WO2005076827A2 - Fbsa compositions for use in promoting fibrinogen aggregation and potentiating fibrin polymerization - Google Patents

Abstract

A composition containing the FbsA protein from Streptococcus agalactiae is provided which is effective in potentiating the polymerization of fibrin or in promoting fibrinogen aggregates so as to be useful as a wound sealant, and a pharmaceutically acceptable vehicle, carrier or excipient. Methods of treating patients by administering an amount of FbsA effective to promote aggregation of fibrinogen so as to promote blood clotting and wound sealing, or an amount effective to potentiate polymerization of fibrin so as to be useful as a fibrin wound sealant or an adjuvant for a fibrin wound sealant are also provided, and said methods are particularly advantageous in treating wounds without the need for sutures or staples in a variety of medical applications.

Description

FBSA COMPOSITIONS FOR USE IN PROMOTING FIBRINOGEN AGGREGATION AND POTENTIATING FIBRIN POLYMERIZATION

Cross-Reference To Related Applications This application claims the benefit of US Provisional Application Ser. No. 60/541,264, filed February 4, 2004, said application incorporated herein by reference.

Field of the Invention The present invention relates in general to the use of FbsA from Streptococcus agalactiae as a wound sealant either directly as a fibrinogen sealant through the promotion of fibrinogen aggregation or as a fibrin sealant or an adjuvant for a fibrin sealant through the potentiation of fibrin polymerization used at a particular wound site. The fibrinogen sealant of the invention is comprised of an FbsA protein in an amount effective to promote fibrinogen aggregation so as to cause clot formation, and the fibrin sealant or adjuvant to be used in conjunction with fibrin comprises an amount of FbsA effective in potentiating the polymerization of fibrin so as to be useful as a fibrin sealant or an adjuvant for a fibrin sealant.

Background of the Invention The fibrin sealants, also known as fibrin glues or fibrin tissue adhesives, are the most successful of the tissue sealants in terms of tissue compatibility, biodegradation and clinical utility. The use of fibrin sealants as opposed to the traditional wound-closing devices such as sutures or staples has been increasingly in demand not only because it can reduce the pain involved in applying and removing these devices, but further can avoid the complications that may arise with suture or staple use such as ischemia, fistualization, granuloma formation and foreign body reaction, which may possibly be associated with partial graft loss. The fibrin sealants have thus been highly advantageous in that they can be used safely and effectively with less discomfort to the patient and with minimal formation of scar tissue. Fibrin sealants have been derived from plasma coagulation proteins. The major components of fibrin sealants are generally fibrinogen, thrombin and calcium chloride, although other constituents may be present, including factor II or XIII and an antifibrinolytic agent such as aprotinin or epsilon-aminocaproic acid. The components of fibrin sealants are typically formulated as two solutions, which are mixed together in equal volumes to mimic the final phase of the coagulation cascade, the conversion of fibrinogen to fibrin, in which a firm clot is produced in minutes. The properties of both the sealant and the clot can be affected by variation in the concentration of fibrinogen and thrombin. The mechanical strength of the fibrin clot is influenced by the proportion of fibrinogen, factor XIII and adhesive proteins (fibronectin) in the fibrin sealant, and an important interaction in the tissue-sealing process is the conversion by thrombin of fibrinogen into fibrin monomers. Higher concentrations of fibrinogen tend to produce stronger clots, whereas sealants containing higher concentrations of thrombin tend to form clots more rapidly and add to clot adhesion. Fibrin sealants are used in a wide range of surgical settings to successfully bring about hemostasis, tissue adhesion and wound healing. In particular, fibrin sealants can be used safely in a wide variety of surgical procedures including: cardiovascular, gastrointestinal, pneumothoracic surgery, neurosurgery, urology, otolaryngology and reconstructive surgery. In the patent arts, there are several patents which disclose various types of fibrin sealants, including US Patent Nos. 6,262,236; 6,268,483; 6,503,527; and 6,699,484, all of said patents incorporated herein by reference. However, the preparation of fibrin sealants has been limited due to the specific materials including blood components that must be obtained in order to obtain the fibrin sealant. In addition, because of problems in combining the fibrinogen and thrombin precursors, there has been a need to develop more effective fibrin sealants which can be prepared from new materials which can be used safely and efficiently. It is thus clear that there is a distinct need to develop additional fibrin sealants which will be safe, effective and easy to use, and which will minimize any undesirable side effects. Summary of the Invention Accordingly, it is an object of the present invention to provide compositions using the FbsA protein from group B streptococci which can be useful as fibrinogen sealants or as adjuvants for fibrin sealants. It is another object of the present invention to provide an FbsA protein in amounts effective to be used in order to promote fibrinogen aggregation and thus be useful as a fibrinogen sealant to seal wounds in a variety of applications. It is another object of the present invention to provide an FbsA protein in amounts effective to be used as a potentiator of fibrin polymerization so as to be useful as a fibrin sealant or an adjuvant to a fibrin sealant. It is further an object of the present invention to provide a method for administering an FbsA composition to a patient in need thereof in an amount effective to potentiate fibrin polymerization and thus be useful as a fibrin sealant or an adjuvant to a fibrin sealant, or in an amount effective to promote fibrinogen aggregation and thus be useful as a fibrinogen sealant.

