Identification of new protein-protein interactions between factor Va and factor Xa by sulfo-sbed cross-linking Khine Win 2 nd year medical student University of Vermont College of Medicine Faculty Mentor: Paula B. Tracy Interim Chairperson and Professor 89 Beaumont Ave. Given C401 Department of Biochemistry University of Vermont College of Medicine Burlington, VT 05405. 1
Introduction and Objectives Circulating platelets play many critical pathophysiological roles in our body, including hemostasis, thrombosis, angiogenesis, inflammation, neoplasia and immunity. Platelets are crucial components of wound healing following tissue injury to prevent excessive bleeding. One of the major roles of platelets is to regulate the production of thrombin by formation of prothrombinase complex on its activated membrane surface (1, 2). Thrombin produced from prothrombinase complex is a major factor involved in all stages of hemostasis and the coagulation cascade. Prothrombinase complex consists of a 1:1 stoichiometric interaction between the serine-protease factor Xa and its cofactor, factor Va, assembled in a Ca 2+ -dependent manner on the activated platelet membrane (3,4). Both factor Va and the activated platelet membrane are essential components of the complex. When associated with the prothrombinase complex, the catalytic efficiency of factor Xa to digest prothrombin to form thrombin is increased by approximately 300,000-fold, compared to its catalytic efficiency without the complex (5). Thus, it is very important to understand how the prothrombinase complex is assembled on activated platelet membrane. Based on the previous studies in our laboratory, factor Va is attached to the membrane via a glycosylphosphatidylinositol (GPI) anchor and required for factor Xa binding to the platelets (9). Many studies for protein-protein interactions between factor Va and factor Xa have used phospholipid vesicles as the membrane surface to assemble 2
the prothrombinase complex (4,10-12). In vitro, factor Xa can bind to the phospholipid vesicle without forming a complex with factor Va. But in vivo, the binding of factor Xa to the activated platelet membrane is absolutely dependent on the presence of bound factor Va which also provides at least part of the binding sites for factor Xa on the platelet membrane (11,16,17). It has been shown that platelet activation is also necessary to mediate the binding of factor Va and Xa, and that the extent of platelet activation is consistent with both maximal complex assembly and expression of function (6). All these data suggest that Prothrombinase complex assembly in vivo is actively regulated by platelets (15). To understand more about prothrombinase assembly on the activated platelet membrane, it is very important to identify protein-protein interaction sites between factor Va and factor Xa assembled on the platelets. The main objective of this research project is to identify and characterize the specific membrane protein-protein interaction sites between factor Va and factor Xa, using the chemical cross-linking approach and mass spectrometric analyses. 3
Methods 1) Labeling of Factor Xa with sulfo-sbed cross-linking reagent Purified human factor Xa (Hematology technologies, Essex Junction, VT.) was labeled with sulfo-sbed (Thermo-scientific), using the manufacture s protocol and all steps were performed in the dark. 1 mg of sulfo-sbed was dissolved in 25 µl of DMSO and diluted with PBS. Then 200 µg of human factor Xa was mixed with five-fold molar excess of sulfo-sbed and incubated at room temperature for 30 minutes. Then the mixture was dialyzed with 200 ml of dialysis buffer containing 50 mm HEPES, 150 mm NaCl ph 7.3 at 4`C overnight. The labeled factor Xa (Xa-SBED) was stored in multiple aliquots at -80`C, protected from light. 