Abstract
Inoculation of Bothrops jararaca snake venom (BjV) induces thrombocytopenia in humans and various animal species. Although several BjV toxins acting on hemostasis have been well characterized in vitro, it is not known which one is responsible for inducing thrombocytopenia in vivo. In previous studies, we showed that BjV incubated with metalloproteinase or serine proteinase inhibitors and/or anti-botrocetin antibodies still induced thrombocytopenia in rats and mice. Thus, herein we identified and characterized BjV toxins responsible for inducing thrombocytopenia. Initially, by filtering BjV on ultrafiltration systems, proteins with molecular masses between 30 and 50 kDa were shown to induce thrombocytopenia in mice, but they were not associated with hemorrhagic or coagulating activities. The 50 kDa ultrafiltrate was chromatographed, and two proteins (named fraction D and fraction E) induced thrombocytopenia in mice. However, neither fraction D nor fraction E induced platelet aggregation in platelet-rich plasma or whole blood from humans or mice. By mass spectrometry analysis, fraction E was identified as jararaca glycoprotein Ib (GPIb)-binding protein. Injection of these fractions caused thrombocytopenia in control or Vwf−/− mice, showing that the axis platelet GPIb – von Willebrand factor is not involved in their biological action in vivo. New studies are necessary to understand how these proteins act in vivo.
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Introduction
Bites by venomous snakes of the family Viperidae cause hemostatic disorders in various regions of the world. In addition to the better-known coagulation disorders, disturbances in platelet count, platelet function, fibrinolysis, and endothelial function are also frequently observed. These hemostatic disorders may be exacerbated by inflammatory disturbances, which are also elicited by the snake venoms1.
Among the snakes in this family, Bothrops jararaca is the primary agent for ophidian accidents in the southeastern region of Brazil. Its venom possesses several anti-hemostatic and inflammatory properties, inducing local and systemic bleeding, consumptive coagulopathy, thrombocytopenia, and platelet function disorders (Fig. 1, blue box). In humans2, dogs3, sheep4, equines5, rabbits6, rats7,8 and mice9,10, inoculation of Bothrops jararaca snake venom (BjV) induces moderate to intense thrombocytopenia. In previous studies aimed at elucidating the mechanisms of action of BjV on hemostasis, we observed that metalloproteinases (SVMP) and serine proteinases (SVSP), the two most abundant enzyme classes present in this venom11, are responsible for coagulopathy, but are not directly involved in thrombocytopenia, indicating that coagulation disorders and thrombocytopenia are independent phenomena8. We also observed that thrombocytopenia is independent of intravascular thrombin generation7. Using rats and mice as experimental models, we have observed that botrocetin, a C-type lectin-like protein (CTLP), is not involved in the development of thrombocytopenia observed during envenomation12. However, BjV aggregates washed human and mouse platelets, evokes secretion of ATP, PF-4, and β-hexosaminidase, but it does not induce a direct cytotoxic effect on the platelets. These effects are independent of platelet cyclooxygenase 1 activity. Eptifibatide, which inhibits the binding of fibrinogen to platelet glycoprotein complex GPIIb-IIIa, inhibited BjV-induced platelet aggregation more intensely in human than in mouse platelets. Moreover, BjV-induced platelet aggregation did not depend on the activity of SVSPs nor SVMPs in mouse platelets, whereas SVSP were rather important for platelet aggregation in humans13.
Blue box - Bothrops jararaca snake venom induces similar clinical and laboratory manifestations in humans and mice. White box - Schematic view of purification and identification of the B. jararaca toxin that accounts for thrombocytopenia in mice.
Various pro-platelet aggregating proteins have already been isolated from BjV. PA-BJ is a SVSP that aggregates washed human platelets and platelets in platelet-rich plasma14. Its action is mediated by the hydrolysis of protease-activated receptor (PAR)-1 and PAR-415. Botrocetin is a CTLP whose activity is dependent on plasma von Willebrand factor (VWF) and platelet glycoprotein (GP) Ibα, and it induces both platelet agglutination in fixed and lyophilized platelets16,17,18, and platelet aggregation and protein phosphorylation in unfixed platelets19,20,21. The administration of botrocetin to dogs, pigs, and rats causes depletion of high and intermediate molecular weight multimers of VWF and coagulation factor VIII, thrombocytopenia, prolonged bleeding time, and the formation of platelet microaggregates in the lungs and spleen, without evidence of platelet activation; however, it is devoid of activity in VWF-deficient animals22,23,24,25. Botrocetin-2 has a high similarity to botrocetin in regard to its platelet agglutinating activity, primary structure, and immunogenicity26,27.
