DD241691A5 - METHOD FOR PRODUCING PROTECTED BLOOD-DISTINGANT PROTEINS - Google Patents

Abstract

The aim of the invention is the provision of new proteins which have anticoagulant properties, without adversely affecting the coagulation properties of the previously known anticoagulants. The method according to the invention is characterized in that blood vessel walls, strongly vascularized tissues or endothelial cell cultures are homogenized and centrifuged differentially and the above liquid is subjected in any order to one or more of the following purification treatments. B) Precipitation with salt C) Affinity chromatography) Ion exchange chromatography) Chromatography a molecular sieve, wherein the products obtained after stages b), c), d) and e), if desired, can be dialyzed.

Description

Field of application of the invention

The present invention relates to a method for producing anticoagulant proteins.

Characteristic of the known technical solutions

Anticoagulant proteins present in most mammals can be divided into three groups. This classification is based on the difference of the protein-action mechanisms.

1. Proteins that form a complex with the coagulation factor and thereby render the coagulation factor ineffective. These include the proteins:

a) Antithrombin III (Thromb. Res. 5,439-452 (1974))

b) α, -Protease Inhibitor (Ann Rev. Biochem., 52, 655-709 (1983))

c) a ₂ -macroglubin (Ann. Rev. Biochem 52, 655-709 [1983])

d) Crn inhibitor (Biochemistry 20, 2738-2743 [1981])

e) Protease Nexin (J. Biol. Chem. 258, 10439-10444, [1983]).

2. Proteins that proteolytically cleave a coagulation factor and thereby deactivate the coagulation factor. The only protein of this type so far described protein C (J. Biol. Chem. 251, 355-363 (1976)).

3. Proteins which shield and / or hydrolyze the negatively charged phospholipids so as to inhibit the phospholipid-dependent reactions of the blood clotting mechanism. So far, only phospholipases isolated from various snake venoms have been described (Eu J. Biochem., 112, 25-32 [1980]).

The gradual coagulation system has been intensively studied in recent years. It is understood to be a reinforcing multistage system of various interconnected proteolytic reactions in which an enzyme converts a zymogen into the active form (see [1980]). The Geschwindigleit this reaction is increased in a decisive manner by the presence of phospholipids and other cofactors such as Factor V _a and Factor VILI. ₃ In vivo, the procoagulant reactions are regulated by various inhibition mechanisms that prevent an explosive thrombotic trauma after a low activation of the coagulation cascade.

The anticoagulation mechanisms can be classified as follows (Rosenberg, R.D., Rosenberg, J.S., J. Clin. Invest., 74, 1-6 [1984]):

1. The serine protease factor X _a and thrombin are inactivated as a result of their binding to antithrombin III or to the antithrombin / heparin complex. Both the prothrombin activation and the formation of fibrin can be inhibited in this way. In addition to anti-thrombin III, there are various other plasma protease inhibitors such as a ₂ -macroglobulin and antitrypsin, the effect of which is time-dependent.

2. The discovery of protein Cs revealed another anticoagulant mechanism. Once activated, protein C acts as an anticoagulant by the selective proteolysis of the protein cofactors Factor V _a and VIII ₃ , which inactivates the prothrombinase and the factor X converting enzyme.

3. Plasmin cleaves monomeric fibrin 1, a product of the action of thrombin on fibrinogen, thereby preventing the formation of an insoluble fibrin (Nossel, H. L, Nature, 291, 165-167 [1981]).

Of the above-mentioned body proteins involved in the coagulation process, z.Z. only the antithrombin III in clinical use. As a significant disadvantage, however, has emerged in the application of this protein increase the bleeding tendency.

All previously used as anticoagulants agent, whether the body's own or synthetic nature in some way, the coagulation factors ineffective and thereby lead to side effects that may adversely affect the coagulation process.

Object of the invention

The aim of the invention is to provide agents which have anticoagulant properties, but without the adverse effects on the coagulation process properties of the previously known anticoagulants.

Explanation of the essence of the invention

The invention has for its object to provide new compounds having the desired properties and processes for their preparation.

Surprisingly, it has now been possible to isolate endogenous proteins that have anticoagulant properties but do not increase the risk of bleeding. As a further surprising feature these proteins lose their inhibitory properties during major bleeding, so that the usual coagulation processes can proceed undisturbed and there is no danger of bleeding.

The subject matter of the present invention are the blood coagulation inhibiting proteins which are referred to below as VAC (VasGular anti-coaguants) and which do not inactivate the coagulation factors. These proteins are capable of inhibiting coagulation induced by a vascular procoagulant or factor X _a , but not clotting induced by thrombin. They do not inhibit the biological and amidolytic activity of factors X _a and II _a .

The invention provides blood coagulation-inhibiting proteins which do not inactivate the coagulation factors and:

The modified prothrombin time trial and / or

The modified, activated, partial thromboplastin time trial and / or

The unmodified prothrombin time trial and / or

The prothrombin activation by the coagulation factor X _a in the presence of negatively charged phospholipids and Ca ²⁺ and / or

The intrinsic X activation by the factor IX _a in the presence of negatively charged phospholipids and Ca ²⁺ and / or

The prothrombin activation of isolated, stimulated platelets and / or

- The coagulation induced by the vessel wall and / or

- inhibit coagulation-dependent platelet aggregation.

The invention also provides blood coagulation-inhibiting proteins which do not inactivate the coagulation factors and whose inhibitory action depends on the amount of phospholipids. The proteins according to the invention induce prothrombin activation inhibition by the factor X ₃ . The inhibition is dependent on the phospholipid concentration and is smaller at high phospholipid concentrations. The phospholipids are not hydrolyzed by the proteins of the invention.

The invention further blood coagulation inhibiting proteins that do not inactivate the coagulation factors and the divalent cations Ca ²⁺ and / or Mn ²⁺ to negatively charged phospholipids, which are found for example in vesicles, liposomes or etherosomes, and / or on the divalent cations bind Ca ²⁺ and / or Mn ²⁺ to negatively charged phospholipids coupled with Spherocil. The binding of the proteins to the negatively charged phospholipids is reversible and can be abolished by ethylenediaminetetraacetic acid (EDTA). The proteins according to the invention are able to displace the factor X _a and the prothrombin from a negatively charged phospholipid surface. The invention relates in particular to blood coagulation-inhibiting proteins which do not inactivate the coagulation factors and have molecular weights of about 70 × 10 ³ , 60 × 10 ³ , 34 × 10 ³ or 32 × 10 ³ , of which the proteins having the molecular weight 34 × 10 ³ 32 x 10 ³ consist of only a single polypeptide chain. The invention preferably blood coagulation inhibiting proteins that do not inactivate the coagulation factors, characterized in that

They are isolated and purified from mammalian blood vessel walls,

- they are not glycoproteins,

- they are not phospholipases,

They have an isoelectric point at pH 4.4-4.6,

The activity of the anticoagulant proteins is thermolabile at 56 ° C,

The activity of the anticoagulant proteins in the citrated plasma remains stable for several hours at 37 ° C.

