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The P-III metalloproteinase is a major component of the venom; it is glycosylated and has a molecular mass of 55 kDa. Mice were sacrificed one h after injection and tissue samples were obtained and routinely processed for embedding in paraffin and further staining with hematoxylin-eosin. Notice prominent hemorrhage in the pulmonary tissue in D arrow. No fluorescence was detected in the control tissues incubated with unlabeled SVMPs.

Control tissues were incubated with the SVMPs without labeling and no fluorescence was detected. Three-dimensional reconstitution of the images and analysis of co-localization were carried out with the IMARIS x64 7. Moreover, no significant differences were observed between co-localization of each SVMPs in the basement membrane of arterioles, capillaries, and PCV.

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Homogenates of the sections of hemorrhagic skin were analyzed by immunoblot for the detection of several ECM components. With regard to the immunodetection of type IV collagen, samples from control skin injected with saline solution showed a predominant band of kDa, with additional faint bands of kDa, kDa, kDa, kDa, and 97 kDa Fig 3A. A conspicuous degradation band of 97 kDa was observed in samples from skins injected with the three SVMPs, together with a reduction in the intensity of the kDa band Fig 3A. After 15 min, mice were sacrificed, their skin was removed, and an area of 12 mm diameter was dissected out.

Tissues of the same group were homogenized and centrifuged, and the supernatant collected. The reaction was detected using an anti-rabbit peroxidase antibody and a chemiluminescent substrate. When laminin was immunodetected in skin homogenates, a predominant band of kDa was observed, with additional bands of kDa and kDa Fig 3C. Samples from skin of mice injected with saline showed a predominant band of kDa and few additional minor bands Fig 3D. PI SVMP induced extensive degradation of nidogen, as evidenced by the disappearance of the kDa band, and the appearance of a 47 kDa degradation band.

Western blot analysis of exudates collected 15 min after injection of the enzymes revealed both similarities and differences between the three SVMPs. A relatively similar pattern was observed in the case of type IV collagen, with the presence of a predominant band of 90 kDa Fig 4A. A highly variable pattern of immunoreactivity was observed in exudates when tested for type VI collagen degradation products Fig 4B.

After 15 min, mice were sacrificed, a 5 mm incision was made in the skin overlying the injected muscle, and a heparinized capillary tube was introduced under the skin to collect the wound exudate fluid; exudate samples from a single treatment were then pooled. Proteins were classified within the following groups, and subgroups: a serum proteins; b proteins of the coagulation cascade; c proteinase inhibitors of plasma; d intracellular proteins; e keratins; f ECM proteins; and g membrane-associated proteins.

S2 — S4 Tables depict the quantitative values of serum proteins, proteins of the coagulation cascade and proteinase inhibitors, respectively. In general, similar values were observed for the vast majority of proteins in exudates collected from mice injected with the three SVMPs, especially in those with greatest quantitative values. Relatively minor differences were observed in the serum proteins S2 Table. Similar amounts of hemoglobin chains occurred in exudates obtained from mice injected with the three SVMPs S5 Table , as expected from the similar extent of hemorrhage induced.

Likewise, similar amounts of many other proteins characterized the three types of exudates. Results on keratins are presented separately S6 Table owing to the relevance of skin damage induced by snake venoms. As shown in Table 2 , no differences were observed in detected BM proteins heparin sulfate proteoglycan and nidogen. In contrast, there were differences in other ECM proteins.

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Identification of the various proteins in ranges of molecular mass lower than the known mass of the native proteins were considered degradation fragments, and the percentage of the total amount of each protein corresponding to hydrolyzed bands was estimated. Lumican was not degraded by any SVMP. Seven membrane-associated proteins were detected in the exudates. With one exception, the amounts of these proteins did not differ more than 3-fold in exudates from mice injected with the three types of SVMPs S7 Table. Since one of the main goals of this work was to gain further insights into the mechanisms of SVMP-induced microvessel damage leading to hemorrhage, the doses of SVMPs injected were standardized as to induce the same extent of hemorrhagic lesions.

