MX2008001587A - Stimulators of factor x activated (fxa) as new topical antihemorrhagic agents - Google Patents

Abstract

The activated coagulation Factor X (FXa) stimulating agents may be used in the treatment of hemorrhages in a subject. Compounds and combinations are described which are particularly useful for the topical treatment of hemorrhaging in healthy subjects or in patients with hemorrhagic diathesis.

Description

STIMULATORS OF FACTOR X ACTIVATED AS NEW ANTI-HEMORRHAGIC AGENTS OF TOPICAL USE FIELD OF THE INVENTION The present invention relates to the treatment of hemorrhages in a subject by the use of FXa stimulators. This invention is based on the discovery that lipidized tissue factor (TF) exerts a new regulatory role by stimulating all the proteolytic activities (prothrombin and amidolytic hydrolytic activity) of both forms of FXa, the soluble and the prothrombinase complex bound.

BACKGROUND OF THE INVENTION 1. Physiology of coagulation Hemostasis is the mechanism by which living beings respond to a hemorrhage and involves the participation of two processes that acquire functionality immediately after an injury and remain active for a long period of time . The first of these is known as primary hemostasis and is characterized by the appearance of vasoconstriction at the point of vascular injury and the formation of platelet aggregate. The second is known as secondary hemostasis, the phase in which the fibrin clot is formed by the action of the different proteolytic enzymes of the coagulation cascade. The platelet aggregate plays a key role in the hemostasis of the capillaries, having special relevance in mucocutaneous hemorrhages. On the contrary, the formation of the fibrin clot is much more important in the hemostasis of the great vessels, acquiring greater relevance in the internal hemorrhages (gastrointestinal, cerebral, etc.). During the formation of the platelet aggregate, the following phases can be distinguished: (i) adhesion of the platelets to the surface of the sub-endothelium exposed by the lesion; (ii) release of the granular content of the platelets, in response to their activation; (iii) aggregation of the platelets with the consequent sequestration and concentration of more platelets at the site of the lesion; and (iv) binding of fibrinogen, as well as other coagulation proteins to the platelet surface, to produce thrombin and form the fibrin clot that will allow the platelets to fuse and consolidate, thereby stabilizing the hemostatic clot. Furthermore, it is well known that the amount of platelets is critical for the formation of the fibrin clot; Amounts of platelets below 20,000 per ID are accompanied by severe bleeding episodes. In the second phase of the blood coagulation process, various proteolytic enzymes and cofactors participate, all referred to as coagulation factors, and consists of several phases that end with the formation of fibrin from the hydrolysis of fibrinogen by the action of the thrombin. Additionally, thrombin production enhances the platelet aggregate by increasing the activation and aggregation of more platelets. Thrombin is previously formed by the proteolytic hydrolysis of an apoenzyme, prothrombin. This proteolysis is performed by the FXa serine protease that binds to the surface of the activated platelets and only in the presence of its cofactor, the activated coagulation factor V (FVa), and calcium ions, this serine protease is able to hydrolyze the prothrombin. FXa can occur through two separate pathways, the intrinsic pathway and the extrinsic pathway. The intrinsic pathway consists of a series of reactions that mainly involve coagulation factor VIII (FVIII), coagulation factor IX (FIX) and coagulation factor XI (FXI), in which each proenzyme is hydrolyzed giving its active form protease (FVIIIa, FlXa and FXIa). In each step, the newly formed proteolytic enzyme will catalyze the activation of the next proenzyme to successively yield the active form. The activation of the different coagulation factors involved in the intrinsic path takes place; therefore, as a cascade, a deficiency of any of the intrinsic pathway proteins blocks the activation of the next step, preventing the formation of the clot and increasing the hemorrhagic tendency. Deficiencies in various coagulation factors, for example, in FVIII, FIX or FXI cause severe hemorrhagic syndromes, such as Haemophilia A, B and C, respectively. In the extrinsic pathway of blood coagulation, the FT exposed by the adventitia cells at the site of injury, binds to coagulation factor VII / activated coagulation factor VII (FVII / FVIIa) to form the FT complex: : FVIIa and, in the presence of calcium, act as a substrate for FX activation. The extrinsic pathway is currently considered the most relevant route in blood coagulation and it is accepted that, in the event of hemorrhage caused by a vascular lesion, coagulation is triggered by the activation of the extrinsic pathway, which involves the interaction of the FT with its ligand, FVII / FVIIa. Another of the roles assigned to the FT :: FVIIa complex in coagulation is to act as a substrate for the activation of FX by FVIIa. As a result, FXa's baseline levels increase (<150pM), which initially are insufficient to generate the formation of the fibrin clot. This increase in the basal concentrations of FXa in the presence of its cofactor, FVa, and of a cellular procoagulant surface, will indeed be capable of producing the thrombin necessary for the formation of the fibrin clot. It is now accepted that platelets, once activated, play a key role in blood coagulation. They provide the procoagulant surface rich in anionic phospholipids and, on the other, expose the FVa and FXa factors stored inside. All this allows the correct assembly of the different agents involved in the coagulation on the surface of their plasmatic membranes forming the well-known prothrombinase complex (which includes FXa, FVa, prothrombin, and a procoagulant platelet phospholipid surface). The theory of the activation of the extrinsic pathway is able to explain how coagulation begins through the role that has been attributed to the FT :: FVIIa complex. An example that illustrates the biological relevance of the FT :: FVIIa complex in the blood coagulation process is Disseminated Intravascular Coagulation Syndrome (DIC). This clinical condition is associated with the intravascular release of TF and can occur in the course of severe clinical conditions (shock, sepsis, cardiac arrest, major trauma, liver disease, major surgery, burns, etc.). Evidently in a context in which FVIIa is not present, as in a congenital deficiency of this factor, coagulation hypothetically will never take place since FX will not activate at sufficient levels and, as a consequence, the hemorrhagic manifestations will be fatal. Murine models confirm this theory and deficiency in FVII is incompatible with life, being accompanied by severe fatal hemorrhages. However, deficiencies of congenital FVII described in humans up to now, are not always accompanied by hemorrhages. Cases of complete FVII deficiency have been reported with no clinical symptoms and occur in healthy individuals without bleeding complications. All this suggests that other independent coagulation trigger mechanisms of FVII must exist in humans. FT is an integral membrane glycoprotein belonging to the class II cytokine receptor superfamily that binds specifically to FVII / FVIIa and plays an important role in the extrinsic pathway of blood coagulation. The physiological roles assigned to TF are well known; on the one hand, specific receptor for the FVIIa and, once the FT :: FVIIa complex has been formed, to act as a substrate for the activation of the FX to take place. Indeed, after a vascular lesion, the TF, which is normally sequestered on the surface of the adventitia cells that externally surround the blood vessels, comes into contact and interacts with its ligand, the FVII present in the blood to form the FT complex :: FVII. Once this complex is formed, the autoactivation of the FVII takes place, rendering its active form FVIIa. Currently there is extensive information on the structure of the FT :: FVII complex. The main binding sites of FVII that participate in the interaction with FT are located in the first domain similar to that of the epidermal growth factor (EGF) and in the protease domain. On the other hand, it has also been described that other binding sites with less relevance are participating (domain rich in 4-carboxyglutamate (domain Gla) and the second domain EGF). The binding sites present in the TF are located in the two domains fibronectin type III and in the intermediate region between both domains. Recent studies have made it possible to identify which Lysine 165 and Lysine 166 FT residues interact with the Gla domain of FX both in the activated form and in the non-activated form. However, unlike what happens with the information regarding the FT :: FVIIa complex, little is known about the FT interaction with the FX and the FXa. First, the data suggest that Lysine residues (165 and 166) act as a substrate for FX activation. On the other hand, it has recently been described that FT can act as a FXa factor for the activation of FVII. That is, the binding of FVII to FT stimulates the autoactivation of FVIIa and the activation of FX. Then, the FXa bound to the FT stimulates the activation of FVII which, in turn, will increase the activation of the FX and as a consequence the hydrolysis of the prothrombin and the formation of the fibrin clot. The inventors have discovered that the postulated role of TF as a cofactor for FXa is much more relevant and becomes critical for hemostasis. Despite the accepted role of FT as a membrane receptor for FVII, the inventors have shown that FT is also a potent stimulator of FXa. The FT acts as a stimulator of the FXa producing a significant increase in its proteolytic activity. "FXa Stimulators" as used in the present description refers to all forms of FXa, such as soluble FXa and FXa bound to the prothrombinase complex. It is well known that FXa at picomolar concentrations is unable to produce any effect on coagulation, even in the presence of its well-known cofactor, the FVa (see Table 9). Therefore, under these conditions the prothrombinase complex is not active. Surprisingly, in the presence of FT (ie, endogenous administration or damage), FXa at picomolar concentrations (i.e., basal physiological concentrations or exogenously administered) causes prothrombin hydrolysis, leading to the formation of the fibrin clot, even in the absence of FVIl / FVIIa (Table 7). In the present patent application the inventors describe that the FT:: FXa interaction is a new trigger mechanism of the coagulation mechanism independent of the FT :: FVIIa complexes and the extrinsic coagulation pathway. Finally, it is well known that there are certain platelet diseases that occur with disorders in platelet aggregation and a greater tendency of hemorrhagic episodes, among which Glanzmann's disease and Bernard-Soulier syndrome stand out, where congenital defects have been described. they affect the fibrogen receptor or the Gplb receptor, respectively. On the other hand, severe hemorrhagic episodes are present in acquired and congenital thrombocytopenic disorders when the amount of platelets drops below 20,000 per ID. In the present patent application, the inventors have demonstrated that lipidized TF is also effective in the treatment of hemorrhages present in acquired, congenital platelet diseases and in severe thrombocytopenic disorders (below 9,000 per ID). 2. Coagulation pathology Congenital deficiencies of each coagulation factor can be associated with the appearance of hemorrhages and generally involve a single protein; thus, for example, hemophilia A is a hereditary hemorrhagic disease that affects FVIII. Acquired coagulation diseases appear in individuals with no previous history of bleeding and may have multiple origins; By way of illustration, the presence of specific inhibitors for coagulation factors can appear in individuals who have undergone many transfusions. Although the deficiencies of acquired coagulation factors are an unknown etiological entity that also cause serious hemostatic problems, they are also one of the most important problems in the polytransfusion to which patients with congenital coagulopathies are subjected. Another important source of acquired coagulation disorders are anticoagulant therapies, such as the drugs heparin and warfarin. A significant percentage of patients (5-10%) treated with anticoagulant drugs have episodes of bleeding, most of them difficult to manage. As previously mentioned, acquired and congenital platelet disorders can also be associated with hemorrhages. Decreases in the number of platelets (below 20,000 per ID) can cause fibrin clot impediment frequently accompanied by severe bleeding episodes. The therapeutic arsenal currently available for mild / moderate or severe / fatal hemorrhage (due to surgery or external trauma) is very limited. There are different hemostatic agents that are able to accelerate blood coagulation and prevent bleeding, for example, (1) blood products derived from humans, such as fibrillar collagen, fibrin glue and prothrombin complex concentrates; (2) recombinant human proteins; (3) antifibrinolytic drugs, such as aminocaproic acid, tranexamic acid; and (4) local hemostatic agents, such as kaolin and silica surfaces. The following critical limitations have been reported: (1) Intravenous administration (2) Requirement of a special device for administration (3) Narrow therapeutic approach (4) Annoying or inappropriate treatment to be administered in specific bleeding episodes (5) Lack of serious effect (6) Instability of the fibrin clot (7) Very dangerous side effects (8) Long-term treatment Blood products derived from humans (coagulation factors and platelet concentrates) The treatment that should always be administered intravenously is very expensive and is rarely available. It has a very narrow therapeutic approach, it is only useful to treat its specific deficiency. It is inappropriate for the topical treatment of any episode of bleeding. It is not useful to treat hemorrhage acutely because it requires prolonged administration protocols to be effective, and above all it is a very dangerous treatment: 20% of hemophiliac patients have developed hepatitis, 5% HIV, and up to 15% they present plasma antibodies against FVIII or FIX (acquired hemophilia) that requires very expensive and special substitution treatments (immunosuppressants, high doses of coagulation factors, plasmapheresis, etc.). For these reasons, public health organizations (WHO, FDA, EMEA, etc.) are very interested in the development of new hemostatic agents better than coagulation factor concentrates. Local hemostatic agents It is an expensive treatment, inappropriate to be administered in some episodes of bleeding (ie, epistaxis), annoying for dental treatment, it forms an unstable fibrin clot, and as coagulation factor concentrates have the same potential side effects dangerous. They should not be used in patients who have never received blood products derived from humans or those who are being treated with recombinant FIX or FVIII due to the potential risk of human viral transmission.

Human recombinant proteins It is the most expensive treatment (an average cost of 6,000 euros) only available for developed countries. As coagulation factor concentrates, it should always be administered intravenously, it has a very narrow therapeutic focus because it is only useful to treat its specific deficiency, it is inappropriate to treat haemorrhages topically, it is not useful to treat a haemorrhage acutely because it also requires prolonged administration protocols to be effective, and although no human viral transmission has been reported, the same percentage of acquired hemophilia has been described (up to 15% have antibodies against FVIII or FIX). As coagulation factor concentrates, public authorities greatly limit their use.

Antifibrinolytic drugs They have a narrow therapeutic focus, this being their most relevant limitation. These drugs require prior formation of the fibrin clot to be effective. Therefore, they are only useful in healthy subjects, however when the fibrin clot is formed inappropriately (ie, congenital coagulopathies, such as hemophilia, FVII deficiency) their therapeutic efficacy decreases dramatically. In addition, they are not useful for acute treatment of hemorrhage because they also require prolonged administration protocols to be effective.