2) Analysis of labeled Factor Xa (Xa-SBED) using SDS-PAGE and Western Blot Labeled factor Xa samples were diluted with 1x sample preparation buffer containing 312.5 mm Tris-HCl, 10% SDS, 50% glycerol, and 0.005% bromophenol blue, ph 6.8, with or without 2% v/v β-mercaptoethanol (β-me). The protein samples were heated at 95`C for 5 minutes, and the samples were run on 10% Tris-glycine gels (Invitrogen). The gel was then transferred to nitrocellulose membrane as described by Towbin et al(8). The membrane was blocked by 5% (w/v) milk in TBST buffer (20 mm Tris, 150mM NaCl, and 0.05% (v/v) Tweene 20, ph 7.4) and probed with Streptavidin-HRP antibody or mouse anti hfx 27-5 antibody. The secondary antibody is HRP-conjugated horse anti-mouse IgG. Antibody binding on the blot was visualized using Western Lightning chemiluminescence reagent (PerkinElmer, Boston, MA). 4
3) Isolation of human platelets and preparation of washed platelets Platelets were isolated from healthy individuals using a modified method of Mustard et al (14). Apyrase is omitted from all washing steps, and 5mM HEPES-buffered Tyrode s albumin, ph ( 0.14 M NaCl, 2.7 mm KCl, 12mM NaHCO 3, 0.42 mm NaH 2 PO 4 -H 2 O, 1mM MgCl 2, 2mM CaCl 2, 5mM dextrose, 0.35% recrystallized bovine serum albumin, 5mM HEPES was used as a final platelet suspension buffer. Platelet concentration was determined using a Coulter Z1 particle counter (Beckman Coulter, Fullerton, CA). 4) Functional characterization of Xa-SBED in prothrombinase assay on activated platelets membrane The enzymatic activity of Xa-SBED was assessed by incorporating it into the prothrombinase complex assembled on activated platelet membranes and analyzing the kinetics of thrombin generation from prothrombin. The reaction mixtures consist of activated platelets, reversible thrombin inhibitor DAPA (dansylarginine N-3-ethyl-1, 5-pentanediyl amide), plasmaderived factor Va (5nM) and 1.39 µm prothrombin. The reactions were initiated by adding either unmodified factor Xa or factor Xa-SBED at room temperature. There were six total reaction mixtures with three different concentrations (0.1 nm, 0.3 nm, 0.5 nm) each for factor Xa and Xa-SBED. Then, 50 µl of reaction mixtures were removed at six different time points (10s, 20s, 30s, 40s, 50s, 60s) and stopped with 50 µl of cold quench buffer (20 mm HEPES, 150 mm NaCl, 50 mm EDTA, 0.1% PEG-8000, ph 7.4). The concentration of α-thrombin generated in each sample was determined using the chromogenic substrate, Spetrozyme TH (0.4 mm). The change in absorbance at wavelength 405 nm was measured using a spectrophotometer and compared thrombin generation from each reaction with the thrombin standard curve (0-200mM). 5
5) Cross-linking of Xa-SBED with factor Va on activated platelets membrane Washed human platelets (1x 10 9 /ml) maximally activated with 50nM thrombin, was incubated in the presence of 5nM purified plasma-derived factor Va for 5 min at ambient temperature. Then factor Xa-SBED was cross-linked to its binding partners on platelets by exposure to long wave UV light (366 nm) at a distance of 2 cm for 30 min at room temperature. Then, the platelets were isolated by centrifugation (1,000 x g) and the supernatant containing unbound factor Va and factor Xa was discarded. The platelet pellet was stored at -20`C before protein purification. 6) Purification and identification of cross-linked proteins The platelet pellet was suspended in lysis buffer (25 mm Tris-HCl, 150mM NaCl, ph 7.4 containing 1% (v/v) NP-40, 1% (v/v) Triton X-100, 5mM EDTA, 100mM β-me and 1X Halt TM protease inhibitor cocktail). The platelets were lysed by incubation at 37`C for 10 min, followed by sonication on ice. The detergent-insoluble fraction was pelleted by centrifugation at 10,000 x g for 10 min at ambient temperature. Biotinylated proteins from the platelet lysates was purified by affinity chromatography using Dynabeads MyOne Streptavidin T1 beads (Invitrogen), following the manufacture s instruction. First, 500 µl of Dynabeads (50% slurry in TBS containing 0.02% sodium azide) were washed three times with 1 ml of wash buffer (PBS). Washed beads were added to the platelet lysates and incubated at room temperature for 30 minutes on a rotator. The beads were pelleted by using a magnetic bar and then washed with 1 ml of wash buffer, six times (first four times with PBS, once with PBST and final wash with distilled water). Then the samples were 6