These and other objects are provided by virtue of the present invention which comprises utilizing the FbsA protein from Streptococcus agalactiae in amounts effective to potentiate the polymerization of fibrin so as to be useful as a fibrin sealant or an adjuvant for a fibrin sealant, or in an amount effective to enhance or promote fibrinogen aggregation so to as be useful as a fibrinogen wound sealant on its own, along with a pharmaceutically acceptable vehicle, carrier or excipient. In addition, a method is provided wherein FbsA may be administered to a patient in need in an amount effective to promote fibrinogen aggregation or potentiate fibrin polymerization so as to be effective in acting on its own as a fibrinogen sealant or as a fibrin sealant or an adjuvant to a fibrin sealant in which case it acts along with thrombin or other fibrinogen-cleaving agent to potentiate fibrin polymerization and promote wound closure and healing.

These embodiments and other alternatives and modifications within the spirit and scope of the invention will become readily apparent to those skilled in the art from reading the present specification and/or the references cited herein. BRIEF DESCRIPTION OF THE DRAWING FIGURES Fig. 1 shows fibrin polymerization in the absence or presence of rFbsA. In the tests reflected in this figure, polymerization was initiated by the addition of thrombin (0.05 U/ml) at time zero to 1 μM (1 micromole/L) fibrinogen and the polymer formation at 25°C was measured as a change in turbidity at 350 nm with time. The influence of FbsA isoforms (FbsA3, FbsA6, FbsA9, FbsA15 and FbsA19) on the assembly of clot was examined by addition of 0.5 μM (0.5 micromole/L) FbsA proteins at the same time with thrombin. Fig. 2 shows the effect of FbsA isoforms on formation of fibrinogen aggregates. In the tests reflected in this figure, polymerization was initiated by the addition of FbsA isoforms (final concentration: 0.5 μM/L) at time zero to 1μM (1 micromole/L) fibrinogen at 25°C in the presence of the indicated amounts of FbsA and measured as reported in Fig. 1. Figs. 3A-3C reflect scanning electron micrographs of regular clot and FbsA-induced fibrinogen aggregates. In the tests reflected in these figures, a regular clot was made incubating at 25°C purified fibrinogen (1 micromole/L) and thrombin (0.05 U /mL) for 45 min (Panel 3A: X 5 000). To form macromolecular fibrinogen complexes, FbsA19 and fibrinogen were incubated at a molar ratio of 1:2 for 45 min (Panels 3B and 3C: X 5 000 and 15 000, respectively). Fig. 4 shows the effect of bacterial fibrinogen-binding proteins on fibrin polymerization. In the tests reflected in this figure, thrombin-induced fibrin polymerization in the presence of 0.5 μM (0.5 micromole/L) ClfA, ClfB, FnbpA and FnbpB from S. aureus and SdrG from S. epidermidis was performed at 25°C as reported in Figure 1. The effect of equimolar concentrations of bovine serum albumin or FbsA 19 is also reported.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In accordance with the present invention, a fibrin sealant is provided which comprises an FbsA composition an amount of FbsA effective in potentiating the polymerization of fibrin so as to be useful as a fibrin sealant or an adjuvant for a fibrin sealant. The FbsA protein from Streptococcus agalactiae (also referred to as Group B Streptococcus or GBS) has been shown to be a fibrinogen binding protein (see Schubert et al., Mol Microbiol. 2002 Oct; 46(2):557-69, incorporated herein by reference), but has heretofore not been disclosed or suggested for use as a fibrin sealant. As shown in Schubert et al., the deduced FbsA protein is characterized by repetitive units, each 16 amino acids in length. In the preferred FbsA protein, this repeat unit has the amino acid sequence GNVLERRQRDAENRSQ (SEQ ID NO:1). Sequencing of the gene coding for the FbsA protein, or fbsA gene, from five different GBS strains revealed significant variation in the number of repeat-encoding units, and the repeat region of FbsA was identified as mediating fibrinogen binding. In Western blot experiments with truncated FbsA-proteins, the repeat region of FbsA was identified as mediating fibrinogen binding. Using synthetic peptides, even a single repeat unit of FbsA was demonstrated to bind to fibrinogen. Accordingly, as set forth herein, the term "FbsA protein" includes the whole FbsA protein as well as FbsA proteins containing one or more repeat units