BjV also contains diverse types of platelet aggregation inhibitors. Disintegrins are low molecular mass proteins that contain the Arg-Gly-Asp sequence and are rich in cysteine. They inhibit platelet aggregation induced by various agonists, as they inhibit the binding of either fibrinogen or VWF to the glycoprotein IIb/IIIa complex (GPIIb/IIIa). Disintegrins isolated from BjV include: jararacin28, jarastatin29, and bothrostatin30, which inhibit human platelet aggregation of human platelets stimulated by different agonists. Jararaca GP-Ib binding protein is a CTLP isolated from BjV, consisting of two polypeptide chains (α = 17.5 kDa and β = 15 kDa)31. It inhibits platelet activation induced by botrocetin and ristocetin. The β chain of this protein functions as a blocker of VWF binding to platelet glycoprotein Ib32. Phospholipases A2, with molecular masses ranging from 12 to 16 kDa, are low abundance enzymes in BjV, and they have been reported to inhibit platelet aggregation induced by collagen, PAF and ADP33,34,35. Nucleotidases are another protein class of platelet aggregation inhibitors. NPP-BJ is a phosphodiesterase that preferentially hydrolyzes nucleoside 5’-triphosphates over nucleoside 5’-diphosphates and inhibits platelet aggregation induced by ADP36. BjV also contains several SVMPs that cause hemorrhages, and jararhagin37 inhibits collagen-induced platelet aggregation, secretion, and phosphorylation by degrading the collagen platelet receptor α2β138,39. Jararhagin-C, a natural fragment of jararhagin, containing a disintegrin-like ___domain, also inhibits platelet aggregation induced by collagen and ADP40.
Those proteins have been typically isolated, and their action on platelets were identified in in vitro assays as pro- or anti-platelet aggregating agents. However, only rarely they have been tested in vivo, and usually their characterization excluded the interaction with other proteins that are present in the venom. Inasmuch as thrombocytopenia has been associated with the severity of envenomation2 and bleeding41, in order to identify the proteins that are involved in BjV-induced thrombocytopenia, we employed an initial protocol to identify by ultrafiltration the fractions from the crude venom that directly cause thrombocytopenia. We purified at least two proteins, one of which has been identified, that are directly involved in the thrombocytopenia evoked by B. jararaca venom in an in vivo model (Fig. 1, white box).
Materials and methods
Venom
Lyophilized venom from adult specimens of B. jararaca snakes was obtained from the Laboratory of Herpetology, Butantan Institute (Sistema Nacional de Gestão do Patrimônio Genético e do Conhecimento Tradicional Associado, SisGen AF375C2).
Characterization of BjV fractions that induce thrombocytopenia
Initially, ultrafiltration was used to identify which venom proteins induced thrombocytopenia in mice. BjV was diluted in saline (1 mg/mL)8,9, and 0.5-mL aliquots were submitted to centrifugation in Amicon Ultra microtubes (0.5-mL centrifugal filter units. Millipore, USA), with cutoff membranes of 30 kDa or 50 kDa, at 14,000 g for 15 min at 4 °C. After this first centrifugation, the ultrafiltrates were removed and maintained in microtubes in ice bath. For the centrifugation 50-kDa units, an additional volume of 375 µL of saline was added, whereas 200 µL of saline was added for 30-kDa ones, and then units were subjected to centrifugation again. The washing process of the membrane was repeated once more, and the ultrafiltrates were pooled and maintained in ice bath. After the third centrifugation, only the centrifugation 30-kDa units was subjected to two additional washes, one time using 200 µL, and the last one using 150 µL of saline. At the end of this first stage, the final volume of the ultrafiltrates of 30- and 50 kDa was 1250 µL. Proteins whose size was larger than the pores, remaining in the upper part of the ultrafiltration systems, were named supernatant proteins (SP), whereas those that passed through the pores were named ultrafiltered proteins (UF). Following manufacturer’s instructions, for the removal of SP from the 50-kDa units (SP50), 0.5 mL of saline was added to top of the filter, and samples were gently homogenized with the micropipette tip to remove the proteins adhered to the membranes, and then the filter unit was inverted and centrifuged at 14,000 g for 15 min at 4 °C. It is important to notice that BjV, UF30 (ultrafiltered proteins < 30 kDa), UF50 (ultrafiltered proteins < 50 kDa) and SP50 proteins contained the same final volume (1250 µL) for biochemical and biological tests. Those fractions were characterized biologically and biochemically.
Protein assay and SDS polyacrylamide gel electrophoresis
Proteins present in fractions were measured by ultraviolet absorption at 280 nm. The amount of proteins present in venom fractions was calculated as described elsewhere42, and expressed in mg/mL. To verify the protein profile of SP and UF fractions, samples (about 10 µg of protein) were subjected to electrophoresis in 12% or 15% SDS-PAGE gels43, under reducing and non-reducing conditions. Gels were silver stained44. The determination of the protein masses of protein bands was performed using the TotalLab TL100 program, version 1.1 (USA).
In vivo tests
Male BALB/c and C57BL/6 were obtained from the Animal Facility of Butantan Institute. Vwf knock-out mice (B6.129S2-Vwftm1Wgr/J, stock No: 003795, Vwf−/−) were obtained from The Jackson Laboratory (Bar Harbor, ME, USA), and bred at the Animal Facility of Butantan Institute. Mice were bred and maintained in a standardized environment, in rooms with defined flow of people, materials and supplies. In addition, they were continuously protected by health status barriers (barrier autoclave, HEPA air filtration system, differential pressure, etc.). In addition, they were surveilled twice a year for the presence of pathogens and ectoparasites, following FELASA recommendations45. Room temperature was maintained at 22 ± 2˚C, and humidity at 55 ± 10%. To control ammonia in the environment, the exhausting system was kept at 15 to 20 air changes per hour at room level. The light cycle was defined as 12-h light: 12-h dark. Mice were maintained in polycarbonate microisolator cages with a floor area of 451-cm2, containing environmental enrichment, and lined with autoclaved wood shavings and corn corb xylan. They had free access to drinking water and commercial pelleted feed: irradiated Nuvilab CR1 (Nuvital1, Quimtia, Brazil). The animal management routine comprised one weekly exchanges of cages, and fresh autoclaved water was changed two times per week. The facility is accredited by Conselho Nacional de Controle de Experimentacão Animal (CONCEA). All procedures involving the use of mice were in accordance with the National Guidelines from CONCEA46. Such procedures were also in accordance with ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines47. All procedures carried out in animals were approved by the Animal Use Ethics Committee of the Instituto Butantan (CEUAIB protocols 5937060618 and 8227240723).
