COMPOUNDS FOR THE ANTAGONISATION OF ANTICOAGULANTS

AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to the inhibition of blood coagulation via anticoagulants, in particular compounds and compositions antagonizing such anticoagulants and reversing their effects.

BACKGROUND

Heparins and heparin related compounds are abundantly used in medicine as immediate inhibitors of coagulation. They are used to treat thromboembolic disorders, to prevent thromboembolism in patients with elevated risk or during procedures in which blood comes into contact with artificial surfaces (catheters, stents, cardiopulmonary bypass devices) which induce clotting of blood leading to embolism or clotting of the extracorporeal device.

In many situations it is desirable to antagonize the inhibitory effect of heparins or heparin-related compounds (also referred to herein as "heparin based anticoagulants"). For example, a need may arise to analyze blood clotting in vitro in a blood sample of a patient who is treated with heparin(s), e.g. for performing thromboelastography or thromboelastometry during heart surgery. Another situation in which the antagonization of heparin and heparin based anticoagulants is desirable is the treatment of bleeding in a patient receiving heparins. Here it is desirable to end the heparin effect in order to restore blood coagulation capability so as to end undesirable bleeding. Also, anticoagulation generally needs to be stopped at the end of procedures that require anticoagulation, e.g. at the end of heart surgery. In this situation the contact of blood with the artificial surfaces of the cardiopulmonary bypass device ends, therefore the anticoagulation of the patient's blood is no longer required. Therefore it is useful to reverse the anticoagulation which also reduces the risk of bleeding which may result in the need for a transfusion or even the need of surgical reexploration of patient(s).

The typical strategy for reversing the anticoagulant effect of heparins and heparin based anticoagulants is the use of protamine sulfate or hydrochloride. However, protamine sulfate or hydrochloride has many shortcomings: as a relatively large animal-derived product it can induce severe allergic reactions. In addition, its

potential to reverse the effect of smaller glycosaminoglycans such as low molecular weight heparin (LMWH) is limited.

Another strategy is the use of polybrene and other polycationic compound(s). However polybrene, due to its toxicity, cannot be used in vivo. Also, its in vitro use has several shortcomings: polybrene has limited solubility and stability in vitro and thus cannot reverse the effect of large amounts of heparin (more than 2 U/ml).

Another strategy is the use of recombinant PF4. The production of this endogenous and quite large compound is expensive as it requires recombinant technology. Also, it is currently not clear what effects PF4 has in vivo apart from heparin neutralization. Thus, the transfusion of large amounts of PF4 might be dangerous.

Another strategy is the use of heparinase, an enzyme of bacterial origin which cleaves heparins into smaller chains. Heparinase is a larger protein of bacterial origin which may induce an immune response and therefore either shorten half-life in repeated applications or induce allergic reactions. In addition, for the antagonisation of unfractionated heparin it is noted that during the process of cleaving the large chains of unfractionated heparin (L)FH) into smaller chains, heparinase produces transiently large amounts of LMWH, which may cause significant bleeding as one molecule of unfractionated heparin (UFH) may be transiently converted into several molecules of LMWH. In this context, it is important to note that heparinase is often used for the production of LMWHs from unfractionated heparin (UFH) in the pharmaceutical industry.

Another strategy which has been proposed to antagonize heparins and related compounds (heparin based anticoagulants) is the use of low molecular weight fragments of protamine. The published data however has shown that low molecular weight protamine is not completely devoid of any protamine related responses. Probably due to those responses such fragments are still not on the market as a heparin inactivating drugs.

The publications and other materials, including patents, used in the following to illustrate the invention and, in particular, to provide additional details respecting the practice are incorporated herein by reference. For convenience, the publications are referenced in the following text by numerals and are listed in the appended bibliography entitled "References".

The serine proteinase inhibitor antithrombin III (ATIII) is the most important plasma inhibitor of activated coagulation factors. It is a key regulatory protein in the intrinsic pathway of blood coagulation. Its most important targets are free FXa, thrombin, FXIa, FXIIa and FIXa. ATIII attains its full biological activity upon binding poly- sulfated glycosaminoglycans, such as heparin [1]. Heparin catalyzes the interaction between ATIII and the coagulation factors, accelerating complex formation up to 1000-fold.

Heparin accelerated binding of ATIII to serine proteases involved in blood coagulation is mediated through a reversible binding interaction. Heparin is not consumed by this interaction and, following formation of the ATIII-coagulation factor complex, heparin dissociates from the antithrombin molecule and is able to repeat the activation cycle, thus acting as a true catalyst [2].

The binding site for ATIII in heparin molecules is a sequence of several acidic sugar molecules. The shortest active chain is a pentasaccharide with a distinct pattern of carboxy and sulphate groups on the glycoside backbone. The presence of the critical pentasaccharide unit is sufficient to mediate accelerated inactivation of factor Xa by ATIII. This domain alone is, however, not adequate to catalyze ATIII inactivation of factors IXa, XIa and thrombin.

Heparin, when administrated as a drug, enhances the inhibitory properties of ATIII, which can result in potent systemic anticoagulation. Heparin is used to treat patients with established thrombosis and to prevent the occurrence in individuals known to be at high risk. It is also used as an adjunct to fibrinolytic therapy.

However, the unfractionated heparin (UFH) which is used as an anticoagulant drug, is characterized by highly variable pharmacokinetics. Bleeding risk as a consequence of anticoagulant overdose necessitates monitoring of the patient's condition. Also, an antidote reversing the strong anticoagulant effects is needed.

The use of other heparin based anticoagulants such as LMWHs does not require routine monitoring of the anticoagulant effect, as the pharmacokinetics are usually less variable. LMWH, containing only a part of the glycosaminoglycan heparin chain, is less active than unfractionated heparin (UFH) and its use is often considered to be safer. But also with LMWH bleeding complications may occur during anticoagulant treatment as suspected and/or demonstrated in cases of impaired clearance of the

drug (e.g. due to renal dysfunction), unusual pharmacokinetics (e.g. children or severely obese patients) or suspected or potential over-dose.