which are capable of binding to fibrinogen. Such "isoforms" of FbsA are well known and can include various numbers of repeat units such as 19 (FbsA19), 15 (FbsA15), 9 (FbsA9), 6 (FbsAδ), or 3 (FbsA3), and such isoforms are useful in the compositions of the invention. In accordance with the invention, it is preferred that the FbsA proteins used in the invention be recombinantly prepared, but it is also possible to use natural versions if so desired. However, the fact that FbsA is a fibrinogen-binding protein yet still potentiates fibrin polymerization and promoted fibrinogen aggregation is quite unusual in that other fibrinogen-binding proteins, such as ClfA, ClfB, FnbpA and FnbpB from S. aureus and SdrG from S. epidermidis, actually inhibit the process of fibrin polymerization. These results are consistent with the role of the C- terminus of fibrinogen gamma chain in both binding of ClfA (or FnbpA/FnbpB) and fibrin polymerization. Likewise, binding of SdrG to the N-terminus of the Bbeta chain results in occupation and steric hindrance of the thrombin binding site and subsequent inhibition of fibrin monomer formation and the protofibril assembly. While it is unclear why an FbsA protein can act as a fibrin or fibrinogen sealant or an adjuvant for a fibrin sealant, it is possible that the potentiating effect of FbsA on fibrin polymerization could be explained if it is assumed that each FbsA molecule does not bind to any of the fibrin(ogen) polymerization sites. Furthermore, the multivalency of FbsA due to a multiplicity of tandem fibrinogen-binding repeat units could allow the formation of complexes which prelude the true fibrin polymerization process when thrombin is added to the mixture. In accordance with the present invention, a composition is provided which will comprise an effective amount of an FbsA protein in combination with a suitable pharmaceutically acceptable vehicle, carrier or excipient. In this context, by "effective amount" is meant that level of use, such as of the particular FbsA protein employed in the composition, that will be sufficient to potentiate fibrin polymerization (when used with a fibrinogen-cleaving agent such as thrombin) or sufficient to induce fibrinogen aggregate formation so as to be useful as a wound sealant promoting clotting to achieve wound closure and begin healing. As such, the present inventions includes FbsA compositions which on their own may be added to fibrinogen to promote wound sealing, or FbsA compositions which can be used as an adjuvant with thrombin to fibrin sealant or be useful as an adjuvant for a fibrin sealant. As would be recognized by one of ordinary skill in this art, the level of FbsA protein needed to be effective in such use as a fibrin or fibrinogen sealant as described above will thus vary greatly depending on the nature and severity of the wound to be treated with sealant, the condition of the patient, and/or the existence of any pre-existing infections. Accordingly, one of ordinary skill in the art would readily be able to determine the effective amount of FbsA necessary for a particular wound-sealing application, and would be able to administer the compositions of the invention so as to assist in wound healing when necessary such as during a surgical operation. As also would be recognized by one skilled in the art, there would be a number of suitable pharmaceutically acceptable vehicles, carriers or excipients that could be used with the FbsA proteins of the invention, and indeed when the compositions of the invention are applied, they may take any of a number of suitable forms, including sprays, gels, ointments, films, etc., as would be readily understood by one of ordinary skill in applying said sealants. In the present invention, the typical composition of the fibrin sealant will also include an effective amount of fibrin, and this is preferably obtained by including effective amounts of fibrinogen and thrombin in the composition as well. Conversely, due to the ability of FbsA to induce fibrinogen aggregates on its own (that is, in the absence of thrombin) an alternative composition would include a suitable mixture of fibrinogen and an amount FbsA effective in promoting the aggregation of fibrinogen so as to be useful as a fibrinogen wound sealant . In the preferred fibrin sealant composition of the invention, the fibrinogen and thrombin are formed into two separate solutions so as to produce fibrin when combined