Inorganic local hemostatic agents The most important restriction for the use of these hemostatic agents is that they are inappropriate to be administered in many types of haemorrhages, such as epistaxis, dental and surgical. In addition, a painful exothermic reaction has been reported, which significantly reduces its use only for mucocutaneous bleeding in critical situations (wars). In conclusion, surprisingly today there are still no useful drugs available for the topical treatment of a simple episode of epistaxis or gingival bleeding after tooth brushing or simply before a daily wound caused by shaving, by the venous puncture of a blood extraction, or by the wound of a fortuitous fall in the street. The problem is even more acute in the case of patients with hemorrhagic diathesis, for example, with congenital coagulopathies of the Hemophilia type or of von Illebrand disease or patients with congenital plaque alterations, of the Glanzmann's type or of the Bernard-Soulier syndrome, or acquired. These patients have serious problems to face the day to day, and before a simple dental extraction or before any small trauma that causes them a bleeding wound, they do not have any medical treatment that improves their quality of life. Obviously the problem is transformed into a much bigger one when these patients suffer an external trauma or a serious bleeding accident since their life is in serious danger. In view of all these situations, only the human plasma containing the deficient factors or the specific human recombinant for each coagulation factor is available as a pharmacological tool. All these therapies involve the use of the parenteral route and, therefore, are not designed to be used with high frequency, as it should be, for example in the face of any daily external bleeding of slight or moderate importance. Finally, it is widely accepted that new local hemostatic agents without the limitations previously described, will represent a significant improvement of the present treatment that will reduce both the cost and the high prevalence of side effects. 3. BACKGROUND OF THE INVENTION Until now it has been accepted that FT is the main element responsible for triggering blood coagulation. For the start of coagulation, the activation of FXa to FXa is absolutely necessary to start prothrombin hydrolysis. The source of this FXa has been attributed mainly to the interaction of FVIIa with its receptor, FT. Although it has been described that FXa is present in the platelet granules and that it can be exposed on the surface when its activation takes place, the physiological concentrations of FXa (<150 pM) present in blood are insufficient to start the formation of thrombin, even in the presence of its cofactor, FVa and a platelet procoagulant surface (figure 1). Therefore, it is now accepted that coagulation can start only when the basal concentrations of FXa increase significantly. The source of the increase in basal FXa concentrations has always been attributed to both the FT :: FVIIa complex and the proteolytic activities of FlXa (figure 2). Recombinant lipidized FT proteins have only been able to accelerate coagulation under in vitro conditions, both in hemophilic and healthy blood samples, attributing this action to a classic role assigned to FT as a FVIIa receptor. However, under the same experimental conditions, non-lipidized TF has shown complete absence of effect, which indicates that lipidation is necessary to achieve the functionality of the TF (section 6.1 of the results). The use of lipidized FT as a topical haemostatic agent has never been described as a unique treatment for lethal, severe or mild bleeding (venous or arterial and traumatic surgical hemorrhages). European patent EP266993 describes the use of non-lipidized TF as a hemostatic agent for the parenteral treatment of hemorrhagic syndromes. However, the same patent describes the important differences in activity between the non-lipidized TF claimed by EP266993 and the lipidized TF that is the subject of the present patent application. In fact, it is well known that lipidized TF is active under in vitro conditions and its parenteral administration immediately initiates disseminated intravascular coagulation with fatal consequences. In contrast, non-lipidized TF is not active under in vitro conditions however it has been claimed (EP266993) for the treatment of coagulopathies by parenteral administration. To date, there is no data on the effect of both lipidized and non-lipidized TF for the individual topical treatment of bleeding episodes. In the present patent application, using the rat tail section model, the inventors have demonstrated that the non-lipidized TF was unable to stop the bleeding. On the contrary, the inventors have shown for the first time that. Lipidized TF is a useful hemostatic agent to treat topically all types of hemorrhages, which include pathological conditions (animals treated with heparin and warfarin) and healthy ones (control rats without alteration of coagulation). Taken together, it indicates that the lipidized and non-lipidized TF are clearly different compounds. As a consequence, the use of lipidized TF as an agent for the individual topical treatment of haemorrhages is not obvious to the person skilled in the art. On the other hand, EP266993 was carried out according to the state of the art which postulates that the FVII serine protease is activated only when it binds to its receptor, FT. Therefore, according to EP266993 and the state of the art, when FVII / FVIIa is not present, the FT should not be active. It was not obvious to think for any expert in the field that FT (lipidized and non-lipidized) could be effective in the treatment of patients deficient in FVII. The inventors have discovered that even in the absence of FVIIa, FT acts as a cofactor for FXa. This discovery is fundamental to understand that unlipidated TF can also act as a parenteral hemostatic agent for bleeding in patients deficient in FVII. International patent application WO94 / 02172 teaches that the temporary inhibition of one or more natural anticoagulants by the systemic administration of an inhibitor of a natural anticoagulant (an antibody) can inhibit microvascular bleeding. Optionally, the inhibitor can be administered in combination with a topical administration of thrombin or lipidized TF. It is important to note that the use of both compounds was always as optional adjuvant treatments and never as an individual treatment. further, WO94 / 02172 claims the use of these synergistic treatments only for capillary bleeding (microvascular bleeding, ie, burns, inflamed visceral surfaces, bleeding liver surface ...) and never for lethal or severe hemorrhages caused by external trauma or surgery which involve venous and arterial damage. However, the inventors of WO94 / 02172 admit that, although they observed a synergistic effect when topically administered thrombin in combination with the systemic treatment, no such synergistic effect was observed when FT is administered topically (WO94 / 02172, Figure 3 and page 22, lines 14-15). In addition, the dose claimed in O94 / 02172 is very high, from 0.1 to 10 mg. Contrary to WO94 / 02172, the present patent application is claiming the individual treatment of venous or mild / moderate to severe / lethal arterial haemorrhage with lipidized TF only by showing examples in which such treatment is effective at an active protein dose of 1, 2 Dg / ml for traumatic hemorrhages (see severe model in tables 24 and 25). Such surprising and extraordinarily effective treatment has not been described so far, because it was not obvious to any person skilled in the art that lipidized TF would act as a stimulator of the proteolytic activity of FXa. The inventors have discovered that in the absence of FVIIa, FT acts as a cofactor for FXa, even in the absence of its well-known cofactor, FVa. This discovery is fundamental to understand that lipidized TF can only act as a hemostatic agent for severe hemorrhages in pathological and healthy conditions.

U.S. Patent 4,721,618 teaches that administration of the synergistic mixture of phospholipids (PCPS) and FXa administered intravenously at high concentrations (0.2 to 0.5 U / Kg) can bypass Factor VIII: C deficiency in a hemophiliac mammal, so that the cascade process of blood coagulation can continue. The suggested high concentrations of FXa are active without the need for lipid vesicles PCPS (which can raise this activity). Also, WO02 / 086118 teaches that compositions that include a mixture of at least one specific phospholipid and at least one blood coagulation factor activated by serineproteses are useful for the treatment of alterations in blood coagulation that decrease the need to administer blood coagulation factors. . In addition, ellagic acid and other accelerators such as zeolite, silica and inorganic oxide materials have been used to increase blood coagulation and are claimed in WO02 / 30479. According to WO02 / 086118, a coagulation factor is defined as a blood coagulation factor activated by serine protease (page 5, lines 13-15). Therefore, FT can not be considered as a blood coagulation factor, because FT is not a serine protease, but a specific cell surface receptor for factor Vlla. In the present patent application, the inventors have shown that phospholipids (phosphatidylserine and phosphatidylcholine at different percentages and molarities) do not increase the procoagulant effect mediated by lipidized TF (Table 21). This indicates that the synergistic effect claimed by US Patent 4,721,618 and O02 / 086118 is exclusive for blood coagulation factors activated by serine protease, but not for membrane receptors such as FT. Surprisingly, when the lipidized TF was combined simultaneously with negatively charged inorganic surfaces (SICN) a significant synergistic effect was observed under experimental conditions in vivo and in vitro. The SICN used in this description is constituted by a mixture of lipids and an accelerator of blood coagulation, ellagic acid. Lipids have no negative charge, which means that the lipid mixture can include neutral or zwitterionic lipids, but must contain a certain amount of negatively charged lipid that confers anionic character to the mixture. By way of illustration, not limitation, examples of negatively charged lipids may be sphingolipids (such as ceramide-1-phosphates, glycosylated phosphatidylethanolamine, hydroxylated or non-hydroxylated sulfatides, gangliosides) and glycerol-based lipids (such as phosphatidylserine, phosphatidylinositol, phosphatidylinositol phosphate, phosphatidic acids, phosphatidylglycerols, cardiolipins). Commercially available SICN such as Dade® Actin® (Dade Behring) brand, for example, DADE® Actin® FS.

In conclusion, the present patent application describes a new local hemostatic agent characterized by the following advantages over commercially available drugs: (1) Easy topical administration (2) Does not require special devices for topical administration (3) Wide therapeutic approach ( deficits of FV, FVII, FVIII, FIX, FX, FXI, FXII and FXIII) (4) Appropriate treatment to be administered in all episodes of bleeding (epistasis, dental bleeding, mucocutaneous, surgical and traumatic hemorrhages) (5) Exerts a potent acute effect (6) Formation of physiological fibrin clot (extreme clot stability) (7) No side effects (8) Low cost treatment The main goal of HO, FDA, EMEA and other public health authorities is to reduce the dangerous side effects associated with the consumption of blood products derived from humans. The present invention has been designed to meet these needs.

COMPENDIUM OF THE INVENTION The role assigned to the FT :: FVIIa complex in coagulation is widely known. The FT :: FVIIa complex acts as a substrate so that FX activation takes place. Recent studies have made it possible to identify that the remains of Lysine 165 and Lysine 166 of the FT interact with the Gla domain of FX, both in the activated and not activated forms. That is, the binding of the FXa to the FT would stimulate the activation of the FVII, which, in turn, will increase the activation of FX. The inventors have discovered that the role of FT as stimulator of FXa is much more relevant, becoming critical for hemostasis (figure 3). It is well known that FXa at picomolar concentrations is unable to produce any effect on coagulation and platelet aggregation. Despite the accepted role of FT as a membrane receptor for FVII, the inventors have shown that FT also acts as a stimulator of FXa to increase its main proteolytic activity, the hydrolysis of prothrombin, leading to the formation of the fibrin clot.

Surprisingly, in the presence of FT (ie, exogenous or damage administration), FXa at picomolar concentrations, causes the hydrolysis of prothrombin, leading to the formation of the fibrin clot, even in the absence of its ligand, FVII / FVIIa. The overall results indicate that FT acts as a stimulator of FXa, and the specific binding of FXa to FT is the first step that triggers blood coagulation. Our data suggest that the rapidity with which coagulation takes place is dependent on the interaction between the FT and in all previously described forms of FXa. In the presence of physiological concentrations of FXa (<150 pM) unable in themselves to initiate coagulation, rapidly lipidized TF is able to initiate thrombin formation, as a result of its action as a cofactor. Surprisingly, these hemostatic effects are independent of the presence of FVII and FVIIa, so the procoagulant effect mediated by the endogenous complex FT:: FXa (FXa < 150 pM) is especially useful and relevant in samples deficient in Factor VII. However, potent hemostatic effects have also been observed in samples of patients with congenital defects of other coagulation factors such as: FVIII (hemophilia A), FIX (hemophilia B), FXI (hemophilia C), FV, FX, FXII and FXIII as well as in individuals with congenital alterations of platelets, such as Bernard Soulier syndrome and Glanzmann's disease and thrombocytopenic alterations. Therefore, the present invention is based on the use of lipidized TF, alone or in combination with FXa and / or with SICN, as a new FXa stimulator, useful for the topical treatment of hemorrhages present in healthy individuals and in patients with hemorrhagic diathesis. The results obtained by the inventors open the door to the use of non-lipidized TF, or a functional fragment thereof, for the treatment of hemorrhages in a subject with a deficit in FVII. Said treatment can be carried out by the use of pharmaceutical dosage forms suitable for the parenteral administration of non-lipidized FT. Therefore, in one aspect, the invention relates to the use of lipidized TF, or a functional fragment thereof, in the manufacture of a medicament for the topical treatment of hemorrhages in a subject.

In another aspect, the invention relates to a product comprising lipidized FT alone or in combination with FXa and / or SICN. The use of said product as a medicament or in the manufacture of a medicament for the treatment of hemorrhages in a subject constitutes a further aspect of this invention. In another aspect, the invention relates to a complex formed by lipidized TF and a compound selected from FXa, a SICN and combinations of both. The use of said complex as a medicament or in the preparation of a medicament for the treatment of hemorrhages in a subject constitutes a further aspect of this invention. In another aspect, the invention relates to a pharmaceutical composition comprising a lipidized TF, together with a pharmaceutically acceptable carrier. In a particular embodiment, said pharmaceutical composition further comprises FXa and / or a SICN. In another aspect, the invention relates to a product comprising said pharmaceutical composition and a support. In a particular embodiment, said pharmaceutical composition further comprises FXa and / or a SICN. In another aspect, the invention relates to the use of non-lipidized TF, or a functional fragment thereof, for the preparation of a medicament for the treatment of hemorrhages in a subject with a deficit in FVII. In a particular embodiment, said medicament is formulated in a pharmaceutical dosage form suitable for the parenteral administration of non-lipidized FT.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a schematic representation showing the basal concentrations and the formation of the fibrin clot. Figure 2 is a schematic representation showing the classical intrinsic and extrinsic pathways of blood coagulation. Figure 3 is a schematic representation showing the novel regulatory effect of the lipidized TF as a stimulator of the FXa according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION The findings described herein demonstrate, for the first time, that lipid FT in the absence of its ligand FVII / FVIIa, acts as a stimulator of FXa causing this enzyme to initiate the hydrolysis of prothrombin and, consequently, coagulation takes place. This new mechanism of action has special relevance at very low levels of FXa concentration (below those considered as physiological 150 pM). It is well known that there are certain platelet diseases that occur with alterations in platelet aggregation and greater tendency to present hemorrhagic episodes. Consequently, the lipidized FT administered exogenously is not only useful in congenital coagulopathies but also, it will allow haemostasis in the congenital and acquired platelet alterations, such as Glazmann's disease, Bernard-Soulier syndrome and thrombocytopenic alterations.

The rapidity with which coagulation takes place is not, therefore, dependent on the formation of the FT :: FVIIa complex and the consequent activation of the FX, as it has been postulated to date, but the interaction of the FT and FXa at concentrations physiological basics (< 150 pM). The most relevant biological consequence of the formation of this interaction is the rapid generation of the start of coagulation and that this only requires the presence of FXa at physiological concentrations present in blood and the interaction with its stimulator, the FT. The process will be amplified immediately, thanks to the action of the thrombin initially formed by the complex and the increased production of FXa either by the FT :: FVIIa complex or by the same thrombin. Therefore, the findings of the inventors clearly show that in the absence of FVIIa and in the presence of FXa at physiological concentrations (<150 pM) unable in themselves to initiate coagulation, the lipidized FT rapidly initiates thrombin formation thanks that its new action as a cofactor of FXa promotes the initiation of the hydrolysis of prothrombin. On the other hand, the inventors' findings show that lipidized TF and FXa at low concentrations (which are not themselves effective in exerting anti-hemorrhagic effects), when administered together, are capable of rapidly causing the onset of clot formation of fibrin. The effects observed with the combination FT :: FXa, in particular with a concentration of FXa < 150 pM, are much better than those detected when only the lipidized TF is administered. The combination of FXa and FT lipidized with SICN can increase the hemostatic effect even more. The results obtained support the idea that in the presence of basal concentrations of FXa present in the plasma (<150 p), incapable of generating the initial procoagulant response by itself, this occurs when FT acts as an FXa stimulator. Consequently, lipidized TF, alone or in combination with FXa and / or SICN, can be used as new topical hemostatic agents useful for the treatment of hemorrhages present in both healthy individuals and patients suffering from hemorrhagic diathesis. Likewise, non-lipidized TF or a functional fragment thereof can also be used as a new hemostatic agent useful for the treatment, preferably parenterally, of hemorrhages present in patients affected by FVII deficiency.

I. Use of Lipidized TF in Topical Hemorrhage Treatment The inventors have found, surprisingly, that the Lipidized TF increases the proteolytic activity of FXa and, consequently, the production of thrombin [see section 1 of the Results of the Example that accompanies this description]. The inventors have also observed that lipidized TF coagulates plasma and blood of healthy subjects and of patients deficient in coagulation factors, including deficient in VF, FVII, FVIII, FIX, FX, FXI, FXII and FXIII, as well as in patients with anticoagulation (by anticoagulant treatment with drugs such as heparin, low molecular weight heparins or coumarin derivatives, these being non-limiting examples) both in vitro [see sections 1 to 5 of the Results of the Example that accompanies this description] as in vivo [see sections 6 and 7 of the Results of the Example that accompanies this description]. Furthermore, the inventors have found that lipidized TF coagulates the blood of patients afflicted with platelet alterations [see section 5 of the Results of the Example that accompanies this description]. These results show that lipidized TF is a useful anti-hemorrhagic agent for the topical treatment of hemorrhages in a subject. Taking into account the state of the art, it is not obvious to think of TF as a topical procoagulant individual treatment in healthy subjects or hemophilic patients (deficient in FVIII, FIX and FXI). It was unlikely to think of patients deficient in FX because the prothrombinase complex can not be assembled; and in heparinized patients because thrombin and FXa are blocked by antithrombin III, and in patients treated with warfarin because the synthesis of all vitamin K-dependent coagulation factors (FVII, FIX, and FX) are inhibited. Therefore, in one aspect, the invention is directed to the use of the lipidized TF, or a functional fragment thereof lipidized, in the preparation of a medicament for the topical treatment of hemorrhages in a subject. FT is a membrane integrating glycoprotein widely distributed in the animal kingdom. The TF protein has a structure in domains, that is, it is a protein with independent functional regions. Each of the human TF apoprotein domains has unique structural and functional characteristics: (1) a signal peptide or a region with a 32 amino acid leader sequence that is processed post-translationally when the protein is processed immature to form mature (2) an N-glycosylated hydrophilic extracellular domain comprising approximately 219 terminal amino acids; (3) a fragment of approximately 23 amino acids, mainly hydrophobic, which are believed to be the amino acids of the transmembrane domain; and (4) the carboxyl terminus of 21 amino acids, which is believed to be the amino acids that are part of the cytoplasmic fragment of the protein. This structure in domains that the human TF protein presents allows the production of, for example, the extracellular domain of the protein or functional fragments thereof. The amino acid sequence of the human TF protein is known and can be consulted in protein databases such as, for example, NCBI (human FT, accession number: P13726). The term "lipidized TF", as used herein, refers to any source of TF, this TF being totally or partially inserted into lipid vesicles or cell membranes. Illustrative, non-limiting examples of sources of "lipidized TF" are: extracts containing lipidized TF (whose isolation can be carried out from various tissues such as brain, placenta and lung tissue, tissue from different animals such as sheep, cows, rabbits , dogs, human beings, etc.); Protein component of purified and (re) lipidized FT (purified from an extract or from recombinant FT), that is to say, that the lipid component has been added after its purification and that can be prepared, by way of illustrative example, not limitative, according to the protocol previously described by Morrisey [see the Example that accompanies this description]. In said protocol, the non-lipidized TF is incorporated into phospholipid vesicles using a non-ionic detergent, such as, for example, N-octyl-beta-D-glucopyranoside. The lipids that can be used in the lipidized FT according to the invention can have any origin (animal, vegetable or synthetic). Virtually any phospholipid can be used in the preparation of the lipidized TF of the present invention. Illustrative, non-limiting examples of lipids that can be used in the preparation of lipidized TF include phospholipids (such as phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, etc.) sphingolipids (such as ceramide, sphingosine-1-phosphate, ceramide inositol phosphate, ceramide mannosyl-inosium phosphate, ceramide, mannosyl-diinositolfosphate, etc.), phosphatidylinositol (such as La-phosphatidylinositol, La-lysophosphatidylinositol, etc.) and phosphatidylinositol phosphates (such as La- [phosphatidylinositol-4-phosphate], 1,2-di-octanol-n-glycero- 3 - [phosphatidylinositol-3, 4, 5-triphosphate], etc). The (molar, weight or volumetric) protein component of the TF: lipid can vary over a wide range, for example, from about 1: 50,000 to about 1: 3,000. The term FT, as used herein, includes variants and wild-type TF mutants that maintain, at least, one of the wild-type TF functions, advantageously, at least one of the wild-type TF functions related to coagulation.

