Marie-Françoise F Calmette
email: marie-francoise.calmette@tse-fr.eu

NCFAP M Kilkenny

C Loustalan

Quality in Open Markets: How Larger Leads to Less

We show how a large country's entrance on world markets can lead to lower and less quality diversity available to consumers rather than more. In our model, autarky quality is directly proportional to the willingness to pay for quality and home market size, and inversely proportional to the cost of quality. We formalize strategically interacting firms, and identify the context in which a low-quality producer can lead, driving high-quality producers out of the market despite the existence of customers willing to pay for higher quality. We discuss the feasibility of this 'predatory strategy' by an emerging country. It is more likely in contexts where the emerging exporter is much larger.

Introduction

Jaguars, Porsches, BMWs…, in 2012 about eleven percent of the new vehicles sold in the USA were luxury cars like these. They were bought by people willing and able to pay for high quality goods. Other people purchased less expensive vehicles produced by other auto makers. The fact that some producers specialize in serving high-end customers while others specialize in supplying low-priced goods has been rationalized by industrial organization and international trade theorists for some time. Recently, however, a new pattern has emerged. In some retail markets, despite the existence of customers willing to pay for quality, luxury goods are no longer available. Although the full spectrum of vertically-differentiated goods was available in the recent past, now only low-quality versions are offered. In this paper we attempt to explain why a market may become dominated by only low-price, low-quality goods despite no reduction in the number of consumers willing to pay for high quality.

Over three decades of literature exists concerning world markets for vertically-differentiated goods, also known as intra-industry trade. A large part of this literature is based on industrial organization, e.g.: [START_REF] Gabszewicz | Price competition, quality and income disparities[END_REF], [START_REF] Shaked | Relaxing Price Competition Through Product Differentiation[END_REF], [START_REF] Motta | Endogenous Quality Choice: Price vs. Quantity Competition[END_REF], [START_REF] Crampes | Duopoly and Quality Standards[END_REF], [START_REF] Fajgelbaum | Income Distribution, Product Quality, and International Trade[END_REF].

A canonical model by [START_REF] Motta | On the Persistence of Leadership or Leapfrogging in International Trade[END_REF] consists of two firms in two countries, each producing a good identified by a quality index, strategically competing for a share of the global market in a two-stage game. Consumers are characterized by their tastes and their willingness to pay for quality. Quality is costly to produce. In the first stage firms simultaneously choose the quality to supply. In the second stage they compete in a noncooperative Cournot or Bertrand game. In autarky, the quality choice that maximises profits depends on the home market size and consumers' willingness to pay for quality relative to the costs of supplying quality.

The literature provides three main implications about what happens when countries open to trade. One, both firms respond to market incentives to change the quality they supply on the global market. Two, in equilibrium the two firms never chose to supply the same quality, as that would drive both firms' profits to zero. Three, there are multiple stable equilibria. It is not possible to predict whether the firm in the country that has the lower quality in autarky will continue to supply low quality globally, or if it will "leapfrog" to supply the high quality product, or vice-versa. These results are consistent with the industrial organisation literature. [START_REF] Motta | On the Persistence of Leadership or Leapfrogging in International Trade[END_REF] assume that the willingness to pay for high quality differs between the countries and that changing quality requires incurring fixed costs. This is why the countries specialize in a different quality good in autarky. When the countries open to trade, intra-industry two-way trade occurs, allowing high quality-loving consumers in both countries to obtain high quality goods, and low price customers in both countries to obtain low price goods. Free trade thus leads to more quality diversity in local markets, not less. And "leapfrogging" on the supply side occurs only if the difference between the two countries' willingness to pay for high quality is sufficiently small. The importance of domestic demand conditions in determining the patterns of international trade has been recognized since [START_REF] Linder | An Essay on Trade and Transformation[END_REF]. Linder hypothesized that suppliers tailor their products to the tastes of domestic purchasers. To the extent that the mix of tastes elsewhere are similar, there will be intra-industry trade with other regions. [START_REF] Krugman | Scale Economies, Product Differentiation, and the Pattern of Trade[END_REF] highlighted the "home market size effect" in a monopolistically competitive context. [START_REF] Porter | America's Green Strategy[END_REF] emphasized the importance of domestic demand for high quality to prepare firms to compete in international markets.

The ability to produce high quality goods has been noted as a condition for export success and economic development (Kremer 1993, Grossman and[START_REF] Grossman | Innovation and Growth in the Global Economy[END_REF]. This could be understood as a prescription for leapfrogging. In contrast, China has become a major exporter to global markets in many product lines without switching to high quality. [START_REF] Husted | China's Fare Share? The Growth of Chinese Exports in World Trade[END_REF] report that initial Chinese exports were labor-intensive, low quality manufactured items such as clothing, footwear, and toys. According to [START_REF] Berger | The Growth of Chinese Exports: An Examination of the Detailed Trade Data[END_REF] the value of Chinese exports more than quadrupled in the eight years between 2000 and 2007. By 2009, China had become the world's largest global exporter. The entrance of China as a low-cost, low-quality supplier in world markets drove the domestic production of low price, low quality goods out of some countries' economies. But no-one expected it to drive high price, high quality goods out of both production and markets as well.

In sum, the existing international trade and industrial organization theories indicate that as long as consumers have differentiated tastes and willingness to pay for quality, firms have incentives to differentiate supplies to satisfy global market demands. The implication that free trade leads to greater quality diversity in each local market is also a consequence of the assumption that firms play a non-cooperative game in two stages, with simultaneous decision making at each stage. But the relative sizes of trading countries today casts doubt on this simultaneous play assumption. In a world where firms in some countries face huge domestic markets, it may be more realistic to assume that such firms assert their ability to lead, forcing smaller country rivals to follow their (quality, price) offer. For example, why should a Chinese firm engage in a non-cooperative simultaneous game on a level-playing field when it exports to the Australian market? Given a Chinese firm's huge domestic market, large economies of scale and low unit production costs, why wouldn't a Chinese firm behave as a leader, forcing the Australian firm to adapt its strategy in response?

This is what we analyze in this paper. We propose a model consisting of two countries, one small but implicitly rich, with a share of consumers willing to pay for higher quality, as well as higher labor costs and lower fixed costs for producing high quality goods. The other country is very large, has low labor costs, low willingness to pay for quality, and a high fixed cost to produce high quality goods. Giving these assumptions, in autarky the large country firm produces a lower quality than the small, rich country firm. The difference in home market size is the first essential difference between our model and the existing literature.

The second essential difference is justified by the first assumption about relative size. Because of the low-quality producing country's large home market size, it can credibly lead. We formalize a Stackelberg-like game. We show that a large country can choose the quality and quantity that forces the small country firm to exit the market. Our model thus rationalizes how a low quality product supplied by a large country can capture an entire market.

We analyze the feasibility of this aggressive predatory strategy, demonstrates that it maximizes profit, and investigate the stability of the equilibria of the game. We show that the autarkic characteristics of the large country are primordial. A large domestic market is essential. Even if countries have the same cost characteristics and face the same willingness to pay for quality, only a sufficiently large country's firm can successfully follow a predatory strategy. If the domestic market is large enough, the low quality-producing country can export its autarky quality, wholly avoiding the costs of adapting quality to compete for just a share of the other country's market. This contrasts with the existing finding in the literature that firms always have incentives to adapt the quality of their supplies to the global market when they open to trade. Furthermore, when the small country/high quality producing firm raises its quality in an attempt to relax the competition on its own market, it's global market revenue is not sufficient to cover costs. High quality production shuts down.

The paper is organized as follows: in the next section we present the model and the autarky situation. Section 3 formalizes the open market situation. Assuming the large country leads, we formalize a Stackelberg game in section 4, focusing on the (quality, quantity) strategy that nullifies the profits of the high quality producer, which we call a predatory strategy. We analyze the feasibility conditions for the predatory strategy in section 5, emphasizing the implications of relative size. In section 6 we compare the profitability of the predatory strategy to the profitability of the equilibria in the non-cooperative simultaneous game. The last section concludes.

The model and the autarky situation

We model two countries, H ("Home") and F ("Foreign"). In country j (j=H,F), consumers indexed by a taste for quality θ are uniformly distributed over the interval [0, b j ] with density S j . On the supply side, firms incur a quadratic production cost 2 j j j q c C = and a fixed cost ju K that is quadratic in quality:

2 j j ju u k K =
. Thus in autarky, in each country j, a monopoly firm produces a single quality u j of a good.

Consumers either buy one unit or none at all. Consumers of type θ will buy one unit only if their net consumer surplus is positive, that is, only if θ u j -p j 0 ≥ , where p j is the price of the good of quality u j . When only one quality u jA is available, consumers with θ higher than JA θ ˆ= jA jA u p buy one unit of the good. In autarky, market demand in each country is thus:

- = jA jA j j jA u p b S D
, j= H,F.

In sum, there are four differences between the two countries:

1) the willingness to pay for quality is lower in Foreign than in Home

H F b b < .
2) Foreign is larger:

H H F F b S b S >
, in contrast with the exiting literature, e.g., [START_REF] Motta | On the Persistence of Leadership or Leapfrogging in International Trade[END_REF]. We normalize 1 = H S .

3) it follows that the marginal cost of production, c j , is lower in F than H. Normalizing that cost to zero implies 0

, 0 > = H F C C .
4) the fixed cost to produce a different quality is lower in Home:

F H k k < < 0 .
These four assumptions describe country H as a relatively small developed country, with higher wages, more sophisticated consumers, and lower product line switching costs (due to, for example, pre-existing R&D or more flexible or productive infrastructure). Meanwhile, country F is a big emerging country, with a relative abundance of labour and thus lower wages, as well as a large population of subsistence consumers.

