©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Prothrombinase Components Can Accelerate Tissue Plasminogen Activator-catalyzed Plasminogen Activation (*)

(Received for publication, April 11, 1995; and in revised form, June 1, 1995)

Edward L. G. Pryzdial (1)(§) Laszlo Bajzr (2) Michael E. Nesheim (2)

From the  (1)Research Department, Canadian Red Cross Society, and the Department of Biochemistry, University of Ottawa, Ottawa, Ontario K1G 4J5, Canada and the (2)Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The enzymatic and cofactor subunits of human prothrombinase, factor Xa (FXa) and factor Va (FVa), respectively, were evaluated as modulators of Glu- and Lys-plasminogen (Pg) activation by tissue plasminogen activator (tPA). The data revealed that both FXa and FVa could accelerate tPA activity by as much as 60-fold for Lys-Pg and >150-fold for Glu-Pg. This function of FVa depended on pretreatment with plasmin (Pn), whereas the FXa fibrinolytic cofactor activity was endogenous. In the native state, FVa was observed to inhibit the acceleration of Pn generation by FXa. These effects were dependent on Ca and procoagulant phospholipid. Interactions between plasminogen and prothrombinase components were quantified. The apparent K for binding to FXa was 35 nM. Strikingly, the affinity between FVa and Pg was increased by approximately 2 orders of magnitude when the FVa was Pn-pretreated (K = 0.1 µM). These data cumulatively suggest a mechanism by which Pn production is coordinated with coagulation and localized to sites where procoagulant phospholipid is exposed on a cell surface.


INTRODUCTION

Solubilization and clearance of fibrin clot requires the generation of plasmin (Pn)() from its inactive precursor, plasminogen (Pg) (Lijnen and Collen, 1994; Robbins, 1982). One of the proteases that mediates Pg activation is tissue plasminogen activator (tPA), which has relatively poor intrinsic reactivity toward Pg. Thus, a fundamental regulatory step in fibrinolysis is the acceleration of tPA through the assembly of a ternary complex between tPA, Pg, and a protein cofactor such as fibrin or a cell surface receptor. The colocalization of Pg and tPA functions to increase the rate of catalysis by 1-2 logs through a K effect and to restrict tPA activity to sites of fibrin deposition. Additionally, the association of Pg with endothelial cell receptors is known to accelerate the conversion of the native NH-terminal Glu-Pg to the truncated Lys-Pg species (Hajjar and Nachman, 1988). The outcome is that Lys-Pg is activated by tPA approximately 10 times more efficiently than Glu-Pg (Holaerts etal., 1982).

Several proteins have been identified as Pg receptors, including: annexin II (Cesarman etal., 1994; Hajjar etal., 1994), a novel 45-kDa endothelial protein (Dudani etal., 1993, 1994), -enolase (Miles etal., 1991), osteonectin (Kelm etal., 1994), and complement component C7 (Reinartz etal., 1995). Through the use of lysine analogues and carboxypeptidase B, these proteins have been shown to have in common a COOH-terminal lysine that may be the only necessary structural feature for binding to Pg (Miles etal., 1991). Most of the Pg receptors have been shown to accelerate tPA-mediated activation of Pg (Cesarman etal., 1994; Kelm etal., 1994; Reinartz etal., 1995) or to bind to tPA and Pg, which is presumed to form the necessary ternary complex for rapid Pn generation (Dudani etal., 1993, 1994).

In the current study, the human blood coagulation factor Xa (FXa), which is known from its cDNA sequence to possess the necessary lysyl moiety (Fung etal., 1985; Messier etal., 1991), was evaluated as a Pg-binding protein and accelerator of Pn generation by tPA. The established function of the serine protease FXa in hemostasis is to produce the final enzyme in the coagulation pathway, thrombin (Krishnaswamy etal., 1994; Mann etal., 1990). To do so, FXa must associate in the presence of Ca with the cofactor Va (FVa) and procoagulant phospholipid (e.g. phosphatidylserine), which synergistically accelerate FXa activity by 5 orders of magnitude (Nesheim etal., 1979). Procoagulant phospholipid is accessible only on the surface of activated cells and therefore restricts prothrombinase assembly to sites of vascular damage (Tracy et al., 1992; Zwaal et al., 1993). Since FVa stabilizes the interaction of FXa with biological membranes, a role for FVa in fibrinolysis was also evaluated. We now report that both FXa and FVa can participate as cofactors for tPA-mediated Pg activation.


