CN101172997B - Oligopeptide or modified substance/derivative thereof and application thereof - Google Patents

Abstract

The present invention provides an oligopeptide RGT or a modification/derivative thereof capable of affecting platelet function, and the oligopeptide RGT or a modification/derivative thereof is useful in the prevention and/or treatment of arterial thrombotic diseases and/or other Application in diseases related to outside-in signal transduction mediated by protein β3. Platelets treated with oligopeptide RGT or its modified/derivatives have decreased functions related to integrin outside-in signal transduction, while inside-out signal transduction is basically not affected; prevent the formation of platelet irreversible aggregation, While reducing platelet activity and preventing stable thrombus formation, it can still retain part of the adhesion and aggregation capabilities of platelets, thereby inhibiting thrombus formation and maintaining part of the normal hemostatic function.

Description

An oligopeptide or its modification/derivative and their applications

technical field

The present invention belongs to the technical field of cell bioengineering, in particular, it relates to an oligopeptide or its modification/derivative, more specifically, it relates to an oligopeptide RGT or its modification/derivative capable of affecting normal platelet function of the human body. Derivatives and their applications.

Background technique

Integrin is a membrane surface adhesion molecule that mediates cell-cell, cell-extracellular matrix adhesion. At the same time, it is also involved in a series of important physiological and pathological processes such as cell signal transduction, cell extension and movement, cell growth, differentiation and apoptosis, inflammation, thrombus formation, tumor metastasis and wound healing.

Integrin β3 can form two forms of complexes with αIIb and αv subunits, namely αIIb/β3 and αv/β3. The former is mainly expressed in megakaryocytes and platelets, while the latter is widely distributed in the body, such as endothelial cells, smooth muscle cells, etc. .

Integrin αIIb/β3 is the main receptor of platelet membrane, not only can participate in platelet adhesion, aggregation, etc., but also mediates platelet signal transduction, so it plays a key role in maintaining the normal function of platelets.

During the process of platelet activation to form a thrombus, platelet agonists (ADP or thrombin, etc.) bind to the corresponding receptors on the platelet membrane, resulting in a change in the configuration of the αIIb/β3 complex, making it have a fibrinogen receptor Activity, promote platelet aggregation and adhesion reaction, this process is inside-out signal transduction. Subsequently, the conformationally changed αIIb/β3 complex causes a series of intracellular biochemical reactions after binding the ligand, such as the increase of calcium ion level, the change of membrane glycoprotein molecular conformation, protein phosphorylation, cytoskeleton Protein recombination, etc., and lead to a series of reactions such as platelet adhesion and stretching, second-phase aggregation, release, and clot retraction. This process is called outside-in signal transmission. The final result is that platelets aggregate with each other and form a stable thrombus, thereby completing the physiological and pathological process of hemostasis and thrombus formation.