In a particular embodiment, the lipidized TF used for the implementation of the invention is lipidized human FT and consists of the TF obtained from tissue extracts; or in the purified protein component of tissue extracts and inserted into a lipid component (with a protein: lipid molar ratio of about 1: 8700, or in recombinant TF (FTr) obtained by a process as described, only as an example illustrative, but not limiting, in US Pat. No. 6,261,803. The obtaining of extracts and purification of the TF can be carried out from various tissues such as brain, placenta and lung, of different animals, such as, sheep, calf, rabbit , dog, human, etc. The term "functional fragment of the lipidized TF" as used in this description includes, but is not limited to, FT-derived peptides, in particular, of the TF protein component, including mutants and variants of the FT. FT wild type protein component, which maintain one or more of the FT functions, preferably functions related to coagulation, for example the ability to bind FXa and / or SICN and develop its function n antihemorrhagic and vessel forming during wound healing. The amino acid sequence of said FT functional fragment may be identical to that of a fragment of the wild-type FT protein component or may present insertions, deletions or modifications of one or more amino acids, provided that they retain at least one of the functions of wild-type TF, advantageously, at least one function related to coagulation. For simplicity, the term "lipidized TF", as used herein, includes any functional fragment of the lipidized TF. The TF protein component used in the practice of the present invention can be part of a fusion protein. In this regard, by way of illustration, not limitation, said fusion protein may contain an A region, constituted by the TF protein or by a functional fragment of said TF protein, bound to a B region constituted by another FT fragment. Said region B is linked to the amino-terminal region of said FT protein or said fragment of the TF protein, or, alternatively, said B region may be linked to the carboxyl-terminal region of said FT or FT protein. said fragment of the TF protein. Both regions A and B can be linked directly or through a spacer polypeptide (linker) between said regions A and B. The fusion protein can be obtained either by chemical synthesis or by gene expression of the nucleotide sequence coding for said fusion protein in appropriate host cells. The term "topical treatment", as used herein, refers to the application of the treatment directly in the place where it is necessary, for example, in discontinuous sections of the skin (cuts, etc.), and of vascular tissue ( rupture of vessels, etc.). According to the present invention, and as demonstrated in the Example that accompanies the present description, the lipidized FT acts as a stimulator of FXa, increasing its proteolytic activity, and, consequently, the production of thrombin and, therefore, , can be used to treat or correct bleeding disorders, in particular, those bleeding disorders associated with hemorrhagic diathesis. The term "hemorrhagic diathesis" refers to the process that causes an alteration in the hemostasis and that, as a consequence, gives rise to the appearance of a hemorrhagic syndrome that may occur, sometimes, with prolonged and excessive bleeding. The hemorrhagic diathesis can be produced by a congenital or acquired coagulopathy and / or by a congenital or acquired platelet alteration or dysfunction.

The term "coagulopathy" refers to an alteration of the coagulation factors. This alteration may be due to a deficiency or specific lack of a coagulation factor, whose consequence will be the appearance of a hemorrhagic syndrome, or by an alteration in a coagulation factor. In general, coagulopathy can be a congenital coagulopathy or an acquired coagulopathy. Illustrative, non-limiting examples of congenital coagulopathies may be mentioned deficiencies in selected coagulation factors between FV, FVII, FVIII FIX, FX, FXII, FXIII and their combinations. The acquired coagulopathies can have different origins. Illustrative examples include synthesis deficit of coagulation factors in severe hepatic insufficiency, anticoagulant therapy (such as heparin, low molecular weight heparins, warfarin, coumarin derivatives, dicoumarins, etc.). An alternative mechanism is based on an exaggerated consumption of the coagulation factors so that before a bleeding lesion, they are not available to form the clot. This mechanism occurs in the syndrome of disseminated intravascular coagulation or consumption coagulopathy that appears in multiple diseases such as in severe sepsis that damage the endothelium of the microcirculation activating platelets and coagulation factors with the formation of multiple microthrombi; in blood invasion by TF such as placental abruption; in the retention of a dead fetus; in polytraumatisms with tissue crushing; in the bites of poisonous snakes, etc. In vasculitis, parietal and endothelial damage releases activators of coagulation. The consumption of coagulation factors is aggravated by the lysis of the fibrin of the numerous microthrombi by plasmin with release of PDF that are antiplatelets and anticoagulants. The term "platelet alteration" refers to an alteration, both in number and functionality, of platelets, the consequence of which is the appearance of a hemorrhagic syndrome. Said platelet alteration can be congenital or acquired. In a particular embodiment, said platelet alteration is a congenital platelet alteration. Illustrative, non-limiting examples of congenital plaque alterations include Glanzamn's disease, Sind Bernard Soulier's disease, Bolin-Jamieson syndrome, Wiskott-Aldrich syndrome, Paris-Trousseau-Jacobsen syndrome, chromosome thrombocytopenia X, Gray's platelet syndrome, Sebastian's syndrome and Fanconi's anemia. In another particular embodiment, said platelet alteration is an acquired platelet alteration. Illustrative, non-limiting examples of acquired platelet alterations include myeloproliferative disorders, such as thrombocythemia, polycythemia, chronic myelocytic leukemia, etc .; in myeloid metaplasia, there are functional platelet disorders with increased bleeding time, retention defect in glass beads, defect of platelet aggregation, abnormal release and defect of platelet factor III. Platelet functional defects have been found in the disproteinemias in scurvy, and in congenital heart disease and cirrhosis. The term "acquired coagulopathy" and "acquired platelet alteration" refer to the origin of the disorder, which may be iatrogenic or secondary to another disease. The term "subject", as used herein, includes any member of an animal species, including the human species; by way of illustration, not limitation, said subject may be a mammal, such as a primate, a domestic animal, a rodent, etc., preferably, said subject is a man or a woman of any age and race. In a particular embodiment, said subject is a human being without a history of alterations of hemostasis, such as an individual who does not present coagulopathies or platelet alterations. In another particular embodiment, said subject is a human being having a history of alterations of hemostasis, such as an individual having a hemorrhagic diathesis, for example, a coagulopathy, such as a congenital or acquired coagulopathy, or a platelet disorder. , such as a congenital or acquired platelet alteration.

Therefore, in a specific embodiment, the invention relates to the use of lipidized TF in the preparation of a medicament for the topical treatment of hemorrhages in a human being without a history of alterations in hemostasis. In another particular embodiment, the invention relates to the use of lipidized TF in the preparation of a medicament for the topical treatment of hemorrhages in a human being that has a hemorrhagic diathesis. For administration to the subject, the lipidized TF will be formulated in a pharmaceutical form suitable for topical administration for the topical (local) treatment of hemorrhages. Illustrative, non-limiting examples of such dosage forms include aerosols, solutions, suspensions, emulsions, gels, ointments, creams, dressings, patches, ointments, mouthwashes, etc. For this, the pharmaceutical formulation comprising the lipidized TF will include the pharmaceutically acceptable carriers and excipients necessary for the preparation of the chosen pharmaceutical dosage form (for further information see the section relating to the "Pharmaceutical composition" of the present disclosure). The dose of lipidized TF to be administered to the subject can vary within a wide range, for example, between 0.01 g of active protein / ml and 100 μg of active protein / ml approximately. The dose of lipidized FT to be administered will depend on numerous factors, including the characteristics of the FT protein used, such as its activity and biological half-life, the concentration of the TF protein in the formulation, the situation clinic of the subject or patient, the hemorrhagic disorder to be treated, etc. (for more information, see the section on "Pharmaceutical composition" of this description).

II. Combination products and applications II.1 Lipid FT + FXa As is known, in the extrinsic pathway of blood coagulation, FT binds to circulating FVII / FVIIa to form the FT :: FVII complex and, in the presence of calcium, act as substrate for the activation of the FX to take place. Activation of the extrinsic pathway involves the interaction of TF with its ligand, FVII / FVIIa. This complex, FT:: FVIIa acts as a substrate for the activation of the FX by the FVIIa. Now, the inventors, surprisingly, have discovered that the lipidized FT together with FXa, even in FVII deficient subjects, induces coagulation, so that the lipidized FT acts as a stimulator of FXa, allowing this serine protease to initiate the hydrolysis of prothrombin. , thrombin is produced, and the origin of coagulation takes place. As used herein, "FXa" refers to a protein, in particular, a serine protease, which, in its active form, is responsible for initiating the hydrolysis of prothrombin and, consequently, leading to the initiation of coagulation. . This proteolysis is effected by the FXa that binds to the surface of the activated platelets and, in the presence of the FVa and ionic calcium, hydrolyzes the prothrombin.

More specifically, the inventors, surprisingly, have found that the combination of lipidized TF and FXa, even at such low concentrations of FXa which by themselves are incapable of inducing coagulation, coagulates the plasma [see, for example, the sections 1 (1.5) and 2 of the Results of the Example that accompanies this description] as in vivo [see sections I and J of the Results of the Example that accompanies this description]. Therefore, in another aspect, the invention relates to a product comprising (i) lipidized TF and (ii) Activated coagulation factor X (FXa). Said product, based on the results mentioned previously, can be used as a medicine, in particular, it can be used in the treatment of haemorrhages, for example, in the topical treatment of haemorrhages, in a subject. Said components (i) and (ii) can be together or separately. In a particular embodiment, the lipidized FT used in the preparation of this product is lipidized human FT. In a particular embodiment, the invention provides a product comprising (i) lipidized TF and (ii) FXa. Both components can be combined and be together in the same composition before their administration to the subject. In another particular embodiment, the invention provides a product comprising, separately, (i) lipidized TF and (ii) FXa. In another particular embodiment, the invention provides a product comprising, separately, (i) lipidized TF and (ii) FXa, as a combination for simultaneous or successive administration to a subject. The combined administration of said components (i) and (ii) to the subject can be carried out simultaneously or sequentially, separated in time, in any order, that is, the lipidized TF can be administered first, and then the FXa , or vice versa. Alternatively, said lipidized TF and FXa can be administered simultaneously.

For administration to a subject the product previously defined is formulated as a dosage form, preferably a pharmaceutical administration form suitable for topical administration, for which the pharmaceutically acceptable excipients and carriers suitable be incorporated for Preparation of the desired pharmaceutical form of administration. Information on said vehicles and excipients, as well as on said suitable administration forms for the administration of said product of the invention can be found in galenic pharmacy treatises. For more information see the section on "Pharmaceutical composition" of the present description. The dose of lipidized TF and FXa to be administered to the subject can vary within a wide range. By way of illustration, the dose of lipidized TF to be administered may be between about 0.01 μg of active protein / ml and 100 μg of active protein / ml. Also illustratively, the dose of FXa administered may be between about 17 w 17 nM (0.001 g of active protein / ml and 10 mg of active protein / ml. In general, doses of FT lipidizado and FXa will be administered will depend on numerous factors, including the characteristics of the protein FT used and the FXa, such as for example, its activity and biological half life, the concentration of the protein TF and FXa in the formulation include the clinical situation of the subject or patient, the hemorrhagic disorder to be treated, etc. (for more information see the section on "Pharmaceutical composition" of the present description).

The weight ratio between the lipidized TF and the FXa present in said product can vary within a wide range; by way of illustration, said lipidized FT weight ratio: FXa is comprised between 106: 1 and 10: 1, although other weight ratios are also possible; in a particular embodiment, said product of the invention comprises lipidized FT and FXa in a weight ratio of 1 to 0.001, ie for each milligram of TF protein, 1 microgram of FXa protein is present in the mixture. The use of the product previously defined as a medicament, in particular, the use of said product in the preparation of a medicament for the treatment of hemorrhages in a subject, especially, for the topical treatment of hemorrhages in a subject, constitute additional aspects of the present invention.

II.2 FT lipidizado + SICN The inventors have also surprisingly found that the combination of FT lipidizado and negatively charged inorganic surfaces (SICN) coagulates plasma and blood of healthy subjects and patients deficient in clotting factors both in vitro [see Sections 1 (1.4), 2 (2.2), 3 (3.4), 4 (4.2) and 5 (5.2 and 5.4) of the Results of the Example that accompanies this description] as in vivo [see section 6.1 of the Results of the Example that accompanies this description]. The term "inorganic surface negatively charged" or "SICN" as used herein, consists of a mixture of lipids and accelerators blood, such as ellagic acid, zeolite, silica, inorganic materials oxido clotting, etc. Lipids have a net negative charge, which means that the lipid mixture can include neutral or zwitterionic lipids, but it must contain a certain amount of negatively charged lipids that confers anionic character to the mixture. A non-limiting illustrative example of charged lipids may adversely be sphingolipids (such as ceramide-l-phosphate, glycosylated phosphatidylethanolamine, sulfatides hydroxylated or not hydroxylated, gangliosides) and lipid-based glycerol (such as phosphatidylserines, phosphatidylinositols, phosphatidylinositol phosphates, phosphatidic acids, phosphatidylglycerols, cardiolipins). In the present patent application, Dade® Actin® commercial products (Dade Behring, brand) were used as a source of SICN. Therefore, in another aspect, the invention relates to a product comprising (i) lipidized TF and (ii) a SICN. Said product, based on the results mentioned previously, can be used as a medicine, in particular, it can be used in the treatment of haemorrhages, for example, in the topical treatment of haemorrhages, in a subject. Said components (i) and (ii) can be together or separately. In a particular embodiment, the invention provides a product comprising (i) lipidized FT and (ii) a SICN. Both components can be combined and be together in the same composition before their administration to the subject. In another particular embodiment, the invention provides a product comprising, separately, (i) lipidized TF and (ii) SICN. In another particular embodiment, the invention provides a product comprising, separately, (i) lipidized TF and (ii) SICN, as a combination for simultaneous or successive administration to a subject. The combined administration of said components (i) and (ii) to the subject can be carried out simultaneously or sequentially, separated in time, in any order, that is, the lipidized TF can be administered first and then the SICN , or vice versa. Alternatively, said lipidized TF and SICN can be administered simultaneously. For administration to a subject the product previously defined is formulated as a dosage form, preferably a pharmaceutical administration form suitable for topical administration, for which the pharmaceutically acceptable excipients and carriers suitable be incorporated for Preparation of the desired pharmaceutical form of administration. Information on said vehicles and excipients, as well as on said suitable administration forms for the administration of said product of the invention can be found in galenic pharmacy treatises. For more information see the section on "Pharmaceutical composition" of the present description. The dose of lipidized TF and SICN to be administered to the subject can vary within a wide range. By way of illustration, the dose of lipidized TF to be administered may be between about 0.01 μg of active protein / ml and 100 μg of active protein / ml. Also, by way of illustration, the dose of SICN (in volume) to be administered may be between about 0.1 and 100 DI per each Dg of protein activated by lipidized TF. In general, the doses of FT lipidized SICN to be administered will depend on numerous factors, among which are included the characteristics of the FT protein used and the SICN, such as, for example, its activity and biological half-life, the concentration of the protein of the FT and SICN in the formulation, the clinical situation of the subject or patient, the hemorrhagic disorder to be treated, etc. (for more information, see the section on "Pharmaceutical composition" of this description). The weight / volume ratio (w / v) of the lipidized FT and the SICN present in said product may vary within a wide range; illustratively said w / v ratio of lipidized FT: SICN is comprised between 1: 1 and 1: 106, although other w / v ratios are also possible; in a particular embodiment, said product of the invention comprises between 0.1 and 100 DI of SICN for each Dg of lipidized active TF protein. The use of the product previously defined as a medicament, specifically the use of said product in the manufacture of a medicament for the treatment of hemorrhages in a subject, particularly for the topical treatment of haemorrhages in a subject, constitute additional aspects of the present invention.