MATERIALS AND METHODS

Chemicals and Reagents

HEPES, EDTA (Sigma), Glu-Gly-Arg chloromethyl ketone (EGRck), 2-guanidinoethylmercaptosuccinic acid (GEMSA), aprotonin, porcine carboxypeptidase B (CPB, Calbiochem), benzoyl-Ile-Glu-(piperidyl)-Gly-Arg-p-nitroanilide (S-2337), Glu-Pro-Arg-p-nitroanilide (S-2366, Helena Laboratories) were obtained commercially.

Proteins

Human coagulation factor X was purified from fresh frozen plasma (Bajaj etal., 1981) or from prothrombin complex concentrate diluted to 1 unit/ml factor X clotting activity (obtained as generous gifts from the Canadian Red Cross Society, Ottawa Collection Centre or from Miles Therapeutics, Inc., respectively). Factor Xa (approximately 1000 units/mg) was generated from factor X (Jesty and Nemerson, 1976; Krishnaswamy etal., 1987) by treatment with the purified activator from Russell's viper venom (Haematologic Technologies) (Jesty and Nemerson, 1976), followed by affinity chromatography using benzamidine-Sepharose (Pharmacia Biotech Inc.) to remove the factor X activation peptide, Russell's viper venom, and residual inactivated factor X (Krishnaswamy etal., 1987). The irreversible inhibition of factor Xa was accomplished by treatment with a 4-fold molar excess of EGRck (to produce EGR-factor Xa) (Pryzdial and Mann, 1991). Complete inhibition of factor Xa (1.5 µM) was confirmed by the lack of detectable conversion of the chromogenic substrate S-2337 (200 µM) over a 15-min period using a kinetic microplate reader (Vmax, Molecular Devices) with full scale set to 0.03 OD. Excess EGRck was removed by Sephadex G-25 gel filtration chromatography (Pharmacia). Human Glu-plasminogen and Lys-plasminogen were purified as described (Nesheim etal., 1990) and passed over benzamidine-Sepharose (Pierce) to remove any trace of serine protease activity. Plasminogen was radioiodinated using Iodogen (Pierce) and chromatographically desalted (Excellulose 5, Pierce) to remove unincorporated I. Human factor Va was commercially prepared (Haematological Technologies) according to established protocols (Fair etal., 1975; Katzmann etal., 1981). The homogeneity of proteins was assessed by sodium dodecyl sulfate polyacrylamide electrophoresis (Laemmli, 1970) and where applicable by autoradiography using X-Omat AR film (Kodak) and Quanta III intensifying screens (DuPont). Purified human plasmin (Diagnostica Stago), single-chain tPA (American Diagnostica), and CNBr-treated human fibrinogen (tPA activator, Kabi Vitrum) were purchased.

Pg Binding

To follow the interaction between prothrombinase proteins and I-Pg, an adaptation of a method employed by Kalafatis et al.(1994) was used. In these experiments, microtiter wells (Immulon 3) were coated with 0.3 µg of a mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) (25%:75%), pure PC or no phospholipid in 100 µl of methanol/well by evaporation in a vacuum desiccator. On the same day, coated wells (and uncoated wells to control for a possible methanol effect) were blocked for 2 h with 200 µl of 5 mg/ml bovine serum albumin (BSA) at room temperature and rinsed once with 20 mM HEPES, 150 mM NaCl, 2 mM CaCl, pH 7.2 (HBS) with 1 mg/ml BSA (HBS/BSA). In 100 µl/well of HBS/BSA, FXa, FVa, or a mixture of the two proteins was equilibrated with I-Pg each at 50 nM for 1 h at 22 °C. In some experiments, the prothrombinase proteins were preincubated for 1 h and then washed three times with HBS/BSA before addition of I-Pg. Aprotonin (150 KIU/ml) and benzamidine (2 mM) were kept throughout to inhibit any potential Pn activity and proteolysis by FXa, respectively. After incubation, the wells were washed four times with HBS/BSA and the I-Pg remaining bound was quantified. To determine the effect of Pn on the binding of FXa or FVa to I-Pg, the FXa and FVa were pretreated for 30 min with Pn (1.0 nanokatal/ml) and then inhibited by addition of a large excess of aprotonin (150 KIU/ml) before introducing the I-Pg into the mixture.

