Robert K. Andrews, Elizabeth E. Gardiner, and Michael C. Berndt. 1. ... activator of von Willebrand factor-dependent platelet aggregation by Read, Shermer,.
20 Snake Venom Toxins Affecting Platelet Function Robert K. Andrews, Elizabeth E. Gardiner, and Michael C. Berndt 1. Introduction Botrocetin from the South American pit viper Bothrops jararaca was described as an activator of von Willebrand factor-dependent platelet aggregation by Read, Shermer, and Brinkhous in 1978 (1). Subsequently, botrocetin has been widely used as an important in vitro modulator in the analysis of von Willebrand factor and platelet aggregation. Botrocetin has since been identified as a heterodimer of the C-type lectin family of snake venom proteins (~25 kDa nonreduced, ~14 kDa reduced), the primary sequence and crystal structure have been determined, and specific binding sites within the A1 domain of von Willebrand factor have been identified (2–8). Interestingly, members of the metalloproteinase-disintegrin family of snake venom proteins—jararhagin, jaracetin, and one-chain botrocetin—are functionally related to two-chain botrocetin, and also interact with the von Willebrand factor A1 domain (4,9,10). Jaracetin and one-chain botrocetin are variably processed forms of jararhagin and are found in the same viper species as the C-type lectin family, two-chain botrocetin. In this regard, recent evidence suggests that C-type lectin proteins and metalloproteinase-disintegrins may be derived from a common gene encoding a much larger precursor protein (11). An increasing number of C-type lectin proteins and metalloproteinases from cobra or viper venoms have been reported that selectively target either von Willebrand factor or its platelet receptor, glycoprotein (GP) Ibα of the GPIb-IX-V complex (some examples are shown in Table 1). These include the cobra venom metalloproteinase-disintegrin, mocarhagin from the Mozambiquan spitting cobra Naja mocambique mocambique (Naja mossambica mossambica) that cleaves GP Ibα within an anionic sequence containing three sulfated tyrosines (19,24,25). Mocarhagin also cleaves the neutrophil receptor PSGL-1 (P-selectin glycoprotein ligand-1) within an analogous sequence, and therefore has antiinflammatory activity in addition to being antithrombotic (18). In contrast, kaouthiagin from Naja kaouthia cleaves von Willebrand factor (21). A variety of C-type lectin proteins including alboaggregin-B (Table 1) bind to GPIbα and inhibit von From: Methods in Molecular Biology, vol. 273: Platelets and Megakaryocytes, Vol. 2: Perspectives and Techniques Edited by: J. M. Gibbins and M. P. Mahaut-Smith © Humana Press Inc., Totowa, NJ
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Table 1 Examples of Snake Toxins that Target Vascular Cell Adhesion Receptors or Ligandsa Protein snake species
Molecular mass
C-type lectin family Two-chain botrocetin ~25 kDa NR Bothrops jararaca ~14 kDa R Bitiscetin ~25 kDa NR Bitis arietans ~14 kDa R Alboaggregin-A ~50 kDa NR Trimeresurus albolabris ~14 kDa R Alboaggregin-B ~25 kDa NR Trimeresurus albolabris ~14 kDa R Convulxin ~85 kDa NR Crotalus durissus terrificus ~14 kDa R Ophioluxin ~85 kDa NR Ophiophagus hannah ~16 kDa R Metalloproteinases Alborhagin ~55 kDa NR/R Trimeresurus albolabris Mocarhagin ~55 kDa NR/R Naja m. mocambique Jararhagin ~55 kDa NR/R Bothrops jararaca Kaouthiagin ~50 kDa NR/R Naja kaouthia Crotalin ~25 kDa NR/R Crotalus atrox Catrocollastatin ~50 kDa NR/R Crotalus atrox
Vascular target(s) von Willebrand factor (A1 domain) von Willebrand factor (A3 domain) GP Ibα (His1-Glu282), and GP VI GP Ibα (His1-Glu282)
Typical yield (mg/g venom) Ref. 5–15
2,3 12
2–5
13
2–5
13
GP VI
14,15
GP VI
16
GP VI, Fibrinogen
2–5
GP Ibα (Glu282/Asp283), 2–5 Fibrinogen Binds von Willebrand factor 2–5 and α2β1 A-type domains von Willebrand factor (cleaves 708/709) GP Ibα; von Willebrand factor Collagen
17 17,18 9,20 21 22 23
a Molecular mass in kiloDaltons (kDa) based on SDS-PAGE (NR, nonreduced; R, reduced). Yields shown are for proteins purified in our laboratory. For additional examples of structurally/functionally related venom proteins, see refs. 24–26.
