(Received for publication, March 27, 1995; and in revised form, August 18, 1995)
From the
The activation of prothrombin is catalyzed by prothrombinase, a
complex of factor Xa and factor Va assembled on a negatively charged
phospholipid membrane. We used a tubular flow reactor to identify the
relative contributions of factor Va, prothrombin, and the negatively
charged phosphatidylserine to the assembly of prothrombinase. Perfusion
of phospholipid-coated capillaries with a mixture of factor Xa, factor
Va, and prothrombin resulted in a steady-state rate of thrombin
production that increased with (i) the phosphatidylserine content of
the phospholipid bilayer, (ii) the factor Va concentration, and, most
interestingly, (iii) the prothrombin concentration of the perfusion
solution. Incorporation of 20 mol % phosphatidylethanolamine, a
phospholipid with poor ability to promote prothrombinase activity, into
a 5 mol % phosphatidylserine membrane also increased the steady-state
rate of thrombin production. Direct measurements of the amount of
prothrombinase in the flow reactor demonstrated that increased
catalytic activities were the result of an increased steady-state
amount of membrane-associated prothrombinase. Thus, similar turnover
numbers of prothrombin activation (3100 min) were
calculated, irrespective of the phosphatidylserine content of the
membrane. We established for membranes with low phosphatidylserine
content (<10 mol%) a linear relationship between the prothrombinase
activity and the arithmetical product of the factor Va concentration in
the perfusion solution and the prothrombin concentration near the
catalytic surface. Our results indicate that, in addition to factor Va,
prothrombin also is essential to the assembly of prothrombinase at
macroscopic surfaces with low phosphatidylserine content. The data
further suggest that the prothrombin concentration near the surface,
controlled by the prothrombinase activity and mass transfer, is an
important regulator of the prothrombinase surface density.
Prothrombinase, the enzyme complex that converts prothrombin
into thrombin, is composed of the serine protease factor Xa, the
protein cofactor factor Va, and phospholipids(1, 2) .
Kinetic studies have indicated that the reversible protein-phospholipid
and protein-protein interactions in the prothrombinase complex all
contribute to the 10-fold increase in the catalytic
efficiency of factor
Xa(3, 4, 5, 6) . It is generally
accepted that the interaction of factor Xa with factor Va enhances the
turnover number of factor Xa about 3000-fold(5) , and that the
interaction of prothrombin with the membrane is responsible for a
dramatic decrease in the K
for
prothrombin activation, namely from
90 µM in the
absence of phospholipid (5) to about 3 nM in the
presence of a planar membrane(7, 8) .
Negatively
charged phospholipids are essential constituents of membranes that
support prothrombin activation(9, 10) . Optimum
prothrombinase activity has been reported for membranes that contain at
least 10 mol %
phosphatidylserine(11, 12, 13, 14) .
It is rationalized that this dependence is the result of the lower
binding affinity of membranes with low phosphatidylserine content for
the protein constituents of the enzyme complex (factor Xa and factor
Va) as well as for its substrate
prothrombin(15, 16, 17) . Consequently,
membranes with a low phosphatidylserine content require higher
concentrations of fluid phase factor Va to incorporate the same amount
of factor Xa into membrane-associated prothrombinase than membranes
with a high phosphatidylserine
content(11, 18, 19) . The reported k values, therefore, do not always reflect the
true catalytic ability(13, 14) .
Because factor Va also interacts with phospholipid-bound prothrombin (20, 21) , one cannot a priori exclude the possibility that prothrombin contributes to the stability of the prothrombinase complex. The consequence of such an effect is that increasing amounts of factor Xa will be incorporated into the prothrombinase complex with increasing prothrombin concentrations(22) . It has been shown, though, that prothrombin does not contribute to the assembly of prothrombinase at membranes with 25 mol % phosphatidylserine, under conditions that were considered to be physiologically relevant with respect to the factor Va and factor Xa concentrations(19) . However, no information is available about the role of prothrombin under conditions that are less optimal for prothrombinase assembly, e.g. low phosphatidylserine content and nonsaturating conditions with respect to reactant concentrations. Such a situation might prevail when coagulation takes place at the surface of activated platelets or other cells in flowing blood(23) . Interestingly, a stabilizing effect of a substrate on its membrane-bound cofactor-enzyme complex has been reported, namely that of factor X on the tissue factor-factor VIIa complex(24) .