Chromogenic Assay for Pg Activation

The tPA (10 nM)-mediated activation of Pg (0.5 µM) was followed at 22 °C in HBS/BSA using a chromogenic assay. At various times, 5 µl of the reaction mixture was combined with 150 µl of chromogenic substrate (S2366, 0.2 µM) and the rate of color development was quantified using a kinetic multiwell plate reader (Vmax, Molecular Devices). tPA was the final component added to initiate the process. The effect of FXa and FVa (in most cases 0.1 µM) was evaluated by including these proteins prior to the Pg. In some experiments the FXa and FVa (0.125 µM) were pretreated with Pn (1.0 nanokatal/ml) for 25 min at 22 °C. The Pn was then inhibited with a small excess of aprotonin (2.5 KIU/ml). To be consistent, the experiments not pretreated with Pn were incubated identically. All reactions and pretreatments were done in the presence of synthetic unilamellar phospholipid vesicles composed of 75% phosphatidylcholine and 25% phosphatidylserine (PCPS, 50 µM) (Krishnaswamy etal., 1994) due to the known acceleratory effect on Pn cleavage of FVa (Omar and Mann, 1987) and requirement for PCPS in prothrombinase assembly (Lim etal., 1977; Mann etal., 1987a; Nesheim etal., 1992).

Electrophoretic Assay for Pg Activation

To confirm the observations made with the chromogenic assay, under identical conditions I-Pg (60,000 cpm/µg) activation was followed by electrophoresis. Samples were reduced using dithiothreitol (20 mM) to resolve the Pg from Pn, then run on 10% acrylamide gels (Laemmli, 1970). After drying, the gels were subjected to autoradiography using X-Omat AR film (Kodak) and Quanta III intensifying screens (DuPont). The Pn bands were excised from the gel to quantify using a -counter (Beckman). To correct for background, a slice from each lane that did not correspond to a band on the autoradiograph was quantified and subtracted from those of Pn.


RESULTS

Initial Observation of a FXa-Pg Interaction

Our first attempt to identify a possible involvement of FXa in fibrinolysis, was to follow the specific interaction of I-Lys-Pg to prothrombinase components that were bound to PCPS-coated microtiter wells (Fig. 1). Selective binding of I-Lys-Pg was observed. However, the preincubation experiment (panelA) was dependent on the presence of both FXa and FVa, whereas in the coincubation system FXa alone or the FXa/FVa mixture facilitated I-Lys-Pg binding. Since FVa is known to stabilize the interaction of FXa with PCPS, this discrepancy is likely the result of the additional washing steps in the former. An additional difference between the two incubation conditions is that considerably less I-Lys-Pg remained bound to the microtiter wells in panelB. This may be the result of solution-phase inhibition by FXa. Both experiments demonstrated that I-Lys-Pg binding is dependent on PS and Ca. The association of I-Lys-Pg was inhibitable by addition of a 100-fold molar excess of unlabeled Lys-Pg to the level observed with BSA alone (data not shown). Cumulatively, these observations are consistent with FXa functioning as a bridge between Pg and PCPS that is stabilized by FVa.


Figure 1: Demonstration of Pg binding to FXa/FVa. 96-well microtiter plates were coated with PCPS, PC, or no phospholipid (0.3 µg) and then blocked with BSA. PanelA, coated microtiter wells were preincubated with FXa and/or FVa (0.05 µM) in HBS/BSA for 30 min, washed four times with HBS/BSA, then incubated for 30 min with I-Lys-Pg (0.05 µM). The counts/min remaining bound after another cycle of washing are presented. PanelB, as in panelA except that the FXa/FVa was coincubated with the I-Lys-Pg. The requirement for Ca was evaluated by adding 10 mM EDTA. The average of triplicates with standard deviation is shown.



Chromogenic Measurement of FXa/FVa as tPA Cofactors

Having demonstrated that FXa is indeed capable of functioning as a receptor for Pg, we next determined whether FXa and/or FVa can accelerate the activation of Lys-Pg by tPA. Although the chromogenic substrate used in these experiments (S2366) is not as specific for Pn as the conventional substrate (S2251), it was selected to increase the assay sensitivity by 3-4-fold. FXa and FVa were used at 0.1 µM, which approximates the K for Pg binding to other receptors (Ganz etal., 1991; Hajjar etal., 1994). As shown in Fig. 2, FXa greatly enhanced the rate of Lys-Pg activation. An initial velocity was estimated from the slope to the first time point (2 min) and compared to the first observation of Pn activity in the absence of FXa (8 min), which suggested a 60-fold increase in the rate of tPA catalysis by FXa. Addition of an equimolar amount of FVa to the FXa did not dramatically alter the kinetics profile, although a modest lag was consistently observed. As a comparison to a well documented fibrinolytic cofactor, the effect of CNBr-cleaved fibrinogen at a weight equal to that of the FVa (0.02 mg/ml) was observed to have approximately 50% of the effect of FXa. The dependence of the reaction on tPA and Pg was confirmed by the lack of chromogenic substrate cleavage after 40 min if either were omitted in the presence or absence of FXa/FVa (data not shown).