Thus, an antidote is needed to reverse the effects of heparin and/or heparin based anticoagulants in the setting of cardiac catheterization, cardiac surgery, hemodialysis, or, if serious bleeding occurs, during heparin anticoagulation [3]. Protamine sulfate or protamine hydrochloride have been used for many years to accomplish this task, but though highly effective, these compounds can cause, due to their antigenicity, hemodynamic changes and other serious side effects. Mixon and Dehmer reported that mild protamine reactions occur in up to 16% of the overall population whereas the incidence of severe reactions varies from 0.2% to 0.3% . Severe protamine reactions were reported in up to 27% of patients previously treated with neutral protamine Hagedom insulin in one study, but a considerably lower percentage of incidences was found in other studies [3]. In addition to those adverse effects, a complete antagonization of LMWH by protamine can not be achieved. Thus, the ideal heparin/heparin based anticoagulant antidote that most clinicians would prefer is a compound that provides all the advantages of protamine, yet lacks anaphylactic potential and preserves hemodynamic stability when being infused [4].

Protamine (sulphate or hydrochloride)

As indicated above, protamine has been used for many years to reverse the anticoagulant effects of unfractionated heparin (UFH) and to alleviate heparin- induced bleeding risks.

The mechanism of heparin neutralization by protamine has been thoroughly investigated. Protamine competes with antithrombin III (ATIII) for binding to heparin and due to its stronger affinity to heparin, protamine dissociates ATIII from the heparin-ATIII complex, reversing the anticoagulant function of heparin [5]. It has been used in the settings of cardiac catheterization, cardiac surgery, hemodialysis, or when serious bleeding occurs, during heparin anticoagulation [3].

Protamine encompasses a family of heterogeneous and highly cationic proteins, obtained from fish sperm. It is a low molecular weight (4.5 kDa) protein rich in basic amino acids such as arginine (nearly 67%). It interacts by high-affinity ionic binding (electrostatic interaction) with the poly-anionic unfractionated heparin (UFH) molecules, thereby counteracting their anticoagulant effect. Paradoxically it also prolongs activated partial thromboplastin time (aPTT), when given in excess. This

effect may be due to direct interactions of protamine with clotting factors, particularly thrombin.

Although protamine is highly effective, it is associated with rare, but clinically significant, adverse reactions, e.g. fatal cardiovascular responses [3], [6]. The combined use of heparin and protamine has been suggested as the major cause of morbidity and mortality in patients undergoing cardiopulmonary bypass [5]. Minor adverse reactions include: pruritus, flushing, urticaria, nausea, leukopenia and thrombocytopenia, while severe ones include: bronchospasm, elevated pulmonary arterial pressure, pulmonary edema, hypotension, cardiac arrest and circulatory collapse, which is associated with a very high mortality rate [3].

Three types of reactions to protamine have been described [3]:

1. hypotension due to rapid administration, which can be, however, avoided by slow administration of protamine,

2. anaphylactic responses due to antibody production, which are rather unpredictable, not preventable, and always life-threatening, and

3. catastrophic pulmonary vasoconstriction of unknown etiology.

The mechanisms of protamine-induced toxicity are complex and not yet completely understood. However, available data indicate that severe adverse reactions result from the immune response against protamine as a non-human protein. Like any foreign protein, protamine possesses immunogenic potential. Thus, at least part of the life threatening protamine induced toxicity is attributed to the immunogenicity and antigenicity of protamine (protamine allergy) [5].

Low molecular weight protamine

It is well recognized that small peptides with molecular weight in the range of 1.5 kDa or below are usually either weak or completely devoid of immunogenicity. It was concluded that small peptidic fragments derived from protamine by chemical or enzymatic digestion might be devoid of, or at least possess markedly reduced, immunogenicity and antigenicity [5], [6]. It was also speculated that the effective binding of protamine to the pentasaccharide sequence in heparin, and displacement of the complexed ATIII via an ionic interaction, may not require the whole protamine molecule, but rather a small fragment encompassing an intact arginine rich sequence which allows favourable electrostatic interaction [4], [5].

Thus, low molecular weight protamine (LMWP) fragments containing an intact arginine sequence were prepared by enzymatic digestion of protamine by thermolysin (the protease that does not cleave the arginyl bonds). Their average molecular weight ranged from 0.7 to 1.9 kDa. The ability of these fragments to neutralize the anticoagulant functions of unfractionated heparin (UFH) and low molecular weight heparin (LMWH) was studied [4], [5], [6], [7].

A typical structural scaffold made up of arginine clusters in the middle and non- arginine residues at the N-terminal of the peptide sequence was observed for all the fragments, which were found to retain the complete heparin-neutralizing function of protamine. It was found that retention of potency similar to that of protamine required the presence of at least two arginine clusters (each containing 4 to 6 arginine residues) in the LMWP fragments; such as the sequence of VSRRRRRRGGRRRR (molecular weight of 1.88 kDa, SEQ ID NO 19). Such a sequence was found to be essential to achieve a binding affinity strong enough to completely neutralize heparin. This finding was further validated by using a synthetic LMWP analogue, an octapeptide, CRRRRRRR (CR ₇ ): it was found that its heparin-neutralizing ability was increased by switching from a monomeric to a dimeric structure of this analogue peptide.

Different experiments were performed to compare the ability of protamine, LMWP and its synthetic analogues to neutralize heparin[4], [5], [6], [7].The in vitro efficacy was examined using an anti-Xa chromogenic assay. It was shown that LMWP was less potent than protamine; nevertheless it competed effectively with ATIII in binding to heparin.

Recombinant Platelet Factor 4 (rPF) [3]

Platelet factor 4 (PF4) is a naturally occurring protein comprising 70 amino acids with a molecular weight of 7.8 kDa. It is synthesized in megakaryocytes and eventually stored for later release in the granules of platelets.

Under normal conditions PF4 released from circulating platelets immediately attaches to the heparan sulphate exposed on the endothelial cell surface. But PF4 plasma levels increase 10- to 30-fold after heparin administration, when PF4 bound to endothelial cell surface is freed.

Although the physiological role of platelet factor 4 (PF4) is not yet fully understood, it has been confirmed that it binds to heparin and thereby effectively antagonizes heparin anticoagulation.

Complete neutralization of heparin was achieved with twice the concentration of rPF4 compared to protamine.