The activity of the anticoagulant proteins of trypsin and / or chymotrypsin is not completely destroyed,

The activity of the anticoagulant proteins of collagenase and / or elastase is not affected,

They bind via the divalent cations Ca ²⁺ and Mn ²⁺ with negatively charged phospholipids found in vesicles, liposomes or etherosomes,

They bind via the divalent cations Ca ²⁺ and Mn ²⁺ with negatively charged phospholipids coupled with spherocil,

The binding of the proteins to the negatively charged phospholipids is reversible and can be abolished by ethylenediaminetetraacetic acid (EDTA),

They displace the factor X _a and the prothrombin from a negatively charged phospholipid surface,

Inhibit the modified thrombin-time-trial,

They inhibit the modified, activated, partial thromboplastin time trial,

- they inhibit the unmodified prothrombin time trial.

They inhibit in vitro prothrombin activation by the coagulation factor X _a in the presence of negatively charged phospholipids and Ca ²⁺ ,

- they do not inhibit the biological and amidolytic activity of factors X _a and II _a ,

They inhibit intrinsic X activation by factor IX _a in the presence of negatively charged phospholipids and Ca ² ,

They inhibit prothrombin activation of isolated, stimulated platelets in vitro,

They inhibit vascular wall-induced coagulation in vitro, and

- The protein-induced prothrombin activation inhibition by the factor X _a is dependent on the phospholipid concentration and is smaller at high phospholipid concentrations.

The invention particularly relates to VAC proteins, substantially free of any animal tissue, preferably in substantially pure form.

Suitable starting materials for the isolation of the VAC proteins are the blood vessel walls and strongly vascularized tissue of various mammals, for example, from cattle, rats, horses and humans as well as endothelial cell cultures of these mammals. In particular, the arterial walls of cattle, rats, equine man as well as human nebula veins and veining are suitable.

The invention also provides a process for the preparation of the protein according to the invention by means of isolation and purification techniques known per se. The following process scheme is particularly suitable:

The homogenized starting material is first subjected to differential centrifugation. The resulting supernatant liquid can then be further treated sequentially in any order as follows. With ammonium sulfate unwanted admixtures can be precipitated. The supernatant is then purified by affinity chromatography, for example via hydroxyapatite, ion exchange chromatography, e.g. B. on DEAE-Sephacel, chromatography on a molecular sieve, such as Sephadex G-100 and immunoadsorbent chromatography, for example, with polyclonal or monoclonal antibodies further purified. Depending on the quality of the starting material, this purification scheme can be modified or other cleaning methods can be used, for example cleaning with the aid of phospholipid vesicles.

In addition to the classic antithrombotic treatment, oral anticoagulation, is now used in the case of a manifest; Thrombosis biosynthetic tissue plasminogen activator administered intravascularly (N. Engl. J. Med. 310, 609-613 [1984]). The proteins of the invention are outstandingly suitable, in particular by the anticoagulant properties simultaneously with the inhibition of coagulation-dependent platelet aggregation of a thrombosis, for example during surgery to prevent.

The present invention therefore also relates to the use of the proteins according to the invention as an antithrombotic agent

Another object of the invention are pharmaceutical preparations containing at least one protein according to the invention. ·

The results of VAC isolation and purification from bovine aortae are listed in Table A. The determination of the amount of VAC activity in the supernatant of the 100,000 xg centrifugation was irrelevant because of the presence of procoagulant activity. The components responsible for this activity could be precipitated with ammonium sulfate at a saturation level of 35%.

It was found that the above solution contained 100% VAC activity after precipitation with 35% ammonium sulfate. For concentration, the solution was added to a saturation of 90% with ammonium sulfate. The precipitate containing the VAC proteins was bound to a hydroxyapatite column in the presence of TBS. After washing, the VAC proteins from this column were eluted with increasing phosphate gradient. At low ionic strength, the VAC proteins were bound to a DEAE-Sephacel column. Elution of the VAC proteins from this column was with an increasing NaCl concentration gradient. At the final purification, the proteins were separated by Sephadex G100 due to their molecular weight by gel filtration. The eluent was a high salt buffer to minimize the interaction of VAC with the Sephadex material. VAC eluted from this column in a volume of about 1.6 times the empty column volume (see Figure 1). The overall yield of VAC activity after this final purification was 35%. According to SDS-PAGE, all G-100 fractions showing VAC activity contain two polypeptides (molecular weight 34,000 and 32,000, respectively). In some cases, a fraction with molecular weight also showed 60000 VAC activity. According to SDS-PAGE, only the peak fractions 138-140 are homogeneous with respect to the two polypeptides. In Table A, purification data from the bovine aortas were summarized. The G-100 fractions, which are homogeneous with respect to the two polypeptides, were used for all further experiments in this specification except for the experiments to characterize the binding of VAC to phospholipid liposomes.

In G-100 fraction 139, 3.4% of VAC activity with a specific activity of 1480 units / mg protein was determined, as determined by the single-step coagulation assay. This fraction contained no detectable amount of phospholipid and from the absorbance at 280 nm and the protein content an extinction coefficient of .gamma. For this purified VAC preparation was determined

i ° = 8.5 calculated.

i cm

Characterization of the VAC

As can be seen in Figure 2, the two polypeptides of molecular weight 34,000 and 32,000 are present in the purified protein material and are attributed to VAC activity. Proteins with a single chain. As noted with Schiff's reagent with basic fuchsin, both proteins contain few carbohydrate groups. In addition, no Gla groups could be detected in either protein. Isoelectric focusing indicates that both proteins migrate in a single band, which corresponds to an isoelectric point of 4.4 to 4.6 (see Fig. 3).

VAC activity was recovered from the PAG plate by elution of this band from the gel. Analysis of this eluate with SDS-PAGE again showed the presence of the two proteins. It was thus confirmed that both proteins migrated in a single band in the pH gradient of the PAG plate. To test the method, human hemoblobin (Hb) was also examined by isoelectric focusing. The value found of 6.8 agrees with the information in the literature (see Fig. 3).

Binding experiments indicated that VAC activity can bind to negatively charged phospholipid membranes. This binding occurs in the presence of Ca ²⁺ and Mn ²⁺ , but not in the presence of Mg ²⁺ and in the absence of divalent metal ions (see Table D). This binding of VAC activity to the liposomes is reversible and can be abrogated by the reagent EDTA.

On the basis of SDS-PAGE it could be shown that both proteins can bind to liposomes in the presence of Ca ²⁺ and that this bond dissociates again on addition of EDTA (see FIG. another indication that the VAC activity is attributable to these two proteins.

When stored in TBS containing 10% glycerol, VAC activity is stable at -70 ° C for at least 3 months, at least 12 hours at o ° C and at least half an hour at 37 ° C. At 56 ⁰ C, the activity disappeared within 2 minutes. , -, .. * ". , .. -

Effect of VAC

VAC prolongs the clotting time after the single-stage coagulation test, triggering coagulation with BTP. Replacement of BTP by purified bovine thrombin or purified bovine factor X _a in this experiment indicates that VAC only prolongs clotting time when factor X _{a is used} to induce coagulation and that clotting induced by thrombin is not affected by VAC. This indicates that VAC directly inhibits Factor X _a activity or that interaction with the prothrombinase complex occurs. For further testing, the amidolytic thrombin-forming experiment with the purified bovine factor X _a and prothrombin was used. Figure 5 shows that when prothrombin is activated to thrombin by Factor X _a in the presence of Ca ²⁺ and phospholipid, VAC inhibits prothrombin activation and the degree of inhibition is dependent on the VAC concentration. In addition, the inhibitory effect of VAC is stronger with a low phospholipid concentration.