Given the fixed cost of quality, to maximize profits firms will produce only one quality. Profits are given by:

ju j jA j jA jA jA j jA K C q S u q u b - - - = ) ( π .
Maximizing jA π with respect to jA q and jA u we find that:

, ) ( 2 + = j j jA j jA j jA c S u b u S q and j j j j j j j j j jA k k c b b k c b S u 16 ) 32 16 ( 2 2 + + - = (1) Given 0 = F c
the autarky quality choice (1) by F simplifies to:

.

8 2 F F F FA k b S u = (2)
Clearly, F's profit-maximizing quality is increasing in S and b, and decreasing in k, the fixed cost of quality This implication contrasts with the usual explanation that emerging economies export low quality goods strictly because their labour is low-priced. Producing lower quality goods is also a rational outcome where domestic consumers have a low willingness to pay for quality, the country is relatively small, and the fixed costs of higher quality are high.

Finally, recall that consumers with θ higher than JA θ ˆ= jA jA u p will buy one unit of a good. Thus for a given population, the firm in the country where consumers have a higher willingness to pay for quality produces a higher quality, sold at a higher price. This is a another significant implication of asymmetric home market size. When there is a difference in the number of consumers in each quality range (density S j ) abstracting from any differences in consumers' willingness to pay for quality (b) nor difference in the fixed cost of quality (K, or k), the autarky quality produced by the firm in the larger country will be higher.

Thus, to formalize the situation in which the larger country produces the lower quality product in autarky, we use ( 1) and ( 2) to obtain the conditions on S F relative to b j , c H , and k j such that in autarky, F produces a lower quality than H:

2 2 2 2 ) 32 16 ( 1 F H F H H H H H H H F b k k k c b b k c b S + + - < < (3)
For the remainder of this paper, we assume parameters values consistent with this condition.

Free Trade

When the two countries open to trade, both low and high quality versions may be available to consumers in each country. In each country j the consumer indifferent between buying the higher or the lower quality is indexed by:

F H F H j u u p p - - = θ ~
Thus in each country, the domestic demand for the higher quality (overscored D) is:

- - - = F H F H j j j u u p p b S D j = H,F (4)
While demand for the lower quality (underscored D) is:

D - - - = F F F H F H j j u p u u p p S j = H,F (5)
When free trade is possible, each firm faces a new set of demands due to the different willingness to pay for quality because The firms may have incentives to adjust quality to supply the global market. But to do so they must pay a fixed cost to change quality. Some authors assume that this adjustment cost depends on the difference between the autarky and open market quality. Unfortunately with this assumption, as Motta, et al, 1997 explain, it is impossible to find analytical results for the equilibria of the game; numerical simulation is required. In models where it is assumed that firms have constant marginal costs, e.g., [START_REF] Venables | The economic integration of oligopolistic markets[END_REF] and [START_REF] Motta | On the Persistence of Leadership or Leapfrogging in International Trade[END_REF], firms segment the market in prices, then maximize the global profit by choosing to supply one quality to both markets. Others, following [START_REF] Brander | A "reciprocal dumping" model of international trade[END_REF], assume that firms choose prices to maximize profits separately on both markets, which can lead to reciprocal dumping.

In order to focus on quality choice we rule out dumping. And our assumption that production costs are quadratic rules out segmenting the market in prices. Because of the fixed cost of quality, firms choose to supply a single quality on both market places. They also choose the quantity to produce that maximizes their global profit.

From (4), the global market demand for high quality products facing the H firm is given by:

- - - + - - - = F H F H F F F H F H H H u u p p b S u u p p b q (6)
given S H normalized to 1, and assuming both qualities are available in both markets. Similarly, from (5), the global market demand for low quality products facing the F firm is:

( ) F F F F H F H F S u p u u p p q + - - - = 1 (7)
The corresponding free trade prices when both versions of the good are transacted are thus:

F F F H F F H H H S u q q b S b u p + - - + = 1 ) ( ( 8
) F F F H F F H F F S u q q b S b u p + - - + = 1 ) ( (9)
Recall that demand for the high quality good in country F is positive only if

F H F H F u u p p b - - > .
Given ( 8) and ( 9), we obtain that ) 1 ( ~,

F H F F H F H F H F H S q b S b u u p p + - + = - - = θ
, and the necessary condition for positive demand for the high quality good in country F is

H F H q b b < - .
It follows that there is difference in the willingness to pay for quality at which two-way trade will not occur, that is, although H may import low quality versions from F, it will not export high-quality versions to F. In the following we assume parameter values consistent with positive demand for high quality goods among country F consumers, i.e., two-way trade.

The Predatory Strategy

Our objective in this paper is to understand how high quality goods could disappear from a developed country's marketplace. We know that this outcome is not rationalized by assuming a situation in which firms play a two-stage non-cooperative game, as in the existing literature.

As we shall show, the observed outcome is rationalized if we assume that the larger country behaves as a Stackelberg leader, to achieve a predatory objective. Taking into account the small country firm's best response, the firm in F commits to supply the quality and quantity on the global market such that when the small country firm adapts quality, its profits are nonpositive, resulting in the ultimate closure of the H-firm.

The game we formalize proceeds in two stages. Similar to the Stackelberg game, anticipating the H-firm's best response, the F-firm commits to supply the quality and quantity that should nullify the H-firm's profits. In the second stage, the H-firm chooses the new quality and quantity that maximizes their profit. As usual, this game is solved by backward induction.

First solve the profit maximization problem of the H-firm, as anticipated by the F-firm.

Assuming that the H-firm will adapt quality 2 (

HA H u u ≠ ), H-firm profit is: 2 )) ( ( H H H H H H H u k q q c p - - = π
With free trade and both goods consumed in both countries, the open market price p H is given by (8). Rearranging, profit is:

( ) 2 1 H H H H F F F H H F F H H H u k q c S u q u q b S b q - - + - - + = π (10)
The H-firm is assumed to choose the quality H u and quantity H q that maximize H π . The first order conditions with respect to H u imply that:

) 1 ( 2 ) ( F H F F H H H H S k b S q b q u + + - = (11)
Expressing H π in terms of this expression for u H we have:

+ - - - + + - + - + = ) 1 ( 4 )) ( 2 2 ) 1 ( 4 ( ) 1 ( 1 2 2 2 2 2 2 F F F H F F H H F F H F H F F H H H H H F H S u q k S b q b S b q S q b S c k b q q S π (12)
The H-firm also chooses the production quantity that maximizes its profit. Let

H F q q λ = ( 0 > λ
). The first order condition with respect to the profit maximizing quantity is:

0 ) 1 ( 2 ) 1 ( 2 3 2 ( ) 3 ( 2 ) 1 ( 4 2 2 2 2 = + + - - + - + + + - = ∂ ∂ F H F F H H F F H H F F F F H F H H H H H H S k S u k q S b b q S b S b q S c k b q q λ π
Of the three solutions for H q , the one consistent with a negative second derivative is:

( )

) 1 ( 16 ) 1 ( 32 2 3 3 4 1 2 2 2 2 F F H F H H F F F F H H F F H H S u k S c k S b S b b b S b b q + + + + + + - + = λ (13)
2 Analyses of this game assuming Home does not adapt results in theoretical open market outcomes inconsistent with the observed outcome we seek to explain: (i) for (b Hb F ) too large, Home serves the domestic market only, but free trade still leads to wider quality choice in Home; (ii) at lower (b Hb F ), there is a threshold S F at which Home also serves only the domestic market, and (iii) at larger S F , there is leapfrogging, also leading to higher quality in the open market than in autarky. Details available from the authors on request.

Equations ( 11) and ( 13) express the H-firm's profit-maximizing response in terms of the quality F u and quantity F q choice variables of the F-firm (and the relevant parameters). Note that one obtains the same results by first maximizing H π with respect to H q . Now, solve the "predatory strategy" problem of the F firm. What pair ) , (

F F u q nullifies H π ? Denote H F q q λ =
and express the value function for H π in terms of H q as given by( 13). This results in an expression for H π in terms of the parameters ( ) , , ,

j H j F k c b S
as well as λ and F's choice of F u . Find the value of λ that nullifies the H-firm's profit by equating

H π ( F j H j F u k c b S , , , , , λ ) to 0: 2 2 2 2 2 ) 1 ( 12 ) ( ) ( ) 1 ( 12 ) (( ) 1 ( 18 1 F H H F F H F F H F H H F F H F H S c k b S b b S b S c k b S b S u k + + + + + + - + + = λ
With this value of λ in (13), the expression for H q is:

2 2 2 2 2 2 ) 1 ( 12 ) ( 8 ) 1 ( 192 17 17 ( 9 9 [ 12 1 F H H F F H F F F H H F F H F F H H S k c b S b b S S k c b S b b S b q + + + + + + + - + = 2 2 ) 1 ( 12 ) ( 8 34 ( F H H F F H F F H S k c b S b b S b + + + + + ] (13') It follows that = = H F q q λ F u G , where + + + + + + - + + = 2 2 2 2 ) 1 ( 12 ) ( ) ( ) 1 ( 12 ) (( ) 1 ( 18 1 F H H F F H F F H F H H F F H F H H S c k b S b b S b S c k b S b S k q G (14)
In sum, this is a game in which the F-firm is able to drive the H-firm profits (12) to zero by offering F q F u = G. A numerical example is given in Appendix 1. Clearly, when the F-firm offers the (q F , u F ) such that F q F u = G + eps, the H-firm's revenues will not cover costs, and it will be driven out of the market.