The major clinically significant adverse reactions caused by protamine after its administration to a heparinized patient were related to protamine-heparin complexes rather than to the protamine alone. Since the PF4 interaction with heparin is quite different, it does not cause complement consumption, haematological or hemodynamic abnormalities, nor is it likely to cause an immunologic response. However, it has been demonstrated that antibodies directed against the heparin-PF4 moiety do indeed form and are more important in the syndrome of heparin-induced thrombocytopenia.

No human study performed so far has shown serious adverse effects related to rPF4; however, several possibilities should be considered.

PF4 has been cloned by gene technology and is available as a recombinant full- length, human-sequence protein and the rPF used for the study was produced by recombinant DNA methods using Escherichia coli as the source of the recombinant protein. The amino acid structure of rPF is identical to that of naturally occurring PF4 from platelet α-granules, but proteins made in bacteria are not glycosylated and could have altered secondary structure leading to neoantigen formation.

rPF4, obtained by an expensive recombinant technology, has also been mentioned as a cofactor in the syndrome of heparin-induced thrombocytopenia.

Although rPF4 was initially evaluated as a clinical alternative to protamine, it has yet to be developed for general clinical use.

Heparinases [3]

Heparinases are enzymes that selectively cleave, via an elimination mechanism, highly sulfated polysaccharide chains containing 1-4 linkages between hexosamines and O-sulfated iduronic acid residues. They are heparin-degrading enzymes that specifically cleave certain sequences of heparin/heparan sulfate. They are extracted from the gram-negative soil bacterium, Flavobacterium heparinum and have also

been purified and used for technical as well as therapeutic purposes. Heparinase-I (Neutralase ™, IBEX Technologies, Montreal, Quebec, Canada) cleaves heparin at its α-glycosidic linkages. Heparinase-lll (IBEX Technologies, Inc., Montreal, Quebec, Canada) degrades heparan sulphate, a related compound. Animal studies demonstrated that heparinase-l normalized ACT levels increased by heparin application without significant hemodynamic effects.

Heparinases, however, obtained by recombinant technology are expensive. They can also degrade unfractionated heparin (UFH) causing the formation of LMWH and increasing the anticoagulant effects.

Synthetic Peptides

WO 2005/014619 describes heparin binding peptides that can be administered to reduce plasma LMWH and heparin levels and to reduce the anticoagulant effects of heparin and LMWH, respectively. These peptides have a high molecular weight and are therefore not only more expensive but also tend to have insufficient biological availability.

Thus, there is a need in the art for improved antagonists of anticoagulants, in particular heparin or heparin based anticoagulants.

SUMMARY OF THE INVENTION

The present invention addresses the above mentioned need by providing compound(s) that allows, in certain embodiments, for the inhibiton/antagonisation of smaller glycosaminoglycans (LMWH) compared to standard compounds currently in use, i.e., protamine hydrochloride. To neutralize the action of 1 U/ml of LMWH synthetic peptides according to the present invention, such as peptide " 20-062" preferably in concentrations of 1.5-0.1 mM (3161.175-210.745 μg/ml), most preferably of 0.3 mM (632.235 μg/ml), may be used.

The synthetically produced peptides of the present inventions bear, in contrast to substances of animal origin, reduced risk of being infected by known or unknown viruses or prions of animal origin. Also the synthesis of small peptides of the present invention can be performed inexpensively on a large scale. In particular, synthesis is inexpensive compared to recombinant technology, which is commonly used, for the production of PF4 and heparinases. Also, compounds produced via recombinant

technology may sensitize patients against mouse or hamster proteins present as a result of production procedures.

The use of small synthetic peptides according to the present invention, when compared to the use of large proteins of animal origin, reduce or exclude the potential for anaphylaxis and allergic responses.

In many embodiments, the substances of the present invention, in particular PLA ₂ and synthetic peptides, have very good solubility in water as opposed to, for example, polybren. Also in contrast to polybren, PLA ₂ showed very good stability. In addition, in certain embodiments of the present invention, certain PLA ₂ S /synthetic peptides were able to reverse the action of high concentrations of anticoagulant, such as 5U of heparin in 1 ml plasma.

In one embodiment, the invention is directed at a compound for antagonizing at least one anticoagulant in a sample having the following formula (I):

R ¹ -(B ¹ ) _m -(B ² ) _n -Tyr-(B ^3a ) _o -(B ^3b )p-Asn _q -Asn _r -Tyr _s -Leu _t -B ⁴ -(Pro) _u -Phe-Abu-B ⁵ -(B ⁶ ) _v -

R ² (SEQ ID NO 4)

wherein R ¹ represents hydrogen or (Ci-C ₈ )-acyl,

R ² represents OH, O-(d-C ₈ )-alkyl, NH ₂ or Ala-Asp-Pro-OH,

B ¹ to B ⁶ represent, independent of one another, a basic amino acid, such as arginine, homoarginine, lysine, ornithine, 2,4-diaminobutyric acid or 2,3-diaminopropionic acid, m,n,o,p,q,r,s,t,u,v represent, independent of one another, zero or 1 , and wherein said compound antagonizes said at least one anticoagulant in said sample.

In another embodiment, the invention is directed at a pharmaceutical composition for antagonizing heparin or heparin based anticoagulant(s) comprising at least one of the compounds of formula (I) and a pharmaceutically acceptable carrier.

DETAILED DESCRIPTION OF THE INVENTION

Surprisingly, during the analysis of the snake venom of the Brazilian Lancehead snake, Bothrops moojeni, it was found that some phospholipases A ₂ bind and inactivate heparin very effectively. In particular, it was found that certain components of the venom inhibit the anticoagulant action of unfractionated heparin (UFH) in plasma. In addition, it was found that small synthetic peptides derived from the

sequence of those phospholipases A ₂ also possess this property. In addition, it has been found that not only unfractionated heparin (UFH) but also the much smaller LMWH can be inhibited by the use of the synthetic peptides. Based on the identified heparin-binding sequence of PLA ₂ , several groups of synthetic peptides could be derived and used for heparin antagonisation.

The above named components of the venom were purified and amino acid sequences of two new phospholipases A ₂ (PLA ₂ S) could be determined.

In addition, a number of peptides, representing the heparin binding site of the new PLA ₂ S (characteristic 115-129 amino acid fragment of the C-terminal region), have been synthesized.

The whole protein, as well as the synthetic peptides, have been shown to bind heparin in vitro in plasma and to inhibit its anticoagulant activity.