Fig. 6 illustrates the Phospholipidabhängigkeit the induced by VAC inhibition of prothrombin activity. It is noteworthy that is not inhibited at a phospholipid concentration of zero, the thrombin activation by factor X _a by VAC. In control experiments it was found that VAC itself does not affect the measuring system.

Incubation of 5μΜ phospholipid (PS / PC; 1: 4 mol / mol) with 107 pg / ml VAC (specific act 1300 units / mg) and 10mM Ca ²⁺ resulted in reduction within 3 min at 37 ° C the procoagulant activity. This showed that VAC has no phospholipase activity.

In contrast to AT-III, VAC has no influence on the amidolytic activity of the purified thrombin and no lasting influence on the factor X _a activity as measured with the chromatogenic substrates S 2238 and S 2237, respectively (see Table C). In this table is also shown that inactivation of factor _Xa and thrombin / AT-III is not amplified by VAC; Heparin, on the other hand, has increased AT-III activity.

The isolation of a novel human anticoagulant protein could be achieved by a basically same procedure from the homogenate of human umbilical arteries.

The anticoagulant was discovered by its ability to prolong the clotting time in a prothrombin time trial. This ability became measurable after a Sephadex G100 fractionation of an arterial homogenate. Due to further isolation steps, it can be assumed that the activity can be attributed to one or more water-soluble substances which carry an overall negative charge at a pH of 7.9.

The analysis of a Sephadex G75 fraction (the last purification step so far) by gel electrophoresis shows a correlation between the intensity of the molecular weight band at 32,000 and the prolongation of the clotting time measured in the MPTT (Figure 15). The relationship between the 32 K band and the anticoagulation activity is unequivocal, as only anticoagulant activity can be eluted from this 32 K band of the polyacrylamide gel. In conjunction with the results that the anticoagulant rapidly loses its activity on incubation at 56 ° C and that proteases can destroy its activity, it must be assumed that the anticoagulant activity is caused by a single protein with an apparent molecular weight of 32,000 daltons.

Trypsin, unlike protease type 1, is a poor inactivator of the anticoagulant. This indicates that the anticoagulant contains only a few lysine and arginine residues that are accessible to trypsin.

The nature of the anticoagulant activity was investigated by triggering the coagulation in several ways. The coagulation triggered either by the vascular procoagulant HTP or by the factor X _a is inhibited by the anticoagulant, but the thrombin-induced coagulation is not. From this it can be concluded that the anticoagulant in thrombin formation but not in the action of thrombin plays a role.

To further elucidate the anticoagulant mechanism of the protein of the invention, prothrombinase was used (2). Under the experimental conditions, the anticoagulant can inhibit the activation of prothrombin by the complete prothrombinase 6 factor X _a , factor V _a , phospholipid, Ca ²⁺ ) and by the phospholipid-bound factor X _a (factor X _a , phospholipid, Ca ²⁺ ) but not the free factor X ₃ (factor X _a , Ca ²⁺ ).

The time course of prothrombin activation in the presence of the anticoagulant indicates the immediate inhibition of prothrombin activation, which remains constant over time. It can therefore be stated that the anticoagulant acts neither by a phospholipase nor by a proteolytic activity. The fact that the activation of prothrombin by factor X _a and Ca ²⁺ is not influenced by the anticoagulant, is a strong indication that the anti-clotting mechanism of vascular protein from the generally known plasma protease inhibitors, such as antithrombin is different III , As shown by Walker et al (8), the activated protein C does not inhibit prothrombin activation by factor X ₁ , Ca ²⁺ and phospholipid; Consequently, the protein according to the invention is also not identical to protein C.

Initial binding experiments indicate that the vascular anticoagulant may interfere with the lipid binding of factor X _a and / or prothrombin. Whether the ability of the anticoagulant to inhibit prothrombin activation is fully responsible for the proliferation effect in the prothrombin time trial remains to be determined.

- Ö - 4IU3I

The fact that the material according to the invention can be obtained from different types of arteries and not from moderately vascularized tissue may indicate that the protein according to the invention is a physiological modulator of hemostasis and thrombosis, active at the vascular level.

Based on the determined properties and effects of VAC, the blood coagulation mechanism under VAC influence can be interpreted as follows:

VAC binds to Ca ²⁺ ions with negatively charged phospholipids resulting from tissue damage and / or stimulated platelets, thereby reducing the binding of certain coagulation factors (vitamin K-dependent coagulation factors) with the negatively charged phospholipid surface responsible for these coagulation factors how a catalytic surface acts (Biochem., Biophys., Acta, 515, 163-205 [1978]), with the result that the phospholipid-dependent blood clotting reactions of VAC are inhibited. Due to the mechanism of action, VAC can be classified in the protein group 3 (see above).

Of crucial importance, however, is the difference with the proteins of this group that VAC does not hydrolyze the phospholipids and thereby can not decompose essential membrane structures!

Furthermore, particularly significant and advantageous are the previously described in none of the known anticoagulants).; ^. * Properties of the VAC:

The anticoagulant effect of VAC is dependent on the amount of phospholipids involved in the coagulation process. This dependence causes the coagulation process, which was triggered for example by a small vessel wall damage and / or low platelet activation, z. B. by a thrombotic process, surprisingly can be inhibited by VAC! On the other hand, however, the coagulation process, which was triggered by a drastic vascular wall damage occur in the phospholipids in high concentration, not inhibited by just these high phospholipid concentrations by VAC! The risk of bleeding when using VAC is therefore surprisingly extremely low.

In contrast to all previously known anticoagulants, VAC offers these superior properties, which render one or more coagulation factors ineffective and thus increase the risk of bleeding!

- VAC surprisingly does not inactivate the coagulation factors themselves! As a result, the coagulation factors continue to be able to fulfill their other tasks, which are increasingly being uncovered. For example, some active coagulation factors have the important nonhemostatic role of Chemotxis for the inffamatory cell. This cell helps to heal a damaged vessel wall!

Surprisingly, VAC does not affect this process!

The present invention for the first time describes a novel class of anticoagulant proteins which do not inactivate the coagulation factors.

embodiment

The invention is explained in more detail below with reference to some examples.

The illustrative examples and the listed properties are not intended to limit the invention in any way. It is possible for a person skilled in the art without inventive performance to obtain further proteins which have anticoagulant properties by means of the method described, without inactivating the coagulation factors. These proteins are also within the scope of the present invention.

