BACKGROUND
after a tissue injury, platelets adhere to the injured blood vessels, which are in direct contact with the exposed collagen in the extracellular matrix.
this induces the release of cytokines, growth factors and numerous pro-inflammatory mediators, leading to platelet aggregation and the activation of intrinsic and extrinsic pathways of coagulation and fibrin clot formation  <cit> .
cellular events that occur during wound healing are initiated, maintained and mediated by a complex series of biochemical events that start with the release of cytokines and growth factors by platelets  <cit> .
there is evidence that the proteins secreted by the platelet α-granules not only produce hemostasis but also induce modulation of inflammation and wound healing  <cit> .
the use of blood-derived products to seal wounds and to stimulate wound healing began with the use of fibrin glues composed of concentrated fibrinogen  and autologous fibrin glues, which are considered the best choice for avoiding viral infection  <cit> .
whitman et al.
<cit>  described the use of platelet concentrates to improve healing and to replace fibrin glues.
autologous platelet concentrate  preparations constitute a relatively new biotechnology in regenerative medicine.
they can be used for the modulation, stimulation and acceleration of tissue healing and bone regeneration in human beings  <cit>  and horses  <cit> .
variations in some of the key properties of the pc, including platelet concentration, the type of clot activator, and leukocyte content, can markedly influence the biological effects of this biodrug  <cit> .
the fibrin network supports the platelet and leukocyte concentrate during its application.
the density of the fibrin network is mainly determined by the concentration of fibrinogen during pc preparation  <cit> .
most protocols for pc preparation result in low-density fibrin gels after activation.
these kinds of gels are convenient for surgical applications, but they lack a true fibrin support matrix.
in contrast, a high-density fibrin network means that the platelet concentrate can be considered a biomaterial  <cit> , and the fibrin matrix itself might have potential healing effects  <cit> .
there is some information available on the electron microscopy characteristics of platelet and fibrin networks in humans  <cit>  and other animal species  <cit> .
however, to our knowledge, there have been few studies on the electron microscopy characteristics of platelet and fibrin networks in clots of pc  from dogs and cats encountered in experimental and clinical settings.
this study describes the electron microscopy characteristics of canine and feline pc activated with a non-proteic activating substance  or the combination of a proteic activating substance  with calcium gluconate .
the aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots  derived from pc collected by the tube method  <cit>  and activated with gc or gcb in dogs and cats.
RESULTS
the average hematological characteristics for dog whole blood were as follows: platelet count 345300/μl, hematocrit  <dig> %, white blood cell count  16600/μl, lymphocytes 5050/μl, monocytes 790/μl, granulocytes 10700/μl, mean platelet volume   <dig>  fl and platelet distribution width   <dig> %.
values for cat whole blood were for platelet count of 412330/μl, hematocrit  <dig> %, wbc 10180/μl, lymphocytes 2950/μl, monocytes 630/μl, granulocytes 6620/μl, mpv  <dig>  fl and platelet distribution width  <dig> %.
the average hematological characteristics for dog pc were as follows: platelet count 529288/μl, hematocrit  <dig> %, white blood cell count  7391/μl, lymphocytes 3390/μl, monocytes 178/μl, granulocytes 3823/μl, mpv  <dig>  fl and pdw  <dig> %.
the average hematological characteristics for cat pc were as follows: platelet count 863312/μl, hematocrit  <dig> %, wbc 4280/μl, lymphocytes 2930/μl, monocytes 120/μl, granulocytes 1230/μl, mpv  <dig>  fl and pdw  <dig> %.
after activation and incubation, all of the canine platelets displayed lysis and loss of their normal architecture, which corresponds to type iii in the classification of platelet morphological changes  <cit> .
platelet morphological changes in cats included full activation in all platelets, an irregular oval shape with centralization of organelles and the extension of pseudopodia   <cit> .
the samples from the cats showed a statistically significant difference between the areas  or percentage areas of platelets and fibrin fibers, as well as the ratios of the minor and major axis and the ratios of the major and minor axes .
the median number of α-granules of platelets activated with cg was  <dig>  , and for platelets activated with cgb the median was  <dig>  .
these differences were statically significant  by the wilcoxon test.
a, denotes statistically significant differences between rows by t-test .
the samples from the dogs showed statistically significant differences between cg and cgb activation  for the intercellular area , percentage of intercellular space, fibrin fiber area , the percentage area of fibrin fibers and the width of fibrin fibers in clots .
ultrastructural characteristics of fibrin clots from canine platelet concentrates activated with calcium gluconate or calcium gluconate plus batroxobin )
a, denotes statistically significant differences between rows by t-test .
discussion
the results of this study prevented a comparison between the platelet gels  obtained from the activated pc of dogs and cats.
the ultrastructural differences observed within each species show that there are differences in both platelet response and fibrin polymerization.
in addition, the platelets from cats and dogs are quite different.
the size of the cat platelets   <cit>  differs from the size of the dog platelets diameter   <cit> , and more complex factors such as glycoprotein receptors and signal transduction pathways might be involved in the structural platelet responses observed in our study  <cit> .
according to sweet et al.
