НЕЙРОХИМИЯ, 2004, том 21, № 2, с. 129-137
ЭКСПЕРИМЕНТАЛЬНЫЕ РАБОТЫ
УДК 577.352
INTERACTIONS OF NEUROPEPTIDES WITH COAGULATION CASCADE COMPONENTS; A POTENTIAL ROLE FOR PROLINE-RICH POLYPEPTIDE IN THE METABOLISM OF FACTOR Xa/ANTITHROMBIN III COMPLEX
© 2004 M. H. Coggin1, R. M. Srapionyan2, A. A. Galoyan2, A. S. Brecher1*
1 Departments of Biological Sciences and Chemistry, Bowling Green State University, USA 2 H. Bunyatian Institute of Biochemistry, Armenian National Academy of Sciences, Yerevan, Republic of Armenia
The effect of the 15 amino-acid residue proline rich polypeptide (PRP) (AGAPEPAEPAQPGVY, 1475 Da) on the interaction in vitro between coagulation factor Xa (FXa) and the primary naturally occurring inhibitor of FXa, antithrombin III (ATIII) was studied via SDS-PAGE. PRP appears to have a biphasic affect on the FXa-ATIII complex. A scheme was proposed to address the transformations of FXaa to FXaP, and transformations of primary (1°) FXa-ATIII complexes to the secondary (2°) and tertiary (3°) complexes, based upon SDS-PAGE and Western Blot identification of protein bands and products. PRP (8 |g) profoundly promoted the transformation of FXaa to FXaP and the further transformation of 1° FXaP-ATIII complex to 3° FXaP-ATIII complex, as well as production of FXay. In contrast, 4 |g PRP inhibited transformation of FXaa to FXaP as well as that of 1° FXaa-ATIII to 3° FXaa-ATIII and of 1° FXaP-ATIII to 3° FXaP-ATIII. A pharmacological role for PRP in the molecular mechanisms of blood clotting has thus been proposed.
Key words: Proline rich polypeptide, factor Xa, antithrombin III, coagulation, neuropeptides.
INTRODUCTION
The proline rich polypeptides (PRP) were discovered by Galoyan and coworkers [1-3]. They were isolated from the bovine neurosecretory granules of neurohypophysis and their primary structures were determined. These new hypothalamic neurohormones are produced by the neurosecretory cells N. Paraventricu-laris and N. Supraopticus. PRP of varying lengths contain 10-15 amino acid residues. Their presence in blood flowing out of the brain has previously been established. The PRP utilized in the investigation reported herein is a 15 amino acid residue polypeptide (AGAPEPAEPAQPGVY; MW 1475).
Factor Xa (FXa) lies at the juncture of the intrinsic and extrinsic coagulation pathways. It has two major coagulatively active forms, FXaa and FXaP [4]. Recently it has been shown that FXaa and FXaP may have some specific functional differences [5, 6]. FXaa is the principal FXa component of the prothrombinase complex [5], while FXaP has a plasminogen binding site and has been shown to accelerate generation of plasmin [6]. The primary inhibitor of FXa is the serpin antithrombin III (ATIII). FXa and ATIII form a 1 : 1 molar complex via a covalent bond. This bond can undergo hydrolysis, producing a modified two-chain version of ATIII (ATIIIm) while allowing FXa to retain its activity [7-10]. Administration of the glycosaminoglycan hep-
*Address for correspondence: Department of Chemistry, Bowling Green State University, Bowling Green, OH. 43403; e-mail: art-brec@bgnet.bgsu.edu; fax: 419-372-9809.
arin has been shown to increase the rate of FXa/ATIII bonding several hundred fold in the presence of Ca2+ [1113], and by at least an order of magnitude alone [14], with concurrent increases of 20-50% in ATIIIM [7, 9].
This laboratory has earlier presented research indicating that low levels of unfractionated heparin promote conversion of 1° FXaP-ATIII complex to 3° FXaP-ATIII complex [15]. Further, additions of both low and moderate levels of protamine sulfate appear to inhibit FXaa-ATIII complex formation, and promote conversion of FXaa to FXaP as well as autolysis to FXay. Similarly to heparin, protamine sulfate appears to promote conversion of 1° FXaP-ATIII complex to 3° FXaP-ATIII complex [15].