Inside-out signal transduction and outside-in signal transduction are two interrelated but distinct signaling pathways. Its signal transduction is mainly realized through the interaction between different molecules (such as talin and Src, etc.) and the intracellular segment of β3 directly or indirectly. For example, during the transduction process of inside-out signal, talin replaces the tail of αIIb subunit and binds to H ⁷²² -K ⁷³⁸ of integrin β3 subunit, thereby activating integrin αIIb/β3 (Ulmer TS, Calderwood DA, Ginsberg MH, et al.Biochemistry, 42(27), 8307-8312, 2003; Garcia-Alvatez B, de Pereda JM, Calderwood DA, et al.Mol Cell, 11(1), 49-58, 2003); signaling protein β3- Endonexin acts selectively on the tail of β3 (N ⁷⁵⁶ -Y ⁷⁵⁹ ), increasing the activity of integrin αIIb/β3 binding to ligand. In outside-in signal transduction, ^{Y 747} -T ^{762 of} β3 subunit regulates intracellular signal transduction after integrin αIIb/β3 binds to ligand; outside-in signal transduction (Liu J, Jackson CW, Gruppo RA, et al. Blood, 105(11), 4345-4352, 2005). In addition, the phosphorylation modification of β3 subunit also has an important regulatory effect on signal transduction (Law DA, Deguzman FR, Heiser P, et al. Nature, 401(6755), 808-811, 1999; Phillips DR, Prasad KS, Manganello J, et al.Curr.Opin.Cell Biol., 13(5), 546-554, 2001; Pasquet JM, Noury M, NurdenAT.Thromb Haemost, 88(1), 115-122, 2002), such as β3 Phosphorylation of ICY domains Y ⁷⁴⁷ and Y ⁷⁵⁹ in the intracellular segment of the subunit is required for stable platelet aggregation (Law DA, Deguzman FR, Heiser P, et al. Nature, 401(6755), 808-811, 1999). The phosphorylation of T ⁷⁵³ and S ⁷⁵² of β3 is involved in outside-in or bidirectional signal transduction by affecting the interaction between Shc, myosin and β3 tail (Law DA, Nanizzi-Alaimo L, Phillips DR.J.Biol.Chem ., 271(18), 10811-10815, 1996; Lerea KM, Cordero KP, Sakariassen KS, et al.J.Biol.Chem.274(4), 1914-1919, 1999; Chen YP, Diaffar I, Pidard D , et al.Porc.Natl Acad Sci.USA, 89(21), 10169-10173, 1992; Chen YP, O'Toole TE, Ylanne J, et al.Blood, 84(6), 1857-1865, 1994) . The selective cleavage effect of calpain on β3 intracellular sequences T ⁷⁴⁷ and Y ⁷⁵⁹ leads to different signal transduction pathways, which further proves the role of the C-terminal sequence T ⁷⁵⁵ -T ⁷⁶² of the β3 subunit in bidirectional signal transduction (Xi X, Fleyaris P, Stojanovic A, et al.J.Biol.Chem., 281(40), 29426-29430, 2006; Xi X, Bodnar RJ, Li Z, et al.J.Cell Biol., 162(2 ), 329-339, 2003). In addition, Src kinase plays a key role in outside-in signal transduction, and the sequence where the β3 subunit interacts with it is located at R ⁷⁶⁰ -T ⁷⁶² (Arias-Salgado EG, Lizano S, Shattil SJ, et al.J.Biol. Chem., 280(33), 29699-29707, 2005).

In recent years, many studies on outside-to-in signal transduction have found that some molecules or links in the signal chain can selectively affect outside-to-in signal transduction, but have little effect on inside-out signal transduction. In the process of platelet activation, the one-phase aggregation of platelets and the formation of primary thrombus are not affected, while the formation of stable thrombus is reduced to a certain extent. For example, compared with normal mice, diYF mice with both mutations of β3Y ⁷⁴⁷ and Y ⁷⁵⁹ had normal initial hemostatic function, but had a more obvious rebleeding tendency (LawDA, Deguzman FR, Heiser P, et al. Nature, 401(6755), 808-811, 1999); in the Gas6-Rs deficient mouse model, pathological thrombus did not occur in the deficient mice, but the bleeding time representing normal hemostatic function did not change significantly (Angelillo-Scherrer A, Burnier L, Flores N, et al . J Clin. Invest 115(2), 237-246, 2005). Therefore, the unidirectional effect of some factors on the outward-inward signal transduction can inhibit the formation of pathological thrombus without causing bleeding symptoms. This may be a new way to prevent and treat thrombotic diseases safely and effectively.

Contents of the invention

One of the objectives of the present invention is to provide an oligopeptide or its modification/derivative that can affect the normal platelet function of the human body.

Another object of the present invention is to provide applications of the oligopeptide or its modification/derivative.

The amino acid sequence of the oligopeptide capable of affecting the normal platelet function of human body is RGT.

According to the present invention, the modification of the oligopeptide is a product obtained by modifying the N-terminus of the oligopeptide RGT, and the modification includes chemical modification, such as myristylation modification, hexadecanoylation modification, etc., and Polypeptide modification with membrane penetration, such as HIV-TAT modification, etc.

According to the present invention, the oligopeptide derivatives refer to a class of compounds whose spatial structure is very close to that of RGT. Due to the similarity of spatial structure, they can play the same role as RGT in vivo.