To determine whether thrombocytopenia-inducing proteins were proteases, the proteolytic activity present in BjV was eliminated by heating it at 70 °C for 10 min48. BALB/c mice, weighing between 30 and 35 g, were injected subcutaneously (s.c.) at the dorsum with saline (negative control), BjV (positive control), heated BjV, UF30, UF50 or SP50, all at a dose of 1.6 mg/kg (dose of crude BjV that causes hemostatic disorders characteristic of B. jararaca envenomation in mice and rats9), using 120 µL of each sample per 30 g of body weight. Aseptic procedures were always employed during blood sampling. After 3 h, mice were anesthetized with isoflurane, and their blood was collected via caudal vena cava and immediately transferred to tubes containing anticoagulants: (a) Na2EDTA plus Bothrops antivenom (SAB, Instituto Butantan, lot 130577) for complete cell blood count2, and (b) CTAD plus SAB for plasma fibrinogen measurement12. The latter sample was centrifuged at 2500 g for 15 min at 4 °C to obtain plasma. Complete cell blood counts were performed in a Mindray automatic cell counter, BC-2800 Vet, and plasma fibrinogen was performed by a colorimetric assay49. After blood collection, mice were maintained anesthetized with isoflurane, and they were euthanized by physical cervical dislocation. Photographic records of the dorsum of mice were made to characterize the presence of hemorrhage at the site of inoculation of BjV, heated BjV, saline and centrifugation fractions.
When necessary, blood samples were also collected from human healthy donors, who did not report the use of any medication affecting hemostasis during the previous 10 days to blood collection. All participants provided accordance to donate blood sample to this study. They signed a written informed consent. All procedures are in accordance with the Declaration of Helsinki and national regulations. This study was approved by the National Human Research Ethics Committee (Plataforma Brasil, CAAE: 51368615.5.0000.0065).
Platelet aggregation
Crude BjV, heated BjV and centrifugation fractions (UF30, UF50 and SP50) were also tested if they exerted any pro- or anti-aggregating properties in human and mouse platelets. Mouse and human platelets were washed as described elsewhere13,50, and kept in a complete Tyrode’s solution at 37 °C in a dry bath throughout the procedure. Final platelet counts of 500 × 109/L and 300 × 109/L were used for the washed platelet suspensions or platelet rich plasma (PRP) of mice and humans, respectively. Aliquots (0.4 mL) of washed platelet suspensions in complete Tyrode’s solution, pH 7.4, were stimulated with 20 µL of BjV (0.4 mg/mL in saline), heated BjV, or aliquots of UF30, UF50 and SP50. As a positive control of platelet aggregation, 10 µL of a solution of 4.1 U/mL bovine thrombin (Sigma, T4648), diluted in saline at 4 °C, was used. Platelet aggregation was performed using the Born’s method51 in a Chrono-log aggregometer (model 560 V), as described elsewhere52. Platelet aggregation tracings were followed for 5 min after the addition of samples at 37° C. For platelet aggregation in whole blood, 400 µL of citrated mouse blood was incubated with 400 µL of saline, and the impedance recorded as previously described52.
Enzymatic activities
Collagenolytic (SVMP), amidolytic (SVSP) and L-amino oxidases (LAAOs) activities in samples of crude BjV, heated BjV, UF30, UF50, SP50 and interest fractions were performed as previously described50. Briefly, collagenolytic activity of samples was determined using a quantitative assay using azocoll as a substrate. Samples (200 µL) were incubated with 50 µL of 5 mg/mL azocoll (Sigma) dissolved in Tyrode buffer (137 mM NaCl, 2.7 mM KCl, 3.0 mM NaH2PO4, 10 mM HEPES, 5.6 mM dextrose, 1 mM MgCl2, 2 mM CaCl2, pH 7.4) for 1 h at 37 °C, taking care to homogenize samples every 5 min during incubation; the reaction was halted by placing samples on ice. After centrifugation for 3 min at 5000 g, the absorbance of the supernatant (175 µL) was measured at 540 nm. Amidolytic activity was assayed in samples (30 µL) incubated with 140 µL of substrate solution (10 mM BAPNA, 50 mM Tris-HCl, 20 mM CaCl2) for 1 h at 37 °C. The reaction was interrupted by the addition of 50 µL of 30% (v/v) acetic acid, and the absorbance was read at 405 nm in a microplate reader. To measure LAAO activity, samples (10 µL) were incubated for 1 h at 37 °C with 90 µL of reactive mixture (5 mM L-leucine, 2 mM o-phenylenediamine, and 0.81 U/ml horseradish peroxidase in 50 mM borax–HCl buffer). The reaction was halted by adding 50 µL of 2 M H2SO4, and the absorbance was read at 492 nm in a microplate reader. In all cases, the activity present in crude BjV was considered as 100%.
