FR2736266A1 - Peptide derived from Korean salmosa viper venom - useful as blood platelet aggregation inhibitor, for the management of thrombosis - Google Patents

Abstract

New peptide (I) that inhibits platelet aggregation, is called salmosin, is derived from the venom the Korean salmosa viper (Agkistrodon halys brevicaudus) and has the amino acid sequence (I): EAGEE CDCGS PGNPC CDAAT CKLRQ GAQCA EGLCC DQCRF MKEGT ICRRA RGDDL DDYCN GISAG CPRNP FHA (I).

Description

INHIBITOR OF PLATELET AGGREGATION AND METHOD FOR
PREPARE THIS
ChamD of the invention
The present invention relates to a novel platelet aggregation inhibitor and method for its preparation, more specifically to a polypeptide inhibiting platelet aggregation and to a method for its preparation from Salmosa venom (Askistrodon halvs brevicaudus ).

Rear Dlan of the Invention
It is known that viper venom contains various peptides or proteins, which have an effect on thrombosis and hemostasis. Thrombosis is of particular significance since thrombi formed by abnormal platelet hyperaggregation can lead to death and disability. In this regard, a variety of peptides or proteins inhibiting or activating platelet aggregation from venoms of various species of vipers have been isolated and their biochemical and biological properties have been characterized.

 It has also been reported that platelet aggregation occurs by binding fibrinogen to its platelet-associated receptors associated with the GPIIb-IIIa complex which is a glycoprotein on the platelet surface membrane or the Arg-Gly amino acid sequence. -Asp fibrinogen is essentially involved (see: Rouslahti and Pierschbacher, Science, 238: 491-497 (1987)). Of course it has been suggested that peptides or proteins containing the Arg-Gly-Asp amino acid sequence are capable of inhibiting platelet aggregation, based on the interference of fibrinogen binding to platelet receptors.

 In the initial stage of research, platelet receptor fibrinogen binding inhibitors isolated from viper venoms were mainly identified as small 5- to 9-kDa proteins (see: Huang et al., J. Biol.

Chem., 262: 16157-1663 (1987)). Later, from the venoms of various species of vipers, several high molecular weight inhibitors were isolated. All inhibitors so far defined have been characterized as cysteine-rich proteins which essentially contain the Arg-Gly-Asp amino acid sequence for the specific binding of the GPIIb-IIIa complex to platelet receptors.

The precise structures of some platelet aggregation inhibitors such as Kistrine have recently been analyzed by NMR spectrometry (see: Adler et al.
Science, 253: 445-448 (1991); Adler et al,
Biochemistry, 32: 282-289 (1993)), Flavoridine (see:
Klaus et al., J. Mol. Biol., 232: 897-906 (1993); Senn and Klaus, J. Mol. Biol., 232: 907-925 (1993)),
Albolabrin (see Jaseja et al., Eur J. Biochem., 218: 853-860 (1993)) and Echistatin (see: Chen et al.
Biochemistry, 30: 11,625-11366 (1991); Cooke et al.
Eur. J. Biochem., 202: 323-328 (1991); Cooke et al.
Protein Eng., 5: 473-477 (1992); Saudek et al.,
Biochemistry, 30: 7369-7372 (1991); Dalvit et al.
Eur. J. Biochem., 202: 315-321 (1991)) In addition, interference in the binding of fibrinogen to the GPIIb-IIIa complex by inhibitors has been clearly demonstrated by in vivo experiment using an animal model. (see: Collen, J. Clin Invest, 76: 101-108 (1985), Gold et al., Circulation, 77: 670-677 (1988), Yasuda et al., J. Clin Invest, 81). : 1,284-1,291 (1988);
Coller et al., Blood, 68: 783-786 (1986); Hanson et al., J. Clin. Invest., 81: 149-158 (1988)).

 Under these circumstances, the present inventors studied 14 species of Korean snakes to screen for a new platelet aggregation inhibitor, and found that a small polypeptide isolated from the venom of a Korean viper, Salmosa (Aakistrodon halvs brevicaudus). ), is a new potent inhibitor of platelet aggregation that has not been reported so far.

Summary of the invention
A main object of the invention is therefore to provide a new peptide inhibiting platelet aggregation from the venom of Salmosa (Aakistrodon halvs brevicaudus).

 The other object of the invention is to provide a method for the preparation of the polypeptide inhibiting platelet aggregation from Salmosa venom.

Brief description of the drawings:
The foregoing and the other objects and features of the present invention will become clearer from the following descriptions given in conjunction with the accompanying drawings, in which: Fig. 1 is a photograph showing the SDS spectrum;
PAGE of a peptide inhibiting aggregation
platelet purified from the venom of
Salmosa; FIG. 2 is a list of the amino acid sequences of
Peptides inhibiting platelet aggregation
according to the present invention (SEQ ID NO: 1
and according to the state of the prior art
(SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and
SEQ ID NO: 5); and FIG. 3 is a MALDI mass spectrum (ionization by
matrix desorption using laser) of the
peptide inhibiting platelet aggregation
according to the invention.