The abbreviations used in the present invention have the following meaning:

VAC: vascular anticoagulant

PFP: platelet-free plasma

TBS: 10 mM NaCl, 50 mM Tris / HCl, pH 7.5

EDTA: ethylenediaminetetraacetic acid

TBSE: TBS with 2 mM EDTA

BTP: thromboplastin from cattle

HTP: thromboplastin from human brains

TBSA: TBS with 0.5 mg / ml human serum albumin, pH 7.9

S 2337: N-benzene-L-isoleucyl-L-glutamyl-L-pipecolyl-glycyl-L-arginine-p-nitroanilide dihydrochloride

S 2238: H-D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride

AT-III: human antithrombin III

S.A .: specific activity

Ole ₂ Gro-P-Cho: i-dioleoyl-sn-glycero-S-phosphocholine

No ₂ GrO-P-SeT: 1,2-dioleoyl-sn-glycero-3-phosposerin

GIa: γ-carboxy-glutamic acid

For the nomenclature of blood coagulation factors, the recommended by Task Force on Nomenclature of Blood Clotting Zymogens and Zymogens intermediates has been used.

materials

The analytical SDS-PAGE and hydroxyapatite HTP chemicals were from Bio-Rad, Sephadex G-100 and G-75, DEAE-Sephacel, and Pharmacia's Low Molecular Weight Calibration Kit, Kabi's chromogenic substrates S2337 and S2238 Vitrum and the Diaflo PM-10 ultrafiltration membrane from Amicon.

example 1

Isolation and cleaning of the VAC

Within half an hour after slaughtering, the aortae were removed from the cattle. The bovine blood was collected in trisodium citrate (final concentration 0.38 wt%) and centrifuged for 10 minutes at room temperature and 2000xg. The low platelet-containing plasma was then centrifuged again (15 minutes at 10000xg). In this way, platelet-free plasma (PFP) was obtained.

The aorta of the animals were rinsed thoroughly with T.BS immediately after their removal. The intima were detached from the aorta and homogenized using a high speed homogenizer such as the Braun MX 32 in TBSE containing the soybean trypsin inhibitor (16 mg / l) and benzamidine (1.57 g / l).

The material homogenized from 8 aortas containing 20% (w / v) solid was centrifuged at 100,000xg for 60 minutes. The supernatant was saturated with solid ammonium sulfate up to 30%. Stirred for 30 minutes and then centrifuged for 20 minutes at 12 000xg. The resulting liquid was saturated with solid ammonium sulfate up to 90%, stirred for 30 minutes and then centrifuged at 12,000xg for 20 minutes.

The solid was suspended in a small volume of TBS and dialysed against benzamidine (1.57 g / L) containing TBS. The dialyzed fraction was applied to a hydroxyapatite column (1 x 20 cm) which had been equilibrated with TBS. The column was washed with 4 bed volumes of TBS. The VAC proteins were eluted from the column with 200 ml of sodium phosphate buffer (pH 7.5) with a linear gradient of 0-50 oMM. The fractions containing VAC were combined and dialysed against 50 mM NaCl, 20 mM Tris / HCl, pH 7.5.

The same buffer was also used to equilibrate a DEAE Sephacel column (3χ5 cm) on which the dialyzed VAC material was chromatographed. The column was washed with 4 bed volumes of the equilibration buffer before eluting the VAC from the column with 200 ml NaCl solution in 20 mM Tris / Hcl, pH 7.5 with a linear gradient of 50-30 oMM.

The fractions containing VAC were collected, dialyzed against 50 mM NaCl, 20 mM Tris / HCl, pH 7.5, and then concentrated in an Amicon concentration cell using a PM-10 ultrafiltration membrane. The 2 ml volume concentrate was applied to a Sephadex G-100 column (3 x 80 cm) equilibrated with 50 mM NaCl and 20 mM Tris / HCl, pH 7.5.

The eluate was collected in fractions of 2 ml each, the active fractions dialysed separately against 10 vol .-% glycerol containing TBS and stored at -70 ° C. All purification was carried out at 0-4 ° C.

Determination of VAC activity

Two different methods were used to determine VAC activity:

a) the single-step coagulation trial (modified prothrombin time trial)

b) a thrombin generation experiment

The single-stage coagulation test was performed as follows:

In a cuvette made of siliconized glass, 175 μl of the fraction to be tested or 175 μl of TBS were stirred as control with 50 μl of PFP and 25 μl of diluted BTP (900 revolutions / min).

After an incubation period of 3 minutes at 37 ° C, coagulation was initiated by the addition of 250 μM buffer containing 8 mM NaCl, 20 mM CaCl _2, and 10 mM Tris / HCl, pH 7.5. Fibrin formation was optically registered using a "Payton Dual Aggregation Module" (Hornstra, G., Phil., Trans Soc R. London, London, 294, 355-371, 1981. The clotting time of the control sample was 65 seconds For the determination of the VAC yield during purification, a unit of VAC activity was defined as the amount of VAC that increased the clotting time in the above-mentioned experiment up to 100% Seconds extended.

In some cases, BTP has been replaced by purified bovine thrombin or the purified bovine factor X _a . In this semi-purified coagulation system, the amounts of thrombin or factor X _{a were} chosen such that the clotting time of the control sample was also 65 seconds.

The thrombin formation experiment was carried out as follows:

20μΙ purified bovine factor X _a (15OmM), 30μΙ CaCI ₂ (10OmM), 30μΙ diluted VAC and 30μΙ PS / PC phospholipid membrane (the final concentrations are given in the legend to the figures) were placed in a plastic cuvette containing 181 μΙ TBSA contained, brought.

The mixture was stirred at 37 ⁰ C for 3 minutes with a Teflon stirrer. Thrombin formation was triggered by the addition of 9μΙ purified bovine factor II (33.33μΜ). After different times, the reaction mixture samples were taken from 50 μΙ, the contents of a plastic cuvette of 1 ml, which was heated by means of a thermostat to 37 ⁰ C and 900 μΙ TBSE and 50 μΙ of the chromogenic substrate S 2238 (5 mM), were added. From the absorbance change at 405 nm measured with a Kontron Unikon 810 spectrometer, the amount of thrombin in the reaction mixture was calculated using a calibration curve prepared from the known amounts of purified bovine trombine. The percentage of inhibition caused by VAC was defined as follows:

% Inhibition = (1- |) x 100 %,

where "a" is the rate of thrombin formation in the absence of VAC in nM Il _a / min and "b" is the rate of thrombin formation in the absence of VAC in nM Il _a / min. is.

proteins

The vitamin K dependent factors prothrombin and factor X _a were obtained by the purification of citrated bovine plasma (see Stenflo J, J. Biol. Chem., 251, 355-363 [1976]). After barium citrate adsorption and elution, ammonium sulfate fractionation and DEAE-Sephades chromatography, there were two protein fractions containing a prothrombin / factor IX mixture, or factor X, respectively. Factor X was obtained by the method of Fujikawa et al. Biochemistry, 11, 4882-4981 (1972) and activated using RVV-X (Fujikawa et al., Biochemistry, 11, 4892-4899 [1972]).

Prothrombin was separated from factor IX by heparin-agarose affinity chromatography (Fujikawa et al. Biochemistry, 12, 4938-4945). The prothrombin-containing fractions from the heparin-agarose column were pooled and further purified by the method of Owens et al., J. Biol. Chem. 249, 594-604 (1974)). The concentrations of prothrombin and factor X _a were determined by the method of Rosing et al., J. Biol. Chem. 255, 274-283 (1980). BTP was prepared by a standard method described by Van Dam-Mieres et al. in "Blood coagulation enzymes. Methods of Enzymatic Analysis "Verlag Chemie GmbH, Weinheim The protein concentrations were determined according to Lwry et al., J. Biol. Chem. 193, (1951).