In particular, during specific fractionation and purification of Bothrops moojeni crude venom, a new Lys49 phospholipase A ₂ was found in one of the HPLC fractions obtained. The following sequences were identified:

1) SLVELGKMIL QETGKNPVTS -YGAYGCNCG VLGRGKPKDA TDRCCYVHKC CYK-

--KLTD C NPKK DRYSYSWKDK TIVC-GENNS CLKELCECDK AVAICLRENL

DTYNKKYKNN YLKPFCKK-A DPC (SEQ ID NO 1)

2) SLVELGKMIL QETGKNPLTS -YGAYGCNCG VGGRGKPKDA TDRCCYVHKC CYK-

--KMTD C DPKK DRYSYSWKDK TIVC-GENNS CLKELCECDK AVAICLRENL

DTYNKKYKNN YLKPFCKK-A DPC (SEQ ID NO 2)

The following sequence is considered to constitute the heparin binding structure of the sequences analyzed:

KKYKNNYLKPFCKK ( SEQ ID NO 3 )

As the person skilled in the art will appreciate, isolated sequences disclosed herein can be modified with respect to amino acid sequence and/or three dimensional structure. In particular, amino acid substitutions, additions and/or deletions are within the scope of the present invention. The sequences are preferably synthesized, for example, but not limited to, by methods such as the ones described in the examples.

Alternatively, the peptides may be produced by recombinant technologies via the respective nucleotide acid sequences, which are also within the scope of the present invention and can be readily determined by a person skilled in the art.

The present invention also includes compounds comprising/consisting of, or consisting essentially of, amino acid sequences that have substantial homology or substantial identity with the entire amino acid sequence of the respective portion of the naturally occurring myotoxic phospholipase A2 (PLA ₂ ) and/or have substantial homology or substantial identity with any of the full or partial amino acid sequences identified herein. The terms "substantial homology" or "substantial identity", when referring to an amino acid sequence of the present invention, indicate that the amino acid sequence in question exhibits at least about 30% identity with the respective portion of the naturally-occurring amino acid sequence, usually at least about 70% identity, more usually at least about 80% identity, preferably at least about 90% identity, and more preferably at least about 95% identity. In particular, any so modified amino acid sequence that antagonized anticoagulants, in particular, heparin and heparin based anticoagulants, are within the scope of the present invention.

The homology of an amino acid sequence of the present invention such as of a myotoxic phospholipase A2 (PLA ₂ ) or any portion thereof, e.g., SEQ ID No. 3, is determined as the degree of identity between two sequences. The homology may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, p. 443-453). Gap with the following settings for polypeptide sequence comparison may be used: Gap creation penalty of 3.0 and Gap extension penalty of 0.1 , the mature part of a polypeptide encoded by an analogous DNA sequence exhibits a degree of identity preferably of at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, more preferably at least 95% or 96%, more preferably at least 97% or 98%, and most preferably at least 99% with the amino acid sequences disclosed herein.

Protein analysis software matches similar sequences using measures of homology assigned to various substitutions, deletions and other modifications.

Based on the above sequence and possible modifications (amino acids content and/or three dimensional structure) different peptides were synthesized (table 1). Their ability to neutralize unfractionated heparin (UFH) and low molecular weight heparin (LMWH) was assessed in vitro.

As a result of this assessment, the following sequence was found to constitute a common denominator for many phospholipases A ₂ isolated. Isolated as well as synthetic analogs of such compounds comprising/corresponding to this general formula are within the scope of the present invention:

R ¹ -(B ¹ ) _m -(B ² ) _n -Tyr-(B ^3a ) _o -(B ^3b ) _p -Asn _q -Asn _r -Tyr _s -Leu _t -B ⁴ -(Pro) _u -Phe-Abu-B ^s -(B ⁶ ) _v -R ²

(SEQ ID NO 4)

wherein R ¹ represents hydrogen or (Ci-CsJ-acyl,

R ² represents OH, O-(C ₁ -C ₈ )-alkyl, NH ₂ or Ala-Asp-Pro-OH,

B ¹ to B ⁶ represent, independent of one another, a basic amino acid, such as arginine, homoarginine, lysine, ornithine, 2, 4-diaminobutyric acid or 2, 3-diaminopropionic acid, m,n,o,p,q,r,s,t,u,v represent, independent of one another, zero or 1 , as well as pharmaceutically acceptable salts of these compounds, which surprisingly interact in vitro with unfractionated heparin (UFH) as well as with low molecular weight heparin (LMWH) neutralizing their anticoagulant properties.

The compounds of formula (I) together with acids can form mono- and/or polyvalent, homogeneous or mixed salts, e.g. with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; or with appropriate organic aliphatic saturated or unsaturated carboxylic acids, e.g. aliphatic mono- or dicarboxylic acids, such as formic acid, acetic acid, trifluoroacetic acid, trichloroacetic acid, propionic acid, glycolic acid, succinic acid, fumaric acid, malonic acid, maleic acid, oxalic acid, phthalic acid, citric acid, lactic acid or tartaric acid; or with aromatic carboxylic acids, such as benzoic acid or salicylic acid; or with aromatic-aliphatic carboxylic acids, such as mandelic acid or cinnamic acid; or with heteroaromatic carboxylic acids, such as nicotinic acid; or with aliphatic or aromatic sulfonic acids, such as methanesulfonic acid or toluenesulfonic acid. Pharmaceutically tolerated salts, in particular salt with acetic acid and/or lactic acid, are preferred and are within the scope of the present invention. These mono- and/or polyvalent, homogeneous or mixed salts can be readily produced by a person skilled in the art.

The amino acids of formula (I) can have an L or D configuration, or represent a mixture of both configurations.

Preferred compounds of formula (I) are those wherein R ¹ represents hydrogen or acetyl, or

R ² represents OH or Ala-Asp-Pro-OH, or

B ¹ to B ⁶ represent independent of one another, arginine or lysine.

The following examples of the table 1 illustrate the invention without limiting its scope.

The compounds of the present invention may be used to antagonize anticoagulants, in particular heparins and heparin based anticoagulants. Antagonization may be performed in vitro using a blood sample or a sample comprising a blood component, e.g., plasma. Antagonzation may also be performed in vivo.