In the present study, we used a tubular flow reactor to assess the contribution of prothrombin to the assembly of prothrombinase and the kinetics of prothrombin activation at low phosphatidylserine membranes. In earlier studies with prothrombinase in a tubular flow reactor, we demonstrated that the kinetics of prothrombinase assembly and the kinetics of thrombin production can be described adequately using a simple model for mass transport for immobilized enzymes(8) . Therefore, the tubular flow reactor may have some clear advantages over the experimental settings using unilamellar phospholipid vesicles. Firstly, the prothrombin concentration near the catalytic surface can be calculated easily from the flow conditions, the concentration of prothrombin in the bulk solution, and the thrombin production in the flow reactor. Secondly, the amount of prothrombinase in the flow reactor can be estimated readily from the amount of phospholipid-bound factor Xa. This paper reports in detail on the relative contributions of prothrombin, factor Va, and the phosphatidylserine content to the assembly of prothrombinase and their implications for the kinetics of prothrombin activation.
1,2-Dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), ()1,2-dioleoyl-sn-glycero-3-phosphatidylcholine
(DOPC), and 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine
(DOPE) were purchased from Avanti Polar Lipids, Inc. S2238, the
chromogenic substrate for thrombin, was obtained from Chromogenix
(Mölndal, Sweden). Bovine serum albumin (BSA) was
from Sigma. Bovine factor V and factor Va were prepared and quantitated
as described previously(18) . Human prothrombin was purified as
described (25) , and the molar concentration was determined
after complete activation with Echis carinatus venom (Sigma)
by active site titration with p-nitrophenyl p`-guanidinobenzoate hydrochloride(26) . Human factor
Xa was prepared by activation of purified factor X (27) with
the factor X activating protein from Russell's viper venom
(Sigma) and isolated as described for bovine factor Xa(28) .
The molar concentration was determined by active site titration with p-nitrophenyl p`-guanidinobenzoate
hydrochloride(29) . Small unilamellar vesicles of mixed
phospholipids were prepared as described before(30) .
Throughout this paper, the mixed phospholipids are described with their
mol % of the negatively charged phospholipid; the remainder of the
phospholipid is DOPC.
where is the mass transfer coefficient, C
is the prothrombin concentration in the bulk solution, and C
is the prothrombin concentration near the
catalytic surface. The mass transfer coefficient is a function of the
geometry of the capillary, the volumetric flow rate, the kinematic
viscosity of the fluid, and the diffusion coefficient of the protein.
For a flow rate of 30 µl/min and the capillary (length 12.7 cm and
inner diameter of 0.065 cm) used in this study, the mass transfer
coefficient,
, for prothrombin equals 0.01 cm
/min (cf. (8) ).
The maximum rate of prothrombin activation,
is thus obtained when the membrane-bound enzymatic activity
causes a total depletion of the prothrombin concentration near the
surface (C = 0). For intermediate
situations when the rate of prothrombin activation is smaller than the
prothrombin mass transfer rate, the prothrombin concentration at the
capillary wall can be calculated from :
Figure 1:
Thrombin generation in a tubular flow
reactor. Capillaries coated with a phospholipid membrane containing 5
mol % DOPS-95 mol % DOPC () and 25 mol % DOPS-75 mol % DOPC
(
) were perfused with Tris buffer containing factor Xa (2
pM), factor Va (50 pM), and prothrombin (100
nM). The perfusion was performed at a flow rate of 30
µl/min (wall shear rate of 20 s
) at 37 °C.
The rate of thrombin production is plotted versus the
perfusion time.
Another interesting
feature of the flow experiment with the 5 mol % DOPS membrane is that,
in spite of a continuous perfusion with a mixture of 2 pM factor Xa, 50 pM factor Va, and 100 nM
prothrombin, not more than 0.06 fmol of prothrombinase could be
assembled. Based on reported binding parameters for the factor
Va-factor Xa complex (cf. (13) ), we estimated that
the binding capacity of the 5 mol % DOPS membrane is at least
10-fold higher. It is apparent that an equilibrium was
established between membrane-associated prothrombinase and the
reactants in the perfusion solution.