The feasibility conditions for a predatory strategy

The predatory strategy by the firm in F is to set F q . F u = G (14). Under what parametric conditions can the F-firm successfully implement this strategy while producing the lower quality version of the good? If the F-firm adapts quality (

FA F u u ≠ ), given our normalizations its profit is 2 ) ( F F F F F u k q p - = π
. Using (9) to express the open market price of the low quality good, we have:

( ) 2 1 F F F F F H F F H F F u k S u q q b S b q - + - - + = π (15)
Alternatively, if the F-firm simply specializes in and exports its autarky quality, then it avoids the cost of adapting quality:

( ) + - - + = F FA F H F F H F F S u q q b S b q 1 π (15')
First alternative: Foreign firm exports its autarky quality Proposition 1:

The emergent countries able to play a predatory strategy by exporting their autarky quality must have a high density of population. The density of the population must be higher when the rich country consumers' willingness to pay for quality is higher.

The F-firm can drive the H-firm out of the market by proposing a pair G u q F F = where G depends on the characteristics of the countries ( 14). The higher is the willingness to pay for quality in the rich country (the higher is H b ) and the lower is the cost of production in the rich country (the lower is c H ), the larger is G, and the more difficult it is for an F-firm to successfully follow a predatory strategy.

When the F firm exports its autarky quality when it behaves as a leader when it opens to trade, it is constrained by the trade-off formalized in ( 14). The lower its autarky quality, FA u , the higher quantity, F q , must be to conform with G u q FA F = . Nevertheless, its production cannot exceed the global market size:

) (

H F F H Max F q b S b q - + = (16)
In consequence, the minimum autarky quality in country F consistent with a predatory strategy is:

min ) ( H F F H FA q b S b G u - + = (17)
From (2) we have that

F F F FA k b S u 8 2 =
. This allows us to express the market density, S F , necessary for a successful predatory strategy by the F firm exporting its autarky quality. By successful predatory strategy, we mean = H π 0 (10), and that the F-firm's profits (15') are higher than under any other strategy. It follows from ( 17) and (2) that in order for the F firm to play the predatory strategy, F must be sufficiently large, i.e., it must be that:

> F S ) ( ) 8 ( 2 H F F H F F q b S b b k G - + . ( 18
)
We illustrate via simulation how the minimum predator size (or density), F S , varies as j b , . At larger size, Foreign's autarky quality (2) exceeds the minimum quality that satisfies (17) for a successful predatory strategy. This underscores our finding that the emerging country must be sufficiently large (about three times larger, depending on the other parameters) to implement the predatory strategy.

The main result is that among emergent countries that have the same cost of quality, cost of production, and their own consumers' willingness to pay for quality, only the large emergent countries--with sufficiently high F S --are able to implement a profit-maximizing predatory strategy while exporting their autarky quality.

Alternatively, when rich country consumers are willing to pay even more for quality (higher values of H b ), the minimum quality of exports from the emergent country ( FA u min ) for a successful predatory strategy must also be higher.

Furthermore, the higher is the willingness to pay for quality in the rich country, H b , relative to b F , the larger the emergent country must be for it to succeed with a predatory strategy. This is illustrated by comparing Figures 2 and1. Figure 2 is generated using the same parameters as Figure 1, except that H b = 7 in Figure 2. Note also that (3) is satisfied through 14 < F S

. Figure 2 shows the higher size of

F ( 5 . 4 = F S
) consistent with a successful predatory strategy in F's autarky quality when the difference in the willingness to pay for quality is larger. The emergent country playing a predatory strategy will chose to adapt quality to the new open market when differences between country willingness to pay for quality and in population are sufficiently small. Nevertheless if differences in population are large, the emergent country firm can successfully corner the market while exporting its autarky quality.

Let us consider now the alternative that the Foreign firm adapts and exports the quality

FA F u u ≠ while satisfying G u q F F = .
In this case the Foreign firm incurs the fixed cost of quality, and F π is defined as in (15). Using the value of λ which nullifies the Home firm's profit, the corresponding value of H q (13'), and setting

= = H F q q λ F u G in (15), the value of F π depends on the parameters ( ) , , , j H j F k c b S
and is only a function of F u . We find the level of quality that maximizes the S the Foreign firm maximises profit by exporting its autarky quality (s the blue curve). Given the parameter values illustrated, the alternatives are equally profitable at F S =2.9. Above that size, exporting the autarky quality is more profitable. Facing a large number of consumers with a low willingness to pay for quality in Foreign, the Foreign firm has insufficient incentives to incur a fixed cost of switching to higher quality to sell to the Home country consumers.

Consider how this result varies with respect to b H . When the willingness to pay for quality in Home, H b , is higher, it is more difficult for the Foreign firm to implement a successful predatory strategy. But there is still a Foreign size at which the predatory strategy dominates. As shown in Figure 4, with H b =7, values of other parameters being the same, the predatory strategy is not profitable at all for 2 < F S

. For 2 < S F < 5.2, the Foreign firm has the incentive to adapt quality and serve more Home consumers. The size effect dominates after S F > 5.2. Above that size the F-firm most profitably exports its autarky quality in a predatory strategy. In this section we have shown that a 'predatory' strategy model rationalizes the observed exclusively low quality goods on an open market following the entrance of a very large, relatively poorer country. The size of the emergent country is key, as is the relative willingness to pay for high quality. When the emergent country is small, as we shall show in the next section, its most profitable strategy is to play a non-cooperative game. That results in the more diverse quality outcomes that have been common in the past on open markets.

When the emergent country is more than thrice the size of its trade partner (depending on other parameters), it maximizes profit by implementing a predatory strategy in which it simply exports its autarky quality when it opens to trade. The threshold size is increasing in the willingness to pay for quality in the Home country, but there is always a sufficiently large size at which the predatory strategy is the most profitable. The best response to the predatory strategy in Home leaves the high quality firm with zero profit (or lower), ultimately driving it out of the marketplace. The ultimate outcome is exclusively lower quality goods after the emergence, compared to before. This is the outcome observed in some markets (e.g., clothing) since China emerged on global markets.

Comparison with the simultaneous non-cooperative Cournot Game

In this section, for purposes of comparison we consider the situation where the monopoly firms in the two countries play a non-cooperative game when they open to trade, as in the existing literature (e.g., Motta, et al (1997)). In the first stage they simultaneously choose qualities u H and u F and in the second stage they compete in quantities q H and q F . According to the existing literature, depending on the values of the parameters j b , j S , j c , and j k , there may be multiple Nash equilibria, including "leapfrog" equilibria (where the low quality producer switches to a higher quality than the high-quality rival).

Assuming both adapt quality to the open market, as in the existing literature, the firms' profits are as usual, given by equations ( 10) and ( 15). We solve by backwards induction. First, find the production quantities that maximize their respective profits. The first order condition solutions consistent with a negative second derivative are:

) 4 ) 1 ( 4 ( ) 2 )( ( F H F H F H F F H H u u S c u u b S b q - + + - + = and ) 4 ) 1 ( 4 ( ) ) 1 ( 2 )( ( F H F H H F H F F H F u u S c u S c b S b q - + + + + + + =
Substituting these expressions into H π and F π we obtain:

( )

2 2 2 2 ) 4 ) 1 ( 4 )( 1 ( ) 2 )( 1 ( ( H H F H F H F F H F H H F F H H u k u u S c S u u S c u b S b - + - + - + + - + + + = π (19) And ( ) 2 2 2 2 ) 4 ) 1 ( 4 )( 1 ( ) 1 ( 2 ( ( F F F H F H F F H H F F H F F u k u u S c S S c u b S b u - + - + - + + + + = π (20)
At the first stage, the firms simultaneously choose the quality to supply to maximize these profit 'functions.' The first order condition solutions consistent with negative second derivatives are the Nash equilibria of the game.

Compare the Foreign firm's profits from a non-cooperative game strategy to its profit from the alternative predatory strategies. The analytical solutions are prohibitively difficult. We can, however, identify superior strategies via simulation, assuming values for the parameters ), and investigate how the strategy rankings vary with respect to the parameters, especially the Foreign market size or density, S F .

In Figure 5 the Foreign firm's profit as a function of F S from both alternative predatory strategies is compared to profits when it participates in a simultaneous non-cooperative game.

Figure 5 shows that at low levels of F S the Foreign firm maximises profit by playing the noncooperative game. When its size or density F S is larger than 1.9, the Foreign firm maximises profit with the predatory strategy alternative in which it adapts quality. At levels of F S higher than F S =2.9, it is most profitable for the F-firm to implement the predatory strategy with a low autarky quality, as shown in the previous section.

As seen, this threshold size is directly proportional to the difference in the willingness to pay for quality. This is illustrated in Figure 6. When H b is raised to 7, all else equal, the predatory strategy is not profitable below 2 . 5 = F S . However, once again, even with a significant difference between the willingness to pay for high quality, the effect of that difference is ultimately overwhelmed by the difference in size. In Figure 6 the size effect dominates after 2 . 5 > F

S

. Above that size the F-firm can most profitably export its autarky quality in a predatory strategy. Note also that when H b =7, the F-firm predatory strategy alternative "adapt" is dominated at all values of F S .

Conclusions

This paper provides theoretical explanations for what has occurred in many developed countries since China emerged on global markets. We have proven how a large country's low-quality exports can dominate an entire market, not only where low-price, low-quality goods are demanded, but also where there is a number of consumers willing to pay for high quality. We have shown how large can lead to less. China's exports of low quality manufactured products such as clothing, footwear, and toys have been documented by [START_REF] Amiti | An Anatomy of China' s Export Growth[END_REF] and [START_REF] Husted | China's Fare Share? The Growth of Chinese Exports in World Trade[END_REF]. Our model shows that an emergent's country quality is inversely proportional to the cost of quality and directly proportional to its size. The finding augments the typical explanation that emerging economies export low quality goods strictly because their labour is low-priced.