at the wound surface, and the addition of an effective amount of FbsA in accordance with the invention will potentiate the fibrin polymerization and promote wound closure and healing. In the compositions of the invention, as would be recognized by one skilled in this art, it is also possible to include other ingredients conventionally used in such fibrin or fibrinogen sealants. For example, the wound sealants of the invention may include calcium chloride as well as other blood factors such as those which also promote coagulation. In this regard, blood coagulation factors such as Factor II and Factor XIII may also be added to the compositions of the invention. Still further, an antifibrinolytic agent such as aprotinin or epsilon- aminocaproic acid may also be added, as well as stabilizers or other biologically active agents as recognized by one skilled in the art. Such biologically active agents may includes, for instance, antibiotics, chemotherapeutics, fibroblastic growth factors, angiogenic growth factors, anti-angiogenic growth factors, antineoplastic agents, cell growth factors and differentiating agents. In general, the properties of both the sealant and the clot are affected by variation in the concentration of fibrinogen and thrombin. Besides thrombin, other factors which will cleave fibrinogen to form fibrin may be used including various forms of snake venom. In the preferred case, the fibrinogen and the thrombin or other fibrinogen-cleaving material are applied to the wound site by any suitable method, e.g., by spraying, sealing or chemical bonding. The mechanical strength of the fibrin clot is influenced by the proportion of fibrinogen, factor XIII and adhesive proteins (fibronectin) in the fibrin sealant, and an important interaction in the tissue-sealing process is the conversion by thrombin of fibrinogen into fibrin monomers. Higher concentrations of fibrinogen tend to produce stronger clots, whereas sealants containing higher concentrations of thrombin tend to form clots more rapidly and add to clot adhesion. In accordance with the invention, the addition of an effective amount of an FbsA protein as described herein will potentiate the polymerisation of fibrin and thus be useful in providing a more effective fibrin sealant. In the preferred operation of the present invention, the compositions of the present invention function as a fibrin glue when placed on a wound site and activated in order to achieve wound closure and begin healing. The compositions of the present invention can be put to a wide range of suitable medical and surgical uses. For example, the FbsA compositions of the invention can be used in hemostasis, as sealants and as adhesives. Additionally, the FbsA compositions of the invention can be used in cardiovascular surgery as a hemostatic, for example, with needle holes, suture lines, diffuse and nonspecific bleeding, friable tissue bleeding, aortic dissections, ventricular ruptures, and fistulas. In otorhinolaryngology (ear, nose and throat, ENT) surgery, they can be used in facial nerve grafts, closure of dural leaks, nasal septal surgeries, and post tonsillectomy hemorrhage. In neurosurgery, they can be used to prevent cerebral spinal fluid (CSF) leakage, peripheral nerve repair, and to anchor dural patches. In plastic surgery, they can be used in a number of procedures relating to skin grafts, including to fix grafts, control oozing and control bleeding. In thoracic surgery, they can be used, for example, in the treatment of pneumothorax and pulmonary leaks. The compounds of the present invention also have a number of other surgical uses including sealing biopsy needle tracks, liver and splenic lacerations, lymphatic fluid leaks, organ resectioning, seroma and hematoma prevention, and gastrointestinal bleeding. Still further, the compositions of the present invention also can be used in conjunction with antibiotics or other biologically active substances which may be desirable to apply to the wound site at the same time the fibrin sealant is being applied. The compositions of the present invention also may serve as a surgical adhesion barrier. As generally used in its desired applications, the fibrin sealant of the invention will be useful in potentiating fibrin polymerization, which is generally the conversion of the fibrin monomer to fibrin polymer (or noncrosslinked fibrin to crosslinked fibrin) "concurrently" with said contacting step means