Figure 2: Effect of FXa/FVa on Lys-Pg activation by tPA. Lys-Pg (0.5 µM) was activated by tPA (10 nM) in HBS/BSA containing PCPS (50 µM) (). The extent of activation was determined at various times using a chromogenic substrate. FXa (0.1 µM, ▾), FVa (0.1 µM, ), FXa + FVa (both at 0.1 µM, ), or CNBr-cleaved fibrinogen (20 µg/ml, ) were added before the Pg or tPA. All reactions were incubated at 22 °C for 25 min before adding the Pg or tPA. The average of triplicates with standard deviation is shown.



When only FVa was included in the Lys-Pg activation mixture, the amount of Pn generated was the same as tPA alone over the first 12-17 min. Following this period, the reaction rate was appreciably accelerated by FVa. Since the proteolysis of FVa by Pn has been reported (Lee and Mann, 1989; Omar and Mann, 1987), we speculated that the FVa may be acquiring fibrinolytic cofactor activity during the experiment by a Pn-dependent feedback mechanism. To test this hypothesis, the FXa and/or FVa were preincubated with Pn. The effect of this treatment (Fig. 3) was to abolish the lag phase observed in the previous experiment when FVa alone was included in the reaction mixture. The FVa fibrinolytic activity remained the same as the system without Pn pretreatment if aprotonin was added before the Pn (data not shown).


Figure 3: Effect of Pn pretreatment of FXa/FVa on Lys-Pg activation by tPA. Experimental conditions were identical to those for Fig. 2except that before adding the Pg and tPA, the various reaction mixtures were incubated for 25 min at 22 °C in the presence of Pn (1.0 nanokatal/ml) and then inhibited with aprotonin (2.5 KIU/ml). No FXa or FVa (), FXa (0.1 µM, ▾), FVa (0.1 µM, ), FXa + FVa (both at 0.1 µM, ), or CNBr-cleaved fibrinogen (20 µg/ml, ).



Pretreatment with Pn was also observed to modulate the effect of FXa on tPA (Fig. 3). This was not as dramatic as observed for FVa and resulted in an approximately 50% decrease of the untreated FXa activity. To our knowledge, evidence for a Pn-sensitive cleavage site on FXa has not been described but would account for our observed loss in activity. We are currently investigating this possibility. As a control, addition of aprotonin before the Pn pretreatment prevented the loss in FXa fibrinolytic activity (data not shown).

The combined acceleratory effect of Pn-pretreated FXa and FVa on tPA activity was approximately the sum of the individual effects. The estimated initial velocity for Pn production was identical to that of untreated FXa, which may indicate that the tPA is saturated under these conditions and the kinetics are approaching V. Pn pretreatment of the CNBr-cleaved fibrinogen appeared to reduce its tPA cofactor activity. Since this was opposite to the effect of Pn on the FXa/Va mixture or FVa alone, it can be inferred that contaminating fibrinogen is not the basis for our observations.

Electrophoretic Measurement of FXa/Va as tPA Cofactors

To confirm our chromogenic assays, we followed conversion of I-Lys-Pg to I-Pn by electrophoresis. These data (Fig. 4) are consistent with the conclusions made from Fig. 2and Fig. 3. 1) FXa and a mixture of FXa/FVa greatly accelerate tPA activity to approximately the same extent (panelA); 2) in the presence of FVa alone, Pn generation undergoes a 12-min lag before the rate is accelerated (panelA); 3) Pn pretreatment of FVa eliminates the lag (panelB); 4) the FXa acceleratory effect is decreased by Pn pretreatment (panelB); and 5) the mixture of Pn-pretreated FXa/FVa has the same initial rate of Lys-Pg conversion as untreated FXa (panelB).


Figure 4: Confirmation of the effect of FXa/FVa on Lys-Pg activation by tPA. Experimental conditions were identical to those for Fig. 2(panelA) and Fig. 3(panelB), except that the extent of Pn production at various times was followed by electrophoresis. No FXa or FVa (), FXa (0.1 µM, ▾), FVa (0.1 µM, ), FXa + FVa (both at 0.1 µM, ).