Production of phospholipid, phosphoipid vesicles and phospholipid liposome

i ^ -dioleoyl-sn-glycero-S-phosphocholine (18: 1 _cis / 18: 1 _C is-PC) and i -dioleoyl-sn-glycero-S-phosphoserine (18: 1 _cis / 18: 1 _cis ) PS) were prepared as described in Rosing et al., J. Biol. Chem. 255, 274-283 (1980). Separate, two-layer phospho-lipid vesicles of PC and PS were prepared using ultrasound as described in Rosing et al., J. Biol. Chem. 225, 274-283 (1980). A stock solution of phospholipid liposomes was prepared by dissolving the required amount of phospholipid in chloroform. The chloroform was evaporated by nitrogen. The remaining phospholipid was suspended in TBS containing 5% glycerol, premixed with a few glass beads for 3 minutes and then centrifuged at 10 000xg for 10 minutes. The above solution was discarded and the residue was carefully resuspended in TBS containing 5% glycerol; in this way the phospholipid-liposome stock solution was obtained. These liposomes were stored at room temperature. The phospholipid concentrations were determined by the phosphate analysis according to Böttcher et al., Anal. Chim. Acta, 24, 20-207 (1961).

SDS-PAGE

Gel electrophoresis on plates in the presence of SDS was carried out according to the method described by Laemmli, Nature, 227, 686-685 (1970) using a gel containing 10% by weight acrylamide, 0.27% by weight N / N ³ -Methylenbisacrylamid and 0.1 wt .-% SDS contained. 5 wt% β-mercaptoethanol was present in gel samples with reduced disulfide bridges. The gels were dyed as follows:

1. with 0.25 wt% Coomassie Bl '; e P-250 in 50 wt% ethanol and 15 wt% acetic acid and decolorized in 10 wt% ethanol and 10 wt% acetic acid

2. Schiffschem reagent prepared from the basic fuchsin according to the method of Segrest et al., Described in "Methods in Enzymology", vol.28, 54-63 (1972) or

3. with silver as described by Merril et al. in Electrophorisis, 3, 17-23 (1982).

electric focusing

The isoelectric pH determinations of proteins were performed on the finished thin film polyacrylamide gels containing Amfoline Carrier Ampholytes (PAG plates, LKB) at a pH in the range 3.5-9.5 according to the manufacturer's instructions. The pH gradient in the gel was determined immediately after electrofocusing by cutting a strip from the gel along a line between the anode and the cathode. The electrolytes were eluted from each strip with distilled water and the pH of the water was measured with a combined glass electrode.

Glutaminsäurebestimmung

The Gla determination was carried out by HPLC on a "Nucleosil 5SB" column (CHROMPACK) according to the method of Kuwada et al., Anal. Biochem., 131, 173-179 (1983).

Example Il The coupling of phospholipids to Spherocil

The required phospholipids were dissolved in chloroform and added to the column material Spherocil in a ratio of 5 mg phospholipid per gram of Spherocil. The chloroform was evaporated with N ₂ -GaS and the dry spherocil phospholipid subsequently washed with the buffer in which VAC had been suspended. VAC binds to Spherocil phospholipid in the presence of Ca ⁺⁺ and / or Mn ⁺⁺ when some of the phospholipids are negatively charged.

Example Ml

50 μΙ citrated / platelet-free plasma was mixed with 200 μΙ buffer (25 mM Tris / HCl pH 7.5, 10 μM NaCl) in which kaolin (artificial surface, which catalyzes the substantial coagulation, in this case ground glass), Inositin (phospholipid source) and VAC were present. This mixture was incubated for 3 minutes at 37 ° C after which 250μΙ Ca ⁺⁺ buffer (20mM Tris / HCl pH7.5.8mM NaCl, 20mM CaCl ₂ ) was added. The clotting time was measured as in Example 1.

Table A

Summary of the purification of VAC from intima of bovine aortae

cleaning stage Protein ⁸ VAC ^b specific yield Reini mg units activities % gungs- Units / mg Degree Above liquid with 35% (NH ₄ J ₂ SO ₄ 630 19,500 31.0 100 1.0 Präzipitätmit 90% (NH ₄ J ₂ SO ₄ 470 19,000 40.4 97 1.3 Hydroxy-apati Group 206 17300 84.0 89 2.7 DEAE fraction 35.8 13 900 388 71 12.5 Sephadex G-100 0.45 666 1480 3.4 47.7

a) The amount of protein was determined by the method of Lowry et al. determined (Lowry, O.H., Rosebrough, N.V., Farr, A.L., and Randall, R.J., J. Biol. Chem. 193 [1951] 265).

b) The VAC units were determined by the one-step coagulation assay described in Example 1 on a series of sample dilutions. The dilution which extends the clotting time of the control sample (65 seconds) to 100 seconds contains a VAC unit as defined.

Table B

The metal-dependent binding of VAC to negatively charged phospholipid liposomes

Cation (10mM) t _c seconds ^al EDTA ^C) above liquid Control (no liposomes) 180 ND ^d) Control (no cation) 174 64.8 CaC ^ 64.2 - · 134 MgCl ₂ 165 N.D. MnCI ₂ 65.1 N.D.

a) the clotting times t _c were determined according to the one-step coagulation test described in Example I.

b) 50 μΙ stock solution of phospholipid liposome (PS / PC; 50/50 mol / ml: 1 mM), 50 μM VAC (250 μg / ml,

S.A. = 700 units / mg) and 100μΙ TBS containing 5% glycerol and the applied cation, pH 7.5, were mixed at room temperature and centrifuged at 15,000xg for 15 minutes, 25μΙ of the above obtained liquid was diluted to 175μΙ with TBS and then tested after the single-stage coagulation test. The residual amount of the above liquid was analyzed by SDS-PAGE (Fig. 4).

c) The liposome precipitate obtained was resuspended in TBS containing 150μΙ 5% glycerol and 10 mM EDTA, pH 7.5. The suspension was centrifuged for 15 minutes at 15,000xg. The VAC activity of the above liquid was analyzed as described under b).

d) N.D. = not determined.

Table C

The effect of VAC on the amidolytic activity of factor X ₃ and factor II _a .

Αίοδ / Μϊη. 10 ³ a VAC

X ₃₁ AT-III X _a , heparin X _a , heparin, AT-III Ha

ll _a, AT-lll ll _a, heparin ll _a. Heparin, AT-III

a) the amidolytic activity was measured as follows: Factor X ₃ or Factor II _a was diluted with the mentioned agents in TBSA. The reaction mixture was stirred with a Teflon-coated stirrer in a plastic cuvette, which was thermostated at 37 ° C, using a thermostat. After 10 minutes, a sample of 100 μΙ (Χ ₃ ) οαβΓ50μΙ (M ₃ ) was removed from the cuvette. The sample was placed in another thermostated plastic cuvette containing 800μΙ TBSE and 100μΙ 32337 (2mM) or 900μΙ S2338 (5mM) The absorbance change at 405nM was measured with a Kontron Spectrophotometer Uvikon 810 was tempered to 37 ^C C by means of a thermostat.

The final concentrations of the various agents in the reaction mixtures were as follows: 18.7 nM factor X ₃ ; 1.5 nM factor M ₃ ; 18.7nM AT-III; 1 unit / ml heparin and 10 ^ g / ml VAC (SA 1300 units / mg).

b) N.D. = not determined.

Example IV

The human blood collected by venipuncture was collected in a trisodium citrate solution (final concentration about 13 mM citrate) and centrifuged for 10 minutes at 2000xg and room temperature. The resulting plasma was again centrifuged at 100,000xg for 15 minutes to obtain platelet-free plasma (PFP). Standard PFP was prepared by mixing plasma from various healthy donors.