The term "heparin" according to the present invention includes, for example, UFH (unfractionated heparin) which is, e.g., available under the trade names LIQUEMIN, THROMBOPHOB, CALCIPARIN and HEPARIN.

The term "heparin based anticoagulant" according to the present invention includes, for example, any substance that comprises at least the heparin pentasaccharide and includes, e.g., fractionated heparin (FH) and low molecular weight heparin (LMWH) such as Certoparin, Dalteparin, Enoxparin, Nadroparin, Reviparin, Tinzaparin, which are also available under the trade names MONO- EMBOLEX, FRAGMIN, CLEXANE, FRAXIPARIN, CLIVARIN and INNOHEP as well as dermatan sulfate and other substances that comprise, optionally sulfated, L- iduronate and GalNAc-4-sulfate as repeating disaccharide units. "Heparin pentasaccharide" refers to a structural unit of heparin having three D-glucosamine and two uronic acid residues. The central D-glucosamine residue contains a unique 3-O-sulfate moiety:

CH ₂ OSO ₃ ^" COCT CH,0SQ ₃ CH ₂ OSO ₅ ^"

NHCOCH ₃ OH NHSO ₃ ^" OSO ₃ ^" N HSO _j "

(See also U.S. Patent Publications 2002/01069143 and 2004/0038932). Also included in this definition are so called heparinoids. The term heparinoids describes a group of substances with a heparin-like effect. These include, for example, sulfated vegetable oligo- and polysaccharides, e.g. polysulfates prepared from alginic acid, pectins, xylans, starches and dextrans, or sulfated animal glycosaminoglycans. Particular mention should be made of pentosan polysulfates, e.g. sodium pentosansulfonate, xylan sulfate, e.g. .beta.-1,4-D-xylan 2,3-bis(hydrogen sulfate),

xylan poly(hydrogen sulfate) and sodium salts thereof, such as dextran sulfates, chitin sulfates, chondroitin polysulfates, as well as so called mucopolysaccharide polysulfates, polyvinylsulfonic acids, also called polyethylenesulfonic acids, e.g. sodium apolate, polygalacturonic acid sulfate (methyl ester methyl glucoside), alginate sulfates, e.g. sodium alginate sulfate and polymannuronic acid sulfate (e.g., danaparoid or Danaparoid-Natrium which is available under the trade name ORAGAN; see also U.S. Patent Application 2003/0161884).

The compounds of the present invention may be used to reverse the effect of anticoagulants at least partially, preferably essentially fully, in vitro and/or in vivo. A set up aimed at the reversal of the effect of an anticoagulant may, for example, include: Blood of a patient that contains an anticoagulant, preferably of a known amount, is sampled and the administration dose required to reverse the anticoagulant's effect is determined. Reversal of the anticoagulant's effect in the context means that the antagomization is sufficient to ensure that both the risk of bleeding due any residual anticoagulant effect as well as the risk of overcoagulation is clinically acceptable.

After such a determination, the compounds of the present invention are administered in the dose calculated for the patient. Thus, the present invention includes administering an effective amount of at least one compound of the present invention/compositions comprising such compound(s) to a patient in need thereof.

Alternatively, with certain anticoagulants, the dose required can be deduced from the anticoagulant and anticoagulant dose employed.

As the person skilled in the art will understand, the compounds of the present invention are generally administered using routes employed to administer the anticoagulant. Thus, the compounds/compositions may be administered transdermal^, intravenously, for example dissolved in a infusion solution, orally, e.g., via loaded microparticles, or via any other acceptable route such as subcutaneously, topically or rectally. Any suitable formulation may be used, such as tablets, coated tablets, pills, granules or granular powder, syrups, emulsions, suspensions, solutions, ointments, transdermal patches or suppositories, optionally together with inert and non-toxic pharmaceutically acceptable carrier, which includes pharmaceutically acceptable excipients and/or solvents.

The compounds of the present invention may be part of kits comprising, in at least a first suitable container, at least one composition comprising at least one compound of the present invention. The kits may also be combination kits comprising, in at least a first suitable container, an anticoagulant; and in at least a second suitable container, at least one composition comprising at least one compound of the present invention. These kits may also comprise further biologically active agents, such as additional anticoagulants, additional coagulants and the like.

Experiments

The following abbreviations are used in the text and in Examples 1-4:

Abu 2-amino-butyric acid

Ac acetyl

AcN acetonitrile

AcOH: acetic acid

Ala alanine

Asn asparagine

Asp aspartic acid

Boc: tert.-butyloxycarbonyl

DMF dimethylformamide

DBU: 1 ,8-diazabicyclo[5]undec-7-ene(1 ,5-5)

DIPEA: diisopropylethylamine

Fmoc: fluorenylmethyloxycarbonyl

Hyp: hydroxyproline

GIy: glycine

Leu leucine

Lys lysine

Phe phenylalanine

Pro: proline

RT: room temperature

TBTU: O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium-tetrafluo roborate tBu: WyI, terf.-butyl

TFA: trifluoroacetic acid

Trt trityl

Tyr tyrosine

Examples 1-4:

The following embodiments 1-4 describe the synthesis of the compounds of formula (I) of the present invention and of salts of such compounds. The eluates and products obtained according to the examples were analysed using HPLC- electrospray MS. The compounds can be manufactured according to known methods described hereinafter (general instructions from M. Bodanszky "The Practice of Peptide Synthesis", Springer, 2 ^nd Edition, 1994). Accordingly, the suitable protected amino acid, e.g. Fmoc-Lys(Boc)-OH, may be bound to a resin at the carboxy terminal end in solid-phase synthesis. The side chain may be protected with, e.g., Boc or t-butyl. If necessary, the protective groups were selectively split off in order to link up the further amino acid derivatives with the reagents commonly used in peptide synthesis until the desired chain was completely built up. Afterwards, the peptide or peptide analogue, respectively, were split off from the resin at the carboxy terminal end with simultaneous removal of all protecting groups.