II.3 Lipidized FT + FXa + SICN The inventors surprisingly have also discovered that the combination of lipidized FT, FXa and SICN coagulates in plasma and blood of healthy subjects and patients deficient in coagulation factors [see sections 1 (1.6) and 2 (2.2) of the results of the Examples included in this description]. Therefore, in another aspect, the invention relates to a product comprising (i) lipidized TF, (ii) FXa and (iii) SICN. Based on the previously mentioned results, said product can be used as a medicament, and can be used in a particular way in the treatment of haemorrhages, for example, in the topical treatment of haemorrhages, in a subject. Said components (i), (ii) and (iii) can be together or separately. Alternatively, two of the components can be together, for example, (i) + (ii), (i) + (iii) or (ii) + (iii), and the third separated component. In a particular embodiment, the invention provides a product comprising (i) lipidized TF, (ii) FXa and (iii) SICN. Said components can be combined and be together in the same composition before their administration to the subject. In another particular embodiment, the invention provides a product comprising, separately, (i) lipidized TF, (ii) FXa and (iii) SICN. In another particular embodiment the invention provides a product comprising, separately, (i) lipidized TF, (ii) FXa and (iii) SICN, as a combination for combined or simultaneous administration to a subject. The combined administration of said components (i), (ii) and (iii) to the subject can be carried out simultaneously or sequentially, separated in time, in any order, that is, the lipidized TF can be administered first, then the FXa and then the SICN, or first the lipidized TF, then the SICN and then the FXa, or first the FXa, then the lipidized TF and then the SICN; or first the FXa, then the SICN and then the lipidized TF, or first the SICN, then the lipidized TF and then the FXa. Alternatively, any two of said components can be mixed in the same composition and administered together while the third component can be added before or after said binary component composition. In another alternative embodiment said lipidized FT, FXa and SICN are administered simultaneously. For administration to a subject, the previously defined product will be formulated into a pharmaceutical administration form, preferably a pharmaceutical administration form suitable for topical administration, to which pharmaceutically acceptable carriers and excipients suitable for the preparation of the form will be incorporated. of pharmaceutical administration desired. Information on said carriers and excipients, as well as on said administration forms appropriate for the administration of said product of the invention, can be found in galenic pharmacy treaties. For more information see the section related to the "Pharmaceutical Composition" of this description. The dose of lipidized TF, FXa and SICN to be administered to a subject can vary within a wide range. By way of illustration, the dose to be administered of lipidized TF may be between about 0, 01 Dg of active protein / ml and 100 Dg of active protein / ml. Also, by way of illustration, the dose to be administered of FXa may be between about 17 pM and 170 pM (0.001 g of protein and 10 g of protein / ml). Additionally, by way of illustration, the dose of SICN (in volume) to be administered may be between approximately 0.1 and 100 μ? per μg of lipidized active TF protein. In general, the doses of lipidized FT, FXa and SICN to be administered will depend on numerous factors, among which are the characteristics of the FT protein, FXa and SICN used, such as, for example, their activity and biological half-life. , the concentration of the FT protein, FXa and SICN in the formulation, the clinical situation of the subject or patient, the hemorrhagic disorder to be treated, etc. (for more information, see the section on "Pharmaceutical composition" of this description). The relationship between the lipidized TF, the FXa and the SICN present in said product may vary within a wide range as previously mentioned in sections II.l and II.2.; however, in a particular embodiment, said product of the invention comprises lipidized FT: FXa: SICN in a ratio of 1: 0.001: 100 (w: w: v). The use of the product previously defined as a medicament, in particular, the use of said product in the preparation of a medicament for the treatment of hemorrhages in a subject, especially, for the topical treatment of hemorrhages in a subject, constitute additional aspects of the present invention.

III. Complexes and applications III.l FT complex:: FXa As previously mentioned (see section II.l of this description), the inventors have found that the combination of lipidized FT and FXa, even at such low concentrations of FXa as by they were unable to induce coagulation, coagulate the plasma. Although it is not desired to be bound by any theory, it is believed that the administration, jointly or separately (in any order), of said lipidized TF and FXa results in the formation of a complex capable of exerting the therapeutic effect (antihaemorrhagic, in particular , antihemorrhagic topical) observed in the place where said therapeutic effect must exert. Therefore, in another aspect, the invention relates to a complex, identified as FT:: FXa in this description, comprising lipidized FT and FXa. Although for simplicity said complex is represented as FT :: FXa, in reality, said complex could be formed by several units of each of said components; all these possibilities fall within the scope of the invention. Said complex, the view of the aforementioned results, can be used as a medicine, in particular, it can be used in the treatment of haemorrhages, for example, in the topical treatment of haemorrhages, in a subject. In a particular embodiment, the lipidized TF used in the elaboration of this complex is lipidized human TF. For administration to a subject, the previously defined complex will be formulated in the form of a pharmaceutical dosage form, preferably a pharmaceutical dosage form suitable for topical administration, for which suitable pharmaceutically acceptable excipients and vehicles will be incorporated for the preparation of the desired pharmaceutical form of administration. Information on said vehicles and excipients, as well as on said suitable administration forms for the administration of said product of the invention can be found in galenic pharmacy treatises. For more information see the section on "Pharmaceutical composition" of the present description. The doses of lipidized TF and FXa present in said complex to be administered to the subject can vary within a wide range. In general, said doses correspond to the doses mentioned previously in section II.1 related to a product comprising lipidized FT and FXa. The weight ratio between the lipidized TF and the FXa present in said FT :: FXa complex can vary within a wide range; although, in general, it corresponds to the one mentioned previously in section II.l related to a product that includes lipidized FT and FXa. The use of the complex previously defined as a medicament, in particular, the use of said complex in the preparation of a medicament for the treatment of hemorrhages in a subject, especially for the topical treatment of hemorrhages in a subject, constitute additional aspects of the present invention.

III.2 Complex FT :: SICN As previously mentioned (see section II.2 of this description), the inventors have found that the combination of lipidized FT and SICN coagulates plasma and blood of healthy subjects and patients with deficits in Coagulation factors both in vitro and in vivo. Although it is not desired to be bound by any theory, it is believed that the administration, jointly or separately (in any order), of said lipidized TF and SICN leads to the formation of a complex capable of exerting the therapeutic effect (antihaemorrhagic, in particular , antihemorrhagic topical) observed in the place where said therapeutic effect must exert. Therefore, in another aspect, the invention relates to a complex, identified as FT :: SICN in this description, comprising lipidized FT and a SICN. Although for simplicity said complex is represented as FT :: SICN, in reality, said complex could be formed by different units of each of said components; all these possibilities fall within the scope of the invention. Said complex, in view of the results mentioned previously, can be used as a medicine, in particular, it can be used in the treatment of haemorrhages, for example, in the topical treatment of haemorrhages, in a subject. In a particular embodiment, the lipidized TF used in the elaboration of this complex is lipidized human TF. For administration to a subject, the previously defined complex will be formulated in the form of a pharmaceutical dosage form, preferably, a pharmaceutical form of administration suitable for topical administration, for which pharmaceutically acceptable excipients and vehicles suitable for administration will be incorporated. Preparation of the desired pharmaceutical form of administration. Information on said vehicles and excipients, as well as on said suitable administration forms for the administration of said product of the invention can be found in galenic pharmacy treatises. For more information see the section on "Pharmaceutical composition" of the present description. The doses of lipidized TF and SICN present in said complex to be administered to the subject can vary within a wide range. In general, these doses correspond to the doses mentioned previously in section II.2 related to a product that comprises the combination of lipidized TF and SICN. The w / v ratio between the lipidized TF and the SICN present in said FT :: SICN complex can vary within a wide range; although, in general, it corresponds to the one previously mentioned in section II.2 related to a product comprising lipidized FT and SICN. The use of the complex previously defined as a medicament, in particular, the use of said complex in the preparation of a medicament for the treatment of hemorrhages in a subject, especially for the topical treatment of hemorrhages in a subject, constitute additional aspects of the present invention.

III.3 Lipidized TF Complex :: FXa :: SICN As previously mentioned (see section II.3 of this description), the inventors have found that the combination of lipidized TF, FXa and SICN coagulates the plasma. Although it is not desired to be bound by any theory, it is believed that the administration, jointly or separately (in any order), of said lipidized TF, FXa and SICN leads to the formation of a complex capable of exerting the therapeutic effect (antihaemorrhagic, in particular, antihemorrhagic topical) observed in the place where said therapeutic effect must exert. Therefore, in another aspect, the invention relates to a complex, identified as FT:: FXa:: SICN in this description, which comprises lipidized FT, FXa and a SICN. Although for simplicity said complex is represented as FT :: FXa :: SICN, in reality, said complex could be formed by different units of each of said components, as well as by any possibility of interactions between said components, for example, FT :: SICN: FXa, FXa :: SICN :: FT, FXa :: FT :: SICN, SICN:: FT:: FXa or SICN:: FXa:: FT; all these possibilities fall within the scope of the invention. Said complex, in view of the results mentioned previously, can be used as a medicine, in particular, it can be used in the treatment of haemorrhages, for example, in the topical treatment of haemorrhages, in a subject. In a particular embodiment, the lipidized TF used in the elaboration of this complex is lipidized human TF. For administration to a subject, the previously defined complex will be formulated in the form of a pharmaceutical dosage form, preferably, a pharmaceutical form of administration suitable for topical administration, for which pharmaceutically acceptable excipients and vehicles suitable for administration will be incorporated. Preparation of the desired pharmaceutical form of administration. Information on said vehicles and excipients, as well as on said suitable administration forms for the administration of said product of the invention can be found in galenic pharmacy treatises. For more information see the section on "Pharmaceutical composition" of the present description.

The doses of lipidized FT, FXa and SICN present in said complex to be administered to the subject can vary within a wide range. In general, these doses correspond to the doses mentioned previously in section II.3 related to a product that comprises the combination of lipidized TF, FXa and SICN. The relationship between the lipidized TF, the FXa and the SICN present in said complex FT :: FXa :: SICN can vary within a wide range; although, in general, corresponds to the one mentioned previously in II.3 related to a product comprising lipidized FT, FXa and SICN. The use of the complex previously defined as a medicament, in particular, the use of said complex in the preparation of a medicament for the treatment of hemorrhages in a subject, especially for the topical treatment of hemorrhages in a subject, constitute additional aspects of the present invention.

IV. Pharmaceutical composition As previously mentioned, lipidized TF can be used as an anti-hemorrhagic agent, in particular, as an anti-hemorrhagic agent for topical application. Therefore, in another aspect, the invention relates to a pharmaceutical composition, hereinafter, the pharmaceutical composition of the invention, comprising a lipidized TF together with a pharmaceutically acceptable carrier. In a particular embodiment, the lipidized FT is lipidized human FT. For administration to a subject, said lipidized TF must be formulated in a pharmaceutically acceptable form of administration, for which said lipidized TF will be formulated with suitable pharmaceutically acceptable excipients and vehicles for the manufacture of the desired pharmaceutical form of administration. Information on said vehicles and excipients, as well as on said administration forms suitable for the administration of the product of the invention can be found in galenic pharmacy treatises. A review of the different pharmaceutical forms of drug administration, in general, and their preparation procedures can be found in the book "Tratado de Farmacia Galenica", by C. Faulí i Trillo, Ia Edition, 1993, Luzán 5, S.A. of Editions. Although, in principle, various pharmaceutical forms of administration of the lipidized TF could be used, in practice it is advantageous to administer said compound topically, whereby said lipidized TF will be formulated in a pharmaceutical form suitable for topical administration. Illustrative, non-limiting examples of such dosage forms include aerosols, solutions, suspensions, emulsions, gels, ointments, creams, dressings, patches, ointments, mouthwashes, etc. For this, the pharmaceutical formulation comprising the lipidized TF will include the pharmaceutically acceptable vehicles and excipients necessary for the preparation of the pharmaceutical form for administering lipidized FT via the topical route. Therefore, in a particular embodiment, the pharmaceutical composition of the invention is a pharmaceutical composition for topical administration of a lipidized TF, comprising lipidized TF and a pharmaceutically acceptable carrier suitable for topical administration of said lipidized TF. The lipidized TF will be present in the pharmaceutical composition of the invention in a therapeutically effective amount. Said amount may vary within a wide range, for example, between about 0.01 active protein / ml and 100 μ active protein / ml. In another particular embodiment, the pharmaceutical composition of the invention comprises:a) a product comprising (i) lipidized TF and (ii) FXa, together with a pharmaceutically acceptable vehicle; or b) separately, (i) lipidized TF together with a pharmaceutically acceptable carrier, and (ii) FXa together with a pharmaceutically acceptable carrier; or c) a product comprising (i) lipidized TF and (ii) a SICN, together with a pharmaceutically acceptable carrier; or d) separately, (i) lipidized TF together with a pharmaceutically acceptable carrier, and (ii) a SICN together with a pharmaceutically acceptable carrier; or e) a product comprising (i) lipidized TF, (ii) FXa and (iii) a SICN, together with a pharmaceutically acceptable carrier; or f) separately, (i) lipidized TF together with a pharmaceutically acceptable carrier, (ii) FXa together with a pharmaceutically acceptable carrier, and (iii) a SICN together with a pharmaceutically acceptable carrier; or g) an FT :: FXa complex together with a pharmaceutically acceptable vehicle; or h) an FT :: SICN complex together with a pharmaceutically acceptable carrier; or i) an FT :: FXa :: SICN complex together with a pharmaceutically acceptable carrier.

The pharmaceutical composition of the previously defined invention contains, as can be seen, lipidized FT alone or combined with FXa and / or SICN or complexed with FXa and / or SICN, as an active ingredient. In a particular embodiment, the lipidized TF present in the previously defined pharmaceutical composition of the invention is lipidized human TF. Also, in a particular embodiment, the pharmaceutical composition of the previously defined invention will be formulated in a pharmaceutical form for topical administration of the active principle (lipidized TF alone or combined with FXa and / or SICN or complexed with FXa and / or SICN). Illustrative, non-limiting examples of such dosage forms include aerosols, solutions, suspensions, emulsions, gels, ointments, creams, dressings, patches, ointments, mouthwashes, etc. For this, the pharmaceutical formulation comprising the aforementioned active principle will include the pharmaceutically acceptable vehicles and excipients necessary for the preparation of the pharmaceutical form for administering said active principle by topical route. The active ingredient will be present in the pharmaceutical composition of the invention in a therapeutically effective amount. The dose of active principle to be administered to a subject will depend, among other factors, on the subject to be treated, on the severity of the pathology suffered by said subject, on the pharmaceutical form of administration chosen, etc. For this reason, the doses mentioned in this invention should be considered only as guidelines for the expert in the field, and he should adjust the doses according to the variables cited above. However, the pharmaceutical composition of the invention can be administered one or more times a day for preventive or therapeutic purposes. The pharmaceutical composition of the invention can be used together with other additional drugs useful in the prevention and / or treatment of a hemorrhagic diathesis (e.g., coagulation factors, human plasmas, etc.) to provide combination therapy. Said additional drugs can be part of the same pharmaceutical composition or, alternatively, they can be provided in the form of a separate composition for simultaneous or sequential administration (sequentially in time) with respect to the administration of the pharmaceutical composition of the invention V. Supported pharmaceutical composition The pharmaceutical composition of the invention can be deposited on a support. Therefore, in another aspect, the invention relates to a product comprising the pharmaceutical composition of the invention and a support. The term "support", as used herein, refers to a substrate of an appropriate material that allows the deposition of the pharmaceutical composition of the invention., its vehiculization and its release in the desired place, for example, in the place where the pharmaceutical composition of the invention must exert its therapeutic effect. Said support may be a solid support or a non-solid support, for example, a liquid support or a gaseous support. Illustrative, non-limiting examples of solid supports include dressings, plasters, compresses, plasters, etc. Illustrative, non-limiting examples of liquid supports include gels, sprays, mouthwashes, etc. Illustrative, non-limiting examples of gaseous supports include air, propellants, etc. In a particular embodiment, the pharmaceutical composition of the invention deposited on said support comprises: (a) (i) a support, (ii) a product comprising lipidized FT and FXa, together with a pharmaceutically acceptable carrier, (iii) a product comprising lipidized TF and a SICN, together with a pharmaceutically acceptable carrier and (iv) a product comprising lipidized TF, FXa and a SICN, together with a pharmaceutically acceptable carrier; or (b) an FT:: FXa complex, together with a pharmaceutically acceptable carrier, or (c) an FT :: SICN complex, together with a pharmaceutically acceptable carrier, or (d) an FT:: FXa:: SICN complex, together with a pharmaceutically acceptable vehicle. In a particular embodiment, the lipidized TF present in the pharmaceutical composition of the invention is lipidized human TF. This product comprising the pharmaceutical composition of the invention deposited on a support can be obtained by conventional methods, for example, by mixing the pharmaceutical composition of the invention and the support. The interaction between the pharmaceutical composition of the invention and the support can be a physical or chemical interaction depending on the nature of the components of the pharmaceutical composition of the invention and the support used.

