JPS5910522A - Protein inhibiting blood platelets from adhering - Google Patents

Abstract

NEW MATERIAL:A protein inhibiting blood platelets from adhering (occurring in membranes of blood platelets; a protein of about 300,000 molecular weight, consisting of 4 subunits of about 75,000 molecular weight; the 4 subunits are connected through disulfide bonds; showing adhesion to collagen of blood platelets). USE:A preventive or remedy for thromboses: it has an action of preventing blood platelets from adhering to collagen in warm-blooded animals including men. PREPARATION:Washed blood platelets or a fraction of blood platelet membranes are suspended in an aqueous solvent such as tris-acid citrate dextorose buffer solution, solubilized with a nonionic surfactant such as isooctylphenyl polyethyoxyalcohol and adsorbed on collagen-Sepharose. The elution is effected using an aqueous solution containing 3-5M urea at 7.35pH to give the objective protein inhibiting blood platelets from adhering.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel platelet adhesion-inhibiting protein that has the ability to inhibit collagen adhesion of platelets, which is one of the triggers of unexploded platelet thrombosis.

Platelets have multiple functions to prevent blood from flowing out of the body due to destruction and damage to blood vessels. That is, it platelets adhere to collagen exposed due to damage to the endothelial cells of the blood vessel wall, and ADP is released, causing platelet aggregation, and the aggregated platelets further regress to form a thrombus. 7
Due to this series of platelet reactions and company policy, a series of plasma proteins present in the blood that are involved in blood coagulation are activated, and eventually the fifturi 7 is converted into fibrin, which further polymerizes. A bath-free polymer with a network structure is formed by this process. Blood vessel damage results in the formation of a strong thrombus through the cooperative action of these two systems, achieving the goal of 1F blood.

By the way, various types of thrombosis, which currently account for the highest mortality rate, are caused by the adhesion and aggregation of platelets on the inner walls of blood vessels for some reason, and further involvement of plasma proteins involved in blood coagulation, resulting in the formation of blood clots within the blood vessels. The phenomenon that triggers the formation of thrombus is the interaction between platelets and the inner wall of the blood vessel, and if a defect occurs in either of them, the platelets will stick to the inner wall of the blood vessel. Thrombus formation when platelets are stationary is assumed to be due to damage to the blood vessel wall, and collagen under the endothelium of the blood vessel wall plays an important role in adhesion.

In addition to mechanical injury, damage to blood vessel walls is due to hemodynamic factors, #4
11 Microorganisms such as M, bacterial endotoxins, viruses, antigens.
It is said to be induced by high levels of metabolites such as antibody complexes and homocystine.

Therefore, if a substance exists that has the ability to inhibit the interaction between Kolaken and platelets, it could potentially be used to prevent thrombus formation.

From this point of view, the "unexploded" person prevents the interaction of Kolaken with platelets: in order to obtain a substance that has the ability,
After repeated research, they learned that such a substance existed in platelet membranes, and succeeded in isolating and purifying this substance. This was named platelet adhesion inhibiting protein (hereinafter abbreviated as F1 inhibiting protein).

Jii relates to this blocking protein, and the properties of the blocking protein are as follows.

(1) Exists in platelet membranes.

(2) Protein v1 with a molecular weight of approximately a o o, o o o
It is.

(3) It is composed of four Zaku units with approximately 75,000 molecular ends.

(4) The above four subunits are linked by disulfide bonds.

(5) There are no special characteristics in the composition of the amino acids that make up the protein.

(Shows adhesion to collagen.

Incidentally, in addition to blocking proteins, the platelet membrane contains fibronectin [P], a substance that exhibits adhesiveness to collagen.
ro, Nat, Acad, USA 75.

5864-5868 (1978)] is known to exist. By the way, this fibronectin has a molecular weight of about 4
60,000, and the molecular weight of the subunit is approximately 220,000, making it a different substance from inhibitory proteins.

The blocking protein can be obtained, for example, as follows.

Washed platelets, platelet membrane fractions, etc. are treated with an aqueous solvent (for example, Tris-Acid C1trate I) exto
rose buffer) and suspended in a non-ionic field [1i + activator, for example, at a final concentration of 05 to 2.0% inoctylphenyl polyethoxyalcohol? Trito
N
Ass, L. F. et al.: J. Lab.

CI in, Med, 8U, 525-584 (
1976)) and an aqueous solution containing 3 to 5 M urea,
For example, Tri,s Ac1d C1t containing 4M urea
It is produced by elution and isolation with raLe Dextorose buffer, pH 7.85.