resuspended in 250 µl of elution buffer containing 0.4% TFA and 50% acetonitrile and incubated at room temperature for 10 minutes. The purified protein samples were evaluated with SDS- PAGE and Western blot analysis. Eluted protein samples were sent for analysis with electronspray ionization liquid chromatography mass spectrometry (ESI LC MS/MS). Results and discussion To characterize the protein-protein interactions between factor Va and factor Xa on the activate platelet membrane surface, we used the sulfo-sbed, a commercially available Biotin Label Transfer Reagent. It is a trifunctional cross-linking reagent, containing amine-reactive N- hydroxysuccinimide (NHS) ester group at one end for labeling a purified bait protein (Factor Xa) through its amine group, a photoreactive phenyl azide group on the other end for crosslinking to interacting prey protein (Factor Va), a biotin handle for affinity purification and detection, and a cleavable disulfide bond in the linker arm for separation of the cross-linked species (Figure 1A and 1B). Based on the Western blot analysis, human factor Xa was labeled with sulfo-sbed crosslinker (Figure 2). Purified factor Xa was seen on the blot as a doublet because the upper band (factor Xa-) can be converted autocatalytically to the lower band (factor Xa-β), because of the loss of 19 residues COOH-terminal peptide from the heavy chain, but it has no effect on its enzymatic activity (12). 7
The functional characterization of Xa-SBED with prothrombinase assays showed that labeled factor Xa has maintained approximately 67% of enzymatic activity compared with unlabeled factor Xa (Figure 3). After cross-linking Xa-SBED with factor Va on activated platelet membranes, the samples were analyzed by Western blot, using Streptavidin-HRP (SA-HRP) and human factor V (anti-hfva hc#17 ) antibodies. First, the blot was probed with SA-HRP, and the biotinylated protein bands were observed (Figure 4A, lanes 2 and 5) and those biotinylated bands are absent in the control lanes 1 and 4. One of the bands was around 105 kda which is about the size of factor Va heavy chain, suggesting that factor Va was cross-linked with factor Xa-SBED and obtained the SBED label from factor Xa. The same blot was reprobed with anti-hfva hc#17 and anti-hfva LC#9, and it showed that the biotinylated protein bands around 100 kda in lanes 2 and 5 of panel A, run similar to the factor Va heavy chain band seen in lane 8 of panel B. There is an intrinsically biotinylated non-specific protein band, about 75 kda, in lanes 1, 2, 4, and 5 of panel A. The factor Va light chain also runs around 74 kda and it is overlapped with the non-specific protein band. So, it is difficult to determine whether factor Va light chain is also biotinylated or not. The platelet lysates, containing the biotinylated proteins from cross-linking experiment, were purified with Dynabeads MyOne Streptavidin beads. The purified protein samples were analyzed by Western Blot (Figure 5A and B). The blot in panel A was probed with SA-HRP and the same blot was reprobed with anti-hfva hc#17. The elution fraction in lane 9 (panel A) has a small amount of biotinylated factor Va heavy chain. But a significant amount of the nonspecifically biotinylated protein band is also present in lane 9 (* band, in panel A) and it might form non-specific signal in ESI LC MS/MS analysis. A detectable amount of smaller protein 8