SAW . Use of non-lipidized TF for the treatment of haemorrhages The results obtained in the tests carried out by the inventors show that FT: increases the proteolytic activity of FXa and, consequently, the production of thrombin; it coagulates the plasma and the blood of healthy subjects and of patients deficient in coagulation factors; it coagulates the blood of patients affected by platelet alterations and stops severe and lethal hemorrhages in vivo in different model animals. Therefore, taken together, these results show that lipidized TF is an antihaemorrhagic agent useful for the treatment of bleeding in healthy subjects and in patients with deficiency in coagulation factors, including FVIII, FIX, FXI, FXIII, FVII , F and FX. European patent EP 266993 describes the use of non-lipidized TF in the treatment of haemorrhages, in particular, in the treatment of haemorrhages due to a deficiency in a coagulation factor, specifically in a coagulation factor selected from FVIII, FIX, FXI or FXIII. However, said patent does not disclose or suggest the possibility of using non-lipidized FT in the treatment of hemorrhages due to a deficiency in the FVII. The discovery now made by the inventors regarding the role of TF, as stimulating agent of the proteolytic activity of FXa independent of whether or not the subject is deficient in FVII, allows establishing the possibility of using the non-lipidized TF, or a functional fragment of the same, not lipidized, in the treatment of hemorrhages in a subject with deficiency in FVII. Therefore, in another aspect, the invention relates to the use of non-lipidized TF, or a non-lipidized functional fragment thereof, for the preparation of a medicament for the treatment of hemorrhages in a subject with FVI I deficiency. The term "non-lipidized TF", as used herein, refers to purified TF (without the lipidized plasma membranes). The term "FT" as used herein includes variants and mutants of wild-type FT that maintain at least one of the functions of the wild-type FT, advantageously, at least one of the functions of wild-type FT related to coagulation. The isolation and purification of the TF can be carried out from various tissues, for example, brain, placenta, lung, etc., of different animals, for example, sheep, calf, rabbit, dog, human being, etc. The FT can also be a recombinant FT (FTr) obtained by a process as described, only as an illustrative, non-limiting example, in the patent US6,261,803. In a particular embodiment, the non-lipidized TF used for the implementation of the invention is non-lipidized human FT and consists of the protein component of an isolated and purified TF of human tissue completely devoid of plasma lipid membranes. The term "non-lipidized TF functional fragment", as used herein, includes, but is not limited to, FT-derived peptides, in particular, of the FT protein component, including mutants and variants of the FT protein component. wild type, which maintain one or more of the TF functions, preferably functions related to coagulation, for example the ability to bind FXa and / or SICN and develop its antihemorrhagic and vessel-forming function during wound healing. The amino acid sequence of said FT functional fragment may be identical to that of a fragment of the wild-type FT protein component or may present insertions, deletions or modifications of one or more amino acids, provided that they retain at least one of the functions of wild-type TF, advantageously, at least one function related to coagulation. For simplicity, the term "non-lipidized TF", as used herein, includes any functional fragment of the non-lipidized TF. Non-lipidized TF can be obtained by conventional methods. To illustrate, the protein component of TF is dissociated from the lipid component by extraction with organic solvents. Illustrative examples of said organic solvents include pyridine, ethanol, heptane-butanol mixtures, etc. The protein component of TF can be purified by conventional chemical methods. Illustrative examples of said chemical methods include treatment with detergents, for example, deoxycholate, Triton X-100, etc., gel filtration and preparative electrophoresis in polyacrylamide gels in the presence of sodium dodecyl sulfate, the use of bound concavalin A to a Sepharose column, the use of affinity columns using antibodies against the protein component (protein) of the TF, etc. For administration to the subject with deficiency of FVII, the non-lipidized TF will be formulated in a pharmaceutical form suitable for administration by any appropriate route. Although practically any pharmaceutical form can be used, in practice the use of pharmaceutical forms for the parenteral administration of said non-lipidized FT is advantageous.

Therefore, in a particular embodiment, the invention relates to the use of non-lipidized TF, or a fragment thereof, which is not lipidized, in the preparation of a medicament for the parenteral administration of said non-lipidized TF or fragment of the same, not lipidized.

In this case, pharmaceutical compositions containing the non-lipidized TF and pharmaceutically acceptable excipients or carriers will be adapted for parenteral administration, in the form of, for example, solutions, suspensions or lyophilized, sterile products, in the appropriate dosage form; in this case, said pharmaceutical compositions will include suitable excipients, such as buffers, surfactants, etc. In any case, the excipients will be chosen according to the selected pharmaceutical dosage form. Information on said vehicles and excipients, as well as on said administration forms suitable for the administration of the product of the invention can be found in galenic pharmacy treatises. A review of the different pharmaceutical forms of drug administration, in general, and their preparation procedures can be found in the book "Tratado de Farmacia Galenica", by C. Faulí i Trillo, Ia Edition, 1993, Luzán 5, S.A. of Editions.

The dose of non-lipidized TF to be administered to the subject with deficiency in FVII can vary within a wide range, for example, between about 0.01 μg of active protein / ml and 100 μg of active protein / ml. The dose of non-lipidized FT to be administered will depend on numerous factors, among which are the characteristics of the FT protein used, such as, for example, its activity and biological half-life, the concentration of the TF protein in the formulation, the patient's clinical situation, the hemorrhagic disorder to be treated, etc.

EXAMPLE In order to evaluate the capacity of the lipidized TF as FXa stimulator, a series of in vi tro and in vivo tests were carried out, namely: 1. In vitro assays that demonstrate that the lipid FT factor (alone or in combination) acts as a direct stimulator of FXa causing fibrin clot formation and blood coagulation in the absence of FVII (caused by absence, deficiency or immunoblocking) 1.1. The lipidized TF increases the proteolytic capacity of FXa and consequently the production of thrombin in the absence of FVII (chromogenic assays in solution and in suspension of washed platelets). 1.2. The lipidized FT is able to coagulate the plasma and blood of patients deficient in FVII (coagulation assays in plasma and in non-anticoagulated total blood) 1.3. Lipidized TF is able to coagulate plasma in healthy subjects in the presence of a monoclonal antibody against FVII (coagulation assays) 1. 4. The combination of lipidized FT and SICN synergistically increases blood coagulation in deficient FVII plasmas (coagulation assays). 1.5. The combination of lipidized FT and FXa increases blood coagulation synergistically in the presence of a monoclonal antibody against FVII (coagulation assays in deficient FX plasmas). 1.6. The combination of FT, FXa and SICN increases coagulation synergistically in the presence of an anti-FVII antibody (coagulation assay in deficient plasma in the FX). In vi tro assays demonstrating that the combination of lipidized TF with FXa at low concentrations (unable to induce any procoagulant effect), produces the coagulation of defective plasmas in FX. 2.1 The lipidized TF acts as a stimulator of FXa when this serine protease is present at low concentrations. 2.2 The combination of lipidized FT with FXa and SICN acts synergistically in stimulating FXa. In vi tro trials that demonstrate that lipidized TF (alone or in combination) produces coagulation in patients with deficiencies in other coagulation factors other than FVII. 3.1 Lipidized TF coagulates the plasma and blood of patients with deficiencies in the coagulation factors FV, FVIII, FVIII, FX, FXI, FXII, and FXIII (coagulation assays in plasma deficient in coagulation factors and in whole blood uncoagulated FVIII and FIX). 3.2 The lipidized FT coagulates whole blood and plasma previously heparinized. 3.3 FT coagulates the plasma of animals treated with warfarin. 3. 4 The combination of lipidized FT with SICN synergistically increases blood coagulation in plasma of patients with deficiencies in the coagulation factors FV, FVIII, FVIII, FIX, FX, FXI, FXIII and FXIII (coagulation assays in deficient plasma in coagulation factors) and in whole blood of hemophilic patients. In vitro studies demonstrating that lipidized FT (alone or in combination) causes blood coagulation in healthy patients. 4.1 Lipidized TF coagulates the plasma and blood of healthy patients. 4.2 The combination of lipidized FT with SICN synergistically increases blood coagulation in plasma and blood of healthy subjects. Absence of synergistic effects when lipidized TF is associated with phospholipids. In vitro assays showing the coagulant effect of lipidized TF (alone or in combination) in plasma of patients with congenital or acquired platelet disorders (thrombocytopenic). 5.1 Lipidized TF coagulates the plasma of patients with congenital platelet disorders. 5.2 The combination of lipidized FT with a SICN increases the coagulation in whole blood of patients with congenital platelet disorders. 5.3 Lipidized TF coagulates in thrombocytopenic samples. In vitro tests demonstrating that TF is a useful agent for topical antihemorrhagic treatment in control rats (applied directly alone or in combination with a SICN in the previously sectioned blood vessel). 6. 1 Lipidized TF (alone or in combination with a SICN) is useful as a haemostatic topical agent in an animal model of severe hemorrhage through the proximal section of rat tails. 6.2 Lipidized TF is useful as a topical haemostatic agent in an animal model of severe haemorrhage previously treated with heparin or warfarin. 6.3 Lipidized TF is useful as a topical haemostatic agent in an animal model of lethal hemorrhage through the proximal section of rat tails.

I. MATERIALS AND METHODS Materials Thromborel® S and Neoplastin® Plus were used as a source of lipidized tissue factor (TF) using recombinant tissue factor (FTr). Specifically, the following commercial preparations were employed: the Human Recombinant Tissue Non-Lipidated Factor (American Diagnostica, USA), relipidated following the method described by Morrissey, lyophilized human placenta thromboplastin containing calcium (Thromborel® S, Dade Behring Inc); freeze-dried rat brain containing calcium (Neoplastin® Plus, Diagnostica Stago-Roche). The procoagulant activity of the FT protein was determined by means of a standard curve using the recombinant lipidized human FT (American Diagnostica, USA) as the reference material and is expressed in the text as active protein Dg / mL and in the tables as Dg / mL .. For simplicity, in this example, the term FTr or FT, unless otherwise indicated, refers to lipidized TF. The commercial compounds of Haematologic Technologies were used as source of FXa, FII (prothrombin) and FVa. As negatively charged inorganic surfaces (SICN), the commercial reagent Dade® Actin® FS (Dade Behring) was used. The deficit plasmas in commercial coagulation factors (FV, FVII, FVIII, FIX, FX, FXI, FXII and FXIII) were obtained from Dade Behring Marburg GmbH.

Methods Method for the relipidation of TF in lipid vesicles using dialysis with octylglucoside (Morrisey method) The non-lipidized TF is incorporated into lipid vesicles using the non-ionic detergent N-octyl-beta-D-glucopyranoside (octylglucoside). Both TF and lipids dissolve in octylglucoside formed micelles. Since octylglucoside has a high critical micelle concentration (CMC = 20 to 50 mM), it can be easily removed from the solution by dialysis. When the octylglucoside is eliminated, the lipids are organized in unilamellar vesicles. FT is embedded in these vesicles by virtue of its transmembrane domain. Normally, 50 to 80% of the FT molecules are arranged looking out of the vesicles.

Buffers and solutions stock Octylglucoside (n-octyl-beta-D-glucopyranoside) Calbiochem. Lipids: Buffers: HBS NaCl 100 mM Hepes / NaOH 20 mM, pH 7.5 Sodium Azide 0.02% (m / v) HBSA (store at 4 ° C) Bovine serum albumin in HBS 0.1% (m / v) ) OG / HBS (prepare at the time) n-octyl-beta-D-glucopyranoside in 100 mM HBS (29.2 mg OG / ml HBS) Preparation of the lipid solution in octylglucoside (OG) For each sample, 2.6 micromoles of total lipids are prepared in a glass tube, using the desired molar radius of lipids. Dry the lipid mixture under a stream of argon or nitrogen. When the tube appears dry, dry in vacuum for another 60 minutes. Add 400 DI of fresh OG / HBS solution (at room temperature) to the tube with the dry lipids.

Relipidación Add the desired amount of FT to the tube containing the 400 DI of OG / lipids and enough HBSA to complete the final volume of 1 ml. Perform this step at room temperature.

Obtaining suspensions of washed platelets The suspensions of washed platelets were prepared according to the method described by Radomski M. et al. (Radomski M, Moneada S., 1983 Thromb Res. This is an improved method for washing of human platelets with prostacyelin 15; 30 (4): 383-9) from blood extractions (sodium citrate 3.15%) of healthy volunteers The processing of the specimens was always carried out immediately after the blood extraction and at room temperature. Previously and during the performance of the assays, the activation status and functionality of the platelets were evaluated by aggregation tests. Activation alone and functionality were estimated by activation with a known agonist (collagen).

Essays in vi tro Chromogenic assays Different chromogenic assays were designed in solution and in suspensions of activated platelets to demonstrate the effect of FTr on the proteolytic activity of Factor Xa.

The amidolytic activity of FXa was determined by a chromogenic assay using S-2765 (Chromogenix) as the chromogenic substrate for FXa, while the thrombin-forming activity was analyzed using S-2238 (Chromogenix) as a chromogenic substrate for thrombin. The chromogenic assays were performed in suspension (in buffer) and using suspensions of washed platelets. In an ELISA plate, the appropriate volumes of each of the factors to be studied were dispensed and, in the case of the use of the washed platelet suspension, an appropriate volume to have in the medium a concentration of 250,000 platelets / μ? . Finally, the addition of the specific chromogenic substrate allowed to quantify the proteolytic activity in question by spectrophotometric reading at 405 nm. In the tests to determine the amidolytic activity only the FXa and the different concentrations of FTr were dispensed, while in the assays to determine the thrombin forming activity it was necessary to add, in addition, Factor II (prothrombin) and FVa (this only in the in the case of the suspended assays, since in those of the washed platelets, they already contain endogenous FVa).

Plasma coagulative assays Spontaneous (unstimulated) procoagulant activity in plasma was measured by a coagulative assay of a given step in a coagulometer (Fibrintimer BFT-II clot-timer Dade-Behring, Germany). Briefly, 50 μ? of platelet-poor plasma to the already tempered cuvettes and 50 μ? of distilled water. This mixture was allowed to incubate for 60 seconds at 37 ° C and immediately 50 μ? of 25 mM calcium chloride and the coagulation time in seconds was determined in the coagulometer verified by the formation of the clot. Each of the samples was tested in duplicate. The platelet poor plasmas were obtained by centrifugation and the number of platelets was determined by Coulter.

Coagulative assays in whole blood The procoagulant activity in total non-anticoagulated blood was determined by a coagulative method. To 1 ml of non-anticoagulated whole blood, the different agents to be studied were added and the coagulation time was measured with a chronometer from the beginning of the extraction to the appearance of the stable and consolidated blood clot. The effect of the different agents was evaluated by the shortening or elongation they have over the time of blood coagulation.

In vivo tests Severe haemorrhage model by proximal section of rat tail 23 male Sprage-Dawley rats weighing 350-450 grams were randomly divided into 3 treatment groups: a control group, consisting of 14 animals that received topical treatment with saline physiological, while the other two groups, also composed of 5 animals, received topical treatment with rFT 1.2 μg / ml, and n = 4 topical treatment with rFT 1.2 μg / ml + SICN (vol: vol 1: 2,) respectively .. All the compounds came into topical contact with the proximal section of the animal's tail to act hemostasically dispensed by a plastic eppendorf pipette. The formation of stable and consolidated clot was evidenced by confirmation of the cessation of bleeding.