DETAILED DESCRIPTION OF THE INVENTION
To isolate the platelet aggregation inhibitor of the invention, the present inventors obtained the venom of SALMOSA (Aakistrodon halvs brevicaudus), a Korean viper. Then, the venom was loaded onto a serine proteinase affinity column to collect the solution that passed through the column without binding, and a series of column chromatographic procedures were performed, namely, anion exchange, filtration on gel and high performance liquid chromatography (HPLC) reverse phase column.

 The peptide inhibiting platelet aggregation (hereinafter referred to as "Salmosin") purified as above was identified as a small protein of molecular weight about 7500 Da, which has not been described. so far. Salmosine-based computer modeling, based on amino acid sequence analysis, teaches that the three-dimensional structure of Salmosine is a hybrid of Kistrin and Echistatin, which are peptides inhibiting platelet aggregation known in the state of the art.

In addition, Salmosin has also been shown to inhibit platelet aggregation much more efficiently than any known peptide inhibiting platelet aggregation. So we propose that the
Salmosine can be developed as a therapeutic agent for the control of thrombosis.

In the description of the amino acid sequence of the proteins and peptides of the present invention, the abbreviated one-letter symbols according to the IUPAC-IUB standards are used as follows:
Amino acid Svmbole
Alanine A
Arginine R
Asparagine N
Aspartic acid D
Cysteine C
Glutamine Q
Glutamic acid E
Glycine G
Histrdine H
Isoleucine I
Leucine L
Lysine K
Methionine M
Phenylalanine F
Proline P
Serine S
Threonine T
Tryptophan W
Tyrosine Y
Valine V
The present invention is further illustrated in the following examples which should not be construed as limiting the scope of the invention.

Example 1: Purification of Salmosine
10 ml of venom obtained from Aakistrodon halvs brevicaudus was diluted by a factor of 10 with 20 mM Tris-HCl (pH 7.5). The diluent was loaded onto a benzamidine-Sepharose column (Pharmacia-LKB, Sweden) (2.5 x 10 cm) pre-equilibrated with 20 mM Tris-HCl (pH 7.5) at a flow rate of 30 ml / hr. The solution which passed through the column was then bound without binding and applied to a DEAE-Toyopearl column (Pharmacia, Sweden) (2.5 x 5.0 cm) at a flow rate of 60 ml / h, and eluted with stepwise salt concentration gradient of NaCl (20 mM, 50 mM, 100 mM and 1M). Active fractions were pooled and concentrated using an ultrafiltration system (Model 8200, Amicon, USA). The concentrate thus prepared was loaded onto an HPLC column.
TSK G2000 (Toyosoda, Japan) (7.5 x 300 mm) at a flow rate of 60 ml / hr and eluted. Active fractions were pooled and concentrated using an ultrafiltration system (Centriprep-3, Amicon, USA).

The active fractions thus concentrated were then applied to a Vydac reverse-phase HPLC column.
C18 (Vydac, USA) (2.5 x 300 mm) equilibrated with 0.1% (v / v) TFA (trifluoroacetic acid), washed with 0.1% TFA (v / v) and with acetonitrile % (v / v) sequentially, and eluted with a linear gradient of acetonitrile concentration of 20% to 60% (v / v). Salmosine was eluted with a retention time of 31.8 minutes.

 The biological activity of Salmosin during the ELISA purification was assayed as follows: Microtitre plates having 98 wells were coated with fibrinogen at 40C overnight.

Then, the fibrinogen was reacted with the receptor
GPIIb-IIIa isolated from human platelets and with solutions containing Salmosine at room temperature for 2 hours. After washing with a buffer of
20 mM Tris-HCl (pH 7.5 containing 0.15 M NaCl, 0.05%
Tween 20 and 0.02% NaN3), the platelets were reacted with anti-human GPIIIa mouse antibody (Tagoimmunologicals, Biosource International, USA) for 2 hours, washed three times with the same buffer and reacted. then with goat anti-mouse IgG-HRP (horseradish peroxidase) (Tagoimmunologicals, Biosource International, USA) for 1 hour. After 3 washes with buffer, a chromogenic substrate, o-phenylenediamine, was added and the absorbance measured at 490 nm.

Example 2: Determination of the purity of Salmosine
To determine the purity of Salmosin prepared in Example 1, the eluted Salmosin solution was treated by C18 reverse phase HPLC chromatography with 100% TCA (trichloroacetic acid) to precipitate Salmosine, etc., and was suspended. Salmosine with a volume of 0.125 M Tris-HCl (pH 6.8, containing 4% (w / v) SDS, 2% (v / v) glycerol and 10% (v / v) mercaptoethanol), and incubated at 1000C for 5 minutes. Salmosine thus prepared was analyzed on
SDS-PAGE using an 18% Tris-glycine gel (Novex, USA) and silver nitrate staining was performed (see: Figure 1). In Fig. 1, column 1 is Salmosin prepared in Example 1, and column 2 is molecular weight markers. As shown in Fig. 1, the SDS-PAGE spectrum shows a single band of Salmosin. Therefore, it is clearly demonstrated that Salmosin was isolated from the venom of (Aakistrodon halvs brevicaudus) in a pure form.