bands (**, around ~50 kda) are also present in the elution fraction (Lane 9, Figure 5A and B). They might be the degradation products of factor Va heavy chain and they are also labeled with SBED. The labeled factor Xa-SBED sample was sent for ESI LC MS/MS analyses to identify the modified lysine residues on factor Xa. It was found that factor Xa was labeled at three lysine residues, K 310, K 316 and K 329. Among those three residues, K 329 is a newly labeled residue, which hasn t been found in previous labeling experiments from our lab. So, it may lead to finding of new interaction site between factor Va and factor Xa on the activated platelet membrane surface. Purified protein samples from factor Va-factor Xa cross-linking experiment are also sent for ESI LC MS/MS analyses to find more interaction sites and the data are still in process. I believe that this project will provide new information about specific protein-protein interaction sites between factor Va and factor Xa on the physiologically relevant activated platelet membrane surface. It will help to improve our understanding of the role of activated platelet membrane surface in prothrombinase complex assembly, which is crucial in many pathophysiological processes such as hemostasis, thrombosis, angiogenesis, inflammation, neoplasia and immunity. The finding of specific interaction sites may also lead to new drug targets in the treatment of many thrombotic disorders. 9
References 1. Mann, K. G., Nesheim, M.E., Church, W.R., Haley, P., and Krishnaswamy, S. (1990) Blood 76 (1), 1-16. 2. Mann, K.G., Butenas, S., and Brummel, K. (2003) Arteriosclerosis, thrombosis, and vascular biology 23 (1), 17-25. 3. Kalafatis, M., Egan, J.O., van t Veer, C., Cawther, K.M., and Mann, K.G., (1997) Critical reviews in eukaryotic gene expression 7(3), 241-280. 4. Rosing, J., Tans, G., Govers-Reimslag, J.W., Zwaal, R.F., and Hemker, H.C. (1980) The Journal of Biological Chemistry 255(1), 274-283. 5. Nesheim, M.E., Taswell, J. B., and Mann, K.G. (1979) The Journal of Biological Chemistry 254(21), 10952-10962. 6. Bouchard, B.A.,, Catcher, C.S., Thrash, B.R., Adida, C., and Tracy, P.B. (1997) The Journal of Biological Chemistry 272 (14), 9244-9251. 7. Nesheim, M.E., Katzmann, J.A., Tracy, P.B., and Mann, K.G. (1981) Methods in enzymology 80 Pt C, 249-274. 8. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proceedings of the National Academy of Sciences of the United States of America 76 (9), 4350-4354. 9. Wood, J. P., Fager, A. M., Silverira, J. R., and Tracy, P. B. (2008) Blood 112, 585. 10. Tracy, P. B. (1988) Seminars in thrombosis and hemostasis 14 (3), 227-233. 11. Tracy, P. B., Eide, L. L., and Mann, K.G. (1985) The Journal of Biological Chemistry 260 (4), 2119-2124. 10
12. Miletich, J. P., Jackson, C.M., and Majerus, P.W. (1978) The Journal of Biological Chemistry 253(19), 6908-6916. 13. Mertens, K. and R. M. Bertina (1980) Pathways in the activation of human coagulation factor X. Journal of Biological Chemistry. 185 (3), 647-658. 14. Mustard, J.F., Perry, D. W., Ardlie, N.G., and Packham, M. A. (1972) Br J Haematol 22(2), 193-204. 15. Nesheim, M.E., Furmaniak-kazmierczak, E., Henin, C., and Cote, G. (1993) Thrombosis and Hemostasis 70 (1), 80-86. 16. Krishnaswamy, S. (1990) The Journal of Biological Chemistry 265 (7), 3708-3718. 17. Krishnaswamy, S., Jones, K. C., and Mann, K. G. (1988) The Journal of Biological Chemistry 263 (8), 3823-3834. 18. Trakselis, M. A., Alley, S. C., and Ishmael, F. T. (2005) Bioconjugate chemistry 16(4), 741-750. 11
Acknowledgements First, I would like to thank my research project mentor, Dr. Paula Tracy, for giving me this research project, allowing me to work in her lab and giving invaluable advices during this project. I also like to thank all lab members, including Dr. Jay Silveria and Jeremy Wood, for guiding me throughout the project and helping me to finish this project in time. Finally, I would like to thank University of Vermont, College of Medicine Summer Research Fellowship Program for giving me an opportunity to perform a research project during the summer and the research program coordinator, Laurie McCrea, for answering all my questions and helping me to get into the summer research program. 12
(A) (B) Figure 1: (A) Structure of sulfo-sbed, and (B) experimental outline for sulfo-sbed biotin label transfer (cross-linking) and analysis by Western blot (Courtesy of www.pierce.net). For this cross-linking experiment, protein 1 represents factor Xa and protein 2 represents factor Va. 13