Figure 2:
The steady-state rate of thrombin
formation as function of the DOPS content of the membrane. Capillaries
with membranes of varying DOPS content were perfused as described for Fig. 1. The lower panel gives the steady-state rate of
thrombin production () and the amount of prothrombinase in the
capillary at the time the steady state of thrombin production was
obtained (
) as a function of the DOPS content of the membrane.
The upper panel gives the turnover number of prothrombin
activation calculated from the rate of thrombin production and
prothrombinase concentration.
To examine the contribution of factor Va to the assembly of an equilibrium concentration of prothrombinase in the flow reactor, phospholipid (5 mol % DOPS)-coated capillaries were perfused with Tris buffer containing 2 pM factor Xa, 100 nM prothrombin, and varying concentrations of factor Va (0.01-2 nM) until a steady state rate of thrombin production was reached. The steady-state rate of thrombin production increased with the factor Va concentration in the perfusion solution until a maximum of 0.9 pmol/min (Fig. 3). This value is close to the transport rate limit of 1 pmol of thrombin/min.
Figure 3: The steady-state rate of thrombin formation as function of the factor Va concentration in the perfusion solution. Phospholipid-coated (5 mol % DOPS-95 mol % DOPC) capillaries were perfused with Tris buffer containing 2 pM factor Xa, 100 nM prothrombin, and varying factor Va concentrations.
Figure 4: The steady-state rate of thrombin formation as function of the prothrombin concentration in the perfusion solution. Capillaries that contained a phospholipid bilayer composed of 5 mol % DOPS-95 mol % DOPC were perfused with Tris buffer containing 2 pM factor Xa, prothrombin at the indicated concentrations, and, from bottom to top, 10, 20, 100, 300, 1000, and 2000 pM factor Va. The steady-state rates of thrombin production are plotted versus the prothrombin concentration in the perfusion solution. The dotted line represents the transport-limited rate of thrombin production under the conditions of the flow experiment.
From Fig. 5it is clearly seen
that the slopes of the lines of Fig. 4do not increase linearly
with the factor Va concentration in the range of 10 to 300 pM (closed squares). However, as noted under ``Experimental
Procedures'' (), the presence of prothrombinase can
easily cause a severe depletion of prothrombin near the catalytic
surface. As a matter of fact, predicts that with a
constant prothrombin concentration in the perfusion solution (C), the prothrombin concentration near the
surface (C
) will decrease in parallel with the
factor Va-dependent increase of the rate of thrombin production. The
slope of the lines shown in Fig. 4, therefore, should be
corrected for the true prothrombin concentration, i.e.
C
. The closed circles in Fig. 5are
the result of this correction. It is now clearly seen that the rate of
thrombin production, normalized for the prothrombin concentration near
the catalytic surface, linearly increased with the factor Va
concentration. The observed linear relationships indicate that for a
constant amount of factor Xa in the perfusion solution, the equilibrium
amount of membrane-associated prothrombinase is proportional to the
arithmetical product of the factor Va concentration in the bulk
solution and the prothrombin concentration near the surface.
Figure 5: Effect of factor Va on the steady-state rate of thrombin production normalized for the prothrombin concentration in the perfusion solution or near the surface. The slopes of the lines of Fig. 4, with the exception of those obtained with 1 and 2 nM factor Va, are plotted versus the factor Va concentration in the perfusion solution (closed squares). The closed circles are the slopes of the same rates of thrombin production plotted versus the corresponding prothrombin concentration near the surface, calculated using .
In
order to substantiate our notion, we measured in similar experiments,
as shown in Fig. 4, the steady-state rates of thrombin
production at membranes with 2 mol % DOPS and 10 mol % DOPS membranes
as a function of the factor Va and the prothrombin concentration. The
amount of prothrombinase was obtained from the steady-state rates of
thrombin production divided by the previously determined (Fig. 2) turnover number of 3100 min. In an
attempt to obtain accurate data, we selected experimental conditions, i.e. factor Va and prothrombin concentrations in the perfusion
solution, that gave less than 70% depletion of the prothrombin near the
catalytic surface in the steady-state phase of the experiment. Fig. 6shows for the different membranes the relationship between
the amount of prothrombinase associated with the membrane and the
arithmetical product of the factor Va concentration in the bulk
solution and the prothrombin concentration near the surface. The slopes
of the lines (± S.E.), obtained by linear regression analysis,
are (1.4 ± 0.1)
10
, (2.9 ±
0.4)
10
, and (3.7 ± 0.5)
10
fmol/nM
for the membranes
with 2, 5, and 10 mol % DOPS, respectively.
