A simple and rapid method to study the association of the contact proteins of blood coagulation

THROMBOSIS

RESEARCH 68; 443-450,1992 $5.00 + .OO Printed in the USA. (c) 1992 Pergamon Press Ltd. All rights reserved.

00493848/92 Copyright

A SIMPLE AND RAPID METHOD TO STUDY THE ASSOCIATION CONTACT PROTEINS OF BLOOD COAGULATION

OF THE

Harold L. Katcher, Mathew Samuel and German B. Villanueva’ Department of Biochemistry and Molecular Biology, New York Medical College Valhalla, New York. (Received

157.1992

accepted in revised form 22.9.1992 by Editor C.W. Francis) (Received by Executive Editorial Office 23.10.1992)

Abstract Native and reduced SDS polyacrylamide gel electrophoresis on the automated PhastSystem (Pharmacia) were used to demonstrate protein-protein binding interactions and structural changes during proteolytic activations of the proteins involved in contact activation. The “mobility shift” assay in native gels has been used to visualize the kinetics of activation of factor XII by dextran sulfate as well as the formation of kallikrein-cleaved high molecular weight kininogen. It shows the formation of prekallikrein-high molecular weight kininogen complexes and factor XII-dextran sulfate complex for the first time in gels. The use of automation makes this procedure fast and reproducible using nanogram amounts of protein in relatively short time.

Prekallikrein (PK) is a single chain glycoprotein isolated in two very similar forms with apparent molecular weights of 85 and 88 kDa (1). High molecular weight kininogen (HK) and factor XII are also single chain glycoproteins with molecular weights of 120 kDa and 80 kDa (2,3), respectively. PK is a zymogen that gives rise to the serine protease, kallikrein. Kallikrein is involved in activation of blood clotting, inflammation and fibrinolysis (4,5). HK is a nonenzymatic protein cofactor that acts by forming a complex with PK or factor XI to facilitate the activation of the latter through a mechanism involving their limited proteolysis (6). HK is also a substrate for kallikrein protease activity. Limited proteolysis converts single chain HK

Key Words: Prekallikrein; high molecular weight kininogen; factor XII. Abbreviations: SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; IEF, isoelectric focusing; STI, soybean trypsin inhibitor; PMSF, phenylmethanesulfonyfluoride; DS-509, dextran sulfate (Mr=500,000). _ ?? Correspondin.g author: Dr. German B. Villanueva, Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA. 443

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to a two chain form and releases the physiologically potent vasoactive peptide, bradykinin (7). When surface-bound factor XII is activated to factor XIIa, it acquires enzymatic activity towards its protein substrates, prekallikrein and factor XI (8,9), which are complexed in vivo, with the contact activation procofactor high molecular weight kininogen (10,ll). Plasma kallikrein produced by the activation of prekallikrein by factor XIIa, in turn, cleaves more factor XII to factor XIIa. This reciprocal activation accounts for the rapid and amplified activation of the intrinsic pathway. Studies on the interactions of these proteins have been limited by the lack of simple and fast assay method to monitor complex formation and their subsequent activation through proteolysis. .