Analysis by mass spectrometry (LC-MS/MS) of UF50
Initially, UF50 samples were digested in solution with trypsin (Sigma), as previously described53. Resulting peptides were dried by vacuum centrifugation (Christ) and dissolved in 15 µL of 0.1% formic acid, and 4 µL of this volume was subjected to analysis by mass spectrometry using the LTQ-Orbitrap Velos spectrometer (Thermo Scientific) coupled to a nanoLC Easy II system (Proxeon). An aliquot of 4 µL was injected into a C18 pre-column (100 μm x 40 mm x 5 μm) (internal diameter x length x particle size), and the peptides were separated in an analytical column containing the Reprosil-Pur resin C18-AQ beads (3 μm, Dr. Maisch GmbH) (75 μm x 150 mm x 3 μm). The separation was performed using a linear gradient lasting 15 min, composed of water (A) and acetonitrile (B) containing 0.1% formic acid, under a flow of 200 nL/min. The system initiated when solution B was at 4%, and reached 35% in 15 min. The MS spectra53 were obtained by the FTMS analyzer with a resolution of 30,000 in the m/z range of 300–2000 (mass range). The fragmentation method chosen was Collission-induced dissociation (CID), and only the ten most intense ions of each MS were selected for fragmentation. For this process, only ions with two or more charges were selected. The collision energy used to obtain fragments (MS/MS spectra) was 35 eV, and the fragments were analyzed in the ion trap. The source voltage was fixed at 2 kV, and the dynamic exclusion time was adjusted to 90 s. A list containing 50 ions was used to decrease the repeated acquisition of the same m/z value. The file from the LC-MS/MS analysis was converted to the MGF format using the RawConverter program54, and then it was submitted to database searching using the Mascot program (version 2.6.2.). The bank used comprised the sequences deposited in Uniprot for snakes (by 07/21/2017; http://www.uniprot.org/; 70,087 sequences) plus 79 complete sequences of toxins obtained by sequencing the transcripts of B. jararaca venom gland55. As variable modifications, oxidation of methionine residues was considered, whereas carbamidomethylation of cysteine residues was established as a fixed modification. The tolerance values for the observed masses were 10 ppm for the precursors selected in MS53 and 0.5 Da for the fragments analyzed in the ion trap. The selected enzyme was trypsin, with the maximum tolerance of 1 missing cleavage site. Criteria for the analysis of identified proteins in the list: false discovery rate less than 1%.
Protein purification and identification
As we observed that thrombocytopenia-inducing proteins were present in the UF50 fraction, we isolated active proteins using chromatographic methods. An amount of 200 mg of B. jararaca venom was dissolved in 20 mL of buffer A (50 mM sodium phosphate buffer, pH 7.0) and subjected to ultrafiltration in a Vivaspin 6 system (50-kDa cutoff polyethersulfone membrane GE Healthcare, USA). Centrifugations were carried out at 3000 g at 4 °C in a 5810R centrifuge (Eppendorf, USA) with a swinging-bucket rotor. After the first ultrafiltration, 1 ml of buffer A was added to the supernatant, and the material was again centrifuged under the same conditions. Thereafter, 1 mL of buffer A was again added to the superior part of the centrifugation system, and the material collected, according to the manufacturer’s recommendations. The UF50 was maintained in aliquots frozen at -80 °C for further purification. The chromatographic procedures were performed at room temperature in an ÄKTA™ pure 25 system (GE Healthcare). The conductivity and absorbance at 280 nm of the eluted material were monitored continuously, and fractions were collected in 96-well Uniplate polypropylene plates. For hydrophobic interaction chromatography, a 5-mL aliquot of UF50 was diluted in 5.0 mL of buffer B (50 mM sodium phosphate buffer, 1.7 M ammonium sulfate, pH 7.0), and applied to a Source 15PHE column (PE4.6/100, GE 17-5071-01), equilibrated with buffer B. The adsorbed material was eluted with a linear gradient of ammonium sulfate 1.7–0.0 M in buffer A, under a flow of 1.0 mL/min, and 1.0-mL fractions were collected. Fractions were tested to verify their ability to induce thrombocytopenia in male BALB/c mice and aggregation in washed platelets, and those that induced thrombocytopenia in mice were pooled, and also analyzed by C8 reversed-phase HPLC by using a YL9100 HPLC System (YL Instruments, Korea). For the identification of fractions from the hydrophobic interaction chromatography that induced thrombocytopenia, male BALB/c mice weighing between 25 and 30 g (n = 2/fraction), were injected subcutaneously (s.c.) with 100 µL of the fractions of the main peaks. To prevent interference from (NH4)2SO4 in the response, fractions were ultrafiltered on 3-kDa pore membranes, and equilibrated in buffer A, prior to administration to the animals. The purity and electrophoretic profile of proteins were monitored by electrophoresis in 15% polyacrylamide gel (SDS-PAGE), under reducing and non-reducing conditions. Protein bands obtained from fractions that induced thrombocytopenia were excised from gels, subjected to in-gel trypsin digestion56, and submitted to identification by mass spectrometry in Lactad (Campinas, Brazil). Briefly, the generated peptides were desalted using Stop-and-go extraction tips (Stage Tip) C-18, and mounted on P-200 tips (Axygen)57. The MaxQuant platform58 was also employed for peptide identification and protein grouping using the same parameters and database as in PEAKS X+. This mechanism is considered highly robust and utilized for both peptide identification and searches for post-translational modifications59.