Based on previous cell models, the inventors found that the intracellular C-terminal sequence ⁷⁶⁰ RGT ⁷⁶² of integrin β3 can effectively affect the signal transduction of integrin αIIb/β3 while hardly affecting the inside-out signal transduction. Achieving inhibition of outside-in signal transduction.

Therefore, the oligopeptide of the present invention or its modification/derivative can be used to selectively block the outside-in signal transduction mediated by integrin β3 and affect the activity of the corresponding integrin receptor, and then can be used to prepare prophylactic And/or drugs for the treatment of diseases related to integrin receptor activity corresponding to integrin β3-mediated outside-in signal transduction, these diseases include: arterial thrombotic diseases such as myocardial infarction, cerebral infarction, and malignant tumor metastasis, etc. .

Integrin αIIb/β3 is the main receptor on the surface of platelet membrane. It participates in the adhesion and aggregation of platelets through bidirectional signal transduction from inside to outside and outside to inside. pathway, and thus are effective specific targets for the regulation of thrombus formation. Different from antagonists that block the binding site of integrin αIIb/β3 extracellular ligand, the present invention focuses on the molecular basis of the signal transduction regulation mechanism during platelet activation. The oligopeptide RGT of the present invention specifically Inhibiting the thrombus formation ability of platelets while basically retaining its activity in normal hemostasis can provide a better solution different from the traditional model for the prevention and treatment of arterial thrombosis diseases or other diseases related to integrin αIIb/β3 function plan.

Observing the role of integrin αIIb/β3 in signal transduction usually adopts the method of cell model, but it is difficult for cell model to completely simulate the real platelet. However, the oligopeptide RGT of the present invention takes normal human platelets as the action object to realize the influence on signal transduction mediated by integrin αIIb/β3 and platelet function. In order to allow the oligopeptide to enter the complete platelet and play a role, the method of myristyl acylation modification is used to make the oligopeptide membrane-permeable, which is different from the special treatment of platelets that changes the permeability of the platelet membrane and is beneficial to the oligopeptide. The entered method avoids the possible influence of the above-mentioned special treatment on platelet function, and obtains the effect of oligopeptide on normal human platelet.

The shortest synthetic oligopeptide previously reported to have an effect on signal transduction of platelets (which retain most of their functions after a treatment that alters membrane permeability) is an octapeptide corresponding to sequence 755-762 of integrin β3. This effect is manifested as a bidirectional blockade of platelet signal transduction, that is, the ability of platelet thrombosis and stabilization is affected, which is basically consistent with the effect of drugs that affect the integrin αIIb/β3 extracellular ligand binding site. The effect of the oligopeptide RGT of the present invention on human platelets is shown to prevent the integrin αIIb/β3 outside-in signal transduction, while basically not affecting the inside-out signal transduction, which is the only one that can selectively affect human platelets so far. Synthetic oligopeptides for outside-in signal transduction and its corresponding functions.

The outside-in signal transduction of integrin αIIb/β3 can be selectively blocked by mutating the tyrosine of integrin β3 or knocking out some downstream signaling molecules such as Gas-6 in the transgenic mouse model, but these mechanisms are not It cannot be observed on normal human platelets, so the oligopeptide RGT of the present invention is currently the only method known to selectively block the outside-to-in signal transduction of normal human platelet integrin αIIb/β3. In addition, molecules known to affect integrin αIIb/β3 outside-in signal transduction are often involved in the regulation of other important signal transduction, so it is difficult to rule out the possibility that they affect other functions of platelets. The oligopeptide RGT of the present invention acts on the final pathway of platelet activation, and will not affect other functions except for the function of integrin αIIb/β3.

Description of drawings

Figure 1A is a schematic diagram of the results of platelets incubated with FITC-labeled peptide (250 μM) for 30 minutes and analyzed by flow cytometry, where the blackened peak represents FITC-labeled myristanoylated RGT peptide (FITC-Myr-RGT )-treated platelets; blank peaks represent FITC-labeled RGT peptide (FITC-RGT)-treated platelets.

Figure 1B is a schematic diagram showing the results of stretching of platelets treated with FITC-Myr-RGT and FITC-RGT on immobilized fibrinogen.