Effect of FXa/FVa on tPA-mediated Activation of Glu-Pg

Fig. 5shows that FXa and FVa also enhance the rate of Glu-Pg activation by tPA. 1) FXa has endogenous tPA cofactor activity (panelA); 2) FVa induced a lag in the endogenous FXa effect (panelA), although this was much more pronounced compared to Lys-Pg (Fig. 2); 3) Pn pretreatment of the FVa converted it into a tPA accelerator; 4) Pn pretreatment decreased the FXa cofactor activity; and 5) Pn-pretreated FXa and FVa combined to increase the rate of Pn generation. As reported in the literature, Lys-Pg is converted more rapidly to Pn than Glu-Pg by tPA. We observed this characteristic in the absence of either prothrombinase constituent where over 30 min of Glu-Pg activation was negligible. As a result, the feedback modulation of FVa by Pn was not observed using Glu-Pg (panelA).


Figure 5: Effect of FXa/FVa on Glu-Pg activation by tPA. Experimental conditions were identical to those for Fig. 2(panelA) and Fig. 3(panelB) except that Glu-Pg was used as a substrate rather than Lys-Pg. No FXa or FVa (), FXa (0.1 µM, ▾), FVa (0.1 µM, ), FXa + FVa (both at 0.1 µM, ).



Regulation of FXa Fibrinolytic Activity by FVa

Since a lag was observed in the rate that FXa-enhanced Pn production when equimolar FVa was present (Fig. 2), we explored the possibility that before cleavage by Pn the FVa was a regulator. Shown in Fig. 6is the effect of increasing the FVa concentration on Pn generation from Lys-Pg at a constant level of FXa. The data show that the extent of the lag phase is dependent on the amount of FVa added and is lengthened to 22 min at the highest concentration of FVa. Following the lag, a rapid rate of Lys-Pg activation is resumed, which is likely contributed to by both FXa and Pn-cleaved FVa fibrinolytic activity. This experiment suggests that within the prothrombinase complex, the participation of FXa as a fibrinolytic cofactor is inhibited until the FVa is feedback-modulated by Pn.


Figure 6: Effect of FVa on the tPA cofactor activity of FXa. Identical experimental conditions as Fig. 2except that the FVa concentration was increased with constant FXa (0.1 µM). No FXa or FVa (); FXa with no FVa (▾); 1:1 FVa:FXa () (data from figure 2); 2:1 (); 5:1 (); 8:1 (); 11:1 (). Averaged duplicates with standard error is shown.



Effect of Other Prothrombinase Components on tPA Acceleration

Having established that FXa and Pn-treated FVa can participate in the acceleration of Pn production by tPA, the contributions of the FXa serine protease active site, Ca, and procoagulant phospholipid were evaluated. Fig. 7(panel A and B) shows the effect of blocking the active site of FXa with a tripeptidylchloromethyl ketone (EGRck). In contrast to native FXa, EGR-FXa did not demonstrate significant tPA cofactor activity alone (panelsA and B). Since Pg is believed to interact exclusively with the COOH terminus of known receptors (Cesarman etal., 1994; Dudani etal., 1993; Kelm etal., 1994; Miles etal., 1991), the effect of blocking the active site of FXa may be on tPA. Without Pn pretreatment (panelA), active site-independent synergy between EGR-FXa and FVa was observed. This effect was not apparent after Pn pretreatment (panelB), which suggests that FXa may function to accelerate the cleavage of FVa by Pn.


Figure 7: Effect of the FXa active site, Ca and PCPS on Pg activation by tPA in the presence of FXa/FVa. Upperpanels (A, C, and E), reactions were not Pn-pretreated (as in Fig. 2). Lowerpanels (B, D, and F), reactions were Pn-pretreated (as in Fig. 3). PanelsA and B, EGRck-inactivated factor Xa was used instead of active FXa; panelsC and D, following the 25-min incubation in the presence of Ca, 10 mM EDTA was added; panelsE and F, PCPS was omitted from the various reaction mixtures. No FXa or FVa (), FXa (0.1 µM, ▾), FVa (0.1 µM, ), or FXa + FVa (both at 0.1 µM, ).