Electrophoresis, like chromatography, consists of several related techniques, each of which explores a different dimension in the characterization of a macromolecule. Of these techniques, SDS-PAGE has become a standard method for determination of protein molecular weight and subunit composition, while IEF-PAGE is a standard procedure used both to determine protein and peptide isoelectric points and to resolve isoenzymes. Native gel electrophoresis separates proteins by a complex function of size, charge distribution and shape (12), hence it can be used to detect changes in protein conformation. Native-PAGE has been .used in protein-DNA (13) and protein-heparin (14) binding studies, but rarely used in the analysis of protein-protein interactions. While standard polyacrylamide gel electrophoresis is, in general, slow and labor intensive, the development of an automated electrophoresis system, the PhastSystem (Pharmacia), which is fast, easy to use and reproducible, justifies its use in developing assays for the multi-dimensional analysis of protein-protein and protein-ligand interactions. When sensitive silver-staining techniques are used together with computer-aided analysis of digitized gel images, only nanogram samples of protein are required to obtain quantitative information, without the use of radioactivity or antibodies. In this paper we use SDS-PAGE and nativePAGE to analyze ‘the protein transformations during the complex formation of HK and PK and , the association of factor XII with dextran~ sulfate. MATERIALS AND METHODS Materials: Human PK, HK and factor XII were purchased from Enzyme Research Laboratories (South Bend, Indiana). Prior to use, the purity of the proteins were verified by gel electrophoresis and are found to be single band on reduced SDS-PAGE. Dextran sulfate (Mr=500,000), soybean trypsin inhibitor (STI), H-D-Phe-Phe-Arg chloromethylketone and phenylmethanesulfonylfluoride were purchased from Sigma Chemicals. The chromogenic substrate, S-2302 (H-D-prolyl-L-phenylalanyl-L-arginine-~-nitroanilide dihydrochloride) was purchased from Helena Laboratories (Beaumont, TX). Electrophoresis was conducted on a Pharmacia PhastSystem. Buffer strips and separation media were obtained from Pharmacia-LKB (Piscataway , NJ). Methods: A. General Methods All reactions involving human PK, HK and factor XII were performed in 50 mM NaCl, 5 mM sodium phosphate buffer, pH 7.4 in microcentrifuge tubes which are coated with polyethylene glycol (PEG 8000) in order to prevent adsorption and autoactivation of the proteins. PEG did not interfere with the activities of factor XII and PK. The protein concentrations were calculated by absorption measurements using c,46 = 7.3 for HK (15), e,% = 11.7 for PK (16), and E,~ = 14.2 for factor XII (17). Amidolytic activity assay of factor XII and PK preparations

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indicate that factor XII contains less than 0.1% factor XIIa and PK contains less than 0.5% kallikrein. These were established by comparison of the amidolytic activity of the protein preparations before and after they were fully activated by kallikrein or factor XIIa. B. Reduced SDS-PAGE Using the PhastSystem. The SDS samples were made by mixing aliquots of proteins from different experiments with 2.5 % SDS, 5% /3-mercaptoethanol and heating on a boiling water bath for 5 minutes. One PL of the SDS sample containing 20 to 40 ng protein is applied to lo-15% gradient gels using sample applicator comb and run as described in the PhastSystem System Guide (Technique File No. 110). The gels are silver stained using the automated development Unit (Technique File No. 210). C. Native Gel Electrophoresis Using the PhastSystem. Samples of the reaction mixtures are applied to gels as above, and run using Native Gel Buffer Strips in place of SDS Buffer Strips (Technique File No. 120). The method used to quantitate the number of factor XII bound to DS500 is described elsewhere (18). RESULTS A. Analysis of Kallikrein-Mediated Proteolysis of HK and PK. HK is incubated with catalytic amounts of kallikrein (100: 1 HKkallikrein) for varying times and analyzed at each time by native and reduced SDS gel electrophoresis. The results are shown in Figures 1A and 1B. It can be seen that on native gel, HK shows a high mobility band (Figure lA, lane 1). When catalytic amounts of kallikrein is added to HK there is a timedependent change in intensity and position of this band. Incubation of HK with kallikrein converts the high mobility band associated with intact HK to a low mobility form, HKa’. 12345678

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FIG. 1 Native and reduced SDS-PAGE of HK and its kallikrein-mediated proteolysis products. HK was incubated with catalytic amount of kallikrein (100: 1 ratio) for various times. Reactions were analyzed concurrently by (A) native gel and (B) reduced SDS gels. Lane (l), HK alone after 60 minutes of incubation at 37°C. Lanes (2-8) are respectively 5, 10, 15, 20, 30, 45 and 60 minutes of similar incubation in kallikrein. PK activated overnight at 25°C by a catalytic amount of factor XIIa was the source of kallikrein.

’ HKa is the form of HK that is cleaved by kallikrein at two points in the polypeptide chain to release the vasoactive peptide, bradykinin.