Severe hemorrhage model by proximal section of rat tail in animals treated with anticoagulant drugs 27 male Sprage-Dawley rats of 350-450 grams were randomized into 5 treatment groups: a control group, consisting of 14 animals that received topical topical treatment with physiological saline. Two groups received 200 U / kg of heparin i.v. 15 minutes before beginning the tail section procedure (to be treated with the FT n = 3 and to be treated with saline N = 5), and two other compound groups received orally 0.1 mg / Kg / day of warfaniña during 3 days before starting the tail section procedure (to treat with FT n = 3 and to be treated with saline, n = 2). Topical treatment with rFT 1.2 g / ml was performed only in one of each treatment group. Thus, it was a control group treated for each anticoagulation treatment. The lipidized TF came into topical contact with the proximal section of the tail of the animal to act hemostasically dispensed by a plastic eppendorf pipette. The formation of stable and consolidated clot was evidenced by confirmation of the cessation of bleeding.

Model of fatal hemorrhage by puncture in rat carotid artery 4 male Sprage-Dawley rats weighing 350-450 grams were randomly divided into 2 treatment groups that each included 2 animals. The animals of the control group received physiological saline as treatment, while the other group received lipidized FT administered topically (at a final dose of 2 dg of active protein). The model of lethal hemorrhage by rat carotid artery puncture was performed following standard procedures. The lipidized TF and the physiological serum were administered directly on the puncture site in a bandage containing 3 ml of each. This was combined with the pressure and contact of the corresponding treatment for two minutes.

II. RESULTS 1 In vitro trials demonstrating that lipidized FT (alone or in combination) acts as a direct stimulator of FXa causing fibrin clot formation and blood coagulation in the absence of FVII (caused by absence, deficiency or immunoblocking) 1. 1 The lipidized FT increases the proteolytic activity of FXa and, consequently, the production of thrombin in the absence of FVII. In order to evaluate the capacity of the lipidized FT, in the absence of FVII, as factor Xa stimulating agent, several in vitro tests. (i) chromogenic assays of amidolytic activity of FXa, in solution; and (ii) chromogenic assays of thrombin-forming activity in washed platelet suspension.

Chromogenic assays of amidolytic activity of FXa Direct assays of the amidolytic activity of FXa in solution using substrate S-2765 specific for FXa, demonstrated that lipidized TF is able to very significantly increase the proteolytic activity of FXa. In the presence of high concentrations of rFT (1 μ / l) an increase in the activity produced by low and high concentrations of FXa was observed (p <0.001). Similar significant stimulatory effects (p <0.001) were observed in the presence of lower concentrations of lipidized TF. Table 1 shows the results obtained in 5 independent experiments.

Table 1 Lipidized TF, in the absence of FVII, increases the proteolytic activity of FXa Without FTr FTr 0.1 FTr 1 FXa 30 nM 550 ± 51 2,108 ± 3,316 ± 61 89 FXa 5 nM 0 341 + 32 505 + 63 Mean ± SEM (n = 5) Chromogenic assays of thrombin formation activity in washed platelet suspensions The procoagulant effect detected was much greater when the platelet suspensions were used as procoagulant surface source and thrombin formation was determined by the specific substrate S-2238 (Table 2). In the absence of exogenous FXa, lipidized TF produced a significant stimulatory effect on thrombin formation (p <0.001). Similar effects were observed in the presence of exogenous FXa at low and high concentrations (p <0.001).

Table 2 Lipidized TF induces the formation of thrombin in platelets washed in the presence of FII but in the absence of FVa (already present in platelets) and FVII without FTr FTr 0.1 FTr 1 μ3 / ta1 μg / l FXa 1,250 421 ± 19 13, 854 ± 14,910 ± pM 145 168 FXa 250 pM 213 ± 21 9,324 ± 9,610 ± 155 114 FXa 125 pM 201 ± 29 7, 802 ± 8,508 ± 113 178 FXa absent 906 ± 89 3,504 ± 0 69 Mean ± SEM (n = 5) 1. 2 The lipidized TF is able to coagulate plasma and blood of deficient patients in FVII (coagulation assays in plasma and in non-anticoagulated total blood) A series of in vitro coagulation assays were performed that showed the coagulant effect of lipidized plasma TF and in non-anticoagulated total blood of patients deficient in FVII and, therefore, the lipidized FT is a useful agent for antihemorrhagic treatment.

Coagulation assays in commercial plasma without FVII and in non-anticoagulated total blood of patients deficient in FVII The effect of lipidized TF on coagulation was investigated by means of coagulation assays using commercial plasma without FVII and non-anticoagulated total blood of two patients with deficiency in the FVII (table 3). The lipidized TF in a concentration-dependent manner will be able to accelerate coagulation significantly in FVII-deficient plasmas. In the same way, non-anticoagulated whole blood from patients deficient in FVII coagulated in the presence of low, medium and high concentrations of lipidized FT, indicating that it is capable of producing a normal coagulation by means of FT even in the absence of FVII. Table 3 shows the results obtained from 5 independent experiments of deficient FVII plasmas.

Table 3 Demonstration of the procoagulant effect of lipidized TF in deficient plasma in FVII Basal coagulation time (s) with FTr 1 μg / ml 0.1 g / ml 0.01 Normal plasma 215.1 ± 11.1 ± 15.3 ± 25.7 ± 24.6 0.2 0_L2 0.4 Plasma > 300 67.7 ± 113.7 ± 202.7 ± deficient in 7.2 28.3 41.6 FVII Mean ± SEM (n = 5) The effect of lipidized TF in the blood of healthy volunteers was significant after the concentration of 0.01 Dg / ml, whereas higher concentrations were needed in patients deficient in FVII (Table 4). Although significant procoagulant effects were detected (p <0.001) after 0.1 Dg / ml, the lipidized TF was able to normalize the clotting time at a concentration of 0.01 Dg / ml, there were no differences between the coagulation times detected in normal subjects under baseline conditions and in subjects deficient in FVII with said concentration of lipidized TF. Finally, although the FT lipidized at 1 Dg / ml was not able to induce the same strong procoagulant effect in plasma without FVII, a very significant effect was observed, indicating that the lipidized FT can normalize hemostasis in these patients.

Table 4 Procoagulant effect of lipidized FT in non-angigoagulated whole blood of healthy and deficient individuals of FVII Basa FTr 1 0.01 0.1 1 10 100 ng / ml ng / ml ng / ml ng / ml ng / ml Sample control no. 1 5.8 4.7 4.4 3.3 2.1 1.3 Sample control no. 2 7.2 6.7 5.5 3.7 2.1 1.0 Patient no. 6 FVII- 11.4 9.5 2.3 n.d. 10.5 5.2 deficient Patient no. 7 FVII- 12.5 10.5 3.1 n.d 11.3 6.3 deficient not determined 1. 3 Lipidized TF is able to coagulate the plasma of healthy subjects in the presence of a monoclonal antibody against FVII (coagulation assays) In vitro coagulation assays were performed showing coagulant effect of TF on plasma of healthy subjects in the presence of an antibody monoclonal against FVII (this antibody is able to block coagulation in normal plasmas).

Coagulation assays in normal plasma lacking FVII by blocking with an anti-FVII The effect of lipidized TF on plasma coagulation was investigated by coagulation assays using plasma from healthy volunteers in the presence of a monoclonal antibody against FVII (Table 5 ). The lipidized TF was able to cause coagulation even in the presence of a monoclonal antibody against FVII, (which is capable of completely inhibiting blood coagulation). Under these experimental conditions (such as FVII-deficient plasmas), lipidized TF is capable of producing plasma coagulation. Table 5 Demonstration of the procoagulant effect of FT in normal plasma in the presence of a monoclonal antibody against FVII With FTr FTr. Mg / ml Normal plasma 126.8 28.1 Normal plasma with anti 216.8 42.5 FVII (400 / zg / ml) 1. 4 Combination of lipidized FT and SICN synergistically increases blood coagulation in FVII deficient plasmas (coagulation assays) A series of in vitro coagulation assays were performed showing the coagulant effect of lipidized FT associated with a negatively charged inorganic surface (SICN ) in deficient plasma of FVII. Said procoagulant effect exceeded that obtained when the lipidized TF was used only as a procoagulant agent. These results show that the effect mediated by lipidized TF is independent of FVII and that it is capable of inducing plasma coagulation even in the absence of its specific ligand. Table 6 shows the results obtained in 5 independent experiments.

Table 6 Procoagulant effect of FTr associated with negatively charged norganese surfaces in heparinized plasmas deficient in coagulation factor Basal coagulation time (s) With FTr Normal plasma 211.1 ± 18.5 18.6 ± 0.1 13 ± 1.2 * Deficient plasma > 300 67 ± 11 32 + 3.4 * in FVII Mean ± SEM (n = 5); * p < 0.001 without SICN versus with SICN; t- Student 1. 5 The combination of lipidized FT and FXa synergistically increase blood coagulation in the presence of a monoclonal antibody against FVII (coagulation assays in FX deficient plasmas) The procoagulant effects of the association of lipidized FT and FXa at low were investigated concentrations (170 pM and 1700 pM) in the absence of FVII in plasma deficient in FXa immunoblocked with an anti-FVII (Table 7).

Table 7 Procoagulant effect of lipidized TF associated with FXa in FXa deficient plasma in the absence of FVII (immunoblocked with anti FVII) Plasma Deficient coagulation time in FXa (s) With FXa (1700 With FXa (170 Without FXa pM) pM) Basal (5 mM> 400 133 ± 18 290 ± 10.2 calcium) With FTr 1 μ9 / p? 1 253.5 ± 83.1 ± 3.2 * 124 ± 6.1 * 11 With FTr 1 μg / ml 234, 5 ± 9 78 ± 2 * 124 ± 6.1 * + anti FVII (400 Mean ± SEM (n = 5); * p < 0.001 without FXa versus with FXa; t-Student 1. 6 The combination of lipidized FT, FXa and SICN synergistically increases blood coagulation in the presence of a monoclonal antibody to FVII (coagulation assays in FX-deficient plasmas) The procoagulant effects of the association of lipidized TF were also investigated, the FXa at low concentrations (170 pM and 1700 pM) and a SICN in deficient plasma of FXa (Table 8).

Table 8 Procoagulant effect of FTr associated with FXa and SICN plasma deficient in FXa deficient plasma Coagulation time in FXa (s) Without SICN (1 With SICN μ?) (2 μ?) Basal (calcium 5 mM) > 400 > 400 290 ± 10.2 FTr 1 μ / t? 1 + FXa 124 ± 6.1 109 ± 4.1 * 84 ± 3 * (170 p) * FTr 1 μg / ml + FXa 83, 1 ± 65.5 ± 5.2 * 45.5 ± 3.2 (1700 pM ) 3.2 * * FTr 1 μg / l + FXa 119 ± 5 * 101 ± 5 * 99 ± 3 * (170 pM) + anti FVII (400 μg / ml) FTr 1 μg / ml + FXa 78 ± 2 27.7 ± 1.2 25 ± 2 (1700 pM) + anti FVII (400 μg / ml) Mean ± SEM (n = 5); * p < 0.001 without SICN versus with SICN; Student 2. In vitro assays showing that the combination of lipidized FT with FXa at low concentrations (unable to induce any procoagulant effect) produces the coagulation of defective plasmas in FX 2. 1 Lipidized FT acts as a stimulator of FXa when this serine protease is present at low concentrations In vivo coagulation assays in FX deficient plasma were performed to show the synergistic effect of FT on FXa (added at low concentrations, 170 pM and 1700 pM, to plasma deficient in FX, so that these plasmas are unable to produce FXa from any coagulation pathway).

The procoagulant effects of the association of lipidized TF and FXa at low concentrations are shown in Table 9.

Table 9 Procoagulant effect of lipidized TF associated with FXa in deficient plasma of FXa Plasma Deficient coagulation time in FX (s) With FXa 1700 With FXa 170 Without FXa pM) pM) Basal (5 mM> 400 133 ± 18 290 ± 10.2 calcium) With FTr 1 253.5 ± 83, 1 ± 3.2 * 124 ± 6.1 * μg / ml 11 Mean ± SEM (n = 5); * p < 0.001 without FXa versus with FXa; t Student 2. 2. The combination of the lipidized TF with FXa and SICN acts synergistically in the stimulation of FXa The procoagulant effects of the association of the lipidized TF, FXa at low concentrations (170 pM and 1700 pM) and SICN in plasma deficient in FX (Table 10).

Table 10 Procoagulant effect of lipid-induced FT associated with FXa and SICN in deficient plasma of FXa Deficient Plasma Coagulation time in FX (s) With SICN (1 With SICN μ?) (2 μ?) Basal (5 m calcium) 1 > 400> 400 290 ± 10.2 FTr 1 μg / l + FXa 124 ± 6.1 109 ± 4.1 * 84 ± 3 * (170 pM)? FTr 1 μ? / T ?? + FXa 83, 1 ± 65.5 ± 5.2 * 45.5 ± 3.2 (1700 pM) 3.2 * * Mean ± SEM (n = 5); * p <0.001 without SICN vs. SICN; t-Student 3. In vitro trials demonstrating that lipidized FT (alone or in combination) causes blood coagulation in patients with deficiencies in other coagulation factors other than FVII 3. 1 Lipidized FT coagulates plasma and blood of patients with deficiencies in FV, FVIII, FVIII, FIX, FX, FXI, FXII and FXIII coagulation factors. The procoagulant effect of lipidized FT (only) in factor-deficient plasma was investigated. of coagulation using commercial plasmas lacking by means of immunoaffinity techniques, as well as in plasma of 3 patients diagnosed with hemophilia A (deficient in FVIII) and 2 diagnosed with hemophilia B (deficient in FIX). Table 11 shows the results. The concentration of 1 Dg / ml of lipidized FT was able to effectively coagulate all plasmas with deficiencies in FV, FVIII, FIX, FX, FXI, FXIII and FXIII. On the other hand, in the absence of the known cofactor of FXa, FVa, the lipidized TF was also capable of producing plasma coagulation, although only at medium and high concentration. In the deficiencies of remaining coagulation factors, lipidized TF was effective in all concentrations used, obtaining excellent results at very low concentrations. Even in the absence of FX (1%) the lipidized TF was able to coagulate but only at high concentrations. All these results were confirmed with samples from patients with hemophilia A and hemophilia B. Table 11 Procoagulant effect of lipidized TF in deficient plasma coagulation factor coagulation factor (s) Without FTr With FTr 0.01 - 1 g / ml 0, 1 μg / ml g / ml Normal plasma 215.1 ± 11.1 ± 15.3 ± 25.7 ± 24.6 0.2 0.2 0.4 Plasma FV-D > 600 84.3 + 123.3 ± 350 ± 15.2 24. 5 29. 5 Plasma FVIII-D > 600 15.5 ± 21.5 ± 32.1 ± 2.3 2.8 2.6 Plasma FIX-D > 600 14.8 ± 22.3 ± 34.6 ± 3.2 2.5 2.4 Plasma FX-D > 600 446.2 ± > 600 > 600 32 Plasma FXI-D > 600 14.3 ± 19.4 ± 27.3 ± 0.5 1.5 0.6 Plasma FXII-D > 600 16.2 ± 22.5 ± 32.5 ± 0.3 1.1 0.5 Plasma FXIII-D > 600 14.2 ± 21.5 ± 30.5 ± 0.3 1.0 0.5 Plasma > 600 12.2 ± 13.8 ± 0.6 21.3 ± Hemophilia A 1.5 0.8 Plasma > 600 12.1 ± 15.3 + 26.8 ± Hemophilia B 0.5 0.4 0.6 Mean ± SEM (n = 5) The procoagulant effect of lipidized FT (alone) in non-anticoagulated human whole blood of people suffering from hemophilia A and B was evaluated using a coagulation assay. The formation of the clot was determined by estimating the time in minutes to consolidate the clot. The effect of lipidized TF in blood of healthy volunteers was significant at concentrations of 0.001 Dg / ml, while higher doses were needed in hemophilic patients, with significant procoagulant effects (p <0.001) detected from 0.01 μg / ml . At a concentration of 0.1 mg / ml the lipidized TF was able to completely normalize the clotting time, there being no differences between the coagulation times detected in normal subjects and in hemophiliacs. Table 12 shows the results obtained from 4 samples from healthy individuals (samples numbers 1-4), 3 from patients suffering from hemophilia A and 2 from hemophilia B.