Willebrand factor binding (13,24,26). Related venom proteins target collagen or its receptors, α2β1 or GPVI on platelets. For example, convulxin and ophioluxin of the C-type lectin family (14–16) and the metalloproteinase, alborhagin (17), are agonists targeting GPVI. Interestingly, the 50-kDa heterotetrameric C-type lectin protein, alboaggregin-A, interacts with both GPIbα and GPVI (27,28). Together, these snake toxins provide both prothrombotic and antithrombotic reagents, and have proven invaluable for analysis of molecular mechanisms underlying platelet function. Although less well characterized, snake toxins such as botrocetin and convulxin have also been used to study megakaryocyte function (29,30).
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Fig. 1. Algorithm for purification of snake venom proteins.
We have used common strategies for isolation of either C-type lectin or metalloproteinase-disintegrin proteins from various species. An algorithm for purification protocols used in our laboratory is summarized in Fig. 1. In addition, many pitfalls and other points to consider when purifying venom proteins are relevant to a variety of snake toxins from different species. This chapter will describe the source, isolation, and use of venom proteins. We will focus on two specific examples: botrocetin of the C-type lectin family that activates von Willebrand factor, and alborhagin that targets GPVI. Finally, imminent new directions in venom protein analysis, such as isolation of selective probes using constitutively activated forms of receptor or ligand as affinity steps, or venomic (venom proteomic) approaches, will be discussed briefly.
2. Materials 2.1. Purification of Venom Proteins Lyophilized crude venom for isolation of alborhagin (Trimeresurus albolabris) or botrocetin (Bothrops jararaca) was from Venom Supplies, Tanunda, South Australia or Sigma, St. Louis, MO, respectively. Regulations pertaining to transportation of products from endangered species may limit acquisition of some venoms internationally. Different suppliers may not always guarantee authenticity of particular species, depending on collection procedures used. In this regard, venoms from snakes and other animals utilize a conserved structural framework such as a C-type lectin, with primary sequence
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Table 2 Chromatography Media and Buffers for Snake Toxin Purificationa Column Heparin-agarose (1.5 × 30 cm) Hydroxylapatite (1.5 × 20 cm) DEAE-sephacel (2.5 × 40 cm) Phenyl-sepharose (1.5 × 30 cm) Sepharose CL-2B (2.5 × 40 cm)
Column/wash buffer
Elution buffer
Ref.
0.01 M Tris-HCl, 0.15 M NaCl, pH 7.4 (TS buffer) 5 mM Na2HPO4, pH 6.8
Linear 0.15–1.0 M NaCl gradient in 0.01 M Tris-HCl, pH 7.4 (total volume, 400 mL) Linear 5–200 mM Na2HPO4, pH 6.8 gradient (total volume, 200 mL) Linear 0.15–1.0 M NaCl gradient in 0.01 M Tris-HCl, pH 7.4 (total volume, 500 mL) Linear 1.2 M to zero (NH4)2SO4 gradient in 0.01 M Tris-HCl, pH 7.4 (total volume, 400 mL) (Gel filtration)
13
0.01 M Tris-HCl, 0.15 M NaCl, pH 7.4 (TS buffer) 0.01 M Tris-HCl, 1.2 M (NH4)2SO4, pH 7.4 0.01 M Tris-HCl, 0.5 M NaCl, pH 7.4
2 2
3
17
a Columns run at 30 mL/h, with samples loaded in the column buffer, and washed until eluate (monitored by A280) returns to baseline before applying the gradient.