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However, at longer incubation time the HK band is again intensified. This is evident when the HK band at 5 minutes (Figure lA, Lane 2) is compared with the HK band at 60 minutes (Figure lA, Lane 8). On the other hand, the corresponding SDS gels show progressive decrease in intensity of the intact 120 kDa HK species. It appears, therefore, that further incubation leads to apparent conversion of HKa to a form which has the same mobility on native gels as intact HK. PK is poorly resolved in native gel system. The kallikrein-cleaved PK is equally poorly resolved, though the intact form was found to have slower mobility than the cleaved PK on the native gel (data not shown). B. Formation and Degradation of the PK-HK Complex. Approximately equimolar amounts of HK and PK (1.7 PM) are mixed and two samples are taken at various time. One was analyzed on native PAGE (Figure 2A) and the other by reduced SDS-PAGE (Figure 2B). After three minutes of incubation, it can be seen in native PAGE that the HK and PK bands are diminished and a new band is formed with a mobility intermediate between that of intact HK and PK bands. There is a change in the intensity and position of the intermediate band with increasing incubation time. The intermediate mobility band grows less intense, and is eventually replaced by a band of slightly faster mobility. Concomitant with these changes, the HK band decreases in intensity. However, the HK band re-intensifies on prolonged incubation (Figure 2, lane 7). As pointed out in Figure 1, this is a behavior that is inherent to HK in the presence of a catalytic amount of kallikrein and is not associated with the formation of the HK-PK complex. Reactions performed under the same conditions but in the presence of kallikrein inhibitors result in inhibition of the formation of the intermediate band with higher mobility as shown in Figure 3. The native PAGE shows that STI is the best inhibitor followed by H-D-Phe-Phe-ArgCH,Cl and PMSF. These results suggest that the intermediate band of low mobility is the PKHK complex while the slightly higher mobility intermediate band is the PK-HKa complex. The 1

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FIG. 2 Native PAGE and reduced SDS-PAGE analysis of the PK-HK complex. 1.7 PM each of PK and HK were mixed and examined by (A) native PAGE and (B) reduced SDS-PAGE. Lanes (1) PK, (2) HK, and (3-7) are PK-HK complexes after 3, 10, 20, 30, 45, 60 minutes of incubation.

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FIG. 3 The effects of kallikrein inhibitors on the degradation of the PK-HK complexes. Lanes (1) PK alone, (2) HK alone, (3) STI, (4) PK-HK complex without inhibitor after 30 minutes, (5) PK-HK plus H-D-Phe-Phe-ArgCH,Cl, (6) PK-HK plus PMSF, and (7) PKHK plus STI. The protein and inhibitor concentrations used are 1.7 PM and 3.4 pM, respectively.

appearance of doublet bands in these complexes is probably due to complex formation of HK with two forms of PKkallikrein of slightly different molecular weights. Addition of ST1 also inhibits the formation of the protein species that leads to the re-intensification of the HK band. C. Analysis of Factor XII-Dextran Sulfate Complex. Factor XII is preincubated in varying amounts of DS500 and analyzed on native-PAGE and reduced SDS-PAGE. The results obtained with native-PAGE are shown in Figure 4A. Factor XII alone can be seen in the separation gel as a low mobility and light staining diffused band (Lane 1). When preincubated in varying amounts of DS500, the factor XII-DSSOO complex migrates only a short distance within the stacking gel and does not enter the separation gel, presumably due to increase in molecular size because of complex formation. The amount of factor XII that is retained in the stacking gel is found to depend on the DS500 concentration. As seen in the reduced SDS-PAGE (Figure 4B) this “band shift” assay correlates well with the proteolytic autoactivation of factor XII. This is demonstrated by its correspondence with the time-dependent cleavage patterns of the uncleaved 80 kDa factor XII to the cleaved species Al

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Bound F XII

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FIG. 4 Native and reduced SDS analysis of the titration of factor XII with dextran sulfate. (A) is a “band shift” assay using native PAGE on a PhastSystem using 12.5% homogeneous gel to visualize factor XII-DSSOO binding. Lanes 1 to 8 correspond to the addition of 0, 2, 4, 6, 10, 15, 25 and 50 pg/ml DS500, respectively. (B) The corresponding SDS gel using a lo-15% acrylamide gel.

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(52 kDa and 28 kDa). The reduced SDS gel also shows decreased fragmentation of factor XII at high DS500 concentration.