The importance of emergent country size, Proposition 1, helps explain why China has managed to dominate some markets while other low-labour cost countries have not. Proposition 2 clarifies how a very large emerging country's lower quality exports can drive high-quality producers out of the market, despite the existence of customers willing to pay for higher quality. These findings are unprecedented in the literature, but widely observed in open markets for some goods. We have shown how free trade can lead to less quality diversity rather than more.

We have shown that a 'predatory' strategy model rationalizes the observed exclusively low quality goods on an open market following the entrance of a very large, relatively poorer country. The size of the emergent country is key, as is the relative willingness to pay for high quality. When the emergent country is not too much larger than its trade partner, we have shown that its most profitable strategy is to play a non-cooperative game (section 6). Thus, when the countries opening to trade are relatively similar in size, the outcome to be expected is more quality diversity, including high quality, on the open market. That outcome has occurred, especially in the past. This also explains why the previous literature analyzed intraindustry trade in vertically differentiated goods assuming similarly-sized countries a noncooperative game market structure.

However, when the emergent country is very large, we have shown that it maximizes profit by implementing a predatory strategy in which it exports its autarky quality when it opens to trade. The best response to the predatory strategy in the relatively smaller country can leave its high quality producers unable to cover costs, ultimately driving them out of the marketplace. Again, the size of the emergent country's domestic market is essential. The size at which a predatory strategy can succeed is directly proportional to the trading partner's willingness to pay for quality, but there is always a sufficiently large size at which the predatory strategy is most profitable for the emerging country. The ultimate outcome is exclusively lower quality goods on the open market, compared to before its emergence. This is the outcome observed in some markets (e.g., clothing) since China emerged on global markets.

While our analyses have provided plausible answers to these questions, we have also opened new lines of inquiry for future research. As noted earlier, since 2009 China has been the world's largest exporter. It may be that no single set of assumptions about size, costs, preferences, and market structure can explain China's success on global markets. For example, in "What's So Special about China's Exports?" [START_REF] Rodrik | What' s so special about China' s exports?[END_REF] noted that although China exported relatively low quality goods twenty years ago, more recently China has been exporting "goods that are three times more sophisticated than what would be normally expected for a country at its income level" [START_REF] Rodrik | What' s so special about China' s exports?[END_REF]. It has dominated in markets for high-tech goods such as cell phones, liquid crystal displays, integrated electronic circuits, and laptop computers. In fact, by 2009, China supplied 85 percent of the imported laptops on the US market [START_REF] Berger | The Growth of Chinese Exports: An Examination of the Detailed Trade Data[END_REF]. Meanwhile there has been an explosion in Chinese demand for these products as well.

Our model provides a point of departure for future research in a few ways. One, domestic market size can be even more important than costs or wages, i.e., incomes, in explaining the quality of goods exported by emerging countries. This is empirically testable. Two, the lower are the costs of switching to higher quality, the more likely is leapfrogging. This is the case in China today with respect to high tech production. Three, in this paper we have abstracted from leapfrogging, because when emerging countries leapfrog the outcome is more quality, not less, which is not the outcome we have sought to explain. We already know that leapfrogging is more likely the more similar are the trading partners with respect to willingness to pay for quality. Clearly it could be interesting to analyze the outcomes of our predatory model allowing for leapfrogging and alternative quality switching costs.

Appendix I. Example of a Successful Predatory Strategy

As an example, consider If the Foreign firm offers a global quantity and a quality such than F q . F u = G (see( 14)), then the best response of the Home firm is to offer a different (higher) quality and the quantity that maximizes its profit, given by equations ( 11) and (13).

Putting these optimal values in (10) with F q . F u = G , we obtain H π =0.

What happens to the Home firm ? It is able to offer, according to (11) , ( 13) and ( 8 . Note that Home firm has raised the quality and decreased the price of its good (relative to autarky) in response to competition from the Foreign firm.

The quantities that the Home firm could offer on each market (see ( 6 ). The resulting profit H π = 0.

Figure 1 .

 1 Figure 1. Autarky quality in Foreign, u FA , compared to the minimum quality choice required for a successful predatory strategy (Min u FA ) with respect to Foreign size S F , assuming b H = 5, b F = 3, c H = 3, k F = 0.8, k H = 0.3.

Figure 2 .

 2 Figure 2. Autarky quality in Foreign, u FA , compared to the minimum quality required for a successful predatory strategy (Min u FA ) with respect to Foreign size S F ; assuming b H = 7 (all else the same as in Figure 1)

ForeignFFigure 3 .

 3 Figure 3. F-firm profits under the predatory the "adapt quality" and "don't adapt" alternatives with respect to density F S , given b H = 5, b F = 3, c H = 3, k F = 0.8, k H = 0.3.

FFigure 4 .

 4 Figure 4. Foreign firm profits under alternative predatory strategies (adapt, not adapt) with respect to S F , given parameters b H = 7, all else equal to Figure 3.

Figure 5 :

 5 Figure 5: Foreign firm profits as a function of S F for both predatory strategy alternatives and the non cooperative strategy, 3 = F b

Figure 6 .

 6 Figure 6. Foreign firm profits with respect to S F under both predatory strategy alternatives and the non cooperative strategy; b F = 3, k H = 0.3, c H = 3, k F = 0.8, b H = 7.

  of the parameters we can see from (1) and (2) that in autarky, the Home firm produces high quality and the Foreign firm produces the lower quality: Foreign firm is able to serve half of its market when the Home firm offers 916

  chosen to adapt quality, the Home firm must pay the fixed cost (

Details of the calculations are available on request to the authors.


Stephen Ferrière

Charlotte Kawecki

Jean-François Ottavi

Cécile V Denis
email: cecile.denis@inserm.fr

Alexandre Kauskot

Olivier D Christophe

Peter J Lenting

A single domain antibody that blocks factor VIIa activity in the absence but not presence of tissue factor

Keywords: factor VIIa, tissue factor, single-domain antibody, inhibitor, hemostasis, coagulation factor 1

A novel sdAb that blocks FVIIa TF-independent activity 2

ESSENTIALS

• Factor VIIa (FVIIa) is an enzyme that has both tissue factor (TF)-dependent and TFindependent activities

• We describe a novel antibody (KB-FVIIa-004) that selectively blocks TF-independent activity of FVIIa

• KB-FVIIa-004 interferes with the action of recombinant FVIIa (rFVIIa) in Factor VIII (FVIII)-deficient mice

• Our data suggest that rFVIIa acts as a FVIII-bypassing agent predominantly in a TFindependent fashion

INTRODUCTION

Human blood coagulation factor VII is a vitamin K-dependent serine protease precursor that is secreted as a 50 kDa-protein consisting of 406 amino acid residues. Structurally, FVII is related to other vitamin K-dependent proteins, such as factor IX (FIX) and factor X (FX): it contains an N-terminal Gla domain enriched in g-carboxylated Glu-residues, two epidermal growth factor-like domains and a C-terminal serine protease domain [START_REF] Broze | Purification and properties of human coagulation factor VII[END_REF][START_REF] O'hara | Nucleotide sequence of the gene coding for human factor VII, a vitamin K-dependent protein participating in blood coagulation[END_REF]. Activation of FVII is achieved via limited proteolysis at Arg 152 , generating a two-chain derivative that is covalently linked via a disulfide bridge.

FVIIa is responsible for the activation of its cognate substrates FIX and FX. Similar to many other vitamin K-dependent serine proteases, the enzymatic activity of FVIIa towards these substrates is relatively poor [START_REF] Pedersen | Inhibition of recombinant human blood coagulation factor VIIa amidolytic and proteolytic activity by zinc ions[END_REF]. Furthermore, the notion that FVIIa also displays low intrinsic activity towards small tripeptidyl-amide substrates suggests that the active site of FVIIa is not fully developed [START_REF] Silverberg | Kinetics of the activation of bovine coagulation factor X by components of the extrinsic pathway. Kinetic behavior of two-chain factor VII in the presence and absence of tissue factor[END_REF]. Indeed, it appears that the N-terminal Ile 153 of the protease domain does not properly fold into the active site pocket, leaving the catalytic triad that consists of His 193 , Asp 242 and Ser 344 immature [START_REF] Rand | Allosteric activation of coagulation factor VIIa visualized by hydrogen exchange[END_REF]. It has been proposed that FVIIa exists in equilibrium between two states, one in which Ile 153 is inserted into the active site pocket and one that is more zymogen-like, with Ile 153 incorrectly inserted.

In order to develop physiologically relevant activity, FVIIa forms a complex with tissue factor (TF). Formation of the FVIIa/TF complex strongly promotes the insertion of Ile 153 into the active site pocket [START_REF] Banner | The crystal structure of the complex of blood coagulation factor VIIa with soluble tissue factor[END_REF]. This does not only result in a functional catalytic triad, but also induces a number of allosteric changes in FVIIa protease domain surface loops, including the S1 entrance frame (residues 365-372) [START_REF] Persson | Allosteric activation of coagulation factor VIIa[END_REF]. Consequently, TF-binding enhances amidolytic activity 20-100-fold, while proteolysis of macromolecular substrates FIX and FX are increased 10 5 -10 6 -fold.