PRP has been shown to prevent erythrocyte hemol-ysis, inhibit aggregation of thrombocytes, and accumulate in lymphocyte and erythrocyte membranes. With the help of monoclonal antibodies, it is possible to establish that PRP is released from the neurohypophysis to the general blood flow and is accumulated to a great amount in lymphocyte and erythrocyte membranes [16, 17]. Because of its involvement with thrombocytes, a critical look at PRP interaction with enzymes of the coagulation cascade was initiated.
In this communication, it is reported that 8 ^g PRP appears to promote both conversion of free FXaa to FXaP and conversion of 1° FXaP-ATIII to 3° FXaP-ATIII, as well as production of FXay. Both 4 and 8 ^g PRP appear to slow conversion of 1° FXaa-ATIII to 3° FXaa-ATIII complex. PRP does not promote the degradation of
200 kDa
116 kDa 97 kDa 66 kDa
31 kDa
Fig. 1. SDS-PAGE of the interaction among 1.5 |lg human FXa, 1.5 |lg human ATIII, and 8 |lg PRP. Lanes: 1 - molecular weight markers; 2 - FXa; 3 - ATIII; 4 - FXa + ATIII; 5 - [(ATIII + PRP), 30 min + FXa], 30 min; 6 - [(ATIII + PRP, 30 min) + FXa ], 30 min; 7 - [(FXa + PRP), 30 min + ATIII ], 30 min; 8 - [(FXa + PRP), 30 min + ATIII ], 30 min; 9 - (FXa + ATIII + PRP), 30 min; 10 - (FXa + ATIII + PRP), 30 min.
FXaP to FXay. PRP 4 |g appears to slow conversion of free FXaa to FXaP and conversion of 1° FXaP-ATIII to 3° FXaP-ATIII, with consequential reduction of FXay production.
MATERIALS AND METHODS
Tris, Sodium laurylsulfate, acrylamide, N,N-Meth-ylene-bis-acrylamide, 3',3'',5',5'' tetrabromophenolsul-fonephthalein, and Coomassie Brilliant Blue were purchased from Sigma Chemical Co. (St. Louis, MO). Methanol, NaCl (enzyme grade), KCl (certified ACS), glycerol (enzyme grade), HCl (certified A.C.S PLUS) and acetic acid (glacial) were purchased from Fisher Scientific (Pittsburgh, PA). Human FXa and human ATIII, were purchased from Haematologic Technologies, Inc (Essex Junction, VT). FXa and ATIII were each diluted to 0.5 mg/ml with 18.5 mM Tris / 56 mM NaCl buffer, pH 8.0 (buffer A). SDS-PAGE molecular weight standards, broad range, were purchased from Bio-Rad (Hercules, CA).
PRP was isolated from neurosecretory granules of bovine neurohypophysis [18, 19]. PRP was reconstituted variously to 0.25 mg/ml, 1 mg/ml, and 2 mg/ml with buffer A.