that such the conversion step and such contacting step occur within a time period of each step so as to form the fibrin clot at the desired site. Thus, concurrently can mean that after the contacting step, the fibrin monomer is converted to fibrin polymer or the noncrosslinked fibrin is converted to crosslinked fibrin. This is preferably carried out by contacting the composition comprising fibrin monomer or noncrosslinked fibrin, after such composition has been applied to the desired site, with the FbsA composition of the invention so as to convert the fibrin monomer to fibrin polymer or the noncrosslinked fibrin to crosslinked fibrin. The conversion step should generally occur within about 0.5 minutes after the contacting step. Otherwise, the composition comprising the fibrin monomer or noncrosslinked fibrin, especially if the noncrosslinked fibrin is a fibrin monomer, may flow away from the desired site. Concurrently can also mean that the contacting step and converting step take place simultaneously. This is carried out by contacting the desired site with the composition comprising the fibrin monomer or noncrosslinked fibrin at the same time that such composition is contacted with a composition that can convert the fibrin monomer to fibrin polymer or the noncrosslinked fibrin to crosslinked fibrin. Finally, and preferably, concurrently can also mean that the conversion step can commence prior to the contacting step, albeit not so far prior to the contacting step that all of the fibrin monomer has been converted to fibrin polymer or all of the noncrosslinked fibrin has been converted to crosslinked fibrin. Otherwise, all of the fibrin monomer will be converted to fibrin polymer or all of the noncrosslinked fibrin will be converted to crosslinked fibrin, prior to the contacting step, which results in a very poor fibrin sealant. This embodiment is carried out by mixing the composition comprising the fibrin monomer or noncrosslinked fibrin with a composition that can convert the fibrin monomer to fibrin polymer or the noncrosslinked fibrin to crosslinked fibrin, prior to the contacting step. Since it takes about 30 seconds for the conversion step to be complete, the conversion step should not begin more than about 30 seconds and preferably not more than about 3 seconds prior to the contacting step. This embodiment is preferred because it ensures that the maximum amount of the composition comprising the fibrin monomer or noncrosslinked fibrin will remain at the desired site and yet also forms an excellent fibrin clot. Conversely, as described in detail above, the FbsA compositions of the invention may be used on their own (e.g., without thrombin), and in this case, the FbsA protein used in the invention will directly cause the fibrinogen to aggregate and promote clotting and wound sealing. In short, the fibrin sealants of the invention may be utilized for any use that is known or to be developed for a fibrin or fibrinogen sealant. The methods, kits or fibrin sealant of the subject invention can be used for connecting tissues or organs, stopping bleeding, healing wounds, sealing a surgical wound, use in vascular surgery include providing hemostasis for stitch hole bleeding of distal coronary artery anastomoses; left ventricular suture lines; aortotomy and cannulation sites; diffuse epimyocardial bleeding seen in reoperations; and oozing from venous bleeding sites, e.g. at atrial, caval, or right ventricular levels. The subject invention is also useful for sealing of dacron artery grafts prior to grafting, sealing tissues outside the body, producing fibrin rafts for cell growth, stopping bleeding from damaged spleens (thereby saving the organ), livers, and other parenchymatous organs; sealing tracheal and bronchial anastomoses and air leaks or lacerations of the lung, sealing bronchial stumps, bronchial fistulas and esophageal fistulas; for sutureless seamless healing ("Zipper" technique), and embolization in vascular radiology of intracerebral AVM's, liver AVM's, angiodysplasia of colon, esophageal varices, "pumping" Gl bleeders secondary to peptic ulcers, etc. The subject invention is further useful for providing hemostasis in corneal transplants, nosebleeds, post tonsillectomies, teeth extractions and other applications. See G. F. Gestring and R. Lermer, Vascular Surgery, 294- 304, September/October 1983, incorporated herein by reference. Also, the fibrin sealant of the subject