Figure 2 shows the effect of Myr-RGT on platelet stretch function, where: A represents platelets incubated with DMSO; B represents platelets incubated with myristic acid and RGT peptide (250 μM); C represents platelets incubated with myristylated GRT peptide (Myr-GRT, 250 μM) incubation; D, E and F represent platelets co-incubated with myristanoylated RGT peptide (Myr-RGT) at concentrations of 62.5 μM, 125 μM and 250 μM, respectively.

Figure 3 shows the quantitative determination of the effect of Myr-RGT peptide on platelet adhesion to solid-phase fibrinogen, where: A, B, and C represent the conditions of the peptide at concentrations of 62.5 μM, 125 μM, and 250 μM, respectively.

Figure 4 shows the effect of the Myr-RGT peptide on the retraction of the fibrin clot, where: A, B, and C represent the conditions of the peptide at concentrations of 62.5 μM, 125 μM, and 250 μM, respectively, and the histogram indicates the volume of the clot The percentage rate of the volume of the reaction system.

Figure 5 shows the effect of Myr-RGT peptide on platelet aggregation induced by ADP (1 μM) or ristocetin (ristocetin, 1.5mg/ml), where: A represents platelets incubated with DMSO, B represents platelets incubated with 10 Co-incubation with tetradecanoic acid and RGT peptide (250 μM), C represents platelets co-incubated with Myr-GRT peptide (250 μM), D, E, F represent platelets co-incubated with 62.5 μM, 125 μM, 250 μM Myr-RGT peptides respectively .

Figure 6 shows the effect of Myr-RGT peptide on the binding function of platelet soluble fibrinogen, wherein, A, B, C, D, E represent respectively platelet and DMSO, RGDS (1mM), myristic acid and RGT peptide (250μM ), Myr-GRT peptide (250 μM) and Myr-RGT peptide (250 μM) incubation results, F is platelets treated with solution without ADP, MnIX represents the average fluorescence intensity.

Figure 7 shows the effect of Myr-RGT peptide on the expression of CD62P on the platelet surface, where: A represents resting platelets without thrombin, B represents platelets incubated only with DMSO, and C represents platelets with myristic acid and RGT Peptide (250 μM) co-incubation, D represents platelets incubated with Myr-GRT peptide (250 μM), E represents platelets incubated with Myr-RGT peptide (250 μM), MnIX represents the average fluorescence intensity.

Figure 8 shows that after platelets were treated with DMSO, myristic acid and RGT, Myr-GRT peptide, and Myr-RGT peptide, the aggregation of platelets was induced by adding thrombin, and then the platelets were lysed with SDS-PAGE sample buffer, and the integrated The results of western blotting of monoclonal antibody (SZ21) to the extracellular domain of β3, anti-β-actin monoclonal antibody, and anti-β3 phosphorylated monoclonal antibody (anti-integrin β3pY747 or anti-pY759).

Figure 9 shows that the platelet lysate was co-immunoprecipitated with the monoclonal antibody (SZ21) against the extracellular segment of β3, or non-specific mouse IgG (as a control), and the co-precipitated platelet lysate was treated with 200 μM (A), 300 μM ( B) RGT peptide or 300 μM (B) GRT peptide was treated, and finally the immunoprecipitate was detected by Western blotting with anti-integrin αIIb/β3 monoclonal antibody (SZ22) or anti-c-Src monoclonal antibody (327) Binding of Src to αIIb/β3 in the precipitate.

Detailed ways

According to the present invention, the so-called "modification of RGT" means that in order to make the oligopeptide RGT have cell membrane penetration, appropriate modifiers, such as chemical modifiers myristylation, hexadecanoylation and membrane penetration The leading peptide modification HIV-TAT, etc., is the product obtained after modifying the oligopeptide RGT.

According to the present invention, the so-called "derivative of RGT" refers to such a compound whose spatial structure is very close to the specific spatial structure of RGT. Because it has a specific spatial structure close to that of RGT, it can perform the same function as RGT in vivo, which is obvious to those skilled in the art.