For FXa or ATIII control preparations, 9 |l of buffer A is added to 1.5 |g FXa or ATIII, respectively, in 3 |l of buffer A and incubated for 30 min at room temperature. In FXa / ATIII preparations, 1.5 |g FXa in 3 |l of buffer A, and 1.5 |g ATIII in 3 |l of buffer A, and 6 |l of buffer A are added at one minute intervals (for an incubation volume of 12 |l) and incubated for 30 min. ATIII (1.5 |g in 3 |l of buffer A) and PRP (8 |g in 4 |l of buffer A) mixtures are incubated for 30 min prior to addition of 1.5 |g FXa in 3 |l of buffer A and addition
of 2 |l of buffer A, which is incubated for a further 30 min. Alternatively, FXa (1.5 |g in 3 |l of buffer A) and PRP (8 |g in 4 |l of buffer A) mixtures are incubated for 30 min prior to addition of 1.5 |g ATIII in
3 |l of buffer A and addition of 2 |l of buffer A, which is incubated for a further 30 min. FXa/ATIII/PRP mixtures are prepared as follows: 1.5 |g FXa in 3 |l of buffer A, 1.5 |g ATIII in 3 |l of buffer A, 8 |g PRP in
4 |l of buffer A and 2 |l of buffer A are added at one minute intervals and incubated for 30 min. Each lane has a constant FXa:ATIII ratio, contact time of 30 minutes and contact volume of 12 |l, and is subsequently raised to a final volume of 20 |l by addition of 4 |l of buffer A and 4 |l stock sample buffer (53% v:v glycerol, 0.1% bromophenol blue, 0.2 M Tris/HCl, pH 6.8) before being boiled at 100°C for 2 minutes and loaded into the gel wells. Mixtures using 1 |g or 4 |g PRP are prepared similarly, using PRP reconstituted to 0.25 mg/ml and 1 mg/ml, respectively. Molecular weight markers are prepared as follows: 2 |l P-mercaptoethanol are added to a 0.5 ml Eppendorf tube containing 2 |l molecular weight markers and 57.6 |l molecular weight marker stock solution and boiled for 5 minutes at 100°C before 12 |l is added to the gel well.
Electrophoresis: A modified Laemli discontinuous gel method for SDS-PAGE was employed [20]. Elec-trophoresis was carried out using the Bio-Rad MiniProtein 3 system (Bio-Rad, Hercules, CA) at R.T. Gels were then electrophoresed at a voltage gradient from 120-140 volts at room temperature for approximately 75 minutes. Following electrophoresis, the gels were stained in 0.1% Coomassie Brilliant Blue, 50% methanol, 12.5% acetic acid for approximately 4 hours and subsequently destained in a 10% (v:v) methanol/10% (v:v) acetic acid solution for approximately 6 hours.
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FXa/ATIII/PRP Mixtures Primary Complex, Alpha Band
FXa/ATIII/PRP
FXa/ATIII ■i
4|g
-Ï-
8|g
(ATIII/PRP) + FXa
(FXa/PRP) + ATIII
1|g
4|g
PRP Level
1ig
4|g
8|g
B
16
e
§ 14
L12 n
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rotei 8 Pr 6 tal 4
Tot 2
^ 0
FXa/ATIII/PRP Mixtures Primary Complex, Beta Band
FXa/ATIII/PRP FXa/ATIII щ.
4|g
(ATIII/PRP) + FXa £
i
1|g
4|g
8|g
PRP Level
(FXa/PRP) + ATIII Î-
■i-
1|g
4|g
8|g
16 14 12 10
otei 8 Pro 6
tal 4
To 2 0
C FXa/ATIII/PRP Mixtures Secondary Complex, Alpha Band
FXa/ATIII/PRP
(FXa/PRP) + ATIII
- FXa/ATIII
(ATIII/PRP) + FXa
1.97 4|g
1.40 8|g
1.42 1|g
1.65 4|g
2.07 1|g
2.05 4|g
1.66 8|g
PRP Level
D FXa/ATIII/PRP Mixtures
Secondary Complex, Beta Band
FXa/ATIII (ATIII/PRP) + FXa
(FXa/ATIII/PRP) (FXa/PRP) + ATIII
Mixture
PRP Levels
«16 an14
e 12 ' e 10
otei 8
Pr 6
tal 4 ot
T 2 0
E FXa/ATIII/PRP Mixtures Tertiary Complex, Alpha Band
FXa/ATIII (ATIII/PRP) + FXa
FXa/ATIII/PRP (FXa/PRP) + ATIII
2.21 3.57
••
2.07 2.39 3.97
4|g 8|g 1^g 4|g
PRP Level
•• ••
1.88 1.75 3.81
1|g 4|g 8|g
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P 6
tal 4 ot
T 2 0
F FXa/ATIII/PRP Mixtures Tertiary Complex, Beta Band
FXa/ATIII (ATIII/PRP) + FXa
FXa/ATIII/PRP (FXa/PRP) + ATIII
FH
3.84
2.93
•• J 6.83
2.02 3.19
4|g 8|g 1^g 4lg 8lg
PRP Level
•• •• 5.80
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