invention is especially suited for individuals with coagulation defects. As indicated above, the desired amount of the FbsA in the sealant compositions of the invention will be those amounts effective in achieving the desired level of fibrin polymerization or fibrinogen aggregation as would be readily determined by one of ordinary skill in this art. Accordingly, the preferred dosage of the composition comprising fibrin monomer and an FbsA protein (fibrin sealant) or fibrinogen and FbsA protein (fibrinogen sealant) will depend on the nature and use of the fibrin sealant composition, but the dosage should be an effective amount for the composition to perform its intended use. Generally, for a composition comprising fibrin monomer that is an aqueous solution, it is believed that from about 3 ml to about 5 ml of such composition is sufficient to be an effective fibrin sealant. However, depending on the use of the composition, the dosage can range from about 0.05 ml to about 40 ml. Also, if a composition comprising noncrosslinked fibrin in polymer form is utilized or the composition is in solid form, then the composition should contain that amount of fibrin that is in such aqueous solution. As indicated above, the FbsA compositions of the invention can be used on their own as a fibrinogen sealant or as an adjuvant for other fibrin sealant compositions. Still further, if so desired, the FbsA sealant of the invention may contain other adjuvants, for example, antibiotics, e.g., gentamycin, cefotaxim, nebacetin and sisomicin, histaminine H.sub.2 -antagonists, e.g., ranitidine, and anticancer drugs, e.g., OK-432. This can be carried out by adding the desired antibiotic to the composition comprising fibrin monomer or noncrosslinked fibrin. See M. C. H. Gersdorff and T. A. J. Robillard, Laryngoscope, 95:1278-80 (1985); A. Ederle et al., Ital. J. Gastroenterol., 23:354-56 (1991); V. Ronfard et al., Burns, 17:181-84 (1991); T. Sakurai et al., J. Control. Release, 18: 39-43 1992); T. Monden et al., Cancer, 69:636-42 (1992); F. Greco, J. of Biomedical Materials Research, 25:39-51 (1991) and H. B. Kram et al., Journal of Surgical Research, 50:175-178 (1991), all of said references incorporated herein by reference. Other adjuvants can also be added, for example, fibronectin, fibrinolytic inhibitors such as aprotinin, alpha-2 antiplasmin, PAI-1 , PAI-2, 6-aminohexanoic acid, 4- aminomethyl cyclohexanoic acid, collagen or keratinocytes. In such cases, when so desired the amounts of the additional adjuvants would be those amounts utilized in conventional fibrin sealants. In addition to its use directly as a fibrin sealant compositions, the FbsA compositions of the invention may also be used in the form of fibrin sealant kits. In one preferred embodiment, the kit may comprise a first component of fibrinogen and a second component comprising an FbsA protein in an amount effective to promote the aggregation of fibrinogen, and such components may be mixed during or just prior to use as a wound sealant. In this case, no thrombin will be necessary to achieve wound sealing. In another embodiment, fibrin sealant kits in accordance with the invention may be provided wherein the kit contains as a first component a composition comprising fibrin monomer and the FbsA composition of the invention as a second component that is capable of polymerizing the fibrin monomer. In this case, the first component can be a composition comprising noncrosslinked fibrin, particularly if the source of fibrinogen utilized to prepare a composition comprising noncrosslinked fibrin is from cell cultures that secrete fibrinogen or recombinant fibrinogen. Still further, the first component can be formed from a combination of thrombin and fibrinogen, ideally combined at or just before application to the wound site. Finally, additional components may be utilized as desired depending on the particular application. For example, additional components of the kit can include blood coagulation factors such as factor II or factor XIII, or any of the other materials which may be added to such sealant compositions as set forth above. In short, the FbsA compositions of the present invention can be used safely and effectively so as to potentiate fibrin polymerization or to promote formation of fibrinogen aggregates and thus be useful as fibrinogen sealants or fibrin sealants or adjuvants for fibrin sealants in the many applications as set forth above.