In the present invention, the oligopeptide RGT is first synthesized, and its N-terminus is modified by hydrophobic myristylation to make it have cell membrane penetration, and then the platelets are treated with the membrane-penetrating oligopeptide (Myr-RGT) , to observe its aggregation, binding to free fibrinogen, adhesion and stretching to solid-phase fibrinogen, fibrin clot retraction, CD62P expression and other functions, as well as the interaction between platelet integrin β3 and Src and Y ⁷⁴⁷ and Y ⁷⁵⁹ The effect of phosphorylation. The results show that the oligopeptide RGT of the present invention or its modification can inhibit the outside-in signal transduction mediated by integrin αIIb/β3 in the process of platelet activation, manifested as two-phase aggregation of platelets and adhesion to solid-phase fibrinogen And stretching, fibrin clot retraction, CD62P expression and other typical indicators decreased; but there was basically no effect on the signal transduction from inside to outside, showing that the ability of platelet one-phase aggregation and binding free fibrinogen remained unchanged. In addition, the oligopeptide RGT regulates the above-mentioned platelet function by affecting the interaction between integrin β3 and Src and inhibiting the classic outside-in signal transduction molecular mechanism such as Y ⁷⁴⁷ and Y ⁷⁵⁹ phosphorylation of integrin β3.

Therefore, the oligopeptide RGT of the present invention or its modification can be used to selectively block the outside-in signal transduction mediated by integrin β3 and affect the activity of the corresponding integrin receptor, and then can be used for the preparation of preventive and/or Or a drug for treating diseases related to integrin receptor activity corresponding to outside-in signal transduction mediated by integrin β3, these diseases include: arterial thrombosis diseases, such as myocardial infarction, cerebral infarction, and malignant tumor metastasis.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that the following examples are only used to illustrate the present invention but not to limit the scope of the present invention.

The concentrations involved in the following examples, unless otherwise specified, are the final concentrations of solutes in the reaction system.

Embodiment 1 , oligopeptide synthesis

Myr-RGT was artificially synthesized with myristyl acylation at the N-terminus to obtain cell membrane permeability. At the same time, artificially synthesized RGT oligopeptide and N-terminal myristylated GRT (Myr-GRT) were used as controls.

In view of the fact that it is very easy to synthesize the above oligopeptides and/or their modifications according to the prior art, and it is also easy to find relevant manufacturers to synthesize them, so the specific synthesis methods will not be described in detail here.

It is easy to understand that although this example only lists the oligopeptide RGT modified by myristylation, it can be modified by other similar modification methods, such as hexadecanoylation modification of the same chemical modification, and membrane penetration The modification of the polypeptide (such as HIV-TAT modification), etc., can also achieve the purpose of obtaining cell membrane permeability, which is obvious to those skilled in the art.

Example 2. Intracellular localization of membrane-penetrating oligopeptides

Take 10ml of healthy human venous blood (ACD 1.6ml anticoagulant), centrifuge at 90×g for 20min to obtain platelet-rich plasma (PRP), centrifuge PRP at 1000×g for 15min, and discard the supernatant. Suspend and wash the precipitated platelets with CGS washing solution (120mM sodium chloride, 13mM sodium citrate, 30mM glucose, pH 6.5), and then use Tyrode's buffer solution (137mmol/L sodium chloride, 2mmol/L potassium chloride, 12mmol/L sodium bicarbonate, 0.3mmol/L sodium dihydrogen phosphate, 2mmol/L calcium chloride, 1mmol/L magnesium chloride, 5.5mmol/L glucose, 5mmol/L hydroxyethylpiperazineethanesulfonic acid, pH 7.4) suspension Platelets, this is the washed platelet suspension, and adjust the platelet suspension to a density of 1.5×10 ⁷ /ml, and let it stand at room temperature for 1 hour.

The intracellular localization of the membrane-penetrating oligopeptide was then observed using the following method:

1) Incubate FITC-labeled Myr-RGT (FITC-Myr-RGT, 250 μM) and FITC-labeled RGT (FITC-RGT, 250 μM) with the resting platelet suspension for 30 min, wash with PBS, and observe with flow cytometry Fluorescence intensity, the results are shown in Figure 1A.