The Kb-blocking protein of the present invention has the effect of inhibiting the adhesion of platelets to collagen in human warm blood cells, and can be used, for example, as a prophylactic or therapeutic agent for thrombosis.

Example 1) Preparation of washed platelets Ac1d C1trate Dextrose (ΔCD
) was added to the sampled cow] [From the 11th solution, the platelet membrane fraction (PRP) was obtained by repeating centrifugation.
'Prepare. Centrifugation is performed in steps 1 to 404 below.

1st step: 8000 x f, ] (perform centrifugation for 1 minute.

This brings plasma to the soil layer, red blood cells to the lower layer, and platelets and white blood cells in between. Remove this middle layer with a dropper and use it for the next general layer.

Second step: Perform centrifugation at 1500X, jp, 5 minutes.

As a result, separation is performed in the same manner as in the first stage, but the proportion of platelets and white blood cells in the middle layer increases.

This layer is uniformly sampled.

8th step: Centrifuge at 200-400Xf for 5 minutes.

In this case, 1. The platelets do not sink, but the red blood cells and white blood cells sink. Collect platelet multifaceted W (PRP) cut into L layer. The centrifugation speed is appropriately selected between 200 and 4 (1 nXf).

Fourth step: The PRP collected in the previous step is centrifuged under the same conditions as the rttt crotch to sediment red blood cells remaining in the PRP.

In this way, the obtained PRP was 1500xf.

Centrifuge for 5 minutes to precipitate and pellet platelets.

Throw away the remaining plasma at the top - use 'l"ris instead of C1
Add ACD buffer and float again. This operation is repeated three times to prepare a washed platelet suspension.

2) Platelet adhesion inhibitory protein IIF washed surface platelet suspension (109-1 (1” cell, s/’45m)
l! 'I'ris-ACI) buffer)
bl2 10% i'ritnnX-100 containing T
Add ris-ACI) buf (er 5 me), stir with a Keyaki stirrer, and leave at room temperature for about 30 minutes. Centrifuge at 50°C for 19 minutes, remove the unsaturated fraction, and add 05% Tri.
ton X-1003qTris-ACI) b
Equilibrate with buffer and apply to collagen-Sepharose.

Corakun Sepharose V: J, ltl bu f
After washing with fer, further Tris-ACI) b
Clean the era name with uffer.

For the elution of protein 11, the buffer was mixed with 4M urea.
'ris -ACr) buf fer (pH7,
85> 1. Performed by Rukokichi. This fraction is 'I
' After dialysis into ris-ACD buffer, @
Store at Mt (-80°C).

Ka<Lf? The molecular level of the blocked blocking protein was measured as follows: (i.e., Sl) S-polyacrylamide gel was used as a support and the molecules were swabbed in the open air. (Gives a medium band to r71m,
After adding 1 and converting this fresh white 1 to ()181β-merecaptoethanol and τ'' return 1-shi, bl L < S
IJ S-Polyacrylic Ami I/Kuru Model γS4i
11, a single bunt 1. λta.

Collagen isolated from a cow's arm was chemically combined with Sepharose 4B using glomerobromide to prepare a -V collagen-defectase complex. Fill this complex with nine columns 2. Q d with various concentrations of 1 (old 1 [i white matter (3
, 0 to 150μ2) to form a bonded state with corakun. Next, add the vanguard platelet suspension (7,8Xl) to the thread.
O pieces) are added, and the small plate or block that originally combines with Koraken is added.
We observed to what extent the presence of Lichiro f1 inhibited the tenacity of the radish. As a result, -C was completely inhibited by the 25 μV blocking can haze, and by 50% in the presence of 6/l F.
Inhibition is observed.

As a result of the above, Irihan's attack stopped the bleeding and prevented him from dramatically blocking Saka's attack on Korakun. The Kolaken exposed due to damage to the endothelium of the blood vessel wall is different from the conditions in the above experiment, and it seems that only a small amount of Kolaken is exposed. It is expected that the effect will be sufficient to suppress this.

Claims (2)

[Claims] (1) Existing in the platelet membrane, it is composed of four subunits with a molecular size of approximately 75,000, and the four subunits are disulfide-bonded proteins with a molecular size of approximately 300,000, which tends to adhere to collagen. Platelet adhesion inhibitory protein. (2) Platelets are solubilized with a nonionic surfactant such as inoctylphenyl polyethoxyalcohol, and the eluate is collected using Coracun Sepharose? 2. The protein according to claim 1, which can be processed by contacting with chromatography and eluting with an aqueous solution containing 8 to 5 M urea.