differences and/or posttranslational modification leading to enormous functional variability (24–26). The large number of isoforms has the advantage of providing extensive functional probes, but at the same time may complicate purification and characterization of particular forms. Batch-to-batch variation of venom profiles may also adversely affect yield. Metalloproteinases are prone to post-translational processing or autodigestion in the venom (during purification or storage), so that variant forms of the same protein may exist in different batches of venom. For example, jaracetin is a disulfide-bridged homodimer composed of one-chain botrocetin subunits (9). In general, it is recommended to monitor fractions throughout the purification, under nonreducing and reducing conditions, and start again using a different batch of venom if the preparation is atypical (see Note 1).
2.1.1. Metalloproteinases: Purification of Alborhagin The steps used for isolation of alborhagin are shown in Fig. 1. Resins, column buffers, and elution conditions are summarized in Table 2. 1. Heparin-agarose resin (Bio-Rad, Richmond, CA) packed into a 1.5 × 20-cm glass column, and equilibrated by washing with at least 10 bed volumes of column buffer (Table 2). 2. Hydroxylapatite (Bio-Rad) washed several times before use by suspending in column buffer in a beaker, and aspirating the upper layer containing fine particles as the bulk of the material settles on standing (see Note 2). It is then packed into a 1.5 × 20-cm column, and washed with 10 bed volumes of column buffer (Table 2). 3. Dialysis tubing (Union Carbide, Chicago, IL) with 100 mM Na2HPO4). 6. Dialyze botrocetin into TS buffer (3 × 2 L), and concentrate using an Amicon ultrafiltration cell fitted with a YM10 membrane (see Notes 7–9).
3.2. Functional Analysis of Venom Proteins 3.2.1. Platelet Aggregation 1. Use a 19-gauge winged infusion kit to collect 27 mL blood into a syringe containing 3 mL trisodium citrate anticoagulant, and mix by gentle inversion. The final concentration of trisodium citrate in the blood is 0.32% (w/v). This concentration of citrate prevents coagulation, but there remains sufficient Ca2+ to enable αIIbβ3-dependent platelet aggregation (refer to step 7 in this section). 2. Spin blood at 100g for 20 min at 22°C in 10-mL plastic centrifuge tubes. 3. Remove citrated platelet-rich plasma (PRP) using a plastic disposable transfer pipet, leaving approx 0.5 cm of PRP above the packed red cells since this layer contains contaminating white cells. There should be minimal turbulent shear stress applied to the platelet sample during these manipulations to minimize the possibility of platelet activation. Pool the PRP into one tube. 4. To prepare platelet-poor plasma (PPP), transfer 0.5–1 mL PRP into an Eppendorf tube, spin in a microcentrifuge for 1 min (10,000g, 22°C) to pellet the platelets, and retain the supernatant. 5. Take 400 μL PPP plus 100 μL TS buffer into an aggregation cuvet, mix, and use this for the “reference” sample in a dual-beam lumiaggregometer. 6. For aggregation measurements, use 400 μL PRP containing a stir bar in the “sample” cuvet; add TS buffer and other additions to make a total volume of 500 μL. Routinely, samples are equilibrated at 37°C, and stirred at 900 rpm. After establishing a baseline, commence the aggregation by addition of agonist. For further details of turbidimetric methods for studying platelet aggregation, see refs. 17,19. 7. For alborhagin purified as described in Subheading 3.1.1., a maximal rate of aggregation is achieved at approx 7.5 μg/mL final concentration. This response is initiated via signaling pathways coupled to GP VI, followed by activation of the integrin αIIbβ3, which binds fibrinogen or von Willebrand factor in a Ca2+-dependent manner. Alborhagin-dependent aggregation of PRP, therefore, is inhibitable by anti-αIIbβ3 monoclonal antibodies, such as CRC64 (10 μg/mL final concentration), or by addition of EDTA (final concentration, 10 mM) prior to alborhagin (17). Alborhagin also induces a platelet shape change prior to the aggregation response (indicated by decreased light transmission through the sample owing to platelets changing from a discoid to a spherical shape). The shape change occurs in the presence of EDTA where aggregation is blocked, suggesting that external Ca2+ is not