2) Add the cells to a 96-well plate coated with fibrinogen (20 μg/ml), incubate at 37°C for 30 min, wash with PBS and fix with 4% paraformaldehyde, use differential interference contrast and fluorescence observation with laser confocal microscope, The results are shown in Figure 1B.

The results in Figure 1A showed that the average fluorescence intensity of platelets treated with FITC-Myr-RGT peptide was higher than that of platelets treated with FITC-RGT peptide; the results in Figure 1B showed that FITC-Myr-RGT peptide penetrated the cell membrane and entered platelets, while FITC- RGT peptide has no such effect. It can be seen that myristylated oligopeptide RGT has the ability to penetrate the cell membrane and enter platelets.

Example 3 , platelet adhesion and stretching

Take Myr-RGT (62.5 μM, 125 μM, 250 μM), DMSO, myristic acid and RGT (250 μM), myristylated GRT peptide (Myr-GRT, 250 μM), at 37 ° C, respectively, with the washed platelet suspension , and incubated for 30min.

Add 100 μl/well of the incubated suspension to the surface of a glass slide coated with fibrinogen (20 μg/ml), and incubate at 37° C. for 1 h. After washing with PBS and fixing with 4% paraformaldehyde, the cells were blocked with 5% skimmed milk powder for 30 min.

Subsequently, it was incubated with anti-integrin αIIb/β3 monoclonal antibody (SZ21, ab19778, Abcam) and FITC goat anti-mouse IgG (15 μg/ml, 66855 Jackson ImmunoResearch Laboratories) at 37°C for 1 h, and then washed 3 times with PBS (10 min each time). ), after dripping anti-fluorescence quenching mounting solution, cover with a clean coverslip to seal, and store in a dark box at 4°C. The fluorescence coloration [0] was observed with the laser confocal microscope at an emission wavelength > 505nm, and the cell outline was displayed by differential interference contrast imaging. The results are shown in Figure 2.

At the same time, the quantitative determination of adhesion was carried out. During the quantitative determination of adhesion, the suspension after incubation was added to a 96-well plate coated with fibrinogen (20 μg/ml) without fixation, and the PNPP method (PNPP p-nitrophenyl phosphate Sodium 3mg/ml, 1% sodium dodecyl sulfate, 50mM sodium acetate pH 5.0 37 ℃ after incubation for 1h, 1N NaOH to terminate the reaction) after color development, read the OD value at 405nm, and respectively take unadhered platelet wells as blank control, The number of adhered platelets was calculated, and the results are shown in Figure 3.

The results in Figure 2 and Figure 3 show that the adhesion and extension of platelets on the solid-phase fibrinogen in the Myr-RGT peptide treatment group are inhibited, and the degree of inhibition is proportional to the peptide concentration. Adhesion and stretching on fibrinogen.

Example 4. Fibrin clot retraction

The preparation of the washed platelets was the same as in Example 2, and the platelet density was adjusted to 3×10 ⁸ /ml with Tyrode's buffer. Take Myr-RGT peptide (62.5 μM, 125 μM, 250 μM), DMSO, myristic acid and RGT peptide (250 μM), Myr-GRT peptide (250 μM), and incubate with the washed platelet suspension at 37°C for 30 min.

Fibrinogen (2mg/ml) and thrombin (1U/ml) were added to the treated platelet suspension, observed and photographed every 30min to record the clot volume, and the test was terminated after 1.5h. The clot volume was calculated with NIH Image 1.67e software (NIH, USA, software website: http://rsb.info.nih.gov/nih-image/ ),

Formula: volume ratio=volume t/volume t0 (t0, t are the volumes of the clot before and after contraction), and the results are shown in Figure 4.

The results in Fig. 4 show that the fibrin clot retraction of platelets in the Myr-RGT peptide treatment group is inhibited, and the degree of inhibition is proportional to the peptide concentration. It can be seen that RGT peptide can inhibit the fibrin clot retraction of platelets.