To determine the role of Ca in the tPA cofactor function of FXa and FVa, excess EDTA was added to the various reaction mixtures. The data show that without Pn treatment (panelC), the cofactor effects of FXa and FVa were abolished in the absence of Ca. The data also suggest that the Ca-dependent association of FXa with PCPS is necessary for tPA cofactor activity. Interestingly, when the FXa and FVa were pretreated with Pn in the presence of Ca (panelD), the subsequent addition of EDTA caused a complete loss of the FXa-dependent tPA activity, but approximately 50% of the initial FVa acceleratory effect on tPA remained. When the entire experiment was conducted in the presence of Ca, but PCPS was completely omitted (panelsE and F), neither FXa nor FVa had an influence on the rate of Lys-Pg activation regardless of whether a Pn pretreatment step was performed.

Binding of I-Lys-Pg to FXa/FVa

The data presented above establish that Pn is capable of altering the participation of FVa and to a lesser extent of FXa in Pg activation by tPA. To determine whether this may be an effect on the ability of FVa and FXa to interact with Pg, the various equilibria were quantified. The binding isotherms shown in Fig. 8, were obtained by coincubating the I-Lys-Pg and prothrombinase constituents. Specific binding was calculated by subtracting the amount of I-Lys-Pg bound to PC-coated microtiter wells from the amount bound to PCPS-coated wells. The apparent dissociation constants (K) and number of moles bound at saturation (B) were derived by fitting the data to a simple rectangular hyperbola, which implicitly assumed a single class of sites (Table 1). Perhaps the most striking conclusion from these data is that Pn treatment of FVa enhances the affinity for Lys-Pg by approximately 2 orders of magnitude. This may account for the acquisition of tPA cofactor activity by FVa when treated with Pn. Similarly the decrease in the tPA cofactor activity of FXa after Pn treatment may be explained by the 10-fold decrease observed in affinity for Lys-Pg. An inconsistency with the kinetic experiments is that prior to Pn treatment, FVa did not influence the binding of Lys-Pg to FXa. Furthermore, after Pn treatment, the FXa and FVa were shown to have a combined effect on Pg activation by tPA (Fig. 3), but FVa had no effect on Lys-Pg binding to FXa. These discrepancies may be reconciled by direct effects on tPA that have yet to be identified.


Figure 8: Quantification of Pg binding to FXa/FVa. Experimental conditions were identical to those for Fig. 1(panelB). Microtiter wells were coated with 0.3 µg of PCPS or as a control for nonspecific binding with 0.3 µg of PC. Panel A, reactions were not Pn-pretreated (as in Fig. 2); panelB, reactions were Pn-pretreated (as in Fig. 3), except that 150 KIU/ml aprotonin was used to inhibit the Pn. I-Pg was titrated in the presence of the following prothrombinase components. FXa (0.05 µM, ), FVa (0.05 µM, ▾), or FXa + FVa (both at 0.05 µM, ). Averaged triplicates with standard error is shown.





Involvement of FXa COOH-terminal Lysine on tPA Acceleration

A possible function for the FXa COOH-terminal Lys was investigated by pretreating the FXa with CPB (Table 2). The data clearly revealed that the tPA cofactor function of FXa is sensitive to CPB, which supports the involvement of a COOH-terminal Lys in FXa. To control for the possibility that the CPB inhibitor GEMSA or residual CPB activity had a direct effect on Pn generation by tPA, the FXa was simultaneously incubated with CPB and GEMSA. A small decrease in the initial velocity compared to no pretreatment of FXa was observed, but was insignificant compared to the loss in FXa cofactor activity when pretreated first with CPB. Similar observations were made when Glu-Pg was used instead of Lys-Pg as a substrate (data not shown).




DISCUSSION

Coagulation and fibrinolysis are opposing hemostatic processes. To effectively coordinate the activation of procoagulant and subsequently of profibrinolytic enzymes, a high degree of molecular cross-talk between these pathways must exist. As an important example, the end product of coagulation, fibrin, communicates to the fibrinolytic pathway by functioning as an accelerator of tPA-dependent Pn generation (Lijnen and Collen, 1994). Consequently, tPA activity is restricted until clot has formed. In the current study, we provide evidence that the protein components of prothrombinase, FXa and FVa, are also capable of signaling fibrinolysis by acceleration of tPA.

A model that summarizes the molecular links between fibrinolysis and prothrombinase is presented in Fig. 9. In previous work, Pn was demonstrated to irreversibly block the FXa cofactor role of FVa in thrombin production (Lee and Mann, 1989; Omar and Mann, 1987). We now report that Pn-modulated FVa acquires tPA cofactor activity, either in the presence or absence of FXa. Treatment of FVa with Pn also correlates with the expression of a cryptic Pg binding site. The apparent K of 0.1 µM is well below the plasma concentration of Pg (2.4 µM) and may be a driving force for the participation of FVa in fibrinolysis. Whether a direct effect on tPA also results after FVa is cleaved by Pn is currently unresolved. Thus Pn induces a functional switch in FVa from a procoagulant in the native state to a profibrinolytic.