Importantly, recombinant FVIIa is used as a therapeutic agent in the treatment of the bleeding disorder hemophilia A. Although clinically effective, its mechanism of action has been a matter of debate for many years, mainly regarding its function being TF-dependent or independent [START_REF] Giansily-Blaizot | Recombinant human factor VIIa (rFVIIa) in hemophilia: mode of action and evidence to date[END_REF]. Based on in vitro and in vivo observations, there is accumulating evidence that this FVIIa activity is localized to the platelet surface, predominantly in a TF-independent manner [START_REF] Monroe | Platelet activity of high-dose factor VIIa is independent of tissue factor[END_REF][START_REF] Feng | FVIIa as used pharmacologically is not TF dependent in hemophilia B mice[END_REF]. However, it cannot be excluded that therapeutic FVIIa also displays TFdependent activity under some conditions [START_REF] Rao | Studies of the activation of factor VII bound to tissue factor[END_REF][START_REF] Butenas | Mechanism of factor VIIadependent coagulation in hemophilia blood[END_REF][START_REF] Shibeko | Unifying the mechanism of recombinant FVIIa action: dose dependence is regulated differently by tissue factor and phospholipids[END_REF].

Soon after the development of monoclonal antibody technology, antibodies against FVII and FVIIa have been generated [START_REF] Broze | Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII[END_REF][START_REF] Coppola | Characterization of a Ca (II)-independent monoclonal antibody that lacks reactivity with Gla-domainless human factor VII[END_REF][START_REF] Biemond | Complete inhibition of endotoxin-induced coagulation activation in chimpanzees with a monoclonal Fab fragment against factor VII/VIIa[END_REF][START_REF] Cheung | Localization of an epitope of a calcium-dependent monoclonal antibody to the N-terminal region of the Gla domain of human factor VII[END_REF][START_REF] Andersen | Antibody-induced enhancement of factor VIIa activity through distinct allosteric pathways[END_REF]. Of note, many of them recognize both the active and zymogen form of FVII. There are nevertheless some examples of FVIIa-specific antibodies.

Philippou et al. generated a polyclonal antibody directed against the C-terminal part of the FVIIa light chain, which displayed >3000-fold greater reactivity to FVIIa than FVII [START_REF] Philippou | A novel specific immunoassay for plasma two-chain factor VIIa: investigation of FVIIa levels in normal individuals and in patients with acute coronary syndromes[END_REF].

Andersen et al. reported on antibodies that show preferential binding to FVIIa and stimulated FVIIa activity [START_REF] Andersen | Antibody-induced enhancement of factor VIIa activity through distinct allosteric pathways[END_REF]. Most recently, a llama-derived single domain antibody (sdAb) has been described that binds to FVIIa but not to FVII [START_REF] Hyseni | Increased mortality in systemic inflammatory response syndrome patients with high levels of coagulation factor VIIa[END_REF]. The antibody described by Philippou remains inhibitory in the presence of TF [START_REF] Philippou | A novel specific immunoassay for plasma two-chain factor VIIa: investigation of FVIIa levels in normal individuals and in patients with acute coronary syndromes[END_REF]. With regard to the FVIIa-specific sdAb, it is not reported whether it is inhibitory or, if inhibitory, if this is affected by the presence of TF.

The aim of the present study was to generate sdAbs against FVIIa that display selectivity to free FVIIa rather and TF-bound FVIIa. Such antibodies could be helpful to gain insight into the role of TF-bound and TF-free FVIIa function.

EXPERIMENTAL PROCEDURES

Animals & ethics statement-FVIII-deficient mice with an exon 16-deletion [START_REF] Bi | Targeted disruption of the mouse factor VIII gene produces a model of haemophilia A[END_REF] Two sources of relipidated recombinant full-length human TF were used during the study (Siemens-Dade, Marburg, Germany & American Diagnostica, Neuville-sur-Oise, France), giving similar results. Soluble murine TF (smTF) was obtained from Novusbio/Bio-Techne (Lille, France). Thrombin was purchased from Kordia (Leiden, the Netherlands). Control sdAb KB-VWF-006bv has been described previously [START_REF] Ayme | A Novel Single-Domain Antibody Against von Willebrand Factor A1 Domain Resolves Leukocyte Recruitment and Vascular Leakage During Inflammation-Brief Report[END_REF]. Bovine serum albumin (BSA) was from Sigma-Aldrich (Saint-Louis, MO). Polyclonal goat anti-FVII/FVIIa antibodies and peroxidase-labeled polyclonal sheep anti-FVII/FVIIa antibodies were from R&D Systems (Minneapolis, MN). Phospholipid vesicles (75% L-a-phosphatidylcholine/25% L-aphosphatidyl-L-serine) were prepared as described [START_REF] Thiec | Role of the Gla and first epidermal growth factor-like domains of factor X in the prothrombinase and tissue factor-factor VIIa complexes[END_REF]. PT and aPTT reagent were from Stago (Asnières, France). Chromogenic substrate CBS-34.47 was from Stago, and S-2222 and S-2238 were from Chromogenix (Mölndal, Sweden).

Isolation of single domain antibodies-

The sdAb repertoire was amplified from lymphocyte cDNA, isolated from llamas immunized with human recombinant FVIIa and cloned into the pHEN6-plasmid. This material was used to transform E. coli TG1 cells to generate a library containing >1x10 8 clones. Via a phage-display approach, this library was used to select sdAbs against FVIIa. From a series of >10 sdAbs, one appeared specific for FVIIa, and was named KB-FVIIa-004. KB-FVIIa-004 was cloned into the pET28-plasmid, and was expressed with an N-terminal His-tag and C-terminal haemagglutinin (HA)-tag. Expression was performed in E. coli SHuffle-C3029H cells (New England Biolabs, Evry, France). Bacteria were grown in 330 ml of LB-medium at 30°C until the culture had reached the logarithmic phase at an optical density between 0.4 and 0.6. Protein expression was then induced at 20°C via the addition of isopropyl-thiogalactoside (0.1 mM final concentration).

Twenty-four hours after induction of protein expression, cells were collected, and soluble proteins were released from the cells via sonication. His-tagged nanobodies were purified via Co 2+ -affinity chromatography using PBS/0.3 M NaCl/0.5 M imidazole as elution-reagent.

Minor contaminants were removed from the preparations via size-exclusion chromatography using 20 mM Hepes (pH7.4)/0.1 M NaCl as equilibrium-buffer. Purified KB-FVIIa-004 displayed >95% homogeneity as assessed via SDS-Page and Coomassie-staining. FVII/FVIIa ELISA-Wells were coated with polyclonal goat-anti FVII/FVIIa antibodies (10 µg/ml) and incubated with various dilutions of human plasma (1/2 to 1/128), purified human FVII (0-50 nM) or with purified FVIIa (0-5 nM) in Tris-buffered saline (pH 7.4)/0.1 % BSA for 2 h at 37˚C. Bound FVII/FVIIa was probed with peroxidase-labeled polyclonal sheep-anti FVII/FVIIa antibodies and detected via hydrolysis of 3-3'-5-5'-tetramethylbenzidine.

Amidolytic activity-FVIIa amidolytic activity was measured via hydrolysis of substrate CBS-34.47 (1.5 mM, unless indicated otherwise). FVIIa amidolytic activity in the absence of TF was performed using 75 nM FVIIa. In the presence of relipidated TF (10 nM), FVIIa concentrations were 7.5 nM. Concentrations of KB-FVIIa-004 were 0-600 nM in the absence of TF, and 0-750 nM in the presence of TF. Substrate hydrolysis was monitored by measuring absorbance at 405nm. FXa (5 nM) and thrombin (5 nM) amidolytic activity were measured in a similar fashion, except that substrates S-2222 (1 mM) and S-2238 (1 mM) were used. All amidolytic activities were measured in 30 mM Hepes (pH 7.4), 125 nM NaCl, 6.5 mM CaCl2, BSA 0.125%.

Schild regression analysis-

The initial rates of CBS-34.47 (0-4 mM) hydrolysis by FVIIa (75 nM) in the presence of KB-FVIIa-004 (0-112.5 nM) were determined. Initial velocity values were then plotted versus log [CBS-34.47], and the pre-programmed EC50-Shift equation (GraphPrism-6, GraphPad Software, San Diego, Ca) was used to determine the dose ratio (dr) for each sdAb concentration. These values were subsequently used for Schild regression analysis [START_REF] Arunlakshana | Some quantitative uses of drug antagonists[END_REF], in which log (dr-1) is plotted versus log [KB-FVIIa-004]. The shape of the curve represents the nature of the inhibitor (a linear curve represents a competitive inhibitor, whereas an exponential curve represents non-competitive inhibition). For a competitive inhibitor, the x-axis intercept of the linear fit corresponds to the Ki of the interaction.

FX activation in absence of TF-FVIIa was preincubated for 60 min in the absence or presence of KB-FVIIa-004, and added to FX and phospholipids. Final concentrations were 25 nM FVIIa, 0.5 µM FX, 40 µM phospholipids and 0-250 nM KB-FVIIa-004. FX activation was terminated after 15 min by the addition of EDTA (50 mM), and samples were analyzed for FXa formation by assessing hydrolysis of substrate S-2222. Assays were performed in 30 mM Hepes (pH 7.4), 125 nM NaCl, 6.5 mM CaCl2, BSA 0.125%.

FX activation in presence of TF-FVIIa and relipidated TF were preincubated for 60 min in the absence or presence of KB-FVIIa-004, and added to FX. Final concentrations were 1 nM FVIIa, 2 nM TF, 0.2 µM FX, 10 µM phospholipids and 0-1 µM KB-FVIIa-004. FX activation was terminated after 15 min by the addition of EDTA (50 mM), and samples were diluted 100-fold. The amount of FXa formed was determined via hydrolysis of substrate S-2222.

Assays were performed in 30 mM Hepes (pH 7.4), 125 nM NaCl, 6.5 mM CaCl2, BSA 0.125%.