Example 1 : H-Lys-Lys-Tyr-Lys-Asn-Asn-Tyr-Leu-Lys-Pro-Phe-Abu-Lys-Lys-OH ^* 7TFA; H-KKYKNNYLKPF-Abu-KK-OH *7TFA

As in a typical solid-phase synthesis protocol, the tetradecapeptide was obtained by repetitive coupling of 1.50 g (1.13 mmol, capacity: 0.75 mmol/g) of commercial available H-Lys(Boc)-2-chlorotrityl resin with 2.0 mmol of the amino acids Fmoc- Lys(Boc)-OH, Fmoc-Abu-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH (2x), Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH (2x), 2.1 mmol TBTU, 4.2 mmol DIPEA and unblocking with 5% DBU in DMF (2 x 8 min). The peptide was cleaved from the resin with simultaneous removal of all protecting groups by 95% TFA. The solution \was added dropwise to 250 ml of an ice-cooled 1 :1 mixture of diethylether and petroleum ether followed by HPLC purification. Yield: 172 mg (66.6 μmol, 5.9%).

Example 2: Ac-Lys-Lys-Tyr-Lys-Asn-Asn-Tyr-Leu-Lys-Pro-Phe-Abu-Lys-Lys-O H *6TFA; Ac-KKYKNNYLKPF-Abu-KK-OH *6TFA

As in a typical solid-phase synthesis protocol, the tetradecapeptide was obtained by repetitive coupling of 1.20 g (0.90 mmol, capacity: 0.75 mmol/g) of commercial available H-Lys(Boc)-2-chlorotrityl resin with 2.0 mmol of the amino acids Fmoc- Lys(Boc)-OH, Fmoc-Abu-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Asn(Trt)-OH (2x), Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Lys(Boc)-OH (2x), 2.1 mmol TBTU, 4.2 mmol DIPEA and unblocking with 5% DBU in DMF (2 x 8 min). With the peptide chain fully assembled, the resin is treated with an acetylating mixture (8ml DMF, 1ml Pyridine and 1 ml acetic anhydride) for 5 minutes. The peptide was cleaved from the resin with

simultaneous removal of all protecting groups by 95% TFA. The solution was added dropwise to 250 ml of an ice-cooled 1:1 mixture of diethylether and petroleum ether followed by HPLC purification. Yield: 116 mg (46.1 μmol, 5.1%).

Example 3: H-Lys-Lys-Tyr-Lys-Asn-Asn-Tyr-Leu-Lys-Pro-Phe-Abu-Lys-Lys-OH *7 AcOH; H-KKYKNNYLKPF-Abu-KK-OH *7AcOH

23.1 mg (8.9 μmol) of H-KKYKNNYLKPF-Abu-KK-OH *7TFA described in Example 1 , was dissolved in 20ml of an 1 :1 mixture of AcN : water and treated with 1.Og of Biorad Ion exchange resin in acetate form for two hours. The resin was filtered off, the solution lyophylized. Yield 17.1 mg (7.8 μmol, 87%).

Example 4: H-Lys-Lys-Tyr-Lys-Asn-Asn-Tyr-Leu-Lys-Pro-Phe-Abu-Lys-Lys-OH *7 HCI; H-KKYKNNYLKPF-Abu-KK-OH *7HCI

23.7 mg (9.2 μmol) of H-KKYKNNYLKPF-Abu-KK-OH *7TFA described in Example 1 , was dissolved in 20ml of an 1 :1 mixture of AcN : water and treated with 1.Og of Amberlite IRA 400 exchange resin in chloride form for two hours. The resin was filtered off, the solution lyophylized. Yield 14.8 mg (7.3 μmol, 79%).

In a similar manner the compounds of the general formula (I) and those of the table 1 can be and were prepared.

In preliminary screening using FXa chromogenic substrate the following sequence was found to exhibit the best neutralizing properties against unfractionated heparin (UFH) and LMWH. Thus it was chosen for further experiments.

Example 5: Chromogenic test.

Materials:

Fxa Hyphen BioMed; France

FXa chromogenic substrate Pefachrome ^® FXa; Pentapharm Ltd.; Basel,

Switzerland Plasma pool unfractionated heparin (UFH) NIBSC; Potters Bar, England

(WHO Standard 97/578)

LMWH (WHO Standard 85/600) NIBSC; Potters Bar, England

Antithrombin III Grifols Germany; Siemensstrasse, 18 ;D-63225

Langen/Hessen Protamine hydrochloride ICN Pharmaceuticals Germany GmbH;

Frankfurt/Main, Germany

Synthetic peptide Pentapharm Ltd.; Basel, Switzerland

Purified PLA ₂ Atheris Laboratories; Geneva, Switzerland

The peptide was tested in a concentration of 0.5 and 0.25 mM, dissolved in 5OmM Hepes buffer, pH 7.5. unfractionated heparin (UFH) and LMWH were used in a concentration of 0.5 U/ml, FXa - in a concentration of 2 μg/ml, FXa chromogenic substrate - in a concentration of 4mM and antithrombin III (ATIII) - in a concentration of 3.75 U/ml. Protamine hydrochloride was taken as a reference substance, in a concentration of 0.5 U/ml. The native, purified phospholipase A ₂ from B. moojeni venom (130 μg/ml), was also included in that test. 5OmM Hepes buffer, pH 7.5 was used as a control.

In the following tables/descriptions peptides and purified phospholipase A ₂ are assigned as samples and protamine hydrochlorid as reference. Following measurements were performed:

The obtained results are shown in the figure 1 and 2.

Example 6: KC4 A micro ball coaqulometer - neutralization of unfractionated heparin (UFH) and LMWH, comparison of the properties of the native PLA?, synthetic peptide 20-062 and protamine hydrochloride.

After the preliminary tests, using a non-physiological system of FXa and its chromogenic substrate, measurements were performed in plasma, on the KC4 A micro ball coagulometer.

Materials:

PiCT ^® activator Pentapharm Ltd.; Basel, Switzerland

Plasma pool unfractionated heparin (UFH) NIBSC; Potters Bar, England

(WHO Standard 97/578)

LMWH (WHO Standard 85/600) NIBSC; Potters Bar, England

Protamine hydrochloride ICN Pharmaceuticals Germany GmbH;

Frankfurt/Main, Germany

CaCI ₂ HemoslL, Instrumentation Laboratory; Milano, Italy

Synthetic peptide Pentapharm Ltd.; Basel, Switzerland

Purified PLA ₂ Atheris Laboratories; Geneva, Switzerland

The experiments were performed as follows:

Clotting was triggered using the PiCT ^® test, which is described in detail in

EP1240528). The test employs a mixture of FXa and RW-V (FV activator from

Daboia russelii russelii venom) in combination with phospholipids. Plasma was spiked with 0.5U/ml unfractionated heparin (UFH) or LMWH.