Figure 9: Molecular links between prothrombinase and Pg activation. The data presented here are consistent with both prothrombinase components, FXa and FVa, being able to accelerate tPA-dependent Pg activation after feedback modulation of FVa by Pn. See text for details. Hatchedlines represent inhibitory pathways.



The FXa moiety of prothrombinase was also observed to accelerate Pn generation by tPA. Unlike FVa, however, the tPA cofactor activity of FXa was endogenous. Evidence that FVa directly regulates the FXa fibrinolytic activity was obtained from experiments showing that addition of native FVa induced a dose-dependent lag before the acceleration of Pn generation was achieved. Following Pn pretreatment, the FVa was found to lose this antagonistic effect. A plausible explanation is that Pn-mediated proteolysis may break the interaction between FXa and FVa in a manner similar to the inhibition of FVa by activated protein C (Guinto and Esmon, 1984; Pryzdial and Mann, 1991). The general conclusion from these data is that Pn-mediated feedback controls the intrinsic fibrinolytic capability of FXa through an indirect route involving FVa. Since we found that FVa did not influence the binding affinity of Pg to FXa, this regulatory mechanism is suggested to include an effect on tPA. The association of Pg with FXa was found to be approximately 10-fold stronger (apparent K = 35 nM) than Pg binding to other receptors. This may make FXa a particularly effective profibrinolytic cofactor.

In this report, we have not attempted to define the kinetics of tPA acceleration by FXa or FVa. However, to compare the efficacy of the prothrombinase components to known tPA cofactors, we have estimated the extent of acceleration relative to tPA alone at a single concentration of reactants. In the presence of FXa or Pn-pretreated FVa, we observed approximately 60-fold enhancement for the initial rate of Lys-Pg activation. This is within the same range as the known tPA accelerators, annexin II (Cesarman etal., 1994) and various fibrin derivatives (Lijnen and Collen, 1994). When Glu-Pg was used as a substrate, the generation of Pn in the presence of tPA alone did not exceed base-line levels over the course of the experiment, which complicates the determination of fold enhancement. Nevertheless, we estimate the increase in observed rate due to FXa to be >150-fold and somewhat less for Pn-pretreated FVa. These values are considerably greater than those reported for other Pg receptors (Kelm etal., 1994; Reinartz etal., 1995) and fibrin derivatives (Lijnen and Collen, 1994). A comprehensive evaluation of the kinetics must be conducted to verify these comparisons.

The role of procoagulant phospholipid as an essential prothrombinase cofactor and as a means to localize thrombin production has been well documented (Bevers etal., 1982, 1983; Mann etal., 1987b; Packham and Mustard, 1984). Calcium is also required for prothrombinase assembly and functions by facilitating the interaction between FVa subunits (Esmon, 1979; Krishnaswamy etal., 1989), the binding of FXa to PCPS (Nelsestuen, 1976; Nesheim etal., 1981) and the binding of FXa to FVa (Pryzdial and Mann, 1991). In the current work, the acceleration of tPA by FXa and FVa was also found to be dependent on PCPS and Ca. This observation may be partially explained by the known increased rate of FVa Pn-mediated proteolysis in the presence of PCPS (Omar and Mann, 1987). However, the modest enhancement that was reported (approximately 5-fold) cannot account for the complete loss of tPA acceleration by Pn-pretreated FVa in the absence of PCPS. Interestingly, when Pn proteolysis of FVa was conducted in the presence of PCPS and Ca, and then EDTA added prior to assaying for tPA activity with PCPS, 50% of the FVa cofactor activity remained. Taken together these data suggested that an interaction between FVa and PCPS is necessary for FVa to express tPA cofactor function and the Ca-independent association of the 30-kDa Pn-derived fragment of the FVa light chain with PCPS may be directly involved. In the case of FXa, we observed that acceleration of tPA was facilitated only when a FXa-Ca-phospholipid complex could form. The interaction of FXa with Ca (Church etal., 1989; Nelsestuen, 1976; Pryzdial and Mann, 1991; Radcliffe and Barton, 1972) and with phospholipid (Krishnaswamy etal., 1992) is known to induce a conformational change that may be required for tPA cofactor activity. Thus the profibrinolytic effect of FXa and FVa is localized to sites only where exposure of procoagulant phospholipid initially triggered the generation of thrombin.