Embodiment 5 , platelet aggregation

Take Myr-RGT peptide (62.5 μM, 125 μM, 250 μM), DMSO, myristic acid and RGT peptide (250 μM) and Myr-GRT peptide (250 μM), respectively, after incubating with PRP at room temperature for 8 min, add the inducer ristocetin (1.5mg/ml) or ADP (1μM), observe and record the aggregation curve, the results are shown in Figure 5.

The results in Figure 5 show that the primary and secondary aggregation of platelets in the DMSO, myristic acid, RGT peptide, and Myr-GRT peptide treatment groups are basically not affected, but the platelet primary aggregation in the Myr-RGT peptide treatment group is not affected. Affected, while the two-phase aggregation is inhibited to varying degrees, and the degree of inhibition is proportional to the concentration of the peptide. It can be seen that the RGT peptide can selectively inhibit the two-phase aggregation of platelets, and has basically no effect on the first-phase aggregation of platelets.

Example 6. Binding of free fibrinogen

The preparation of the washed platelets was the same as in Example 2, and the platelet density was adjusted to 3×10 ⁸ /ml with Tyrode's buffer. Take Myr-RGT peptide (250 μM), DMSO, myristic acid and RGT peptide (250 μM), Myr-GRT peptide (250 μM), RGDS peptide (1 mM), and incubate with the washed platelet suspension at 37 ° C for 30 min, add FITC Labeled fibrinogen (10 μg/ml) and inducer ADP (2 μM) were co-incubated at room temperature for 15 minutes (while resting platelets plus the same amount of PBS was used instead of ADP as a blank control), washed with PBS and observed on a flow cytometer. The fluorescence intensity was used to determine the changes in the amount of bound free fibrinogen, and the results are shown in Figure 6.

The results of flow cytometry fluorescence analysis in Figure 6 show that the soluble fibrinogen binding function of platelets in the RGDS peptide treatment group was inhibited, while the soluble fibrinogen binding function of platelets in the Myr-RGT peptide treatment group and other treatment groups was not affected , it can be seen that the RGT peptide has no effect on the inside-out signal transduction of platelets.

Embodiment 7 , CD62P expression

The preparation of the washed platelets was the same as in Example 2, and the platelet density was adjusted to 3×10 ⁸ /ml with Tyrode's buffer. Add Myr-RGT peptide (250 μM), DMSO, myristic acid and RGT peptide (250 μM), Myr-GRT peptide (250 μM) to the platelet suspension respectively, and after incubation at 37°C for 30 min, add thrombin (0.1 U/ml, The blank control is the same as above 2.5). Activated platelets were fixed with 4% paraformaldehyde and labeled with FITC-anti-CD62P antibody (1164, Immunotech) (the expression of CD62P antigen on the surface of platelets is a sign of platelet activation), and the flow cytometer detected FITC fluorescence at 515 nm to analyze the expression of CD62P. The results are shown in Figure 7.

The results of flow cytometry fluorescence analysis in Figure 7 show that the expression of CD62P in platelets in the Myr-RGT peptide treatment group is reduced after thrombin activation. It can be seen that RGT peptide can inhibit the expression of CD62P in platelets after thrombin induction, that is, inhibit the activation function of platelets .

Example 8. Detection of Tyrosine Phosphorylation of Platelet β3 Subunit by Western Blot

The preparation of the washed platelets was the same as in Example 2, and the platelet density was adjusted to 3×10 ⁸ /ml with Tyrode's buffer. Add Myr-RGT peptide (250 μM), DMSO, myristic acid and RGT peptide (250 μM), Myr-GRT peptide (250 μM) to the platelet suspension respectively, add 0.05 U/ml thrombin after incubation at 37°C for 30 min and Stir (1000 rpm, 1 min) to induce aggregation of platelets. Platelets were lysed with SDS-PAGE sample buffer (containing PMSF 1 mM, Protease Inhibitor Cocktail Set III 0.1%, EDTA 5 mM, Na ₃ VO ₄ 1 mM), and boiled at 100° C. for 10 min. Protein samples were treated with anti-integrin β3 extracellular domain monoclonal antibody (SZ21, ab19778, Abcam) and anti-actin monoclonal antibody (CloneAC-74, A5316, Sigma), anti-integrin β3 phosphorylated casein at position 747 or 759 Monoclonal antibody (anti-integrin β3pY ⁷⁴⁷ or anti-pY ⁷⁵⁹ ) (phospho Y785, ab5191, Abcam; phospho Y 773, ab5190, Abcam) was used for Western blotting, and the results are shown in FIG. 8 .