Plasma was incubated 180s with all the needed reagents and then recalcified with

CaCI ₂ used in a concentration of 25 mM. The peptide was tested in a concentration of 0.5 and 0.25 mM, dissolved in 5OmM Hepes buffer, pH 7.5. Protamine hydrochloride was taken as a reference substance, in a concentration of 0.5 U/ml and the native phospholipase A ₂ from B. moojeni venom - in a concentration of 130 μg/ml. 5OmM Hepes buffer, pH 7.5 was used as a control.

50 μl Plasma with or without UFH/LMWH

25 μl Sample or reference

25 μl PiCT ^® activator

3 min. Incubation at 37°C

50 μi CaCI ₂ to start coagulation

The clotting times (CT) obtained are presented in the table 2.

Tab.2

* CT measured in plasma without unfractionated heparin (UFH) (30.85s) represents

100% of CT for UFH measurements.

** CT measured in plasma without LMWH (29.05s) represents 100% of CT for LMWH measurements.

Example 7: KC4 A micro ball coaqulometer - neutralization of 1U LMWH, comparison of the properties of the synthetic peptide 20-062 and protamine hydrochloride.

Experiments were performed as in the example 2. LMWH was added to plasma to obtain an end concentration of 1 U/ml. The obtained results are shown in the figure 3.

Example 8: Automated Coagulation Test System BCS (Behrinq Coagulation System).

The Automated Coagulation Test System BCS was used to evaluate the properties of the tested substances to neutralize the anticoagulant activity of unfractionated and low molecular weight heparin. PiCT ^® assay was performed as described in example 2.

Materials:

PiCT ^® activator Pentapharm Ltd.; Basel, Switzerland

Plasma pool unfractionated heparin (UFH) NIBSC; Potters Bar, England

(WHO Standard 97/578)

LMWH (WHO Standard 85/600) NIBSC; Potters Bar, England

Protamine hydrochloride ICN Pharmaceuticals Germany GmbH;

Frankfurt/Main, Germany

CaCb HemoslL, Instrumentation Laboratory; Milano, Italy

Synthetic peptide Pentapharm Ltd.; Basel, Switzerland

Purified PLA2 Atheris Laboratories; Geneva, Switzerland

Clotting was triggered using the PiCT ^® test, which employs a combination of FXa and RW-V (FV activator from Daboia russelii russelii venom) in combination with phospholipids. Plasma was spiked with 0.5U/ml of unfractionated heparin or with 0.5U/ml of LMWH. Plasma was incubated 180s with all the needed reagents and then recalcified with CaCI ₂ used in a concentration of 25 mM. The peptide was tested in a concentration of 0.5 and 0.25 mM, dissolved in 5OmM Hepes buffer, pH 7.5. Protamine hydrochloride was taken as a reference substance, in a concentration of 0.5 U/ml and the native phospholipase A ₂ from B. moojeni venom - in a concentration of 130 μg/ml. 5OmM Hepes buffer, pH 7.5 was used as a control. The obtained results are shown in the figures 4 and 5.

Example 9: Rotem system (Thromboelastoqraphv measurements) with the native PLA?.

Materials:

DiaPlastin Pentapharm Ltd.; Basel, Switzerland in-TEM ^® Pentapharm Ltd.; Basel, Switzerland star-TEM ^® Pentapharm Ltd.; Basel, Switzerland

ROTROL N Pentapharm Ltd.; Basel, Switzerland

Synthetic peptide Pentapharm Ltd.; Basel, Switzerland

Purified PLA ₂ Atheris Laboratories; Geneva, Switzerland

Heparin not only affects the onset time of clotting, i.e. the beginning of clot formation, but also the clot formation kinetics. Therefore we examined the effects of heparin inhibition by the technique disclosed herein in addition to with a method which continuously quantifies the clot formation kinetics.

So-called ROTEM ^® analysis was applied, which is described in detail in US5777215. In essence the system quantifies the clot formation. The clotting time represents the time of the onset of clotting, the clot formation time, the time from onset of clotting till the formation of a clot of defined strength and the maximum clot firmness defines the maximum clot stability.

The experiments were performed as follows:

20 μl star-TEM ^® , 0.2 M buffered CaCI ₂ solution, pH 7.4

20 μl in-TEM ^® , ellagic acid solution an organic activator of the contact phase, resulting in activation of factors

XII, Xl, IX, VIII, V, X and thrombin or ex-TEM, a tissue factor solution derived from rabbit brain, activating hemostasis by the extrinsic pathway, i.e. by factors VII, X, V and thrombin 20 μl Purified PLA ₂

ROTROL N, lyophilized, standardized plasma 200 μl produced from pool donor plasma (+/-unfractionated heparin (UFH))

The activity of purified PLA ₂ was evaluated. The purified PLA ₂ was tested in a concentration of 10 mg/ml.

The results obtained are shown in figure 6 and 7.

In both measurements good heparin neutralizing properties of the purified PLA ₂ could be measured, even for high concentrations of unfractionated heparin (UFH) (5 and 10 U/ml), where no clotting could be observed in the performed tests.

Example 10: Accelerated stability testing of the native PLA ₂ .

An accelerated stability testing of the native, purified PLA ₂ , dissolved in deionized water (10 mg/ml), was performed in Rotem ^® system (Thromboelastography measurements). Experiments were performed as in the example 4.

20 μl star-TEM

20 μl in-TEM or ex-TEM)

20 μl Purified PLA ₂

200 μl ROTROL N (+/-unfractionated heparin (UFH))

The PLA ₂ was stored at 37°C during a period of 2 weeks and at 2-8°C during a period of 3.5 weeks. In both cases very good stability data was obtained. The obtained results are shown in the figure 8.

Example 11 : Rotem system (Thromboelastographv measurements) with synthetic peptide 20-062.

Similar experiments were performed with the synthetic peptide 20-062, tested in a concentration of 5 mM, dissolved in deionized water. The experiments were performed as follows:

20 μl star-TEM

20 μl in-TEM or ex-TEM

5 or 20 μl Purified PLA ₂

300 μl ROTROL N (+/-unfractionated heparin (UFH))

The results obtained are shown in figure 9 and 10.