It was hypothesized that, in these experimental conditions, the ECM proteins whose hydrolysis is directly responsible for microvessel damage should be degraded to a similar extent by the three enzymes. It has been proposed that one of the basis for the higher hemorrhagic activity of PIII SVMPs, as compared to PI enzymes, has to do with the ability of the former to locate in specific sites in microvasculature of tissues [ 9 , 13 , 18 ].

This has been demonstrated for the case of jararhagin, a PIII SVMP of the venom of Bothrops jararaca , where selective binding to microvessels and a pattern of co-localization of jararhagin and type IV collagen was described [ 8 ]. In addition, the Dis-like domain of jararhagin might contain sequences that mediate its binding with different types of collagen [ 9 ]. Our observations on the tissue localization of the three SVMPs in an ex vivo model conclusively demonstrate, using a quantitatively morphometric approach, the different pattern of distribution of PI and PIII SVMPs, since the former shows a more widespread pattern, whereas the latter preferentially bind to the microvessels and clearly co-localizes with type IV collagen.

Hence, the dimeric PII SVMP, containing only metalloproteinase and disintegrin domains, is preferentially located in the microvasculature. The possible pathological effects of SVMPs on these components of the microvasculature, in addition to capillaries, has not been studied and deserve consideration in order to fully understand the vascular pathology in snakebite envenoming. These observations suggest that even though the high hemorrhagic activity of PII and PIII SVMPs largely depends on their ability to selectively bind to microvessels, other factors also determine the hemorrhagic potential of these enzymes.

Differences in the turnover rate of hydrolysis of relevant substrates in the BM, especially of type IV collagen, may play a key role. Alternatively, PII and PIII SVMPs might present differences in the exosites in the Dis, Dis-like and Cys-rich domains, which determine subtle variations in the localization of these enzymes in the relevant substrates, with the consequent functional effects related to the proteolysis-induced mechanical destabilization of BM structure. Another possible explanation has to do with differences in the stability of these enzymes in the tissues, with more stable enzymes exerting a higher hemorrhagic effect.

This subject deserves further investigation. Since the hemorrhagic activity of SVMPs is likely to depend on the hydrolysis of BM components [ 8 , 18 , 22 ], particular attention was placed in this work to the analysis of degradation of BM proteins. Proteomics analysis of exudate collected in the vicinity of the hemorrhagic areas only detected perlecan and nidogen, and no differences were observed between the three SVMP classes regarding BM proteins.

No protein fragments of laminin and type IV collagen were detected in this analysis.

Extracellular Matrix Degradation

However, more sensitive immunochemical assessment of skin and exudates revealed subtle variations which might shed light on the mechanisms of hemorrhagic activity. Different patterns of hydrolysis were observed regarding nidogen, laminin and type VI collagen. In contrast, there were evident similarities between the three enzymes concerning hydrolysis of type IV collagen. This has interesting implications because a previous study identified type IV collagen as a likely candidate to play a key role in the onset of hemorrhagic activity, since hemorrhagic and non-hemorrhagic SVMPs differ in the extent of hydrolysis of this collagen [ 22 ].

Our present findings are therefore compatible with the hypothesis that degradation of type IV collagen is critical for microvessel damage and hemorrhage. This in turn agrees with the known role of this type of collagen in the mechanical stability of the BM [ 24 — 27 , 38 ], mostly owing to the presence of a covalently-linked network formed by this BM component [ 24 ]. In contrast to type IV collagen, hydrolysis of nidogen, laminin and type VI collagen by the three SVMPs showed differences both in the degradation patterns and in the intensity of the bands observed by immunoblotting of exudates, where the PI enzyme showed a more extensive degradation of these substrates.

These observations, in the context of a similar extent of hemorrhage by the three SVMPs, suggest that the hydrolysis of nidogen, laminin and type VI collagen might not be directly associated with the onset of microvessel damage leading to hemorrhage but rather may be a general by-product of microvessel damage. On the other hand, there were notorious differences in the amounts of other ECM proteins, which are not BM components, in the proteomic analysis of exudates collected from mice injected with the three toxins.