Table 12 Procoagulant effect of lipidized FT in non-anticoagulated whole blood of healthy subjects and hemophilic patients Basa 1 FTr 0.001 0.01 0.1 Control sample no. 1 5. 8 3.3 2.1 1.3 Sample control no. 2 7. 2 3.7 2.1 1.0 Sample control no. 3 7. 2 4.2 2.1 0.9 Sample control no. 4 7. 5 3.8 2.0 1.0 Patient no. 1 13 .3 8.0? 6 ±. C 1. O Hemophilia A Patient no. 2 17.3 9.0 5 g 1.0 Hemophilia A Patient no. 3 15.3 12.4 1.5 7.0 Hemophilia A Patient no. 4 20.5 11.1 1.5 6.1 Hemophilia B Patient no. 5 16.3 11.3 1.6 5.5 Hemophilia B Clotting time (expressed in minutes): the time the clot needs in a non-anticoagulated blood sample 3. 2 Lipidized TF coagulates whole blood and previously heparinized plasma The lipidized TF was able to coagulate plasmas (Table 13) and non-anticoagulated whole blood (Table 14) previously treated with high concentrations of heparin, demonstrating that its effect on FXa is independent of the inhibitory effect mediated by this anticoagulant drug and, even in the presence of heparin inhibitors.

Table 13 Effect of lipidized TF in heparinized plasma (coagulation time in seconds) Without lipidized TF With lipidized TF Control 320.4 ± 160 17.2 ± 1 Heparin 3 U / ml > 600 58 ± 12 Table 14 Effect of lipidized FT in heparinized whole blood (coagulation time in minutes) Without lipidized TF With lipidized TF Control 3. 96 ± 0.54 0, .4 ± o, .28 Heparin 0.25 12 .45 ± 0.69 15.7 ± o, .22 U / ml Heparin 1 U / ml 26.6 ± 5.76 4.5 ± 2.38 3. 3. Lipidized TF coagulates the plasma of warfarin-treated animals The lipidized TF was able to coagulate plasmas from rats treated with warfarin following the protocol specified in the methods. The results shown in Table 15 indicate that even when the synthesis of all the vitamin K-dependent coagulation factors is inhibited, the lipidized TF is able to coagulate, demonstrating that its effect on FXa is independent of the inhibitory effect mediated by these. anticoagulant drugs. Table 15 Effect of lipidized plasma TF of animals treated with warfarin (coagulation time in seconds) without TF With lipidized lipidized TF (1 μg / ml) Control 49 ± 10.2 10.9 ± 2.2 Plasma treated 60, 1 ± 5.6 12, 8 + 3.2 with warfarin (0.1 mg / kg for 3 days) 3. 4 The combination of lipidized FT with SICN synergistically increases plasma blood coagulation in patients with deficient coagulation factors FV, FVIII, FVIII, FIX, FXI, FXIII and FXIII (coagulation assays in coagulation factor-deficient plasma) and in whole blood of hemophiliac patients A series of in vitro coagulation assays were performed showing the effect of lipidized TF associated with SICN in plasma of patients deficient in coagulation factors (FV, FVII, FVIII, FIX, FXI, FXIII and FXIII). Said procoagulant effects exceeded those obtained when the lipidized TF was used, only as a procoagulant agent. Even at low concentrations (1 Dg / ml) with which the lipidized FT was not able to coagulate plasma deficient in FV (plasma FV-D), the combination of both agents produced a significant procoagulant effect. Likewise, similar synergistic effects were obtained when plasma deficient in FVII (plasma FVII-D) was used. These results show that the effect mediated by the lipidized TF is independent of FVII and is able to induce plasma coagulation even in the absence of FV. Similarly, the combination (lipidized TF + SICN) also produced potent procoagulant effects in plasma samples from hemophilic patients (A and B). Table 16 shows the results obtained in 5 independent experiments. Table 16 Procoagulant effect of FTr associated with SICN in coagulation factor-deficient plasmas Coagulation time (s) Without FTr With FTr 1 g / ml 1 g / ml + SICN normal plasma 281. 1 ± 12.5 11. 1 ± 0 .2 9.1 ± 0. 4 * plasma FV-D >; 600 84. 3 ± 15 .2 55.2 + 2 .3 * plasma FVIII-D > 600 15. 5 ± 2 .3 10.1 ± 0 .5 * plasma FIX-D > 600 14. 8 ± 3 .2 10.3 ± 0 .2 * plasma FX-D > 600 446; .2 ±: 32 253.5; ± 19 * plasma FXI-D > 600 14. 3 ± 0.5. 11.5 ± 0.6 * plasma FXII-D > 600 16. 2 ± 0 .3 12.9 ± 1.5. Plasma FXIII-D > 600 14. 2 ± 0 .3 10.8 ± 0.9 * Plasma Hemophilia > 600 12. 2 ± 1 .5 10.4 ± 0.9. * Plasma Hemophilia > 600 12. 1 ± 0 .5 10.1 ± 0. 8 * B Mean ± SEM (n = 5); * p < 0.001 without SICN versus with SICN; t- Student The procoagulant effect of lipidized FT and the combination SICN in non-anticoagulated whole blood were also investigated by coagulation assays using blood from patients suffering hemophilia A (3 patients) and B (2 patients). The procoagulant effect of the association (FTr + SICN) was significantly higher (p <0.001) (Table 17) compared to the trials in which the lipidized TF was alone (Table 12).

Table 17 Procoagulant effect of FTr together with SICN in whole blood Without SICN With SICN Sample control 7.1 ± 0.6 6 .0 ± 1.4 * FTr 0.1 μg / ml 1.1 ± 0.9 0 .5 ± 0.1 * Basal FTr 0.1 μg / ml + SICN Patient no. 1 13. 3 6.8 * Hemophilia A Patient no. 2 17. 3 7.2 * Hemophilia A Patient no. 3 15. 3 9.1 * Hemophilia A Patient no. 4 20. 5 8.5 * Hemophilia B Patient no. 5 16. 3 8.6 * Hemophilia B Clotting time (expressed in minutes); the time the clot needs to consolidate in a non-anticoagulated blood sample 4. In vitro tests demonstrating that lipidized TF (alone or in combination) causes blood to clot in healthy subjects 4.1 Lipidized TF coagulates plasma and blood Healthy subjects A series of in vitro coagulation assays were performed showing the coagulant effect of lipidized plasma FTT and non-anticoagulated whole blood of healthy volunteers with no history of haemostatic alterations. Table 18 shows the results obtained from 5 independent experiments in healthy plasmas and Table 19 in total blood not anticoagulated.

Table 18 Procoagulant effect of plasma FT in healthy subjects Normal plasma Coagulation time (s) Basal 281.08 ± 12.5 FTr 0.0001 μg / ml 201 ± 12.2 FTr 0.0001 μg / ml 184 ± 5.8 FTr 0.01 μg / ml 73.5 + 2.3 FTr 0.1 μg / ml 28.6 ± 0.1 FTr 1 μg / l 16.4 ± 0.9 Mean ± SEM (n = 5); the non-lipidized TF at the same concentration was not able to modify the coagulation time Table 19 Procoagulant effect of the TF in non-anticoagulated blood of healthy subjects Basa FTr Sample no. 1 5 .8 3.3 2. 1 1. 3 Sample no. 2 7 .2 3.7 2. 1 1. 0 Sample no. 3 7 .2 4.2 2. 1 0. 9 Sample no. 4 7 .5 3.8 2. 0 1. 0 4. 2 The combination of lipidized FT with SICN synergistically increases blood coagulation in plasma and blood of healthy subjects. Absence of synergistic effects when lipidized TF is associated with phospholipids A series of coagulation assays were performed in vi tro showing the coagulant effect of FTr associated with SICN in plasma of healthy volunteers. Table 20 shows the results obtained from 5 independent experiments. The procoagulant effect of the association (FTr + SICN) was significantly higher (p <0.001) in each and in all the concentrations used compared to the trials in which FTr was alone.

Table 20 Procoagulant effect of lipidized FT associated with SICN in normal plasma Normal plasma Coagulation time (s) Without SICN With SICN Basal 281.08 ± 167 ± 10 * 12.5 FTr 0.0001 μg / ml 201 ± 12.2 69 ± 0.8! * 'FTr 0.001 μg / ml 184 ± 5.8 44.9 ± 3.2 * FTr 0.01 ^ g / ml 73.5 ± 2.3 30.6 ± 2.5 * FTr 0.1 / xg / ml 28.6 ± 0.1 16 ± 4.2 * FTr 1 μg / ml 16.4 ± 0.9 12.8 + 0.5 * Mean ± SEM (n = 5); * p < 0.001 without SICN versus SICN: t-Student Table 21 shows the absence of synergistic effects when lipidized TF is associated with phospholipids (0.66 m FC / FS, phosphatidylcholine / phosphatidylserine at different molar ratio). Table 21 Effect of phospholipids on the procoagulant activity of lipidized TF Lipidized TF Time of coagulation (s) (0.1 / xg / ml) Without FC / FS 37.9 with FC / FS 44 90/10 80/20 39.7 70/30 41.7 60/40 40.1 50/50 43.5 40/60 34.6 30 / 70 41.5 20/80 44.3 10/90 41.9 . In vi tro trials demonstrating the coagulant effect of lipidized FT (alone and in combination) in the blood of patients with congenital and acquired platelet (thrombocytopenic) alterations . 1 Lipidized FT coagulates the whole blood of patients with congenital platelet alterations The procoagulant effect of lipidized FT on blood coagulation in patients with platelet alterations was investigated by coagulation tests using the blood of two patients suffering from Glanzmann's disease and the Syndrome Bernard. The FTr was able to accelerate very significantly, and in a concentration-dependent manner, blood coagulation in individuals suffering from Glanzmann's disease and Bernard's syndrome (Table 22); FTr is therefore a useful agent for the treatment of these individuals. Table 22 Procoagulant effect of TF in patients with platelet alterations Basa FTr 1 0.01 μg / vl 0.1 1 μg / l 9 / ml Control sample no. 15.8 3, .3 2, .1 1, .3 Patient no. 6 13.6 7. .3 2. .9 3, .4 Glanzmann's dis. Patient no. 7 S. 11.9 7.1 3.1 4.1 Bernard-Soulier 5.2 The combination of lipidized FT with SICN increases the coagulation in whole blood of patients with congenital platelet alterations A series of in vitro coagulation tests were carried out showing the coagulant effect of the FTr associated with SICN in whole blood of patients with congenital platelet alterations. Table 23 shows the results obtained from two patients with Glanzmann's disease and Bernard-Soulier syndrome. Comparing with the trials with the FTr alone, the procoagulant effect of the association (FTr + SICN) was significantly higher in each patient (p <0.001).

Table 23 Pro-coagulant effect of FTr together with SICN in whole blood Without SICN With SICN Patient no. 6 13.6 4.7 * Glanzmann Patient no. 7 S. 11.9 4.6 * Bernard-Soulier Clotting time (expressed in minutes): time the clot needs to consolidate in a sample of blood that is not anticoagulated. * p < 0.001 without SICN versus with SICN; t- Student . 3 Lipidized TF coagulates thrombocytopenic samples A series of in vitro coagulation assays were carried out showing the coagulant effect of lipidized TF in plasmas with different number of platelets. Table 24 shows the results obtained from normal platelet number at thrombocytopenic conditions.

Table 24 Effect of FT tetrapidemic in thrombocytopenic samples Number of Without FT With Lipidized Lipid FT Platelets (1 / xg / mL) 350, 000 / μ1 226.3 21.9 150, 000 / μ1 232.6 22.8 50, 000 / μ1 253.9 22.4 9, 000 / μ1 321.2 21.3 < 1,000 / μ? > 400 21.3 6. In vivo assays demonstrating that FT is a useful agent for topical antihemorrhagic treatment in control rats (by direct application in the previously sectioned blood vessel) 6. 1 Lipidized TF is useful as a topical haemostatic agent in an animal model of severe hemorrhage by proximal section of rat tails In vivo tests were performed showing that lipidized TF administered alone or in association with SICN is a useful agent for topical antihemorrhagic treatment. The use of lipidized TF as a topical haemostatic agent administered alone or combined with SICN was evaluated using an animal model of severe hemorrhage by proximal section of rat tails. The results obtained are shown in Table 25. As can be observed, in said animal model of severe hemorrhage, the bleeding coagulated spontaneously in the control animals (control SSF, treated with physiological saline solution) at 18.1 ± 5.98 minutes; however, topical administration of the lipidized FTr (alone) produced a significant reduction (11.1 ± 5.54 minutes, p <0.001). When the FTr was administered in combination with a SICN the procoagulant effect was even higher (5.0 ± 1.1 minutes, p <0.001).

Table 25 Severe hemorrhage model by proximal section of rats. Bleeding coagulation time Bleeding clotting time Saline control group 18.16 ± 5.98 (n = 14) Lipidized treatment group FT 11.1 ± 5.5 (n = 5) Lipidized TF + SICN 5.0 ± 1.1 (n = 4) The results are expressed as the time in minutes to reach a consolidated coagulation The non-lipidized TF was evaluated under the same experimental conditions (n = 3). No effects were observed and the clotting time of bleeding was similar to that of the control animals. These results indicate that unlipidated TF is not useful for the topical treatment of bleeding episodes. 6. 2 Lipidized TF is useful as a topical haemostatic agent in an animal model of severe haemorrhage previously treated with heparin or warfarin In vivo tests were performed showing that lipidized TF administered alone is a useful agent for topical antihemorrhagic treatment under anticoagulant conditions (Table 25 ). The use of lipidized FT as a topical haemostatic agent administered only by using an animal model of severe hemorrhage was evaluated by the proximal section of rat tails previously treated with 200 u / kg of heparin i.v. 15 minutes before beginning the procedure of the tail transection. The results obtained are shown in Table 26. As can be seen, in this severe hemorrhage model, the saline control group, treated with physiological saline solution, coagulated spontaneously at 18.1 ± 5.98 minutes. The control group treated with heparin did not coagulate the bleeding spontaneously (> 90 minutes). The group treated with warfarin coagulated spontaneously at 41.6 ± 8.5 minutes. Table 26 shows that topical administration of lipidized FT (alone) produces a significant reduction in all treatment groups.

Table 26 Animal model of severe hemorrhage by proximal section of rat tails in animals treated with anticoagulant. Bleeding coagulation time Bleeding coagulation time (min.) Saline control group (n = 14) 18.16 ± 5.98 Control group treated with > 90 heparin (n = 5) Control group treated with 41.6 ± 8.5 warfarin (n = 2) Group treated with heparin 26.3 ± 2.5 (n = 3) + Lipid FT Group treated with warfarin 4.5 ± 2.5 (n = 3 ) + Lipidized TF The results are expressed as the time in minutes to reach a consolidated coagulation 6. 3 Lipidized TF is useful as a topical haemostatic agent in an animal model of lethal hemorrhage by puncture in the carotid artery In vivo tests were performed showing that the administered rTF is only a useful agent for the topical antihemorrhagic treatment directly applied to the blood vessel. The use of FTr administered only by the use of an animal model of lethal hemorrhage by puncture of the carotid artery was evaluated. The results obtained are shown in Table 27 and were very significant. As can be observed, in the group of control animals, (control SSF, treated with physiological saline solution), all animals died by bleeding, while in the lipidized TF group no animals died and the section could be sealed and coagulate satisfactorily in all cases. The treatment was effective in terms of the time necessary to achieve stable coagulation and sealing of the puncture wound.

Table 27 Model of lethal hemorrhage by carotid artery puncture of rats Control SF FTr animal dead animal no. 3 125 no. 1 animal dead animal no. 4 135 no. 2 Mean ± 130 ± 7.1 DE The results are expressed as the time in seconds to achieve a consolidated coagulation and the sealing of the puncture wound. The FTr administered as indicated in the text at a dose of 2 Dg of active protein In conclusion: The results of Example number 1 clearly demonstrate that: 1) in the absence of its ligand, FVII, the lipidized FT is able to directly interact with the FXa significantly increasing its proteolytic activated (both, amidolytic and thrombin-forming), these results show first time a new role for the lipidized TF, acting as a new cofactor for the FXa (independent of the well known FVa). 2) Lipidized TF is able to coagulate FVII-deficient plasmas. Therefore, lipidized TF is a good alternative for the treatment of these patients (currently the only treatment is the expensive recombinant FVIIa). 3) The lipidized FT acts synergistically with SICN and FXa (at low concentrations unable to produce coagulation).