Example 3 Analysis of the amino acid sequences of the
Salmosine
The purified Salmosin was digested with CNBr and its amino acid sequence was determined using an automatic amino acid sequence analyzer. The amino acid sequences of FIG.
Salmosin and several known peptides inhibiting platelet aggregation (SEQ ID NO 1, SEQ ID NO 2, SEQ
ID NO 3, SEQ ID NO 4 and SEQ ID NO 5). In Figure 2, underlining indicates amino acids distinct from each other when compared to Halysin (see: Huang et al., Biochem. Pharmacol., 42: 1209-1219 ( 1991a)), a known peptide inhibiting platelet aggregation isolated from the venom of a Japanese viper, Mamushi (Aakistrodon halvas), which, in terms of taxonomy, is identical in species to a Korean viper,
Salmosa (Aakistrodon halvs brevicaudus). As shown in Figure 2, Salmosin is a new peptide inhibiting platelet aggregation, which has an amino acid sequence different from that of known inhibitory peptides.

EXAMPLE 4: Determination of the molecular mass of
Salmosine
The value of the molecular weight of Salmosine was determined to be 7,473.6 Da using MALDI
MS (laser desorption ionization mass spectrometry, Kratos Kompact Mold II, Kratos
Analytical, Manchester, United Kingdom) (See Figure 3).

Example 5 Analysis of the Three-Dimensional Structure of Salmosine
From the amino acid sequence of the
Salmosine determined in Example 3, the three-dimensional structure of Salmosine was analyzed by means of a computer modeling system which uses
Indigo2-XZ (Silicon Graphics Corp., USA) and Insight
II / Discover and Homology (Biosym Corp., USA), as hardware and software, respectively. Computer modeling analysis teaches that the three-dimensional structure of Salmosine is a hybrid of
Kistrin and Echistatin, which are peptides inhibiting platelet aggregation already known in the state of the art.

Example 6 Comparison of Activation Inhibition of Platelet Aggregation
In order to compare the activities of Salmosin with known peptides inhibiting platelet aggregation, platelet aggregation inhibition activity was measured by two different methods as follows:
(1) The ELISA Method of Example 1
(2) The human platelet aggregation assay:
225 μl of human plasma rich in s
platelets (3 x 105 platelets / yl plasma)
with 25 Ctl peptide inhibiting aggregation
platelet in PBS (0.1 μg / ml) and
incubated in an Agrégometer (Chromo-Log Corp.,
USA) at 250C for 5 minutes and measured
turbidity after the addition of an agent
aggregation, namely, the ADP.

Table 1 shows platelet aggregation inhibition activities of Salmosin and known peptides inhibiting platelet aggregation, namely, Kistrine, t-Trigramine (see:
Proc. Natl. Acad. Sci., USA, 87: 2471 (1989)),
Echistatin and a synthetic peptide (GRGDSP), measured using the methods described above. As can be seen from Table 1, it is clearly demonstrated that Salmosin has platelet aggregation inhibitory activity superior to any known platelet aggregation inhibitor.

Table 1:
Comparison of aggregation inhibition activity
platelet

<tb><SEP> 1C50 <SEP> (nM) <SEP>
<tb> Peptide <SEP> inhibiting <SEP> Method <SEP> ELISA <SEP> Assay
<tb><SEP> Aggregation <SEP> Aggregation
<tb><SEP> platelet platelet platelet
<tb><SEP> human
<tb> Kistrine <SEP> 2.7 <SEP> 128
<tb> -Trigramine <SEP> 2.2 <SEP> 240
<tb> Echistatin <SEP> 2.7 <SEP> 560
<tb> Salmosin <SEP> - <SEP><SEP> 0.7 <SEP> 50
<tb> GRGDSP <SEP> 300 <SEP> 230 <SEP> 000
synthetic peptide whose amino acid sequence is Gly-Arg-Gly-Asp-Ser-Pro.

 As illustrated and demonstrated clearly above, the present invention provides a novel platelet aggregation inhibiting peptide, Salmosin and a method for preparing same from the venom of a Korean viper, Salmosa (AaXistrodon halvs). brevicaudus). Salmosin inhibits platelet aggregation more efficiently than any other known peptide platelet aggregation inhibitor. It is suggested that Salmosine can be developed as a therapeutic agent for the control of thrombosis.

Claims (2)

 1. Peptide (Salmosin) platelet aggregation inhibitor, derived from Salmosa venom (Askistrodon halvs brevicaudus), which consists of the amino acid sequence represented by (SEQ ID NO: 1):  EAGEE CDCGS PGNPC CDAAT CKLRQ GAQCA EGLCC DQCRF40  MKEGT ICRRA RGDDL DDYCN GISAG CPRNP FHA 73  A process for the preparation of the platelet aggregation inhibiting peptide (Salmosin) according to claim 1 from Salmosa venom (Askistrodon halvs brevicaudus), which comprises the steps of: obtaining Salmosa venom; loading the venom onto a serine proteinase affinity column to collect the solution having passed through the column without binding; and subjecting the solution to a series of operations, anion exchange, gel filtration and chromatography HPLC in reversed phase column.