Human umbilical cords were obtained 15 minutes after birth. The arteries were immediately rinsed with ice-cold TBS buffer, then dissected free of Warton's Jelly (Warton's gel) and homogenized in TBS using a Braun MX32 homogenizer A 10% homogenate (w / v) was then fractionated.

The fractionation of the supernatant of the homogenate centrifuged at 10000 × g with Sephadex G100 gave a reproducible specific elution profile (see FIG. 7).

110.5 110.5 80.0 81.5 110.5 109.0 47.5 ND ^b 7.5 7.5 5.4 5.6 7.5 7.1 0.56 N.D.

The fractions that affect the coagulation system when measured in the MPTT are shown in FIG. A procoagulant activity eluted with the forerun. This activity can only be detected in MPTT in the presence of factor VII by experiments using human congenital factor VII-deficient plasma. Consequently, this procuagulant must be considered a tissue thromboplastin.

Certain fractions had pronounced anticoagulant activity. These fractions were pooled and further purified by DEAE Sephacel chromatography (Figure 8A). The anticoagulant had bound to DEAE-Sephacel at a salt concentration of 50 mM NaCl and 50 mM Tris / HCl pH 7.9. Elution of the activity with a linear NaCl gradient at pH 7.9 was performed with 150-16 oMM NaCl. The DEAE fractions containing anticoagulant activity were pooled and subjected to gel filtration with Sephadex G-75. The column (1.5 x 50 cm) was equilibrated with TBS. The activity in the fractions having a molecular weight of 30,000-60,000 daltons. The MPT was used as a quantitative assay for determining the amount of anticoagulant activity (Figure 9). One unit of anticoagulant activity was defined as the amount that prolongs the clotting time in the MPTT, using HTP as a coagulation inducing agent (final concentration of 95 μg protein / ml), from the control value of 65s to 100s. Using this assay, it was calculated that from 10g of wet arterial tissue about 2 mg of protein was obtained with approximately 1200 units of anticoagulant activity.

Modified Prothromfein Time Attempt (MPTT)

The modified prothrombin time trial (MPTT) was performed as follows. In a cuvette of siliconized glass, 50 μΙ PFP were stirred at a temperature of 37 ° C with 150 μΙ TBS, 25 μΙ a standard HTP dilution and 25 μΙ TBS (control) or with 25 μΙ a fraction of the homogenized arterial material. After incubation for 3 minutes, coagulation was initiated by addition of 250 μM Ca ²⁺ buffer (8 mM NaCl, 20 mM CaCl ₂ and 10 mM Tris / HCl pH 7.9). Fibrin formation was registered using a Payton Dual Aggregation Module (13). Was used in the MPTT for triggering the coagulation factor X _a, HTP was omitted and 25 μΙ purified factor along with the amount of 250 μΙ Ca ⁺⁺ buffer ² added to the diluted PFP.

Modified thrombin time trial (MTT)

The modified thrombin time trial (MTT) was carried out in the same manner as described above, except that the X _a preparation was replaced by 25 μΙ purified thrombin.

proteins

Protease type I and trypsin (EC 3.4.2.1.4.) Were from Sigma. HTP was named after Van Dam-Mierasetal. (14) described method of human brains. Factor X _3, prothrombin and thrombin were from citrated bovine blood as Rosing et al. (2), cleaned. Factor V was after at Lindhoutetal. (13) obtained from bovine blood. Factor V ₃ was obtained by incubation of factor V with thrombin (15). The prothrombin concentration was calculated from M _r = 72000 and Alto = 9.6 (17). Factor X ₃ and thrombin concentrations were determined by titration of active sites (2). The remaining protein concentrations were determined as in Lowryetal. (18).

Production of phospholipids and phospholipid vesicles

Ole ₂ Gro-P-Cho and Ole ₂ -P-Ser were prepared by the method of Rosingetal. (2) produced. Single bilayer vesicles were prepared by ultrasound from 0Ie ₂ Gro-P-Ser / Ole ₂ Gro-P-Cho (20:80 molar ratio) (2).

Phospholipid concentrations were determined by phosphate analysis according to Böttcher et al. (19) determined.

Measurement of prothrombin activation

The time course of prothrombin activation was examined with various concentrations of the anticoagulant.

The mixtures of (X ₃ , Ca ²⁺ ), (X ₃ , phospholipid, Ca ²⁺ ) or (X ₃ , V ₃ , phospholipid, Ca ²⁺ ) were mixed with various amounts of anticoagulant at 37 ° C in 50 mM Tris / HCI, 175 mM NaCl, 0.5 mg / ml human serum albumin stirred at pH 7.9. After 3 minutes, the reaction was triggered by the addition of prothrombin. Samples of 25 μΙ were placed in a cuvette (at 37 ° C thermostated) at various time intervals containing TBS, 2 mM EDTA and 0.23 mM S2238 (the total volume was finally 1 ml). From the absorbance change at 405 nm, measured with a Kontron spectrophotometer Uvikon 810, and a plot made from known amounts of purified thrombin, the amount of thrombin formed was calculated at various concentrations of anticoagulant.

The phospholipid was added as a vesicle of Ole ₂ Gro-P-Ser and 0Ie ₂ GrO-P-ChO with a molar ratio of 20:80.

characterization

Various fractions of the G-75 chromatography were checked in the MPTT and analyzed by SDS-PAGE. The results (see Fig. 10) indicate that the anticoagulant has a molecular weight of about 32,000. The relationship between the anticoagulant activity and the 32 K band was confirmed by cutting the polyacrylamide gel and then eluting the proteins from the strip with TBS containing 0.5 mg / ml bovine serum albumin. Anticoagulant activity is present only in the band attributable to the 32K band. In addition, it was found that this activity is thermolabile at 56 ⁰ C and has a similar dose-response with MPTT as the starting material. The G75 fractions showing the highest anticoagulation activities were pooled and used for further labeling of the anticoagulant. Incubation of the anticoagulant at 56 ° C gives a rapid decrease in activity until after 2 minutes no activity can be measured (see Figure 16). The anticoagulant loses its activity completely within 2 hours upon incubation at 37 ° C with Type I protease, while trypsin hardly inactivates the anticoagulant within a 3 hour incubation period (see Figure 11). The concentration of protease type I and trypsin used in these experiments inactivates 2.5 nM thrombin within 15 minutes. The amounts of protease type I and trypsin transferred from the reaction mixtures in the MPTT did not affect the control clotting time.

The MPPT is extended both in the presence of the anticoagulant (see FIG. 12) and in the activation with HTP and when triggered with the factor X ₃ . However, thrombin-induced coagulation is not inhibited.

Based on these experiences, the effect of the anticoagulant on the conversion of prothrombin into thrombin was examined by factor X ₃ , factor V ₃ , phospholipid and Ca ²⁺ . Under the experimental conditions mentioned, thrombin formation is inhibited in a dose-dependent manner by the anticoagulant (see Figure 13A). Activation of prothrombin by factor X _a , phospholipid and Ca ²⁺ in the absence of factor V _a can also be inhibited by the anticoagulant (see Figure 13B). However, this inhibition is not observed when activation occurs in the absence of phospholipid (see Figure 13C).