While the invention has been described above with regard to preferred embodiments, it is clear to one skilled in the art that there will be additional embodiments, compositions and methods which fall within the scope of the invention which have not been specifically described above. The following examples are provided which exemplify aspects of the preferred embodiments of the present invention. However, it will be appreciated by those of skill in the art that the techniques disclosed in the example which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. Moreover, those of skill in the art will also appreciate that in light of the present specification, many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention

EXAMPLES

EXAMPLE 1: A turbidometric method to assay fibrin polymerization. Measurements of light scattering (turbidity) at 350 nm over the time of fibrin clot formation allow monitoring of different stages of polymerization. In the initial lag phase, monomeric fibrin forms long two-stranded protofibrils, which do not affect light scattering. In a second phase, fiber growth occurs by lateral association and branching of protofibrils, which is accompanied by a rapid raise of turbidity. In a third plateau phase, fibrin assembly is nearly completed; therefore turbidity either stabilizes or continues to increase slowly.

EXAMPLE 2: Influence of FbsA on fibrin polymerization and fibrinogen aggregate formation. FbsA proteins from different S. agalactiae strains are composed of varying number of repetitive units, each of 16 amino acids in length (FbsA isoforms). The influence of these isoforms on the assembly of a clot was examined by addition of FbsA to a fibrinogen/thrombin mixture. As shown in Figure 1, incorporation at time zero of 0.5 μM (0.5 micromole/L) FbsA19, FbsA15 or FbsA9 proteins into the reaction mixture containing 1 μM (1 micromole/L) fibrinogen and 0.05 U/ml thrombin induced a substantial reduction of lag phase, a significant increase in the rate of turbidity rise and maximum turbidity as well. Conversely, FbsA6 and FbsA3 had a more reduced effect on fibrin polymerization. In the absence of thrombin, a strong increase of turbidity was observed when fibrinogen was incubated with FbsA19 or FbsA15, suggesting that FbsA containing 19 or 15 repeats had the ability to induce the formation of large fibrinogen aggregates. FbsA9 and FbsA6 had still a significant but more moderate effect on the aggregate formation, whereas FbsA3 had no effect (Figure. 2). To further substantiate this finding, fibrin clot and FbsA19-induced fibrinogen aggregate ultrastructures were compared by scanning electron microscopy (SEM). Clots made from fibrinogen/thrombin incubation mixture exhibited a very different architecture compared with aggregates made of fibrinogen incubated with FbsA 19. As shown in Figure 3A, the fibrin clot had thin fiber strands growing in different directions in space, whereas FbsA-promoted fibrinogen aggregates were organized in a 3D, infinite network structure having the macroscopic dimension of the sample (Figure 3B). Moreover, in fibrinogen gels the fiber strands were thick and the liquid spaces large. Figure 4 shows the specificity of FbsA activity on fibrin polymerization. As a matter of fact, bovine serum albumin included in the incubation mixture had no effect on fibrin polymerization. Conversely, ClfA, ClfB, FnbpA and FnbpB from S. aureus and SdrG from S. epidermidis, all exhibiting fibrinogen-binding activity, inhibited the process. These results are consistent with the role of the C- terminus of fibrinogen gamma chain in both binding of ClfA (or FnbpA/FnbpB) and fibrin polymerization. Likewise, binding of SdrG to the N-terminus of the Bbeta chain results in occupation and steric hindrance of the thrombin binding site and subsequent inhibition of fibrin monomer formation and the protofibril assembly. It is also possible that the potentiating effect of FbsA on fibrin polymerization could be explained if we assume that each FbsA molecule does not bind to any of the fibrin(ogen) polymerization sites. Furthermore, the multivalency of FbsA due to a multiplicity of tandem fibrinogen-binding repeat units could allow the formation of complexes which prelude the true fibrin polymerization process when thrombin is added to the mixture. In summary, the present examples confirm that FbsA has a potentiating effect on fibrin polymerization or induces fibrinogen aggregate formation and thus can be used in compositions and methods wherein a fibrinogen sealant, or a fibrin sealant or an adjuvant of a fibrin sealant, is desired.

