purified HLE and thus almost identical to that obtained with monocytes. Even though on a per cell basis neutrophils expressed 25-40 times the activity observed with monocytes (data not shown), they did not contribute significantly to factor V cleavage and activation in our system.


DISCUSSION

The assembly and function of a competent prothrombinase complex at the monocyte/macrophage surface requires the participation of the nonenzymatic cofactor factor Va(10, 11, 21) , which is produced by limited proteolysis of the procofactor factor V(12) . In this report, we demonstrate that addition of factor V to a suspension of freshly isolated whole blood monocytes leads to factor V cleavage and the generation of factor Va cofactor activity. Proteolysis occurs through a novel cleavage pattern when compared with those that result from activation by the well described activators-thrombin and factor Xa (15) , and may explain the observation that the cofactor activity generated by the monocyte-bound proteases is just slightly less than that obtained with factor Va. However, the novel cofactor generated remains associated with the monocyte membrane such that in the presence of added factor Xa, a functional prothrombinase complex is assembled at its surface. Based on the data depicted in Fig. 3, pathophysiologically relevant concentrations of thrombin may be produced quite rapidly; for example, 1 NIH unit/ml (approxl0 nM) can be produced within 1 min of the addition of a plasma concentration of factor V to a monocyte suspension (5 times 10^6 cells/ml). Furthermore, within 5 min, 70% of the cofactor binding sites on the monocyte membrane surface are occupied and competent to support the factor Xa-catalyzed activation of prothrombin. However, the activation of factor V catalyzed by the monocyte-associated proteases proceeds to completion and is thus capable of generating a substantial concentration of active cofactor in the surrounding milieu.

The proteolytic activity observed appears to be due in large part, if not completely, to membrane-bound forms of elastase and cathepsin G. Immunohistochemical evidence indicates that human peripheral blood monocytes contain enzymes antigenically similar to leukocyte and cathepsin G(36) . While monocytes appear to be heterogeneous with respect to enzyme content, the antigens are present predominantly in peroxidase-positive cytoplasmic granules(36) , although membrane-bound forms of elastase have been reported(35) . Other studies have demonstrated that human leukocyte elastase binds to monocytes and remains in an active form at the membrane surface for as long as 24 h (37) . Since these proteases are susceptible to inhibition by the plasma protease inhibitors alpha(1) anti-proteinase inhibitor, alpha(2)-macroglobulin, or alpha(1)-antichymotrypsin, we suspect that the proteases would not be active in plasma under normal conditions. This notion was confirmed by demonstrating that resuspension of proteolytically active monocytes in autologous, normal plasma completely abrogated their ability to activate factor V (data not shown). Therefore, we propose that the monocyte-associated proteolytic activity may play a significant role in thrombin generation primarily at extravascular tissue sites, as well as in clinical settings where extensive depletion of plasma protease inhibitors occurs. Examples might include alpha(1)-proteinase inhibitor deficiency, disseminated intravascular coagulation, and thrombolytic therapy. Finally, since our initial studies of plasma inhibition by autologous plasma were done using cells in suspension, those studies must now be done with an adherent cell population since enzyme expression may be different, possibly modifying the ability of plasma inhibitors to regulate monocyte-associated proteolysis. In fact, several studies have been reported indicating that cell- or matrix-bound proteases expressed by, or released from, adherent monocytes/macrophages appear to be protected from their respective plasma inhibitors(38, 39, 40) .

The functional significance of monocyte/macrophage-derived thrombin is underscored by the fibrin deposition and fibroproliferative responses that accompany wound repair, chronic inflammation, and atherosclerosis, processes in which these cells are known to participate. As cells that support procoagulant enzymatic reactions, monocytes/macrophages are unique in that they provide the appropriate membrane surface required for the assembly and function of all the coagulation enzyme complexes required for thrombin generation in vivo. Through the provision of the membrane-bound enzymes, elastase and cathepsin G, which can effect the generation of an essential cofactor in this process, these cells appear to enhance their procoagulant phenotype substantially.

Continued maintenance of this procoagulant phenotype may be accomplished through additional elastase- and cathepsin G-mediated events. For example, elastase can proteolytically inactivate proteins C (41) and S(42) , which would effectively abrogate activated protein C-catalyzed inactivation of factor Va(43) . In addition, while platelets are not activated by elastase directly, elastase enhances the platelet activation-dependent responses induced by low concentrations of cathepsin G, indicating that both enzymes may function synergistically to activate platelets(44, 45) . Further, elastase can proteolytically inactivate tissue factor pathway inhibitor(46, 47) , which would ordinarily limit thrombin production through inhibition of factor Xa and a factor Xa-factor VIIa-tissue factor complex.

Elastase and in some cases cathepsin G have been demonstrated to proteolytically inactivate several inhibitors of coagulant proteases including antithrombin III(48, 49) , heparin cofactor II(50) , chlorine inactivator(51, 52) , and alpha(2)-antiplasmin(51, 53) . The decreased concentration of active inhibitor would slow the protease inhibition, thereby increasing the local steady state level of the target protease. In such circumstances, the inactivation of potential inhibitors would contribute to the local progression of coagulation at inflammatory sites. In the case of tissue factor pathway inhibitor, elastase cleavage leads to regeneration of factor Xa and tissue factor activity from previously formed inhibitory complexes. These collective observations suggest that monocyte-associated elastase and cathepsin G would play a stimulatory role in coagulant events and would begin to more firmly establish the monocyte procoagulant phenotype and its relationship to both intra- and extravascular thrombin generation and fibrin deposition.

In stark contrast, elastase and cathepsin G, have been demonstrated to proteolytically inactivate coagulant zymogens(54, 55, 56, 57, 58) , proteases(59) , and cofactors (19, 60) and thus may produce an anticoagulant effect. However, these studies have all been done with ``free'' proteolytic inactivators, while our studies explored the effect of membrane-bound proteases. For example, whereas the ability of cathepsin G to activate factor V is a new finding, other investigators have shown that purified neutrophil elastase, in solution, will first activate factor V then rapidly inactivate the formed cofactor(19) . However, as shown in these studies, elastase associated with the monocyte membrane surface rapidly activated factor V, but neither inactivated the formed cofactor nor exogenously added factor Va. The factor Va bound to the monocyte surface was further cleaved, but these additional cleavages were without effect on its cofactor activity. These combined data would suggest that the monocyte membrane surface is in some way modulating the inactivation event. The three-dimensional conformation of the enzyme, the substrate, or both may be different when associated with monocyte membrane. In our studies with purified neutrophils, a similar observation was made. However, when studies were performed with purified human leukocyte elastase, factor V, and synthetic phospholipid vesicles, we found that the presence of a synthetic lipid membrane did not prevent the inactivation event; it merely slowed the rate of both factor V activation and factor Va inactivation (data not shown). Thus, it appears that the presence of a cell membrane, with possible attendant carbohydrate or protein receptors, may be required for full biological regulation.


FOOTNOTES

*
This work was supported by Grants HL-34863 and HL-46703 (Project 4) from the NHLBI, National Institutes of Health, and the Department of Biochemistry, University of Vermont College of Medicine. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: The Department of Biochemistry, University of Vermont College of Medicine, Given Bldg. C201, Burlington, VT 05405. Tel.: 802-656-1995; Fax: 802-862-8229.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; HLE, human leukocyte elastase.


ACKNOWLEDGEMENTS

The Blood Drawing Services of the General Clinical Research Center (RR-109) of the Medical Center Hospital of Vermont are greatly appreciated.


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