Example V Polygonal antibodies to VAC

Polygonal antibodies against bovine VAC were stimulated in rabbits. Cattle VAC, purified by the method described above, was mixed with equal proportions of complete Freund's adjuvant. The mixture was injected subcutaneously into the rabbit. After 4 weeks, a refresher was performed by re-cleaning the rabbit cattle VAC s.c. was injected. This booster injection was repeated twice at intervals of 2 weeks. Ten days after the last booster injection, the rabbit was bled and the blood coagulated to obtain serum. Immunoglobulins (Ig) were isolated from the serum according to the following scheme.

a) the serum was heated to 56 ° C for 30 minutes.

b) Subsequently, the serum was applied to DEAE-Sephacel which had been equilibrated with 50 mM Tris, 100 M NaCl, pH 8.2.

c) The unbound protein is precipitated with (NH ₄ I ₂ SO ₄ at 50% saturation with (NH ₄ J ₂ SO ₄ .

d) The precipitated proteins were pelleted by centrifugation, the pellet was resuspended in 50 mM Tris, 10 mM M NaCl pH 7.9 and dialyzed extensively against the same buffer.

e) The resulting protein mixture contains the Ig.

From bovine aorta, bovine lung, rat and equine aorta and human umbilical artery, protein fractions were isolated according to the described methods which show VAC activity. The proteins were electrophoresed on a polyacrylamide gel in the presence of dodecyl sulfate and under non-reducing conditions, the proteins were transferred from the gel to nitrocellulose strips as described by Towbin et al. (21) described, transferred. The strips were incubated with the anti-VAC-Ig and, after extensive washing, incubated with goat anti-rabbit Ig coupled with horseradish peroxidase. This is visualized with diamine-benzidine-tetrahydrochloride, a substrate for the proxidase. Upon completion of the procedure described, a brown ribbon on the nitrocellulose strip indicated the presence of a goat rabbit Ig. In addition, anti-VAC-Ig and proteins to which the anti-VAC-Ig bind were present on this spot. Immunoblots of proteins having VAC activity isolated from bovine aorta, bovine lung, rat and equine aorta, and human umbilical artery are shown in FIG.

These results show the following: Using the isolation scheme described, a protein fraction having VAC activity can be obtained from bovine aortas, bovine lungs, rat and equine aortas, and human umbilical arteries. Furthermore, the isolated protein fractions with VAC activity contain proteins with molecular weights of ± 32,000, ± 34,000, and ± 700,000, which react with anti-VAC-Ig stimulated against purified bovine VAC in rabbit.

Example Vi Purification step for VAC using bulky phospholipid vesicles

Large volume phospholipid vesicles (LVV) composed of 1,2-dioleoyl-sn-glycero-3-phosphoserine (PS) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (PC) were prepared as described by P. van de Waart et al. (20). LVV containing PS: PC (molar ratio 20:80) was used for the purification step. However, other molar ratios should be used, with the proviso that negatively charged phospholipids must be present. Likewise, the chain length of the fatty acids in the phosolipids can be varied

 Method:

LVV, ± 1 mM phospholipid in 50 mM Tris / HCl, 10 mM NaCl, pH 7.9, was mixed with an equal volume of a protein fraction containing VAC activity. The proteins are dissolved in 50 mM Tris / HCl, 100 mM NaCl, 10 mM CaCl ₂ , pH 7.9. The mixture is kept at room temperature for 5 minutes. The mixture is then centrifuged for 30 minutes at 20,000 xg. The pellet is resuspended in 50 mM Tris / HCl, 10 mM NaCl, 10 mM CaCl ₂ , pH 7.9 and recentrifuged. The resulting pellet is then resuspended in 50 mM Tris / HCl, 10 mM ethylenediaminetetraacetic acid (EDTA), pH 7.9, and recentrifuged. The resulting supernatant contains the VAC activity. The method described above is an efficient purification step in the process of obtaining purified VAC.

figure description

Figure 1 Gel filtration of VAC on Sephadex G-100.

The column (3 x 80 cm) was prepared at a pressure of 60 cm and equilibrated with 50 mM NaCl and 20 mM Tris / HCl, pH 7.5. The VAC-containing fraction obtained after DEAE chromatography was concentrated to 2 ml and then passed onto the Sephadex G-100. The pressure height of 60 cm was maintained. The empty volume was 245 ml (fraction 70).

Thereafter, the fractions of 2 m were collected. The fractions were dialysed against TBS containing 10% glycerol, after which they were tested for VAC activity following the single step coagulation assay as described in Example I. The clotting times were obtained at dilutions of 1:10 of the G-100 fractions with TBS. The clotting time in the absence of VAC was 65 seconds.

Figure 2 Analytical SDS-PAGE of VAC.

SDS-PAGE with gels containing 10 wt .-% acrylamide, 0.27 wt .-% N, N-methylenebisacrylamide ³ and 0.1% SDS was carried out according to Laemli (Laemli, UK, [1970] Nature 227, 680-685). The meaning of the numbers on the x-axis is as follows:

1. Standard molecular weight materials in which the disulfide bonds are reduced,

2. 25 μg reduced VAC,

3. 25 pg unreduced VAC.

The gel was stained with Coomassie Blue and destained in the manner described in Example I.

Figure 3 The determination of the isoelectric pH of VAC

Electrofocusing was performed with PAG plates in the pH range of 3.5-9.5 (see Example I). 200 μg of human Hb ¹ and 20 μg of VAC were placed on the gel after the pH gradient was formed in the gel. Human _Hb served as reference with the known isoelectric point at pH 6.8. Before the gel was stained with Coomassie Blue, the gel was fixed for 30 minutes with 0.7 M trichloroacetic acid.

Figure 4 Analysis of the binding of VAC to negatively charged phospholipid liposomes by SDS-PAGE SDS-PAGE was performed on Laemli (Laemli, U.K., [1970] Nature 227, 680-685) on the same platelets as described in Figure 2. The analyzed samples were obtained from the binding experiments as mentioned in the explanation in Table B. The meaning of the numbers on the x-axis is as follows:

1. Materials with standard molecular weight, in which the disulfide bonds are reduced,

2. above liquid obtained after centrifuging a VAC preparation in the absence of liposomes,

3. above liquid obtained after centrifuging VAC in the presence of liposomes,

4. above liquid obtained after centrifuging VAC in the presence of liposomes and Ca ⁺⁺ and

5. The above liquid obtained after centrifuging the liposome precipitate of 4, which was resuspended in TBS containing 10 mM EDTA.

Figure 5 The effect of concentration of VAC on inhibition (%) of thrombin formation. The concentrations of VAC mentioned are the final concentrations present in the experimental systems. Thrombin formation was measured on 1μΜ prothrombin, 1OnM factor X _{a, and} 0.5M (A-A) or 5M (x) phospholopid membrane (PC / PS; 4: 1, mol / mol) in TBSA containing 10 mM CaCl ₂ . The reaction mixture was treated with the above-mentioned amounts of VAC (SA. 1300th '• inl.ieiten / mg) was stirred for 3 minutes at 37 ⁰ C without prothrombin. The addition of prothrombin to the mixture resulted in the formation of thrombin as in Example I where the rate was measured. The rate of thrombin generation in the absence of VAC was 3.3 nM Il _a / min. (A - A) or 10.9 nM Il _a / min. (· - ·).