Statistical analyses
Data from platelet counts and fibrinogen levels were checked for normal distribution and homoscedasticity using Stata™ 14.1 software. Statistical analyses for comparison of mean groups were performed using the SPSS software, version 22, using one-way analysis of variance (ANOVA), followed by the Bonferroni test. Differences with p < 0.05 were considered statistically significant. Data were expressed as mean ± standard error of the mean.
Results
Initial characterization of BjV fractions that induce thrombocytopenia
In order to identify and characterize BjV protein(s) responsible for causing thrombocytopenia, diverse methodological approaches were employed, so that we could identify the molecular masses and biochemical properties of toxins of interest. Thus, BjV was either heated or ultrafiltered and then in vitro and in vivo tests were carried out. As most of authors first purify toxins, and then search for biological effects of interest in vivo, we inverted the order of experiments, so that we purified toxins based on their in vivo and not on in vitro activities.
As expected, at 3 h after s.c. administration of BjV in mice, it was noticed a reduction around 60% in platelet counts (p < 0.001), a drop in plasma fibrinogen levels (p < 0.001) and the development of local hemorrhage (Table 1). Heating or ultrafiltration was used to initially differentiate the defibrinogenating from the platelet pro-thrombocytopenic activities. As shown in Table 1, heating resulted in denaturation of proteins, as evidenced by the opaque appearance of solution (data not shown) and apparently increased protein content at 280 nm; SVSP activity was moderately preserved, but SVMP and LAAO activities were completely annihilated by heating (Table 1). In vivo tests showed that heating preserved the defibrinogenating activity, but completely destroyed the pro-thrombocytopenic activity (Table 1), corroborating previous findings showing that coagulation disturbances and thrombocytopenia are independent events7,8,12. On the other hand, ultrafiltration of BjV samples using 10-kDa (data not shown) or 30-kDa (UF30) pore membranes (Table 1) did not allow the passage of thrombocytopenia-inducing proteins, defibrinogenating proteins, nor hemorrhagins (P-III SVMPs). However, ultrafiltration through 50-kDa pores membranes (UF50) allowed the passage of most thrombocytopenia-inducing proteins (p < 0.001), and some proteins that caused defibrinogenation (p < 0.05), as compared to control BjV, but no local hemorrhage was noticed (Table 1). UF50 contained low activity of SVMP, SVSP and LAAO. Hemorrhage-inducing proteins were mostly present in SP50 (Table 1 and Figure S1).
Profiles of electrophoretic mobility (Fig. 2) of these venom samples were also determined by SDS-PAGE, under reducing and non-reducing conditions. As expected, samples under reducing conditions (Fig. 2a) showed a greater number of bands than the samples that were not reduced (Fig. 2b). The heated BjV sample was able to minimally enter the gel under non-reducing conditions, demonstrating that heating caused denaturation and aggregation of proteins. Non-treated BjV, heated BjV and SP50 revealed the presence of a major band with a molecular mass of 50 kDa, whereas this band was not observed in the samples of UF30 and UF50; all reduced samples showed protein bands with molecular mass below 30 kDa. However, the 50-kDa supernatant still showed contamination with proteins with a molecular mass less than 50 kDa.
Thus, thrombocytopenia-inducing proteins had between 30 and 50 kDa. The in vivo tests determined that the hemorrhage caused by the venom contributes minimally to thrombocytopenia, since the animals injected with UF50 showed no signs of bleeding, corroborating previous findings8,60. Furthermore, blood coagulation activation also seems to have little participation in thrombocytopenia, since animals injected with heated venom did not have a decrease in the number of circulating platelets, despite the consumption of fibrinogen, which was similar to that caused by crude BjV (Table 1).
Electrophoretic profiles of crude BjV, heated BjV, UF30, UF50 and SP50. Samples (10 µg) were submitted to electrophoresis on 12% SDS-PAGE, under non-reducing (a) and reducing (b) conditions. The molecular mass markers are shown in lane 1. Proteins were silver-stained44.
As controls of platelet aggregation, addition of BjV or thrombin into either mouse or human washed platelet suspensions (Fig. 3) induced platelet aggregation, as previously described13. In human platelets (Fig. 3), UF50 induced platelet aggregation, but not SP50 nor UF30. On the other hand, only SP50 induced platelet aggregation in mouse platelets (Fig. 3), although it did not induce thrombocytopenia in vivo (Table 1). UF30 and heated BjV induced minimal platelet aggregation. Although UF50 caused no platelet aggregation in mice, it induced intense thrombocytopenia in vivo. To determine whether UF50 depended on plasma proteins, red blood cells or white blood cells, we evaluated platelet aggregation in mouse whole blood (Fig. 2), but it continued having no direct platelet aggregating activity.