Inactivation of FVIIa by PPACK-FVIIa (0.1 µM) was incubated with various concentrations of PPACK (0-10 µM) for 60 min at 37˚C. Samples were then analyzed in parallel for binding to KB-FVIIa-004 and for amidolytic activity.

Bleeding time-Mouse tail-bleeding time was performed using the tail clip-bleeding assay.

Briefly, immediately after tail vein-injection, mice were anesthetized using ketamine (100 mg/kg)/xylazine (10 mg/kg) solution. Once anesthetized, the 3 mm of the distal tip of the tail was amputated. The amputated tail was immersed in prewarmed physiological saline (37˚C) and blood was collected for 30 min. After 30 min, the mixture of blood and physiological saline was centrifuged at 1500g. The red blood cell pellet was then lysed in H2O and the amount of hemoglobin was obtained by reading the absorbance at 416 nm. The volume of blood lost in each sample was calculated from a standard curve, which is obtained by lysing defined volumes (20 µl, 40 µl, 60 µl, 80 µl and 100 µl) of mouse blood in H2O to extract hemoglobin.

Statistical analysis-Statistical analysis was performed using One-way ANOVA with

Dunnett's or Tukey's correction for multiple comparisons. P-values below 0.05 were considered significant.

RESULTS

Selection of FVIIa specific sdAb KB-FVIIa-004-sdAb libraries were constructed from lymphocytes obtained from a llama immunized against FVIIa. FVIIa-specific sdAbs were selected via phage-display screening using purified recombinant FVIIa. After two rounds of panning, 18 unique sequences were obtained. Following expression in E. coli SHuffle cells, one sdAb was identified that seemed specific in interacting with human FVIIa. This nanobody, designated KB-FVIIa-004, was chosen for further analysis in this study. We first analyzed the capacity of KB-FVIIa-004 to recognize FVII/VIIa in plasma. Serial dilutions of normal plasma were added to immobilized polyclonal anti-FVII/FVIIa antibodies or KB-FVIIa-004. Whereas a dose-response was observed when plasma samples were incubated with polyclonal anti-FVII/VIIa antibodies, no signal above background levels could be detected for sdAb KB-FVIIa-004 (Fig. 1A). In a second series of experiments, binding to purified FVII to KB-FVIIa-004 was tested. FVII efficiently bound to polyclonal anti-FVII/FVIIa antibodies, whereas no binding to KB-FVIIa-004 was observed for FVII concentrations up to 50 nM (Fig. 1B). In contrast, binding of FVIIa to KB-FVIIa-004 was already detected at 300-fold lower concentrations 0.16 nM, and a dose-dependent binding was established using FVIIa concentrations up to 5 nM (Fig. 1C). Apparently, KB-FVIIa-004 displays preferential binding to FVIIa over FVII.

Effect of KB-FVIIa-004 on coagulation assays-

To further characterize KB-FVIIa-004, we tested the effect of this sdAb on FVIIa-dependent and FVIIa-independent clotting assays, the prothrombin time (PT) and activated partial prothrombin time (aPTT), respectively. Plasma was incubated with varying concentrations of KB-FVIIa-004, control sdAb KB-VWF-006bv (which is directed against von Willebrand factor; ( 22)) or affinity-purified polyclonal goat anti-FVII/FVIIa antibodies (PT only). As expected, control sdAb KB-VWF-006bv left both PT and aPTT unaffected (Figs. 2A-B). Furthermore, no changes in clotting time were observed when KB-FVIIa-004 was tested in the aPTT, which functions independently of FVIIa (Fig. 2A).

Polyclonal anti-FVII/FVIIa antibodies efficiently prolonged PT clotting time, in agreement with the FVIIa-dependent nature of this assay. The clotting time was prolonged from 12.4±0.5 sec to 29.3±0.5 sec and 48.3±1.0 sec at concentrations of 0.05 µM and 0.15 µM polyclonal antibodies, respectively (n=3-6; Fig. 2B). A prolongation of the PT clotting time was also observed for KB-FVIIa-004, with the longest clotting time being prolonged less than 10 sec (from 13.1±0.6 sec to 22.7±1.6 sec (n=9)) in the presence of 10 µM KB-FVIIa-004 (Fig. 2B).

These data suggest that KB-FVIIa-004 partially reduces the activity of FVIIa in the PT-assay. However, it is doing so in a rather inefficient manner compared to polyclonal anti-FVII/FVIIa antibodies, since even a 200-fold higher sdAb concentration (10 µM sdAb compared to 0.05 nM polyclonal antibody) is unable to achieve a similar prolongation of the clotting time.

Inhibition of FVIIa amidolytic activity by KB-FVIIa-004-

To better understand the inhibitory capacity of KB-FVIIa-004 towards FVIIa, we next performed inhibition assays using purified FVIIa. First, the effect of KB-FVIIa-004 on the amidolytic activity of FVIIa was assessed.

Whereas control sdAb KB-VWF-006bv did not interfere with substrate conversion, KB-FVIIa-004 efficiently inhibited amidolytic activity of FVIIa (Fig. 3A-B). When plotted as residual FVIIa activity versus molar ratio sdAb/FVIIa, half-maximal inhibition was obtained at a molar ratio of 0.7±0.2 (Fig. 3B). Of note, no inhibition of thrombin or FXa amidolytic activity could be detected in control experiments (Fig. 3C). To analyze the nature by which KB-FVIIa-004 inhibits FVIIa amidolytic activity, a Schild regression analysis was performed [START_REF] Arunlakshana | Some quantitative uses of drug antagonists[END_REF]. When the log (dose ratio-1) was plotted against log [KB-FVIIa-004] in this analysis, a straight line was obtained (Fig. 3D), compatible with KB-FVIIa-004 acting as competitive inhibitor. In addition, the intercept of this line on the X-axis at Y=0 corresponds to the log (Ki) of the interaction, and the ensuing Ki was found to be 45±2 nM in this analysis.

Given the competitive nature of KB-FVIIa-004, we were also able to calculate a Ki-value from the half-maximal inhibition value of the data presented in Fig. 3A-B Effect of active site inhibitors on the interaction between FVIIa and KB-FVIIa-004-We next investigated whether binding of KB-FVIIa-004 is affected by the presence of the irreversible inhibitor D-Phe-Pro-Arg-chloromethyl ketone (PPACK). To this end, FVIIa was incubated with PPACK, and samples were subsequently tested in two separate assays. One portion of the sample was used to measure residual FVIIa activity, while a second portion of the sample was used to determine binding to immobilized sdAb KB-FVIIa-004. As expected, incubation with PPACK resulted in a dose-dependent loss of amidolytic activity (>90% loss at 10 µM PPACK; Fig. 4, right Y-axis), compatible with PPACK forming a covalent bond with the active-site serine. When simultaneously tested for binding to immobilized KB-FVIIa-004, a similar dose-dependent decrease in binding to this sdAb was observed (Fig. 4; left Y-axis).

This indicates that inhibition of the FVIIa active site by PPACK coincides with a loss of binding to KB-FVIIa-004. It seems possible that the epitope for KB-FVIIa-004 is close to or within the active site of FVIIa.

Effect of KB-FVIIa-004 on FX activation-Having observed that KB-FVIIa-004 interferes with

FVIIa amidolytic activity, we were interested to analyze the effect of this antibody on FVIIamediated activation of the macromolecular substrate FX. FX was incubated with FVIIa and phospholipids and varying concentrations of KB-FVIIa-004. Activation was stopped by the addition of EDTA and FXa activity was monitored by following S-2222 proteolysis. Using this approach, we observed a dose-dependent inhibition of FXa formation in the presence of KB-FVIIa-004 (Fig. 5A). Half-maximal inhibition was obtained at a sdAb/FVIIa molar ratio of 1.9±0.7 (Fig. 5C). Also for this inhibition, a Ki could be calculated from the Cheng-Prusoff equation, by using a KM of 0.6 µM for FVIIa-mediated FX activation [START_REF] Nielsen | Engineering of a membrane-triggered activity switch in coagulation factor VIIa[END_REF]. This approach generated a Ki value of 26±10 nM. Apparently, KB-FVIIa-004 displays a similar efficacy in blocking FVIIa activity towards small and macromolecular substrates.

Effect of TF on KB-FVIIa-004 mediated FVIIa inhibition-In view of the notion that FVIIa activity is strongly enhanced by its cofactor TF, it was of interest to reassess both amidolytic and FX-activating activity of FVIIa in the presence of relipidated TF and varying concentrations of KB-FVIIa-004. Little inhibition of FXa generation (<15%) was detected at a 1000-fold molar excess of KB-FVIIa-004 over FVIIa (Fig. 5B). With regard to amidolytic activity, some minor reduction in FVIIa activity was observed, that reached a maximum of 20-25% at a 50 to 100-fold molar excess of KB-FVIIa-004 (Fig. 6A-B). These data suggest that the presence of TF eliminates or strongly reduces the inhibitory activity of KB-FVIIa-004. This was also explored in another experiment, in which the ability of TF to reverse KB-FVIIa-004-mediated inhibition of FVIIa was investigated. FVIIa (7.5 nM) was incubated with the amidolytic substrate CBS-34.47 and various concentrations of KB-FVIIa-004. Despite the inefficient hydrolysis of CBS-34.47 under these conditions, a clear dose-dependent inhibition of FVIIa activity was observed, with >85% inhibition at a 100-fold molar excess of KB-FVIIa-004 over FVIIa (Fig. 6C-D). Upon the addition of relipidated TF, a rapid increase of substrate conversion was observed both in the absence and presence of KB-FVIIa-004 (Fig. 6C-D).