DISCUSSION The PhastSystem Gel Electrophoresis combines the advantages of digitally controlled electrophoresis and the use of standardized pre-cast separation media with a protein sensitive silver staining module. The result of this is that as many as twenty-four samples containing nanogram quantities of proteins may be reproducibly electrophoresed and silver-stained within 2 hours with minimal manipulation time. Coupled with such techniques as the mobility-shift assay in native gels and concurrent reduced SDS analysis, they can provide quantitative information about dynamic processes. In this work, native gel electrophoretic analysis is used to study the interactions of proteins involved in contact activation. When examined by native-PAGE, HK, a very acidic protein @1=4.5), forms a densely staining, compact band with high mobility (Figure 1A). Under these conditions, HKa, formed by kallikrein cleavage of HK is found to have slightly lower mobility than the single chain HK. PK, a basic protein @I = 8.5-9), is seen as a diffused band with low mobility, in part because electrophoresis takes place at a pH very close to its p1. It can be seen that, for these proteins, the pH of the gel buffer system (pH 8.8) is a more important determinant of mobility than molecular weight, as HK was far more mobile than PK though its molecular weight is almost 50% greater. It is noteworthy that the kallikrein-cleaved PK has slightly higher mobility than the intact PK in the native gel system. Since no peptide is released under this condition because cleavage occurs within a disulfide bridge, this suggests altered protein conformation upon cleavage. When equimolar quantities of HK and PK are mixed and analyzed by electrophoresis on native gel, a new band appears with a mobility midway between that of PK and HK (Figure 2A, lane 3). Because it is known that in plasma, PK and HK exist as a dimeric complex (7), it is concluded that this new intermediate band represents the PK-HK complex. On continued incubation, the intermediate band is converted to a new band with slightly higher mobility. Evidence that the new intermediate band is a complex of PK with cleaved HK (HKa) is provided by four lines of evidence: (1) Incubation of a PK-HK mixture shows that as HK is degraded, the new intermediate band grows in intensity, while the PK-HK complex band fades (Figure 2A). By this time reduced SDS analysis reveals that very little intact HK is left, the low mobility PKHK complex band disappears and the new high mobility intermediate band is at its maximum intensity (Figures 2A and 2B). (2) None of the kallikrein-mediated proteolysis products of isolated HK and PK proteins produce bands at the intermediate positions. (3) Incubation of the PK-HK mixture in the presence of protease inhibitors prevents the formation of the new intermediate mobility band, while the PK-HK band remains at constant intensity and position (Figure 3). (4) Concurrent reduced SDS analysis shows that only HK experiences significant proteolytic cleavage. This means that if the new intermediate band represents a complex of one protein with a proteolysis product of the other, the HKa would be the cleaved partner. These evidences taken together allow for the conclusion that the protein represented by the new intermediate mobility band is the PK-HKa-complex. This study clearly demonstrates that both HK and HKa can form complexes with PK and also, that the PK-HK complex once formed can convert to PK-HKa complex.

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On prolonged incubation of the PK-HK mixture the band corresponding to free HK regains intensity in the native gel but reduced SDS-PAGE shows that there is almost complete disappearance of intact HK under these. conditions. We interpret this to mean that a cleavage product of HKa is formed which has the same mobility in native gel as the intact HK. It is not a fragment of PK since this is also present when HK is incubated with a catalytic amount of kallikrein . Native-PAGE can be used also to analyze the mechanism of the autoactivation of factor XII in DS500. The data on native-PAGE (Figure 4A) can be quantitated by densitometric analysis to calculate free and bound factor XII, thus allowing for the determination of binding parameters. By this method we have demonstrated in our earlier studies (18) that one DS500 molecule can bind 192 + 20 factor XII molecules. The reappearance of the low molecular weight complex in the native gel (Figure 4A, lane 8) and the diminished fragmentation of 80 kDa form of factor XII at high DS500 concentrations are in agreement with the earlier observation of the inhibition of autoactivation of factor XII at high dextran sulfate concentration (19). It is conceivable that at high DS500 concentration each DS500 molecule binds to fewer factor XII molecules to form a “low molecular weight complex”. In these complexes, fewer factor XII molecules are in contact with each other in the DS500 chain to induce autoactivation. This can explain the reduced fragmentation of factor XII. Previously, the PK-HK complex was only demonstrated by time-consuming gel filtration analysis (10,ll). The identification of HK and HKa was shown only by radiolabelled ligand blotting techniques (20). We have developed a fast and simple electrophoretic analysis utilizing the automated PhastSystem which should facilitate further investigations of the proteins of the contact activation system. This technique, when combined with immunological methods, potentially has the means to demonstrate these complex formations in plasma. This is currently being investigated in our laboratory. ACKNOWLEDGEMENTS We thank Dr. Elsamma Samuel for the critical reading of the manuscript. This work is supported by NIH Grant No. HL 43252. REFERENCES 1.

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