Annexin II is the only other known cellular receptor for Pg that may also be localized to sites where procoagulant phospholipid is expressed. This is through the preferential interaction of annexin family proteins with procoagulant phospholipid (Andree etal., 1990; Meers etal., 1991; Tait etal., 1989). However, unlike FXa and FVa, the fibrinolytic cofactor activity of annexin II does not depend on the association with procoagulant phospholipid (Cesarman etal., 1994) and consequently may not be as strictly localized as FXa and FVa.

FXa and FVa can associate with a variety of stimulated cells and may function to generate Pn at these sites either independently or with other Pg receptors. Activated platelets are considered to provide the most abundant physiological source of procoagulant phospholipid (Dahlbach and Stenflo, 1978; Miletich etal., 1978; Nesheim etal., 1981; Tracy et al., 1981) and are a significant constituent of clots. As a result, prothrombinase is concentrated within a clot and may locally participate in concert with fibrin to accelerate fibrinolysis. At this time, the concentration of FXa and FVa at these sites is unknown. Since the concentration of fibrin is high, it is probable that the largest individual effect of prothrombinase constituents on fibrinolysis will be on susceptible cells that are not trapped by fibrin.

While other Pg receptors may exist on platelets, annexin II is not found in association with these cells (Eldering etal., 1993). The fibrinolytic activity of annexin II has been identified on the endothelial surface (Hajjar etal., 1994), which is also an important site for prothrombinase assembly (Rodgers and Shuman, 1985). Therefore annexin II, FXa, and FVa may act together to stimulate fibrinolysis on the endothelium. A functional difference is suggested from the possibility that annexin II may exist on the surface of resting cells (Hajjar etal., 1994), whereas recruitment of FXa and FVa requires cell stimulation. At least one other endothelial cell receptor for Pg has been identified (Dudani etal., 1993), but whether the expression of this 45-kDa protein is modulated following cell stimulation is unknown.

The formation of prothrombinase has also been described in association with monocytes (Mcgee and Li, 1991; Tracy etal., 1985) and carcinoma cells (Sakai etal., 1990). In previous studies, Pg binding to these cells and generation of Pn on the surface of monocytes was observed to be enhanced by Pn pretreatment (Gonzalez-Gronow etal., 1991). This is similar to our current observation with FVa. Certain carcinoma cells are known to secrete FV (Sakai etal., 1990), but whether these reports are due to FV/FVa Pn-mediated modulation or other Pg receptor(s) is unknown. In addition to a fibrinolytic function, the possibility that FVa and FXa may accelerate Pn generation at the surface of monocytes or carcinoma cells suggests a role in tissue remodeling (Kwaan etal., 1991).

The interaction of Pg with fibrin (Lijnen and Collen, 1994), various cells (Dudani etal., 1993; Hajjar etal., 1994; Holaerts etal., 1982; Miles etal., 1991), or collagen matrices (Kelm etal., 1994) is known to depend on the accessibility of specific Lys residues in the receptor protein. Like many Pg receptors, the FXa-mediated acceleration of Pg activation by tPA was observed by us to involve a COOH-terminal Lys in FXa. This suggests that in addition to the control of FXa fibrinolytic function by FVa, a recently identified thrombin-activated plasma protein having carboxypeptidase B activity (TAFI) (Bajzr et al., 1995) may be involved. Whether the acquisition of FVa fibrinolytic activity following Pn treatment is the result of exposing a new COOH-terminal Lys is under investigation.


FOOTNOTES

*
This work was supported by Canadian Red Cross Society Research and Development Grants 924 and 926. 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: Canadian Red Cross Society, Research Dept., 1800 Alta Vista Dr., Ottawa, Ontario K1G 4J5, Canada.

The abbreviations used are: Pn, plasmin; Pg, plasminogen; tPA, tissue plasminogen activator; PS, phosphatidylserine; PC, phosphatidylcholine; BSA, bovine serum albumin; EGRck, Glu-Gly-Arg chloromethyl ketone; CPB, carboxypeptidase B; GEMSA, 2-guanidinoethylmercaptosuccinic acid; FXa, factor Xa; EGR-FXa, EGRck-inactivated FXa; FVa, factor Va.


ACKNOWLEDGEMENTS

We thank G. Kessler and Y. Coulombe for purification of proteins, T. Raynor for reading the manuscript, and Dr. A. Dudani for helpful suggestions. We also thank Dr. S. Krishnaswamy and Dr. P. Lollar for critical evaluation of the initial data.


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