Although some residual inhibition remained under these conditions, this was less than 15% at the highest concentration tested (i.e. a 100-fold molar excess of KB-FVIIa-004 over FVIIa). Finally, we tested the direct effect of TF on binding of FVIIa to immobilized KB-FVIIa-004 in an immunosorbent assay (Fig. 6E-F). The presence of relipidated TF dosedependently interfered with binding of FVIIa to KB-FVIIa-004 (Fig. 6E). Interestingly, also recombinant soluble ectodomain of murine TF (smTF) reduced FVIIa binding to this antibody, albeit to a lesser extent (20±5%; p<0.0001) and at higher concentrations (Fig. 6F).

Effect of KB-FVIIa-004 on in vivo activity of FVIIa in FVIII-deficient mice-To apply KB-FVIIa-

004 in vivo, we generated a variant in which this sdAb was fused to human albumin. KB-FVIIa-004 has a molecular weight of approximately 15 kDa, and will thus be rapidly eliminated via renal clearance. Fusion to albumin was anticipated to improve its circulatory half-life. This fusion-protein, designated 004-HSA, was identical to KB-FVIIa-004 in its inhibitory action towards FVIIa, both in terms of efficacy and specificity to inhibit free FVIIa but not TF-bound FVIIa. For the in vivo experiments, FVIII-deficient mice were divided in 5 groups: one group receiving vehicle, one group receiving low-dose FVIIa (0.6 mg/kg), one group receiving high-dose FVIIa (3 mg/kg), one group receiving high-dose FVIIa (3 mg/kg) with a 3-fold molar excess of 004-HSA (5.3 mg/kg) and one group receiving high-dose FVIIa (3 mg/kg) with a 10-fold molar excess of 004-HSA (16 mg/kg). Five minutes following intravenous injection, the distal 3 mm of the tail was amputated, and blood was collected for 30 min. Blood loss in vehicle-treated mice was 805±83 µl (mean±SD; n=7; Fig. 7). Mice treated with low-dose FVIIa alone had mildly reduced blood loss (634±71 µl (n=5; p=0.0325 vs vehicle), while those treated with high-dose FVIIa had strongly reduced blood loss (139±79 µl; n=7; p<0.0001 vs vehicle). Interestingly, the presence of low-dose 004-HSA increased blood loss in mice treated with high-dose FVIIa to the same amount as found for low-dose FVIIa: 546±168 µl (n=4; p=0.0016 vs vehicle and p=0.62 vs 0.6 mg/kg FVIIa).

Moreover, high-dose FVIIa in combination with high-dose 004-HSA resulted in blood loss that was indistinguishable from vehicle-treated mice (773±42 µl; n=4; p=0.80 vs vehicle).

Apparently, the activity of FVIIa in vivo is inhibited by the presence of 004-HSA, suggesting that its functioning under these conditions is predominantly independent of TF.

DISCUSSION

Single-domain antibodies (sdAbs) have been shown to be useful tools for research, diagnostic and therapeutic applications, partially because they can associate with epitopes that are inaccessible to regular monoclonal antibodies. For example, we have previously identified sdAbs that specifically recognize the active, platelet-binding conformation of Von Willebrand factor, while others have developed sdAbs to monitor conformation changes of G Protein-coupled receptors [START_REF] Hulstein | A novel nanobody that detects the gain-of-function phenotype of von Willebrand factor in ADAMTS13 deficiency and von Willebrand disease type 2B[END_REF][START_REF] Groot | The presence of active von Willebrand factor under various pathological conditions[END_REF][START_REF] Manglik | Nanobodies to Study G Protein-Coupled Receptor Structure and Function[END_REF]. In this study, we have generated a sdAb, designated KB-FVIIa-004, that interferes with the enzymatic activity of FVIIa in the absence but not presence of its cofactor TF. KB-FVIIa-004 was isolated from a sdAb-library isolated from a FVIIa-immunized llama via standard procedures. Binding experiments using human plasma, purified FVII or FVIIa revealed that the antibody recognizes FVIIa but not FVII (Fig. 1). Interestingly, the ability of FVIIa to hydrolyze the small synthetic substrate CBS-34.47 was inhibited in a dosedependent manner by KB-FVIIa-004 (Fig. 3). By using the Schild-regression analysis, we determined that KB-FVIIa-004 inhibited FVIIa activity in a competitive manner. The efficacy by which KB-FVIIa-004 inhibits activity of FVIIa was determined via two independent approaches. First, the Schild regression analysis, which is based on activity assays in which the concentration of the substrate CBS-34.47 is varied. Second, by varying the KB-FVIIa-004 concentration at a fixed substrate concentration, which allows the calculation of the halfmaximal inhibition, and subsequently the Ki by applying the Cheng-Prusnoff equation.

Ultimately, three Ki values were obtained (two for amidolytic activity and one for proteolytic activity), which were all in the same range: 45±2 nM, 28±8 nM and 26±10 nM.

As mentioned, FVIIa requires interactions with TF to achieve full enzymatic activity, and this cofactor enhances both amidolytic activity and proteolytic activity of FVIIa [START_REF] Lawson | The evaluation of complex-dependent alterations in human factor VIIa[END_REF][START_REF] Neuenschwander | Importance of substrate composition, pH and other variables on tissue factor enhancement of factor VIIa activity[END_REF]. When KB-FVIIa-004 was tested for its inhibitory capacity towards FVIIa in the presence of TF, little if any inhibitory potential was observed (Figs. 5 &6). Even at a 100 to 1000-fold molar excess over FVIIa, inhibition was less than 15% towards FX and less than 25% towards CBS-34.47. The poor inhibitory capacity towards TF-bound FVIIa was confirmed in two control experiments. First, TF was added to KB-FVIIa-004-inhibited FVIIa, resulting in the reappearance of FVIIa amidolytic activity (Fig. 6). Second, a prolongation of the clotting time in the plasma-based PT assay (which depends on FVIIa/TF activity to initiate coagulation) was only observed when using a >300-fold molar excess of KB-FVIIa-004 over the endogenous FVIIa concentration (Fig. 2). It seems conceivable therefore that the binding of FVIIa to TF relieves FVIIa from the inhibitory effect of KB-FVIIa-004. To our knowledge, this is the first antibody that displays specific inhibition of FVIIa in the absence of TF. Several antibodies specific for FVIIa in comparison to FVII, with or without the capacity to modify FVIIa activity have been reported. However, none of these interferes with FVIIa activity in the absence but not presence of TF.

Considering the specificity of KB-FVIIa-004 for FVIIa in combination with its capacity to block FVIIa activity in a competitive manner, it is likely that this sdAb binds within or close to the active site of FVIIa. This would be compatible with the observation that inhibition of FVIIa by the active site-inhibitor PPACK, which covalently interacts with the active site serine-residue, coincides with loss of FVIIa binding to KB-FVIIa-004 (Fig. 4). However, at first sight this may seem incompatible with the notion that TF competes with the sdAb for FVIIa binding (Fig. 6E). A reasonable explanation for this finding is that KB-FVIIa-004 binds to a cryptic epitope close to the active site that is exposed only in the zymogen-state of FVIIa. When FVIIa engages in binding either to a substrate or to TF, it undergoes structural changes to adopt the active protease conformation. In this active conformation, the cryptic epitope for KB-FVIIa-004 is no longer exposed, and results in loss of antibody binding. This mode of action (illustrated in Fig. 8) would be compatible with the mutually exclusive binding of TF and the antibody to FVIIa, and would appear to be competitive inhibition in conventional binding assays. Definitive insight into the binding epitope for KB-FVIIa-004 will be obtained via cocrystallization studies, which have been initiated recently in our laboratory.

The unique characteristic of KB-FVIIa-004 to block FVIIa activity in the absence but not presence of TF could be helpful to better understand the physiology of FVIIa activity under various conditions. In particular, it has been a matter of debate whether the activity of FVIIa to restore hemostasis in FVIII-deficient plasma involves a TF-dependent or a TFindependent process. However, relatively high concentrations of FVIIa are required to achieve efficient hemostatic activity in humans and even more in mice: therapeutic dosing in humans is 90-270 µg/kg, resulting in FVIIa plasma levels of about 25-70 nM when assuming 50% in vivo recovery. Final plasma levels in mice are about 600 nM, when dosing 3 mg/kg and assuming 50% recovery. These concentrations exceed FVII plasma concentrations 2.5-7-fold and 60-fold, respectively. It was thought therefore that high FVIIa concentrations are needed to compete with endogenous FVII for binding to TF [START_REF] Rao | Studies of the activation of factor VII bound to tissue factor[END_REF][START_REF] Augustsson | In vitro evidence of a tissue factor-independent mode of action of recombinant factor VIIa in hemophilia[END_REF]. The need for FVIIa for TF-binding was further compatible with the observation that thrombin generation in FVIIainfused primates could be neutralized in the presence of TF-inhibitors [START_REF] Ten Cate | The activation of factor X and prothrombin by recombinant factor VIIa in vivo is mediated by tissue factor[END_REF]. However, other studies have contradicted these results, finding that FVIIa restores hemostasis in the absence of FVIII in a TF-independent manner. Evidence included efficient thrombin generation on TF-deficient platelets and the restoration of hemostasis in mice expressing minimal amounts of TF [START_REF] Monroe | Platelet activity of high-dose factor VIIa is independent of tissue factor[END_REF][START_REF] Hoffman | Human monocytes support factor X activation by factor VIIa, independent of tissue factor: implications for the therapeutic mechanism of high-dose factor VIIa in hemophilia[END_REF][START_REF] Lisman | Recombinant factor VIIa enhances platelet adhesion and activation under flow conditions at normal and reduced platelet count[END_REF][START_REF] Keshava | Pharmacological concentrations of recombinant factor VIIa restore hemostasis independent of tissue factor in antibody-induced hemophilia mice[END_REF]. We therefore used KB-FVIIa-004 to further gain insight into the in vivo mode of action of FVIIa in FVIII-deficient mice. As expected, a dose of 3 mg/kg FVIIa efficiently corrected bleeding in FVIII-deficient mice (Fig. 7). Correction was less efficient in mice receiving 0.6 mg FVIIa/kg, a dose corresponding to 20% of the efficacious 3 mg/kg dose. Pre-incubation of FVIIa with a 3-fold molar excess of KB-FVIIa-004 was anticipated to result in a 80%-inhibition of FVIIa. Indeed, mice receiving 3 mg/kg FVIIa in the presence of KB-FVIIa-004 displayed similar blood loss as mice that received 0.6 mg/kg (Fig. 7). In addition, a 10-fold molar excess of 004-HSA over FVIIa resulted in full reversion of FVIIa activity, with FVIII-deficient mice having similar blood loss as vehicle-treated mice (Fig. 7). It is important to mention that human FVIIa interacts poorly with murine TF [START_REF] Fang | Activation of factor X by factor VIIa complexed with human-mouse tissue factor chimeras requires human exon 3[END_REF][START_REF] Petersen | Characterization of recombinant murine factor VIIa and recombinant murine tissue factor: a human-murine species compatibility study[END_REF], suggesting that the contribution of murine TF in this FVIII-mouse model is limited.