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required for this response to alborhagin. If aggregation occurs in the presence of anti-αIIbβ3 antibody or EDTA, then the “alborhagin” may be an analog functionally related to jararhagin, and may induce plasma von Willebrand factor binding to platelet GP Ibα, a pathway which may be independent of αIIbβ3 and external Ca2+. 8. For botrocetin purified as described in Subheading 3.1.2., a maximal rate of aggregation is achieved at approx 2.5 μg/mL final concentration. Botrocetin-dependent aggregation under these conditions is strictly dependent on von Willebrand factor binding to platelet GP Ibα, and may agglutinate platelets without the requirement for platelet activation and αIIbβ3 activity. Botrocetin activity, therefore, may not be blocked by anti-αIIbβ3 antibodies or EDTA, but may be confirmed by pretreating PRP with mocarhagin (see Note 3) at a final concentration of 10 μg/mL for 6 min at 37°C prior to the addition of botrocetin. The mocarhagin used for this test should be confirmed as active by showing that it inhibits aggregation of PRP induced by ristocetin (final concentration, 1.5 mg/mL). As an alternative to mocarhagin, inhibitory anti-GP Ibα monoclonal antibodies (for example, AK2) can be pre-equilibrated with PRP at a final concentration of 10 μg/mL prior to the addition of botrocetin. AK2 can also be confirmed as functional by showing that it inhibits ristocetindependent aggregation. If botrocetin does not induce aggregation of PRP at 2.5–5 μg/mL, then the concentration should be increased to 50 μg/mL to assess whether it is an active form of botrocetin with lower activity (see Note 7). If there is still no aggregation, then the “botrocetin” may be inactive (see Note 6). On the other hand, if botrocetin induces aggregation of PRP that is not inhibitable by mocarhagin and/or AK2, then the “botrocetin” is not typical, and the aggregation could be owing to a contaminating thrombin-like serine protease, for example, or may represent an analog that targets another platelet receptor, rather than activating von Willebrand factor to bind GP Ibα (refer to Subheading 2.1.). 9. Finally, specific methods suitable for more detailed analysis of alborhagin or other venom proteins (13–17,27,28) involving measuring of platelet activation events such as secretion, determining expression of surface activation markers such as P-selectin, or protein kinasedependent phosphorylation of cytosolic signaling molecules are described elsewhere in this book (see Chapters 7–9, 19, vol. 1 and 10 and 12, vol. 2).
3.2.2. Digestion of Fibrinogen 1. Dissolve fibrinogen in TS buffer at a stock concentration of approx 300 μg/mL. 2. Microcentrifuge the sample for 2 min at 10,000g, if necessary, to remove any insoluble precipitate. 3. To confirm metalloproteinase activity of alborhagin, digest fibrinogen at approx 100 μg/mL (final concentration) in TS buffer with a final concentration of 10 μg/mL alborhagin for 5–30 min at 22°C. Digestion should be carried out in the presence of either EDTA (10 mM, final concentration) or CaCl2 (10 mM, final concentration), in a total volume of 200 μL. 4. Stop the reaction at various times ranging from 0–80 min by adding an equal volume of SDS-PAGE loading buffer. 5. Analyze samples of fibrinogen without added alborhagin, alborhagin alone, and the digests in adjacent lanes on SDS-7.5%-polyacrylamide gels to reveal cleavage of alpha, beta, and/or gamma chains that run as a closely spaced triplet at about the midpoint of the gel under reducing conditions. For alborhagin, there should be time-dependent cleavage of the alpha chain in the presence but not the absence of Ca2+, and appearance of a major digestion fragment that runs below the gamma chain on SDS-PAGE. Maximum cleavage should occur after approx 80 min at 22°C (17).