Figure 6: Dependence of prothrombinase density at membranes with varying DOPS on the arithmetical product of the factor Va concentration in the perfusion solution and the prothrombin concentration near the surface. Capillaries with 2, 5, and 10 mol % DOPS membranes were perfused with 2 pM factor Xa, 5-100 pM factor Va, and 50-200 nM prothrombin. The amounts of prothrombinase at the steady-state phase of thrombin production and the prothrombin concentration near the surface were calculated from the steady-state rates of thrombin production and prothrombin concentration in the perfusion solution, respectively. Further details are given in the text.
Figure 7:
The dependence of the steady-state rate of
thrombin production on the presence of phosphatidylethanolamine in 5
mol % DOPS membranes. Capillaries were coated with phospholipid
bilayers composed of 5 mol % DOPS-95 mol % DOPC (), 5 mol %
DOPS-20 mol % DOPE-75 mol % DOPC (
), 25 mol % DOPS-75 mol % DOPC
(
), and 20 mol % DOPE-80 mol % DOPC (
). Perfusion was
performed with Tris buffer containing factor Xa (2 pM), factor
Va (50 pM), and prothrombin concentrations as indicated. The dotted line represents the transport-limited rate of thrombin
production under the conditions of the flow
experiment.
The relative contributions of protein-phospholipid interactions and protein-protein interactions to the overall stability of the prothrombinase complex and their consequences for the catalytic efficiency of the enzyme have been studied extensively (14, 34, 35 and references therein). The experimental design with a flow reactor, as presented here, enables, however, a straightforward study of prothrombinase assembly and kinetics of prothrombin activation. The clear advantage over a system with small unilamellar phospholipid vesicles is the readily controlled delivery of the individual components of the prothrombinase complex, factor Xa and factor Va, and the substrate prothrombin to the planar (macroscopic) phospholipid bilayer and, most importantly, the possibility of a direct determination of the amount of prothrombinase assembled on the macroscopic surface.
Initially, we wanted to determine the kinetics of prothrombin activation by known amounts of assembled prothrombinase as a function of the DOPS content of the membrane as described previously(8) . However, pilot experiments revealed that perfusion of capillaries with mixtures of factor Va and factor Xa, in case the membrane contained a low DOPS content, resulted in surprisingly low and unstable levels of membrane-associated prothrombinase. In contrast, when prothrombin was added to the factor Xa-factor Va mixtures, increasing amounts of thrombin appeared at the outlet of the capillary until a steady-state was reached. We hypothesized that, with decreasing affinity of factor Xa and factor Va for DOPS membranes, prothrombin by virtue of its affinity for both factor Va and factor Xa significantly promotes the stability of the prothrombinase complex (cf. (15, 16, 17) and 36).
To verify our notion, we examined the assembly
of prothrombinase at membranes with varying DOPS content from perfusion
solutions that contained factor Xa (2 pM), factor Va (50
nM), and varying concentrations of prothrombin. We observed in
all perfusion experiments that the thrombin production in the flow
reactor reached a steady-state rate well below the transport limit.
From direct prothrombinase measurements we learned that the amount of
membrane-associated prothrombinase in the flow reactor dramatically
decreased with the DOPS content of the membrane. Increasing prothrombin
concentrations in the perfusion solution, however, did increase the
membrane-bound amount of prothrombinase. Although the turnover values,
calculated from the steady-state rate of thrombin production and the
corresponding amount of prothrombinase ( Fig. 2and Table 1), varied between 2500 and 3900 min, it
is reasonable to conclude that no gross differences were observed for
the catalytic activities of the prothrombinase assembled at the
membranes that varied in DOPS content from 2 to 10 mol % and
prothrombin concentrations that varied between 50 and 200 nM.