Nevertheless, the high concentration of FVIIa (600 nM) would allow for some interaction with murine TF. In experiments using smTF, we did observe some inhibition (15-20%) of the FVIIa-antibody interaction by this protein. It seems plausible therefore that the in vivo data are compatible with the view that FVIIa generates its therapeutic effect in the absence rather than presence of TF.

All together, we have isolated a novel sdAb that interferes with FVIIa in the absence but not presence of its cofactor. This sdAb could be used to investigate the TF-independent activities of FVIIa in vitro and in vivo. Following conversion of FVII into FVIIa, the activated derivative exists in equilibrium between an inactive zymogen-like state and an active conformation (enzyme-like state). This equilibrium is forced into the active conformation upon binding of FVIIa to TF or via the incorporation of a substrate into the FVIIa active site. KB-FVIIa-004 does not interact with FVII or with TF-bound or PPACK-inactivated FVIIa. It seems possible therefore that KB-FVIIa-004 selectively binds to the zymogen-like state of FVIIa, and that its binding impairs conversion of FVIIa into the active conformation. Additional studies are needed to determine the exact binding epitope for this antibody.

LEGENDS

  have been backcrossed (>10 times) on a C57Bl/6 background. Males and females were used throughout the study (8-12 weeks old, 20-25g). Housing and experiments were performed in accordance with French regulations and the experimental guidelines of the European Community. This project was approved by the local ethical committee of Université Paris-Sud (comité d'éthique en experimentation animale n˚26) under the APAFIS number #4400-2016021716431023-V5. Materials-D-Phe-Pro-Arg-chloromethyl ketone (PPACK) was obtained from Santa Cruz Biotechnology (Dallas, TX). Purified recombinant FVIIa was from NovoNordisk (Malov, Denmark). Purified plasma-derived FVII was from Haematologic Technologies, Inc (Essex Junction, VT), and traces of FVIIa (less than 2%) were inactivated by incubation with PPACK and benzamidine. Purified plasma-derived FX was from Haematologic Technologies, Inc.

  , by applying the Cheng-Prusoff equation (Ki=IC50/(1+[S]/[KM]); (25)). Using a KM of 1.6 mM for FVIIa-mediated hydrolysis of CBS-34.47 (determined in separate experiments), the resulting Ki was 28±8 nM, which is in the same order of magnitude as the Ki value obtained from the Schild regression analysis.

Figure 1 :

 1 Figure 1: Binding of KB-FVIIa-004 to FVIIa. Microtiter wells coated with polyclonal anti-FVII/FVIIa antibodies (red symbols) or KB-FVIIa-004 (blue symbols; both 10 µg/ml) were incubated with various dilutions of human plasma (panel A) or various concentrations of purified FVII or FVIIa (panels B and C, respectively). Bound FVII/FVIIa was probed with peroxidase-labeled polyclonal anti-FVII/FVIIa antibodies and detected via hydrolysis of 3-3'-5-5'-tetramethylbenzidine. Plotted is the absorbance at 450nm versus FVII/FVIIa concentration. Data represent mean±SD of three experiments.

Figure 2 :

 2 Figure 2: Prolongation of PT clotting time by KB-FVIIa-004. Panels A-B: Human plasma spiked with various concentrations of KB-FVIIa-004 (blue symbols), control sdAb KB-VWF-006 (red symbols; both 0-10 µM) or polyclonal anti-FVII/FVIIa antibodies (0-0.4 µM; panel B only) was analyzed in an aPTT (panel A) or PT (panel B). Plotted is clotting time versus antibody concentration. Data represent mean±SD of three experiments.

Figure 3 :

 3 Figure 3: Inhibition of FVIIa amidolytic activity by KB-FVIIa-004. Panel A: FVIIa (75 nM) was incubated in the absence (red line) or presence (blue lines) of various concentrations of KB-FVIIa-004 (19-600 nM) for 60 min at 37°C. CBS-34.47 (1.5 mM) was added and its hydrolysis was monitored for 30 min by measuring absorbance at 405 nm. A typical experiment is shown in which absorbance at 405 nm is plotted versus time (s). Panel B: Presented is the percentage of residual FVIIa amidolytic activity relative to activity in the absence of sdAb as function of the molar ratio sdAb/FVIIa. Data representing mean±SD of 3-10 experiments is shown for KB-FVIIa-004 (blue symbols) or KB-VWF-006bv (grey symbols). Panel C: Hydrolysis of substrates S-2222 by FXa (5 nM; black symbols) or S-2238 by thrombin (5 nM; green symbols) in the absence or presence of KB-FVIIa-004 was determined. Shown is percentage residual FVIIa amidolytic activity relative to activity in the absence of KB-FVIIa-004 versus the molar ratio KB-FVIIa-004/FVIIa. Data represent mean±SD of three experiments. Panel D: Schild regression analysis of KB-FVIIa-mediated inhibition of FVIIa amidolytic activity. Plotted is the log [dr -1] versus log [KB-FVIIa-004]. dr = dose ratio. Data represent mean±SD of three experiments. X-axis intercept (Y=0) was calculated to be 1.65, corresponding to 44 nM KB-FVIIa-004.

Figure 4 :

 4 Figure 4: PPACK-mediated inhibition of FVIIa coincides with loss of binding to KB-FVIIa-004. FVIIa (0.1 µM) was incubated with various concentrations of PPACK (0-10 µM) for 60 min at 37°C. Samples were then analyzed in parallel for residual binding to immobilized KB-FVIIa-004 (left Y-axis, red symbols) and residual amidolytic activity (right Yaxis; blue symbols). The percentage residual binding and activity relative to the absence of PPACK is plotted versus PPACK concentration. Data represent mean±SD of 3-4 experiments.

Figure 5 :Figure 6 :

 56 Figure 5: Inhibition of FVIIa-mediated FX activation by KB-FVIIa-004. Panels A-B: FVIIa was incubated in the absence (red line) or presence (blue lines) of KB-FVIIa-004 for 60 min at 37°C, and samples were then added to a mixture containing relipidated TF, FX and phospholipids. FX activation was stopped after 15 min by the addition of EDTA. The presence of FX was measured by monitoring hydrolysis of substrate S-2222 (1 mM) for 5 min. Concentrations used were: Panel A: FVIIa: 25 nM, KB-FVIIa-004: 0-2.5 µM, FX: 0.5 µM, phospholipids: 40 µM. Panel B: FVIIa: 1 nM, relipidated TF: 2 nM, KB-FVIIa-004: 0-1 µM, phospholipids: 10 µM. Depicted are hydrolysis curves of a typical experiment in the absence or presence of relipidated TF. Hydrolysis curves were generated using undiluted samples for panel A, and samples diluted 100-fold for panel B. Panel C: Presented is percentage residual FVIIa activity relative to activity in the absence of KB-FVIIa-004 as function of the molar ratio sdAb/FVIIa. Shown are data representing mean±SD of 3-5 experiments.

Figure 7 :

 7 Figure 7: KB-FVIIa-004/albumin fusion protein interferes with FVIIa activity in FVIIIdeficient mice. FVIII-deficient mice were given vehicle or FVIIa (0.6 or 3 mg/kg) via intravenous tail vein injection. Two series of mice received FVIIa (3 mg/kg) that was preincubated with a 3-or 10-fold molar excess of KB-FVIIa-004 fused to human albumin (004-HSA; 5 and 16 mg/kg, respectively) prior to infusion. Five minutes after injection, the distal 3 mm segment of the tail of anesthetized mice was removed. The tail was immersed in a prewarmed physiological salt solution (37°C), and blood was collected for 30 min. Plotted is the amount of blood loss for each group. Bars represent mean±SD, and symbols individual mice. Statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test.

Figure 8 :

 8 Figure 8: Possible mode of action of KB-FVIIa-004.

ACKNOWLEDGEMENTS

Financial support of the Société Française d'Hématologie (Stephen Ferrière) is gratefully acknowledged.

CONFLICT OF INTEREST

The authors declare that they have no conflict of interest with the contents of this article. AUTHOR CONTRIBUTIONS SF, CK, JFO: performed experiments and analyzed data; CVD, AK, ODC and PJL: designed research, analyzed data, and supervised the study. PJL wrote the manuscript with input of all authors.