This observation may be due to two factors: since a higher absolute amount of this enzyme was injected, owing to its lower hemorrhagic activity, there was a higher proteolytic activity in the tissue, thus resulting in higher hydrolysis. The higher extent of hydrolysis in the case of the PI SVMP observed by immunoblotting of exudates supports this hypothesis.

In contrast, the PI SVMP, being devoid of such exosites, would have less restriction to hydrolyze an ample spectrum of ECM substrates, as observed in our proteomics results.

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As would be expected, the majority of ECM proteins detected in exudates corresponded to proteolytic fragments, on the basis of their molecular mass. The different pattern of hydrolysis by the SVMP of various ECM components, as detected by Western blot analysis, might be due to variations in the cleavage site preferences among these enzymes. Alternatively, this might depend on the presence of exosites in the non-metalloproteinase domains, which target these enzymes to different substrates in the ECM including BM or to different sequences in particular substrates, as observed by Serrano et al.

In addition to ECM proteins, proteomic analysis of exudates allows the detection of serum, intracellular and membrane-associated proteins, and these findings may shed light on the pathological action of SVMPs from a broader perspective. Similar quantitative patterns of plasma-derived proteins were detected in exudates from mice injected with the three toxins.

Extracellular matrix degradation by metalloproteinases and central nervous system diseases.

This seems logical, as the presence of these proteins is largely a consequence of overt microvessel damage by the action of these enzymes. Hence, extravasation of blood results in similar amounts of serum plasma proteins, and of hemoglobin as well. An exception to this general trend was observed with hydrolysis products of fibrinogen, which were in higher amounts in exudates from animals injected with the PI SVMP.

Since this enzyme has fibrinolytic activity [ 30 ] and was injected in a higher dose than the other two SVMPs, this may have resulted in hydrolysis of the fibrin formed as a consequence of extravasation and clot formation. This suggests that these enzymes induce a higher cytotoxic activity in various cell types in the tissue.

The three SVMPs induced a similar extent of skeletal muscle damage, as revealed by the similar amounts of the cytosolic muscle cell marker creatine kinase in exudates; it has been suggested that hemorrhagic SVMPs induce myotoxicity as a consequence of tissue ischemia [ 41 ]. The higher amounts of several intracellular markers in exudates collected from PII and PIII SVMPs-injected mice may be due to the targeting of these enzymes, through exosites present in the additional domains, to sites in the plasma membrane or in the vicinity of cells, a hypothesis that remains to be investigated.

Furthermore, immunochemical results support the hypothesis that hydrolysis of type IV collagen is likely to be a key event in SVMP-induced microvessel damage and destabilization leading to hemorrhage. This work was performed in partial fulfillment of the requirements for the PhD degree for Cristina Herrera at Universidad de Costa Rica. Abstract Snake venom hemorrhagic metalloproteinases SVMPs of the PI, PII and PIII classes were compared in terms of tissue localization and their ability to hydrolyze basement membrane components in vivo , as well as by a proteomics analysis of exudates collected in tissue injected with these enzymes.

Author Summary Local and systemic hemorrhage are typical manifestations of envenomings by viperid snakes. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files.

Introduction Zinc-dependent enzymes of the M12 reprolysin family of metalloproteinases are abundant components in the venoms of snakes, especially from species classified in the family Viperidae [ 1 ]. Ethics statement All in vivo experiments were performed in CD-1 mice. Experiments in vivo Immunochemical detection of ECM proteins in the skin and exudate. Download: PPT. Fig 1. Table 1.

Fig 2. The characteristic helical diffraction peak at a spacing of 0. The scatter, centred at 0. Integration of the peaks allowed analysis of the collagen:gelatin ratio. During the time course of treatment, gelatinisation occurred within the tissue samples treated with both MMP—enriched media and prostaglandin.

This process appeared to be more acute in human sclera compared to porcine: occurring after 24 hours of incubation in human tissue and over a period of 72 hours in porcine tissue. The same degree of gelatinisation was not observed in control media. The targeting of MMP activity has great potential for a future therapeutic intervention in glaucoma.

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