Figure 6 Effect of phospholipid concentration on inhibition (%) of thrombin generation by VAC. Thrombin formation was measured at 1 μΜ prothrombin, 1OnM factor X ₃ , 10.7pg / ml VAC (SA 1300 units / mg) and various concentrations of phospholipid membrane (PC / PS; 4: 1, mol / mol) in TBSA. Factor X ₃ , VAC and phospholipid were stirred for 3 minutes at 37 ⁰ C in TBSA. Thrombin formation was triggered by the addition of prothrombin to the reaction mixture. The rate of thrombin formation was measured as described in Example I. Percent inhibition of thrombin generation (···) was measured for each phospholipid concentration at the appropriate rate of thrombin formation in the absence of VAC (A-A).

Figure 7 The gel filtration of a 10000xg supernatant from an umbilical artery homogenate on Sephadex G100 2 ml of a 10000xg supernatant was applied to a Sephadex G-100 column (1.5 x 80 cm) pre-equilibrated with TBS. The column was eluted with TBS. Aliquots of the fractions obtained were tested in the MPTT. Certain fractions (□) show procoagulant activity and initiate coagulation in the MPTT without the addition of HTP, Factor X _a or thrombin. Other fractions (□) prolong the clotting time in the MPTT when using HTP as a trigger of coagulation.

These fractions were collected and further fractionated.

Figure 8 Chromatography of the anticoagulant on DEAE-Sephacel (A) and Sephadex G-75 (B) The pool of the Sephadex G-100 column containing the anticoagulant was placed on DEAE-Sephacel. Elution was performed with 200 ml of a linear gradient of 50mM-30molM NaCl (- (fractions (4ml) were collected on A ₂ so

() and anticoagulant activity in MPTT using HTP (final concentration 95 pg protein / ml) as a starter for the

Coagulation (·) checked. The fractions with anticoagulant activity were collected, concentrated, and then on

Sephadex G-75 (B) brought. 2ml fractions were collected and checked for A ₂ so () and anticoagulant activity (·). V ₀

identifies the void volume of the column

 FIG. 9 Dose dependency of the anticoagulant in the MPTT

Varying amounts of anticoagulant were added to the MPTT. Coagulation was triggered by HTP (final concentration 95 μg protein / ml). The control clotting time was 65 seconds

 Figure 10 Gel electrophoresis of various fractions of the G-75 eluate

A certain amount of different fractions of the G-75 eluate were subjected to gel electrophoresis as described. The gels were prepared as described by Merril et al. (12), stained with silver (12). Column 1: reduced low molecular standards; Columns 2-6: unreduced aliquots of G-75 fractions numbers 35, 39, 41, 43, and 50. Figure 11 Effect of proteolytic enzymes on anticoagulant activity

The anticoagulant was incubated at 37 ° C with protease type I (final concentration 0.11 units / ml). Trypsin (A, final concentration 88 BAEE units / ml) and without proteolytic enzymes (·) incubated. At indicated times, 5μΙ, containing 6μg of anticoagulant protein, were removed from the reaction mixture and added to the MPTT. The coagulation was triggered with HTP (final concentration 18 protein μg / ml). Control clotting time was 110s The indicated units for the proteolytic enzymes are derived from the manufacturer's instructions

Figure 12 Effect of Vascular anticoagulant on clotting time, which was in the M (P) TT either by HTP, factor X _a or thrombin induced

The concentration of the coagulation initiators (HTP 18 μg protein / ml 1.5 nM factor X ₃ or 0.4 nM thrombin) were chosen so that the control clotting time was about 110 s (open bars). Using Factor X _a , phospholipid vesicles (final concentration 10μΜ) composed of O ₂ Gro-P-Ser / Ole ₂ Gro-P-Cho (20:80 molar ratio) were added to the reaction mixture. Coagulation times determined by the indicated agents in the presence of 3.5 μg anticoagulant protein are shown as shaded bars

FIG. 13 Effect of the anticoagulant on prothrombin activation by (X _a , V ₃ , phospholipid, Ca ²⁺ ), (X _a , phospholipid, Ca ²⁺ ) and (X ₃ , Ca ²⁺ )

The reaction mixtures contained: (A) 1 μM prothrombin, 0.3 nM X ₃ , 0.6 nM V ₃ , 0.5 μM phospholipid and 10 mM CaCl ₂ with 12 g / ml anticoagulant (EJ), 4.8 μg / ml anticoagulant ( A) and no anticoagulant ·). (B) 1μΜ prothrombin, 1OnM X ₃ , 0.5μΜ phospholipid and 1mM CaCI ₂ with 2 ^ g / ml anticoagulant (3), 0.48μg / ml anticoagulant (A) and no anticoagulant (·). (C) 1 μM prothrombin, 75 nM X ₃ and 10 mM CaCl ₂ with 120 μg / ml anticoagulant (A) and no anticoagulant (×). Samples were taken at the indicated times and the thrombin determined as described.

FIG. 14 Immunoblots

The blots were obtained as described above.

Column 1: Protein fraction with VAC activity isolated from bovine aorta.

Column 2: Protein fraction with VAC activity isolated from bovine aorta.

Column 3: Protein fraction with VAC activity isolated from bovine lung.

Column 4: Protein fraction with VAC activity isolated from human umbilical artery.

Column 5: Protein fraction with VAC activity isolated from rat aorta.

Column 6: Protein fraction with VAC activity isolated from equine aorta.

Figure 15 The gel electrophoresis (A) and anti-coagulant activity (B) of the different fractions of the eluate from G-75. Certain amounts of the different fractions of the eluate from G-75 were subjected to gel electrophoresis as described in the foregoing. The bands were stained by silver according to the method of Merril C.R. Goldman D., & Van Keuren M.L. (1982) Electrophoresis 3.17-23. Electrophoresis lane 1: low molecular weight standard material; Electrophoresis lanes 2-6: unreduced equal amounts of G-75 fractions, with increasing volumes of elution. Specific amounts of G-75 fractions analyzed by gel electrophoresis were tested in the MPTT (see description above) using HTP to induce clotting. The control clotting time is shown by the open bar. The numbers under the hatched bars correspond to the numbers of the electrophoretic sheets in Fig. 15 A.

Figure 16 Heat Inactivation of the Vascular Anticoagulant The anticoagulant was incubated at 56 ° C, and after the various incubation times, a 5μΙ sample containing 3.6μ of protein was removed, immediately cooled with ice and then submerged in the MPTT Use of HPT checked as coagulation trigger. The clotting time of the control sample was 110 seconds.

Claims (4)

Invention claim: 1. A process for the preparation of anticoagulant proteins, characterized in that the blood vessel walls, highly vascularized tissue or endothelial cell cultures a) are homogenised and centrifuged by differential means and the above liquid is exposed in any order to one or more of the following purification treatments: b) precipitating with salt c) affinity chromatography d) ion exchange chromatography e) Chromatography over a molecular sieve, wherein the products obtained according to steps b), c), d) and e) can, if desired, be dialyzed. 2. The method according to item 1, characterized in that a further purification by means of a Immunoadsorptionsschromatographie is performed. 3. The method according to item 1 or 2, characterized in that the proteins are purified by means of Phoepholipid Vesikin. - 4. Method according to any one of items 1-3, characterized in that for the precipitation of Sufeb) ammonium sulfate, for the chromatography of stage c) hydroxyapatite, for the chromatography of stage d) DEAE Sephacel and for the chromatography of stage e) SephadexG -100 or G-75 is used. For this 14 pages drawings