Altogether, these results showed that thrombocytopenia was caused by proteins present in UF50, but thrombocytopenia in mice was not directly associated with platelet pro-aggregating proteins.
Representative tracings of platelet aggregation induced by BjV, heated BjV, or ultrafiltration fractions of BjV in washed platelet suspensions from humans or BALB/c mice, or in mouse whole blood. Bovine thrombin (0.1 U/mL, final concentration) or collagen (12 µg/mL, final concentration) were used as positive controls. Aliquots (20 µL) of the fractions (Table 1) were applied to washed platelet suspensions. In the case of aggregation in mouse whole blood, 40 µL of UF50 was applied instead. The asterisk indicates the addition of the corresponding agonist. These results are representative of two different experiments.
Mass spectrometry analysis of UF50
To initially identify which proteins were present in UF50, and further guide protein purification, we submitted the UF50 sample to mass spectrometry analysis. A total of 162 proteins were identified (Supplementary Table 1), and among these, 30 showed to be more abundant with a score of 200 to 800 (Table 2). The main families of toxins present in UF50 were SVMP (37%), CTLP (33%), SVSP (17%), phospholipases A2 (17%), and other ones (3%).
Isolation and identification of UF50 proteins that induce thrombocytopenia
Taking into consideration that one of the most abundant proteins present in UF50 was jararaca GPIb-binding protein (BP)31,32,63, and that SVMP were not directly involved in promoting thrombocytopenia8,12,60, UF50 was initially submitted to hydrophobic interaction chromatography, as previously published31 (Fig. 4a). Two peaks were obtained with thrombocytopenic activity, as determined by injection into mice. These fractions decreased platelet counts by about 80% (red line) and 90% (green line) in relation to the reference values of healthy BALB/c mice, while maintaining fibrinogen levels within the normal reference values and causing no bleeding. The fractions that made up the second peak (herein called fraction E protein, green asterisk, Fig. 4a) had a single band in SDS-PAGE and a single peak in HPLC (Fig. 4a-c). Moreover, this fraction had no collagenolytic or amidolytic activity. The reduced protein bands excised from the gel were submitted to mass spectrometry analysis, and they were identified as jararaca GPIb-BP32 (Fig. 5). Fractions that made up the peak denoted with a red asterisk (Fig. 4a), herein called fraction D, had several bands between 18 and 30 kDa under non-reducing conditions in 15% SDS-PAGE gels (results not shown), suggesting that fraction D was a mixture of different proteins, at least one showing thrombocytopenic activity.
(a) Purification profile for UF50 chromatographed on a Source 15PHE column. Adsorbed proteins were eluted with a decreasing linear gradient of (NH4)2SO4 (blue line). The peaks were injected in mice (right panel), and those that induce thrombocytopenia were marked with red and green asterisks. mAu, milli-units of absorbance at 280 nm. (b) Protein purification profile of fraction E analyzed by reversed-phase HPLC. A Venusil XBP C8 column (5 μm, 100 Å, 4.6 × 250 mm, Agela Technologies) was equilibrated with solvent A (0.1% trifluoroacetic acid, TFA, in ultrapure water), and the protein was eluted with a 0–100% increasing linear gradient of solvent B (0.1% TFA/90% acetonitrile) and monitored at 280 nm. (c) Electrophoretic profiles of fraction E in 15% SDS-PAGE, under reducing and non-reducing conditions. Protein E had a single 27.5 kDa chain under non-reducing conditions, and two chains, 15.4 kDa and 12.9 kDa, under reducing conditions. Molecular mass markers are shown in the left side. Gel was silver stained44.
Identification by mass spectrometry of peptides present in fraction E. Percertanges of coverage of α- and β-chains of GPIb-BP were 95.8% and 100%, respectively. NCBI protein code: Q9PSM6: Snaclec GPIB-BP subunit alpha; Q9PSM5: Snaclec GPIB-BP subunit alpha. Graph was generated by PEAKS Studio 64-bit (Matrix Science, London, UK).
Platelet aggregation
As shown in Fig. 6, although fraction D and fraction E induced an intense decrease in platelet count in vivo, both proteins did not directly induce platelet aggregation in whole blood (Fig. 6a, b) or platelet-rich plasma (Fig. 6c, d,e and Fig. S2), from either humans or mice. Once fraction E was identified as jararaca GPIb-BP by mass spectrometry analysis, we also observed that it inhibited ristocetin-induced platelet aggregation in platelet rich plasma (Fig. 6c), as previously reported31. However, fraction D could not inhibit platelet aggregation induced by ristocetin, demonstrating that it was a different protein from jararaca GPIb-BP. To decrease the interference of low calcium levels in platelet aggregation tests, we also tested proteins in whole blood anticoagulated by PPACK, a thrombin inhibitor, but either the fraction D or E was shown to induce no platelet aggregation. Washed platelets were not either stimulated by fraction D or fraction E (data not shown).