The results in Figure 8 show that after platelets treated with DMSO, myristic acid, RGT, and Myr-GRT peptide were induced by thrombin, both β3 subunits Y ⁷⁴⁷ and Y ⁷⁵⁹ were phosphorylated, while platelets treated with Myr-RGT peptide Phosphorylation of β3 subunits Y ⁷⁴⁷ and Y ⁷⁵⁹ of platelets induced by thrombin and without thrombin was not found. It can be seen that RGT peptide inhibits the phosphorylation of β3 subunit Y ⁷⁴⁷ and Y ⁷⁵⁹ during platelet activation, that is, inhibits the outside-in signal transduction mediated by β3 subunit during platelet activation.

Example 9. Detecting the binding of platelet β3 subunit and Src by co-immunoprecipitation technique

Take RIPA lysis buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.1% Protease Inhibitor Cocktail Set III, 1 mM Na ₃ VO ₄ ) The platelets were lysed and washed, and the protein in the lysate was quantified by the BCA method. Take 0.5ml of the lysate (protein content 600 μg), add 10 μg of anti-integrin β3 extracellular fragment antibody SZ21 (ab19778, Abcam) or non-specific mouse IgG (sc -2025, Santa Cruz Biotechnology) 10 μg, incubated at 4°C with rotation for 1 hour, then added 20 μl of Protein G agrose beads (25%, sc-2002, SANTA CRUZ BIOTECHNOLOGY), and incubated at 4°C with rotation for 6 hours. The immunoprecipitate was centrifuged at 3500rpm and 4°C for 3min. After the supernatant was discarded, 1ml RIPA lysis buffer was added for washing, and the washing was repeated 3 times to obtain 100 μl of immunoprecipitate. Add RGT peptide (200M or 300M), DMSO, GRT peptide (300M), incubate at 4°C with rotation for 1h, dissolve in SDS-PAGE sample buffer, and boil at 100°C for 10min. Protein samples were detected by Western blotting with anti-integrin αIIb monoclonal antibody (SZ22, ab19687, Abcam) or anti-c-Src monoclonal antibody (Clone 327, Ab 16885, Abcam), and the results are shown in Figure 9 Show.

The results in Figure 9 show that the combination of Src and β3 subunits in the DMSO and GRT peptide treatment groups is not affected, while the combination of Src and β3 subunits in the RGT peptide treatment group (200 μM or 300 μM) is reduced, showing that RGT inhibits the binding of Src and β3 Combination of subunits.

In summary, the oligopeptide RGT of the present invention or its modification/derivative can selectively block the outside-to-in signal transduction mediated by integrin β3, but basically has no effect on the inside-to-out signal transduction, therefore It can be used to prevent and/or treat diseases related to integrin receptor activity corresponding to integrin β3-mediated outside-in signal transduction, these diseases include: arterial thrombosis diseases such as myocardial infarction, cerebral infarction and malignant tumors transfer etc.

Claims (5)

1. An oligopeptide, characterized in that the amino acid sequence of the oligopeptide is RGT. 2. A modification of the oligopeptide as claimed in claim 1, wherein the modification of the oligopeptide is a product obtained after modifying the N-terminus of the oligopeptide RGT, and the modification is membrane penetration chemical modification, the membrane penetrating chemical modification is myristylation modification or hexadecanoylation modification. 3 . The modified oligopeptide according to claim 2 , wherein the membrane-penetrating chemical modification is myristylation modification. 4 . 4. The application of the oligopeptide according to claim 1, characterized in that it is used to prepare a drug that selectively blocks the outside-in signal transduction mediated by integrin β3 and affects the activity of the corresponding integrin receptor. 5. The application of the modified oligopeptide according to claim 2 or 3, characterized in that it is used to selectively block the outside-in signal transduction mediated by integrin β3 and affect the corresponding integrin receptor active drug.

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