(A) SA-HRP (B) Anti-hFXa kda 1 2 3 4 1 2 3 4 100 75 50 FXa-α-SBED FXa-β-SBED FXa-α FXa-β 37 25 Figure 2: Western blotting analyses of labeling of factor Xa with sulfo-sbed. Lane 1 and 2 show unlabeled factor Xa before and after first dialysis with PBS, respectively. Lane 3 and 4 show labeled factor Xa before and after overnight dialysis with label transfer buffer respectively. Western blot analysis with SA-HRP is shown on the left (A) and analysis with Anti-hFXa is shown on the right (B). Both labeled and unlabeled factor Xa-α and Xa-β can be seen around 50 kda. The concentration of FXa-β is increased after labeling. 14
Vo (nm IIa/s) [IIa] (nm) (A) FXa vs FXa-SBED Prombinase Assay 25 20 15 10 5 0.1 nm FXa 0.3 nm FXa 0.5 nm FXa 0.1 nm FXa-SBED 0.3 nm FXa-SBED 0.5 nm FXa-SBED Linear (0.1 nm FXa) Linear (0.3 nm FXa) 0 0 20 40 60 80 Time (s) (B) 0.45 0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 FXa vs FXa-SBED, Prothrombinase assay y = 0.7403x + 0.0167 R² = 0.9963 y = 0.4938x - 0.0046 R² = 0.9929 0 0.1 0.2 0.3 0.4 0.5 0.6 [Enzyme] (nm) FXa FXa-SBED 15
Figure 3: Functional Characterization of labeled factor Xa-SBED vs. unlabeled factor Xa using Prothrombinase assay. Graph A represents thrombin generation curves from six prothrombinase assays, stopped at different time points. Graph B was plotted using the slopes of thrombin generation curves against the three different concentration of unlabeled factor Xa or labeled factor Xa. Based on the slopes of the curves on graph B, the catalytic efficiency of labeled factor Xa-SBED is approximately 67% of the unlabeled factor Xa. 16
(A) SA-HRP (B) Anti-hFVa HC#17 and Anti-hFVa LC#9 kda 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 250 150 100 75 FVa-HC- SBED * FVa-HC FVa-LC 50 FXa-SBED 37 Figure 4: Western Blotting Analysis of factor Xa-SBED cross-linking with plasma-derived factor Va on activated platelet membrane. The blot on panel A was probed with SA-HRP antibody and the blot on panel B was probed with factor Va-heavy chain and light chain antibodies. Lanes 1, 2, 4 and 5 contain the mixture of factor Xa-SBED, factor Va and activated platelets. Lanes 1 and 4 have the samples before UV cross-linking, and lanes 2 and 5 have the samples after UV crosslinking. Lanes 1 and 2 are run without adding β-me, and lanes 4 and 5 are run with the addition of β-me. Lane 3 contains the standard molecular weight markers (Bio-Rad). Lanes 6, 7 and 8 have unlabeled factor Xa, labeled factor Xa-SBED and plasma-derived factor Va, respectively. Labeled factor Xa-SBED proteins (~48kDa) are visible in lanes 1, 2 and 7 of panel A. A small amount of factor Va heavy chain- SBED (~105kDa) is also visible lanes 2 and 5 of panel A. 17
There is an intrinsically biotinylated non-specific protein band (*), about 75 kda, in lanes 1, 2, 4, and 5 of panel A blot. The factor Va light chain also runs around 74 kda in the blot in panel B and it overlaps with the non-specific protein band (*) on the blot. 18
(A) SA-HRP (B) Anti-hFVa-heavy chain kda 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 150 100 75 FVa-HC-SBED * FVa-HC- SBED 50 ** ** 37 Figure 5: Purification of cross-linked protein mixtures from platelet lysates using Dynabeads MyOne Streptavidin T1. Lanes 1and 2 contain the platelet lysates before and after mixing with Dynabeads, respectively. Lane 3 has standard molecular weight markers (Bio-rad). Lanes 4 through 8 have the samples collected after each washing step. Lane 9 contains the eluted protein fraction. The elution fraction contains a small amount of factor Va heavy chain with SBED label (~104 kda) and a significant amount of (*) protein band which is the non-specifically biotinylated proteins from the platelets. (**) protein bands are also present in the elution fraction. They might be the degradation products of factor Va heavy chain, and they are also labeled with SBED. 19