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3.3. Future Directions The general approach to venom protein purification by a series of simple separation techniques (Fig. 1) has realized numerous useful probes, as well as significant new findings concerning the nature of venoms, and the function of vascular adhesion receptor targets. It would be expected in the future, however, that more selective affinity isolation procedures will play a much greater part in screening for, and isolating, venom proteins with a particular desirable function. For instance, for antithrombotics, it may be preferable to isolate venom proteins that selectively inhibit shear-induced thrombosis. Recent evidence suggests specific sites on activated GPIbα or von Willebrand factor may be critical for initiating shear-induced platelet aggregation (33). It could be envisaged that functional domains of GPIbα or the von Willebrand factor A1 domain containing gainof-function mutations may be useful for affinity purification of venom proteins that selectively recognize these active conformers. Second, in combination with such separation methods, the occurrence of multiple, closely related isoforms in venoms would suggest that they could be analyzed using proteomic approaches. Although full genomic sequences are lacking, the viability of “venomics” will continue to be enhanced as structure-function relationships of functionally different isoforms are deciphered, together with increased availability of crystal and model structures for C-type lectinlike proteins and metalloproteinase-disintegrins (25). In summary, the plethora of functional isoforms in snake venoms, and considerable batch-to-batch variability, is a complexity when it comes to isolation of particular forms of metalloproteinase-disintegrins and C-type lectin-like proteins. However, investigation of unexpected or unusual fractions has frequently led to discovery of novel probes for analysis of vascular cell adhesion receptors or ligands. New technologies promise exciting new developments in venom protein analysis.
4. Notes 1. Toxicity of crude lyophilized venoms toward humans may not have been established. They should therefore be considered potentially hazardous and handled with care, avoiding inhalation, especially on opening vials or containers. Dispose of contaminated materials and side fractions carefully to avoid the possibility of accidental envenomation. 2. Using sodium phosphate rather than potassium phosphate avoids precipitation when adding to SDS-PAGE sample buffer. This is particularly a problem for samples eluted later in the gradient, when the potassium concentration would be relatively high. 3. For isolation of mocarhagin, dissolve 0.5 g Naja mocambique mocambique venom (Sigma) in 10 mL TS buffer. Load at approx 30 mL/h onto a 1.5 × 20-cm heparin-agarose column, and wash with TS buffer. Elute with a linear 200-mL 0.15–1 M NaCl gradient in 0.01 M Tris-HCl, pH 7.4. Analyze approx 5-mL fractions on 5–20% SDS-PAGE under nonreduced and reduced conditions and pool fractions containing mocarhagin (~55 kDa, nonreduced and reduced). The main contaminant is a smeary band at approx 20 kDa that resembles an upsidedown “U.” Concentrate to approx 5 mL by Amicon ultrafiltration using a YM30 membrane, and load at approx 30 mL/h onto a Sepharose CL-6B column in 0.01 M Tris-HCl, 0.5 M NaCl, pH 7.4. Collect approx 5-mL fractions and analyze on 5–20% SDS-PAGE, nonreduced and reduced. Pool fractions containing mocarhagin, and dialyze into TS buffer. 4. Purified venom proteins may be stable at 4°C for several months. For long-term storage, freeze aliquots at –70°C to avoid repetitious freeze-thawing. Metalloproteinases such as
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Andrews et al. alborhagin or mocarhagin are susceptible to autodigestion on storage (17), and if storing at 4°C for >1 wk, 1 mM EDTA (ethylenediaminetetraacetic acid, tetrasodium salt) should be included in the storage buffer. Depending on the type of functional assay, it may be necessary that Ca2+ in the sample buffer be increased in order to compensate for Ca2+ binding to EDTA added along with the metalloproteinase. Changes of pH resulting from EDTA⬊Ca2+ binding when adding Ca2+ to PRP are possible, but usually do not affect platelet aggregation under these conditions. The concentration of purified protein is estimated using the BCA (bicinchoninic acid) protein quantitation method with bovine serum albumin (BSA) as standard according to the manufacturer’s instructions (Pierce). An inactive analog of botrocetin elutes early in the gradient (