The results of Example number 2 clearly demonstrate that: 1) The lipidized FT produces physiological concentrations of FXa, unable to produce any significant procoagulant effect, trigger the hydrolysis of prothrombin and consequently the formation of thrombin takes place. 2) The lipidized TF has a new role acting as a cofactor for all the proteolytic activities of all forms of FXa (soluble and bound to the prothrombinase complex). 3) Even in FX-deficient plasmas (which contain traces of FX) they can be coagulated by the lipidized TF. Therefore, lipidized TF is a good alternative for the treatment of these patients, 4) Lipidized TF acts synergistically with a SICN in the stimulation of FXa activity.

The results of Example number 3 clearly demonstrate that: 1) The lipidized TF is capable of producing blood coagulation in hemophilic patients (FVIII, FIX and FXI). Therefore, lipidized TF is a good alternative for the treatment of these patients. 2) The lipidized TF is capable of producing blood coagulation even in the absence of VF. These results clearly show that the lipidized FT produces a strong stimulatory effect in the FXa, because in the absence of its cofactor (plasmas deficient in FV), the lipidized FT acts as a cofactor causing the same stimulatory effect. 3) The lipidized TF is able to produce blood coagulation even in FX-defective plasmas (which contains traces of FX). Therefore, lipidized TF is an alternative for the treatment of these patients. 4) Lipidized TF is capable of producing blood coagulation in heparin and warfarin-treated plasmas, indicating that lipidized TF interferes with the effect of antithrombin III and probably through its stimulatory effect at basal FXa concentrations can even coagulate under conditions of warfarin treatment. 5) Finally, the lipidized FT acts synergistically with SICN in the procoagulant effect observed in all the deficiencies in coagulation factors.

The results of Example number 4 clearly show that: 1) The lipidized TF is capable of producing blood coagulation in healthy subjects. Therefore, lipidized TF is a good alternative for the treatment of bleeding episodes in healthy subjects.

Lipidized TF acts synergistically with SICN but not only with phospholipids in the procoagulant effect observed in healthy subjects.

The results of Example number 5 clearly demonstrate that: 1) Lipidized TF is capable of producing blood coagulation in subjects with platelet alterations, such as congenital and acquired (eg, thrombocytopenia). Therefore, lipidized TF is a good alternative for the treatment of bleeding episodes in patients with alterations in the number and / or functionality of platelets.

The results of Example number 6 clearly demonstrate that: 1) The lipidized FT administered topically is capable of stopping bleeding in an animal model of severe hemorrhage (total proximal tail transection). 2) Topically administered lipidized FT is able to stop bleeding in an animal model of severe hemorrhage (total proximal tail transection) complicated by anticoagulation therapy (heparin or warfarin). 3) The lipidized FT administered topically is able to stop bleeding in an animal model of lethal hemorrhage (carotid puncture in the carotid artery).

Claims (44)

CLAIMS 1. Use of Lipidized Tissue Factor (TF), or a functional fragment thereof, in the preparation of a medicament for the topical treatment of hemorrhages in a subject. 2. Use according to claim 1, wherein said subject is a healthy subject or a subject having a hemorrhagic diathesis, wherein said hemorrhagic diathesis comprises a coagulopathy and / or a platelet alteration. 3. Use according to claim 2, wherein said coagulopathy is a congenital coagulopathy or an acquired coagulopathy. 4. Use according to claim 3, wherein said congenital coagulopathy is a coagulopathy based on a deficiency in a clotting factor selected from coagulation factor V, coagulation factor VII, coagulation factor VIII, coagulation factor IX, coagulation factor X, Coagulation factor XI, Coagulation factor XII, Coagulation factor XIII and combinations thereof. 5. Use according to claim 4, wherein said congenital coagulopathy is a coagulopathy based on a deficiency in a coagulation factor selected from the coagulation factor V. 6. Use according to claim 4, wherein said congenital coagulopathy is a coagulopathy based on a deficiency in a coagulation factor selected from Coagulation Factor VII. 7. Use according to claim 4, wherein said congenital coagulopathy is a coagulopathy based on a deficiency in a coagulation factor selected from Coagulation Factor X. 8. Use according to claim 2, wherein said subject has an acquired coagulopathy produced by an anticoagulant treatment with anticoagulants. 9. Use according to claim 8, wherein the anticoagulants are heparin, low molecular weight heparins, warfarin, coumarin derivatives or dicoumarins. 10. Use according to claim 2, wherein said subject presents a congenital or acquired platelet alteration. 11. Use according to claim 10, wherein said congenital platelet alteration is selected from Glanzamn's disease, Sind Bernard Soulier's disease, Bolin-Jamieson syndrome, Wiskott -Aldrich, Paris-Trousseau-Jacobsen syndrome, thrombocytopenia of the X chromosome, Gray's platelet syndrome, Sebastian's syndrome and Fanconi's anemia. 12. Use according to claim 10, wherein said acquired platelet alteration is selected from a myeloproliferative disorder, such as thrombocythemia, polycythemia, or chronic myelocytic leukemia; myeloid metaplasia; disproteinemias in scurvy, in congenital heart disease and in cirrhosis. 13. Use according to claim 1, wherein said lipidized FT is lipidized human FT. 14. A product comprising (i) lipidized TF and (ii) Activated X coagulation factor (FXa). 15. A product comprising, separately, (i) lipidized TF and (ii) activated FXa. 16. A product comprising, separately, (i) lipidized TF and (ii) FXa, as a combination for simultaneous or successive administration to a subject. 17. A product comprising (i) lipidized TF and (ii) a negatively charged inorganic surface (SICN). 18. A product comprising, separately, (i) lipidized TF and (ii) a SICN. 19. A product comprising, separately, (i) lipidized TF and (ii) a SICN, as a combination for simultaneous or successive administration to a subject. 20. A product comprising (i) lipidized TF, (ii) FXa and (iii) a SICN. 21. A product comprising, separately, (i) lipidized TF, (ii) FXa and (iii) a SICN. 22. A product comprising, separately, (i) lipidized TF, (ii) FXa and (iii) a SICN, as a combination for simultaneous or successive administration to a subject. 23. Product according to any of claims 14 to 22, wherein said lipidized FT is lipidized human FT. 24. Product according to any of claims 14 to 23, as a medicine. 25. Use of a product according to any of claims 14 to 23, in the manufacture of a medicament for the treatment of hemorrhages in a subject. 26. Use of a product according to any of claims 14 to 23, in the preparation of a medicament for the topical treatment of haemorrhages in a subject. 27. A complex (FT :: FXa) comprising lipidized FT and FXa. 28. Complex according to claim 27, as a medicine. 29. Use of a complex according to claim 27, in the preparation of a medicament for the treatment of hemorrhages in a subject. 30. Use of a complex according to claim 27, in the preparation of a medicament for the topical treatment of haemorrhages in a subject. 31. A complex (FT :: SICN) comprising lipidized FT and a SICN. 32. Complex according to claim 30, as a medicine. 33. Use of a complex according to claim 31, in the preparation of a medicament for the treatment of hemorrhages in a subject. 34. Use of a complex according to claim 31, in the preparation of a medicament for the topical treatment of haemorrhages in a subject. 35. A complex (FT:: FXa:: SICN) comprising lipidized FT, FXa and a SICN. 36. Complex according to claim 35, as a medicine. 37. Use of a complex according to claim 35, in the preparation of a medicament for the treatment of hemorrhages in a subject. 38. Use of a complex according to claim 35, in the preparation of a medicament for the topical treatment of haemorrhages in a subject. 39. A pharmaceutical composition comprising: a) a product according to claim 14, together with a pharmaceutically acceptable carrier; or b) separately, (i) lipidized TF together with a pharmaceutically acceptable carrier, and (ii) FXa together with a pharmaceutically acceptable carrier; or c) a product according to claim 17, together with a pharmaceutically acceptable carrier; or d) separately, (i) lipidized TF together with a pharmaceutically acceptable carrier, and (ii) a SICN together with a pharmaceutically acceptable carrier; or e) a product according to claim 20, together with a pharmaceutically acceptable carrier; or f) separately, (i) lipidized TF together with a pharmaceutically acceptable carrier, (ii) FXa together with a pharmaceutically acceptable carrier, and (iii) a SICN together with a pharmaceutically acceptable carrier; or g) an FT :: FXa complex according to claim 27, together with a pharmaceutically acceptable carrier; or h) an FT :: SICN complex, according to claim 31, together with a pharmaceutically acceptable carrier; or i) an FT complex:: FXa:: SICN, according to the claim 35, together with a pharmaceutically acceptable vehicle. 40. Pharmaceutical composition according to claim 39, in a pharmaceutical dosage form for topical administration. 41. A product comprising a pharmaceutical composition according to any of claims 39 or 40 and a support. 42. A product comprising a pharmaceutical composition comprising: (i) a support, (ii) a product according to the claim 14, together with a pharmaceutically acceptable carrier, (iii) a product according to claim 17, together with a pharmaceutically acceptable carrier and (iv) a product according to claim 20, together with a pharmaceutically acceptable carrier; or an FT :: FXa complex, according to claim 27, together with a pharmaceutically acceptable carrier, or an FT :: SICN complex according to claim 30, together with a pharmaceutically acceptable carrier, or an FT:: FXa:: SICN complex, according to claim 34, together with a pharmaceutically acceptable carrier. 43. Use of the non-lipidized TF, or a functional fragment thereof, for the preparation of a medicament for the treatment of hemorrhages in a subject with a deficit in Coagulation Factor VII. 44. Use according to claim 43, for the preparation of a medicament for the parenteral administration of non-lipidized FT or fragment thereof. CLAIMS

1. Use of Lipidized Tissue Factor (TF), or a functional fragment thereof, in the preparation of a medicament for the topical treatment of hemorrhages in a subject.

2. Use according to claim 1, wherein said subject is a healthy subject or a subject having a hemorrhagic diathesis, wherein said hemorrhagic diathesis comprises a coagulopathy and / or a platelet alteration.

3. Use according to claim 2, wherein said coagulopathy is a congenital coagulopathy or an acquired coagulopathy.

4. Use according to claim 3, wherein said congenital coagulopathy is a coagulopathy based on a deficiency in a clotting factor selected from coagulation factor V, coagulation factor VII, coagulation factor VIII, coagulation factor IX, coagulation factor X, Coagulation factor XI, Coagulation factor XII, Coagulation factor XIII and combinations thereof.

5. Use according to claim 4, wherein said congenital coagulopathy is a coagulopathy based on a deficiency in a coagulation factor selected from the coagulation factor V.

6. Use according to claim 4, wherein said congenital coagulopathy is a coagulopathy based on a deficiency in a coagulation factor selected from Coagulation Factor VII.

7. Use according to claim 4, wherein said congenital coagulopathy is a coagulopathy based on a deficiency in a coagulation factor selected from Coagulation Factor X.

8. Use according to claim 2, wherein said subject has an acquired coagulopathy produced by an anticoagulant treatment with anticoagulants.

9. Use according to claim 8, wherein the anticoagulants are heparin, low molecular weight heparins, warfarin, coumarin derivatives or dicoumarins.

10. Use according to claim 2, wherein said subject presents a congenital or acquired platelet alteration.

11. Use according to claim 10, wherein said congenital platelet alteration is selected from Glanzamn's disease, Sind Bernard Soulier's disease, Bolín-Jamieson syndrome, Wiskott -Aldrich, Paris-Trousseau-Jacobsen syndrome, thrombocytopenia of the X chromosome, Gray's platelet syndrome, Sebastian's syndrome and Fanconi's anemia.

12. Use according to claim 10, wherein said acquired platelet alteration is selected from a myeloproliferative disorder, such as thrombocythemia, polycythemia, or chronic myelocytic leukemia; myeloid metaplasia; disproteinemias in scurvy, in congenital heart disease and in cirrhosis.

13. Use according to claim 1, wherein said lipidized FT is lipidized human FT.

14. A product comprising (i) lipidized TF and (ii) Activated X coagulation factor (FXa).

15. A product comprising, separately, (i) lipidized TF and (ii) activated FXa.

16. A product comprising, separately, (i) lipidized TF and (ii) FXa, as a combination for simultaneous or successive administration to a subject.

17. A product comprising (i) lipidized TF and (ii) a negatively charged inorganic surface (SICN).

18. A product comprising, separately, (i) lipidized TF and (ii) a SICN.

19. A product comprising, separately, (i) lipidized TF and (ii) a SICN, as a combination for simultaneous or successive administration to a subject.

20. A product comprising (i) lipidized TF, (ii) FXa and (iii) a SICN.

21. A product comprising, separately, (i) lipidized TF, (ii) FXa and (iii) a SICN.

22. A product comprising, separately, (i) lipidized TF, (ii) FXa and (iii) a SICN, as a combination for simultaneous or successive administration to a subject.

23. Product according to any of claims 14 to 22, wherein said lipidized FT is lipidized human FT.

24. Product according to any of claims 14 to 23, as a medicine.

25. Use of a product according to any of claims 14 to 23, in the manufacture of a medicament for the treatment of hemorrhages in a subject.

26. Use of a product according to any of claims 14 to 23, in the preparation of a medicament for the topical treatment of haemorrhages in a subject.

27. A complex (FT :: FXa) comprising lipidized FT and FXa.

28. Complex according to claim 27, as a medicine.

29. Use of a complex according to claim 27, in the preparation of a medicament for the treatment of hemorrhages in a subject.

30. Use of a complex according to claim 27, in the preparation of a medicament for the topical treatment of haemorrhages in a subject.

31. A complex (FT :: SICN) comprising lipidized FT and a SICN.

32. Complex according to claim 30, as a medicine.

33. Use of a complex according to claim 31, in the preparation of a medicament for the treatment of hemorrhages in a subject.

34. Use of a complex according to claim 31, in the preparation of a medicament for the topical treatment of haemorrhages in a subject.

35. A complex (FT:: FXa:: SICN) comprising lipidized FT, FXa and a SICN.

36. Complex according to claim 35, as a medicine.

37. Use of a complex according to claim 35, in the preparation of a medicament for the treatment of hemorrhages in a subject.

38. Use of a complex according to claim 35, in the preparation of a medicament for the topical treatment of haemorrhages in a subject.

39. A pharmaceutical composition comprising: j) a product according to claim 14, together with a pharmaceutically acceptable carrier; or k) separately, (i) lipidized TF together with a pharmaceutically acceptable carrier, and (ii) FXa together with a pharmaceutically acceptable carrier; or 1) a product according to claim 17, together with a pharmaceutically acceptable carrier; or m) separately, (i) lipidized TF together with a pharmaceutically acceptable carrier, and (ii) a SICN together with a pharmaceutically acceptable carrier; or n) a product according to claim 20, together with a pharmaceutically acceptable carrier; or or) separately, (i) lipidized TF together with a pharmaceutically acceptable carrier, (ii) FXa together with a pharmaceutically acceptable carrier, and (iii) a SICN together with a pharmaceutically acceptable carrier; or p) an FT:: FXa complex, according to claim 27, together with a pharmaceutically acceptable carrier; or q) an FT :: SICN complex, according to claim 31, together with a pharmaceutically acceptable carrier; or r) an FT :: FXa :: SICN complex, according to claim 35, together with a pharmaceutically acceptable carrier.

40. Pharmaceutical composition according to claim 39, in a pharmaceutical dosage form for topical administration.

41. A product comprising a pharmaceutical composition according to any of claims 39 or 40 and a support.

42. A product comprising a pharmaceutical composition comprising: (i) a support, (ii) a product according to claim 14, together with a pharmaceutically acceptable carrier, (iii) a product according to claim 17, together with a pharmaceutically acceptable carrier and (iv) a product according to claim 20, together with a pharmaceutically acceptable vehicle; or an FT :: FXa complex according to claim 27, together with a pharmaceutically acceptable carrier, or an FT :: SICN complex according to claim 30, together with a pharmaceutically acceptable carrier, or an FT:: FXa:: SICN complex, according to claim 34, together with a pharmaceutically acceptable carrier.

43. Use of the non-lipidized TF, or a functional fragment thereof, for the preparation of a medicament for the treatment of hemorrhages in a subject with a deficit in Coagulation Factor VII.

44. Use according to claim 43, for the preparation of a medicament for the parenteral administration of non-lipidized FT or fragment thereof.