Platelet aggregation tracings induced by fractions D or E. (a) Mouse whole blood: 400 µL of citrated BALB/c mouse blood was diluted in 400 µL of saline. (b) Human whole blood: 400 µL of human citrated blood was diluted in 400 µL of saline. At the time indicated by the asterisk, 20 µL of PBS (control), protein fraction E (61 µg/mL) or fraction D (35 µg/mL) were added. Collagen (Col, final concentration 4.9 µg/mL for human platelets, or 12 µg/mL for mouse platelets) was used as a positive control. (c–e) Platelet aggregation in human PRP induced by the platelet agonists ristocetin ((c) 1.5 mg/mL, final concentration), collagen ((d) 4.5 µg/mL, final concentration), or PAR-1 agonist ((e) SFFLRN, 30 µM, final concentration). Aliquots of 40 µL of fraction E (61 µg/mL), fraction D (35 µg/mL), or saline (control) were added to human PRP, and after incubation the platelet agonist was added. These results are representative of two different experiments.
Injection of fraction D or fraction E in vWF −/−mice
When either fraction D or E were inoculated in Vwf−/− mice, it was noticed that the absence of VWF in circulation did not block the development of thrombocytopenia (Fig. 7), similarly as observed earlier for the crude BjV12. These results evidence that even though fraction E (jararaca GPIB-BP) binds to platelets and inhibits ristocetin- and botrocetin-induced platelet aggregation31, its mechanism of action to induce thrombocytopenia in vivo does not involve the presence of VWF.
Platelet counts and fibrinogen levels at 1 h after i.v. administration of either fraction D or E in Vwf−/− or C57BL/6 mice. Either fraction D or fraction E induced intense thrombocytopenia in both mouse strains, showing that they cause thrombocytopenia regardless the presence of VWF in circulation. Note that fibrinogen levels showed no alteration. Results were expressed as mean ± s.e.m.
Discussion
Considering that thrombocytopenia is an overlooked but significant risk factor for both local and systemic complications of patients bitten by B. jararaca2, herein we isolated at least two toxins that induced thrombocytopenia in envenomed mice. Fractions D and E showed no direct platelet aggregating activity, either in the absence (washed platelet suspension) or presence of plasma (platelet-rich plasma), or even in the presence of other blood cells (whole blood), but they evoked intense thrombocytopenia in mice. Fraction E contained only one protein, and when analyzed by mass spectrometry, it showed to be identical to jararaca GPIb-BP (Fig. 1, white box). Our results confirmed previous findings that GPIb-BP does not directly induce platelet aggregation, but only impaired ristocetin-induced platelet agglutination31. Previous studies also reported that jararaca GPIb-BP did not induce platelet secretion19,31,70,71, and blocked GPIb-induced tyrosine phosphorylation of FcRγ-chain71.
Despite previous results showing that the inhibition of ristocetin-induced platelet agglutination evoked by jararaca GPIb-BP depended on the presence of VWF31, our results demonstrated that the extent of thrombocytopenia in control and Vwf−/− was similar, indicating that the in vivo mechanism of action of jararaca GPIb-BP is independent of the presence of VWF. Similar results had been previously demonstrated for the crude BjV, showing that the presence of VWF in circulation is irrelevant for the occurrence of thrombocytopenia in mice or rats12.
Jararaca GPIb-BP63, echicetin (isolated from Echis carinatus snake venom72), tokaracetin (isolated from Protobothrops tokarensis snake venom73), agkistin and agkicetin-C (isolated from Deinagkistrodon acutus snake venom74,75) are all antagonists of GPIb, and also induce thrombocytopenia in animal models. They are CTLP that competitively inhibit the binding of VWF to GPIb, and inhibit ristocetin- and botrocetin-induced platelet agglutination. They have already been reported to induce rapid and reversible platelet depletion in vivo in animal models. Interestingly, echicetin does not bind to rabbit platelets72. Sakurai et al. (1998) had also observed the same extent of thrombocytopenia in splenectomized guinea pigs after intravenous injection of jararaca GPIb-BP76, suggesting that the spleen is not the site of platelet sequestration. Besides, the formation of platelet microaggregates by jararaca GPIb-BP was not evidenced76,77.
In other Bothrops sp. venoms, aspercetin has been ascribed as the responsible for thrombocytopenia induced by Bothrops asper snake venom78,79. Like botrocetin, aspercetin activity depended on the presence of GPIb and VWF. However, BjV contains not only botrocetin, which promotes platelet aggregation by the binding of GPIb to VWF, but also jararaca GPIb-BP, which inhibits both ristocetin- and botrocetin-induced platelet agglutination. Previously, we could not demonstrate the involvement of botrocetin in experimental envenomation induced by BjV in mice and rats12. As shown in Table 2, jararaca GPIb-BP preponderate in comparison with botrocetin in BjV obtained from Instituto Butantan, as also observed elsewhere for other venom lots80. On the other hand, the thrombocytopenia induced by Bothrops caribbaeus snake venom seems to be due to a CTLP with direct platelet aggregation/agglutination activity, that binds to GPIb81.