Example 12: Cytotoxicity study [191

Cytotoxicity study was performed using normal human fibroblast cells (NHF03) and the MTT-assay. MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is a yellow, water-soluble tetrazolium dye that is reduced by living, but not dead, cells to a purple formazan product that is insoluble in aqueous solutions. The measurement of the MTT reduction is an indirect method of the viability assessment.

The amount of MTT-formazan product was determined spectrophotometrically once the MTT-formazans crystals have been dissolved in a suitable solvent. The cytotoxicity was measured after a supplementation period of 72h. Sodium dodecyl sulfate (SDS) was used as a control. No cytotoxic effects were observed with the synthetic peptides up to the highest concentration tested 100 μM, which corresponds to 200 - 350 μg/ml, depending on the peptide.

Conclusions

The properties of some phospholipases A ₂ purified from the venom of B. moojeni snake were analyzed. Although it is well known that different PLA2S from snake venoms influence blood the coagulation system, here the novel use of those substances or their fragments to antagonize the anticoagulant effect of heparin in vivo or in vitro is proposed.

Heparin is known to interact in a non-covalent, charge-dependent way with basic myotoxic phospholipases A ₂ (PLA ₂ S), leading to inhibition of their enzymatic and biological activities [20]. It was reported that heparin was used for neutralization of the myotoxic effects caused by some snake venom PLA ₂ S [11], [12], [21], [22], [8], [23], [13], [24], [10], [15], [20], [25].

Lomonte et al. [8], [23] first described that heparin binds to a site comprising residues 115-129 of the Lys49 myotoxin Il (MT-II) from B. asper, resulting in the neutralization of its toxic actions. In the same paper it was also reported that the synthetic peptide 115-129 of this protein mimics the cytotoxic effects of the whole protein in vitro. Thus due to heparin binding properties of that region, the toxic activity could be reduced or completely abolished.

Here, heparin is not used to combat the effect of myotoxic phospholipases A ₂ (PLA ₂ S) in snake venom. Rather, such PLA ₂ S, fragments thereof and compounds derived therefrom are used to antagonize/reverse the effect of heparin, heparin based as well as other anticoagulants in vitro and in vivo.

It will be appreciated that the methods of the instant invention can be incorporated in the form of a variety of embodiments, only a few of which are disclosed herein. It will be apparent to the artisan that other embodiments exist and do not depart from the spirit of the invention. Thus, the described embodiments are illustrative and should not be construed as restrictive.

Brief description of the figures

Fig.1 Neutralization of anti-FXa activity of unfractionated heparin (UFH) assessed using FXa chromogenic substrate. Best neutralizing activity was measured for the crude PLA2 purified form B. moojeni venom (95%). Two concentrations of the synthetic peptide, corresponding to the heparin binding sequence of the PLA2, showed weaker neutralization of unfractionated heparin (UFH): 68% and 67%. In comparison, protamine hydrochloride in the therapeutical concentration neutralized 54% of anti-FXa activity of unfractionated heparin (UFH).

Fig.2 Neutralization of anti-FXa activity of LMWH assessed using FXa chromogenic substrate. Best neutralizing activity was measured for the crude PLA2 purified form B. moojeni venom (63%). Two concentrations of the synthetic peptide, corresponding to the heparin binding sequence of the PLA2, showed weaker neutralization of LMWH: 43% and 48%. In comparison, protamine hydrochloride in the therapeutical concentration neutralized 45% of anti-FXa activity of LMWH.

Fig.3 Synthetic peptide 20-062 neutralized 1 U LMWH more efficiently than protamine hydrochloride. The normal CT in plasma without LMWH is expressed as 100%. The concentration of 1 U LMWH/ 1 ml plasma prolongs CT up to 527%. Best inhibition was obtained with the concentration of 0.3 mM of the 20-062 peptide (176%), while 3U and 5U of protamine hydrochloride were not as efficient (223% and 226% respectively).

Fig.4 All tested substances neutralized the anticoagulant activity of unfractionated heparin (UFH) in plasma. The clotting times obtained were very similar for all three substances

Fig.5 The anticoagulant activity of LMWH was neutralized well by protamine hydrochloride and slightly better by the synthetic peptide. Interestingly, B. moojeni PLA2, that showed good neutralizing properties against unfractionated heparin (UFH), did not neutralize LMWH. In the contrary to expected effects, it prolonged the clotting time. Thus, best neutralizing properties of LMWH could be established in plasma for synthetic peptide 20-062.

Fig.6 Using the in-TEM test and heparinized plasma the effect of B. moojeni PLA ₂ on unfractionated heparin (UFH) was measured in the intrinsic pathway of blood coagulation. However clotting time prolongation could be observed in the

measurements performed in plasma without addition of unfractionated heparin (UFH) ₁ very good neutralization of unfractionated heparin (UFH) was obtained even for the concentration of 10 U unfractionated heparin (UFH) in 1 ml plasma.

Fig.7 Using the ex-TEM test and the heparinized plasma the effect of β. moojeni PLA ₂ on unfractionated heparin (UFH) was measured in the extrinsic pathway of blood coagulation. A slight clotting time prolongation could be observed in the measurements performed in plasma without any addition of unfractionated heparin (UFH). Also in the extrinsic pathway very good neutralization of unfractionated heparin (UFH) was obtained even for the concentration of 10 U unfractionated heparin (UFH) in 1 ml plasma.

Fig.8 The purified B. moojeni PLA ₂ shows very good stability during the period of 2 weeks at 37°C, as well as during the period of 3.5 weeks at 2-8°C. All performed measurements gave the same, high level of neutralization of the anticoagulant activity of unfractionated heparin (UFH).

Fig.9 The synthetic peptide 20-062 neutralized anticoagulant activity of unfractionated heparin (UFH), measured in the in-TEM test.

Fig.10 Concerning the better neutralization of unfractionated heparin (UFH) obtained with β. moojeni PLA ₂ in the extrinsic system of blood coagulation, two concentrations of synthetic peptide 20-062 were checked in the ex-TEM test. Both showed good, concentration dependent neutralization of unfractionated heparin (UFH).

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