The turnover numbers obtained here are close to the previously reported k
value of 3600 min
for 25
mol % phosphatidylserine membranes(8) .
The observation that
similar kinetics of thrombin production were found with a limited
steady-state amount of membrane-associated prothrombinase indicates
that the prothrombinase complex remains assembled only at the membrane
of the flow reactor when it is fully occupied with its substrate
prothrombin. That is, prothrombinase that is not acting on its
substrate will dissociate from the membrane. Consequently, under these
conditions, the kinetic parameter K, defined as
the prothrombin concentration that is required to obtain
half-saturation of the enzyme, loses its meaning.
Regarding the relative contributions of factor Va and prothrombin to the stability of the complex, we present evidence that with a constant amount of factor Xa in the perfusion solution, the amount of membrane-associated prothrombinase is proportional to the arithmetical product of the factor Va concentration in the perfusion solution and the prothrombin concentration close to the catalytic surface (Fig. 5). The proportionality constant of this relationship, which probably reflects the protein-protein and protein-phospholipid affinities, decreases 2000 fold when the DOPS content of the membrane increases from 2 to 10 mol %. Only a 10-fold difference was seen between 5 mol % and 10 mol % DOPS membranes.
Prothrombinase-catalyzed conversion of prothrombin into thrombin is a multistep reaction rather than a single product (thrombin) reaction. It has been reported (37) that the prothrombin concentration and the composition of the phospholipid membrane has an effect on the relative amounts of thrombin and meizothrombin formed. That is, higher prothrombin concentrations and higher DOPS content favor the formation of meizothrombin. The question has thus to be raised whether a change in the ratio of meizothrombin over thrombin also could affect the stability of the prothrombinase complex. However, it is rather difficult to see how meizothrombin is produced in the absence of a fully assembled prothrombinase. We also like to point at an other item of the present study that needs to be considered in the extrapolation of our results to the physiological situation. Throughout this and our previous study(8) , we have used bovine factor Va. However, pilot experiments in which we used human factor Va (a kind gift of Dr. Jan Rosing), instead of bovine factor Va, showed no marked differences in the amount of prothrombinase assembled on a 5 mol % DOPS membrane.
It was recently reported that phosphatidylethanolamine (DOPE) incorporated into phospholipid vesicles containing DOPS and DOPC dramatically enhances activated protein C inactivation of factor Va (38) . Smirnov and Esmon (38) also stipulated that the incorporation of DOPE into DOPS-DOPC vesicles did not affect the kinetics of prothrombin activation by prothrombinase. However, it should be stressed that their conditions of prothrombin activation were almost optimal with respect to the procoagulant surface (high DOPS content), factor Va, and prothrombin concentration. Our observations regarding the role of prothrombin in the assembly of prothrombinase and the proposed modulating role of DOPE stimulated us to examine the effect of DOPE on prothrombinase assembly under the conditions of the present study. It is of interest to see that the incorporation of phosphatidylethanolamine (20 mol %) into a membrane that contained 5 mol % DOPS had a dramatic effect on the assembly of the prothrombinase complex (Fig. 7). Prothrombinase densities were obtained that resulted in transport-limited catalysis for the prothrombin concentrations used (50-200 nM) and became indistinguishable from membranes that contained 25 mol % DOPS. When the same experiment was performed with membranes that contained 20% DOPE but no DOPS, no detectable amounts of thrombin could be measured. Apparently, phosphatidylethanolamine is an important regulator of the procoagulant activity in membranes with low phosphatidylserine content. It is apparent that only for low DOPS membranes with limiting amounts of factor Va and prothrombin, DOPE contributes to the assembly of prothrombinase which in turn results in an increased catalytic efficiency of prothrombin activation.
In summary, studies on the prothrombinase assembly from a perfusion solution to planar phospholipid membranes that contained varying mol % DOPS have provided evidence that the steady-state prothrombinase surface density is a function of the prothrombin concentration. Because the prothrombin concentration near the surface is controlled by convection and diffusion and the catalytic activity of that surface, prothrombinase seems to regulate its assembly via the prothrombin concentration near the surface.