Claims

What Is Claimed Is:

1. A composition comprising an FbsA protein from Streptococcus agalactiae in an amount effective in potentiating the polymerization of fibrin or in promoting formation of fibrinogen aggregates so as to be useful as a wound sealant or an adjuvant for a wound sealant, and a pharmaceutically acceptable vehicle, carrier or excipient.

2. The composition according to claim 1 wherein the FbsA protein comprises at least 3 FbsA repeat units.

3. The composition according to claim 1 wherein the FbsA repeat unit has the sequence of SEQ ID NO:1

4. The composition according to claim 1 wherein the FbsA protein is selected from the group consisting of FbsA, FbsA19, FbsA15, FbsA9, FbsA6 and FbsA3.

5. The composition according to claim 1 wherein the composition comprises an amount of FbsA effective to potentiate fibrin polymerization and further comprises a fibrin monomer.

6. The composition according to claim 5 wherein the fibrin monomer is formed by the combination of fibrinogen and thrombin or a fibrinogen-cleaving snake venom.

7. The composition according to claim 1 further comprising a material selected from the group consisting of blood coagulation factors, anti-fibrinolytics, stabilizers, biologically active agents and combinations thereof.

8. The composition according to claim 7 wherein the blood coagulation factors are selected from the group consisting of Factor II and Factor XIII

9. The composition according to claim 7 wherein the anti-fibrinolytics are selected from the group consisting of aprotinin and epsilon-aminocaproic acid.

10. The composition according to claim 7 wherein the biologically active agents are selected from the group consisting of antibiotics, chemotherapeutics, fibroblastic growth factors, angiogenic growth factors, anti-angiogenic growth factors, anti-neoplastic agents, cell growth factors and differentiating agents.

11. A method of potentiating the polymerization of fibrin or promoting the formation of fibrinogen aggregates in an application wherein wound sealing is desired comprising administering to a patient in need an effective amount of an FbsA protein from Streptococcus agalactiae.

12. The method according to claim 11 wherein the application is selected from the group consisting of dermatology, otorhinolaryngology, neurosurgery, general surgery, cardiovascular surgery, thoracic surgery or a treatment of an internal medical pathology.

13. A method of treating a wound site of a patient in need thereof during a medical procedure comprising applying to said wound site an effective amount of the composition of claim 1.

14. A method for preparing a fibrin sealant for application to a site in need thereof comprising the steps of: (a) preparing a component containing a fibrin monomer; and (b) adding to said component an FbsA protein from Streptococcus agalactiae in an amount effective to potentiate polymerization of the fibrin monomer.

15. The method of claim 14 wherein the fibrin sealant is prepared prior to the application to a site.

16. The method of claim 15 wherein the FbsA is added to the fibrin monomer at said site, thus forming a fibrin polymer at said site.

17. A kit for sealing wounds comprising (a) a first component comprising a fibrin monomer; and (b) an FbsA protein from Streptococcus agalactiae in an amount effective to potentiate polymerization of the fibrin monomer.

18. The kit according to claim 17 further comprising a component selected from the group consisting of blood coagulation factors, anti-fibrinolytics, stabilizers, biologically active agents and combinations thereof.

19. A kit for sealing wounds comprising (a) an FbsA protein from Streptococcus agalactiae in an amount effective to promote formation of fibrinogen aggregates; and (b) fibrinogen.

20. The kit according to claim 19 further comprising a component selected from the group consisting of blood coagulation factors, anti-fibrinolytics, stabilizers, biologically active agents and combinations thereof.