Platelet glycoprotein Ib (GPIb) is an essential component of the platelet membrane that plays a pivotal role in hemostasis by mediating the initial adhesion of platelets to the site of vascular injury. This adhesion is a key step in the formation of a hemostatic plug, which prevents excessive bleeding. GPIb is a part of the GPIb-IX-V complex, a receptor complex that includes GPIbα, GPIbβ, GPIX, and GPV subunits. The extracellular ___domain of GPIbα contains the binding site for von VWF, a crucial ligand that anchors platelets to the exposed subendothelium at the injury site, especially under high shear stress conditions found in arterial circulation. Structurally, the GPIb-IX-V complex is organized in a supramolecular assembly where GPIbα covalently associates with GPIbβ, and non-covalently with GPIX, and GPV, forming a functional receptor unit on the platelet surface. GPIbα is the largest subunit and contains a leucine-rich repeat (LRR) region that is essential for ligand binding82. Even though we only explored herein the axis GPIb-VWF for the in vivo action of jararaca GPIb-BP, using Vwf−/− mice, other ligands for GPIb have also been described in the literature, such as thrombin83, protein C and activated protein C84, protein-disulfide-isomerase (PDI)85, thrombospondin-186,87, semaphorin 7 A88, P-selectin89, leukocyte integrin Mac-1 (αMβ2)90, reelin91, high molecular weight kininogen92, factor VIIa86, factor XI93, factor XII94. However, it has been already demonstrated that jararaca GPIb-BP does not compete with thrombin for the binding in GPIb31,63. Anfibatide, an antagonist of GPIb isolated from Deinagkistrodon acutus snake venom, although induced no thrombocytopenia95,96, inhibited thrombosis in Vwf−/− mice, suggesting that it has additional antithrombotic effects beyond its inhibitory role in GPIb-VWF axis97,98. The discovery of new ligands for GPIb is crucial as it expands our understanding of platelet function and the regulation of hemostasis, potentially leading to novel therapeutic targets for thrombotic and bleeding disorders. Future studies are required to understand if any of these ligands are involved in the in vivo action of jararaca GPIb-BP or even if other cells are involved in this action. Studies are now being carried out to understand the mechanisms of action of jararaca GPIb-BP and the role it plays in inducing thrombocytopenia during snakebite envenomation by B. jararaca.
Fraction D, which also caused thrombocytopenia in mice, neither exhibited platelet pro-aggregating activity nor direct anti-aggregating activity in vitro, in both human and mouse platelets. HPLC analysis of CTLP-D showed the presence of at least two different proteins. As the proteins in fraction D are minor components of BjV, they are still being isolated and characterized.
In conclusion, jararaca GPIb-BP and proteins found in fraction D are the main triggers of thrombocytopenia induced by BjV in mice, without involving the consumption of coagulation factors. These results confirmed previous results that thrombocytopenia, consumptive coagulopathy are independent events during the B. jararaca snakebite envenomation2, and that the development of thrombocytopenia is not strictly due to hemorrhages or generation of intravascular thrombin7,8.
Data availability
The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation. Please contact: [email protected].
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Acknowledgements
We thank the staff of the Life Sciences Core Facility (LaCTAD), from State University of Campinas (UNICAMP), for the Proteomics analysis.
Funding
This study was supported by the São Paulo Research Foundation (FAPESP, www.fapesp.br), grants #2013/25177-0 (MLS), 2013/07467-1 (SMTS), 2016/16935-7 (DAS), 2017/00715-0 (DTS), 2016/50411-5 (MLS, MEP), 2018/26015-8 (ATAS), 2012/03657-8 (AST), 2019/03779-5 (AST) and 2019/07618-6 (MLS); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, www.cnpq.br), grants #312469/2018-7 and 309980/2021-6 (MLS), 302703/2017-9 (AST), 309551/2021-8 (AST), and INCT-Entomologia Molecular (AST); Fundação Butantan (www.fundacaobutantan.org.br), grant FB n°. 001/0708/000.223/2024; Agencia I + D + i (https://www.argentina.gob.ar/ciencia/agencia), grant PICT-2020-SERIEA-02027; CONICET (https://www.conicet.gov.ar/), grant for the International Cooperation Project CONICET/FAPESP 2016; Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, https://www.gov.br/capes/pt-br) (JGR). The funding sources had no involvement in relation to the study design, collection, analysis and interpretation of data, writing of the report and decision to submit the article for publication.
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Marcelo Larami Santoro: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. Ana Teresa Azevedo Sachetto: Methodology, Validation, Visualization, Writing – review & editing. Jaqueline Gomes Rosa: Methodology, Validation, Visualization, Writing – review & editing. Ricardo José Soares Torquato: Methodology, Validation, Visualization, Writing – review & editing. Débora Andrade-Silva: Methodology, Validation, Visualization, Writing – review & editing. Dilza Trevisan-Silva: Methodology, Validation, Visualization, Writing – review & editing. Cynthia Zaccanini de Albuquerque: Methodology, Validation, Writing – review & editing. Solange Maria de Toledo Serrano: Funding acquisition, Supervision. Vânia Gomes de Moura Mattaraia: Resources, Writing – review & editing. Aparecida Sadae Tanaka: Funding acquisition, Writing – review & editing. Maria Elisa Peichoto: Methodology, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing.
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Santoro, M.L., Sachetto, A.T.A., Rosa, J.G. et al. Jararaca GPIb-binding protein causes thrombocytopenia during Bothrops jararaca envenomation. Sci Rep 14, 31769 (2024). https://doi.org/10.1038/s41598-024-81851-8
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DOI: https://doi.org/10.1038/s41598-024-81851-8