<cit> , platelet shape is approximately elliptical, and the ratio between the minor and major axes is  <dig> .
mody and king  <cit>  thought that, in general, the elliptical shape is determined by the ratio between the minor and major axes, which is always < <dig>  but when this shape becomes more elongated, the ratio is reversed so that the determination is being made by the ratio between major and minor axes, which is always > <dig>  <cit> .
in our study, the platelets from cats activated with calcium gluconate show a ratio between minor and major axes of  <dig> , and they show a ratio between major and minor axes of  <dig> .
the cat platelets activated with batroxobin show a ratio between minor and major axes of  <dig>  and a ratio between major and minor axes of  <dig> , which puts them closer to the elliptical shape.
this suggests a greater change of the elliptical shape in platelets activated with calcium gluconate.
cat platelets activated with cgb occupied a greater percentage of total area than platelets activated with cg, and they also had a greater number of α-granules in their cytoplasm.
these findings, along with the relationships between the axes, suggested a moderate degree of platelet activation  <cit> .
platelet gel from dogs activated with cg presented fibrin fiber widths of  <dig>  nm and  <dig>  nm.
these findings are within the range described for thick fibers in horses , human beings , monkeys , oryxes , sheep , penguins  and sea turtles   <cit> .
however, pretorius et al.
used scanning electron microscopy and did not provide information about the ultrastructural characteristics of the platelets contained in the prp.
pretorius et al.
<cit>  also described the fine and intermediate fibrin fibers for the aforementioned species.
however, these kinds of fibrin fibers were not observed in our study.
this may indicate that canine platelet gel from pc activated with either cg or cgb produces only fibrin fibers of higher quality compared with other species .
however, our results show that cgb produces thinner fibrin fibers than those from human prp activated with thrombin  <cit> .
our results clearly demonstrate that the activating substance influences the quality of the resulting biomaterial .
the smaller area and percentage area of intercellular space observed in the fibrin clots from canine pc activated with calcium gluconate suggest greater cell aggregation.
these findings, in conjunction with the greater area and percentage of area of fibrin fibers and the greater width of the fibrin fibers, indicate a strong degree of platelet activation with rapid formation of fibrin clots, and the fibers in these clots are thicker than those from pc activated with cgb.
it is important to consider that platelet activation induced by cg is the result of two different signaling events: the direct effect of ca2+ on platelets  <cit>   and the effect of autogenously generated thrombin via ca2+ dependent activation of the coagulation pathway  <cit> .
we are not aware of reports that describe the ultrastructural characteristics of platelet gels from dogs and cats, but the general findings of this study indicate that, regardless of the substance used for pc activation, cat platelets require a longer incubation time than dog platelets to reach the type iv morphological classification  <cit> .
the differences observed between dog and cat clots activated with either cg or cgb could depend on the mechanisms of action of the two activating substances.
it is known that batroxobin does not activate platelets trapped within the fibrin network  <cit> .
in this study, however, batroxobin was reconstituted with calcium gluconate, and the amount of calcium was proportionally less when pc was activated only with calcium gluconate.
the results observed in this study corroborate the fact that, when batroxobin is used for pc activation, there is no massive release of the contents of platelet alpha granules, in contrast with the effect produced by the calcium salts alone  <cit> .
CONCLUSIONS
the findings of this study suggest that activation with calcium gluconate produces a massive release of alpha granules, at least during the study period .
on the other hand, batroxobin prevented early platelet degranulation  <cit> .
calcium gluconate also produced a better fibrin network than cgb in both species.
the results of this study suggest that pc activated with cg or cgb produces different rates of polymerization, which could cause different speeds of incorporation of the circulating cytokines into the fibrin meshes .
therefore, the configuration of the fibrin mesh implies differences in the lifespans of these cytokines  <cit> .
this could mean that pc activated with cg produces a thicker fibrin biomaterial and a faster release of gf and cytokines and that pc activated with cgb produces a slow release of growth factors .

 after a tissue injury, platelets adhere to the injured blood vessels, which are in direct contact with the exposed collagen in the extracellular matrix. this induces the release of cytokines, growth factors and numerous pro-inflammatory mediators, leading to platelet aggregation and the activation of intrinsic and extrinsic pathways of coagulation and fibrin clot formation. cellular events that occur during wound healing are initiated, maintained and mediated by a complex series of biochemical events that start with the release of cytokines and growth factors by platelets. there is evidence that the proteins secreted by the platelet α-granules not only produce hemostasis but also induce modulation of inflammation and wound healing. the use of blood-derived products to seal wounds and to stimulate wound healing began with the use of fibrin glues composed of concentrated fibrinogen and autologous fibrin glues, which are considered the best choice for avoiding viral infection. whitman et al. described the use of platelet concentrates to improve healing and to replace fibrin glues. autologous platelet concentrate preparations constitute a relatively new biotechnology in regenerative medicine. they can be used for the modulation, stimulation and acceleration of tissue healing and bone regeneration in human beings and horses. variations in some of the key properties of the pc, including platelet concentration, the type of clot activator, and leukocyte content, can markedly influence the biological effects of this biodrug. the fibrin network supports the platelet and leukocyte concentrate during its application. the density of the fibrin network is mainly determined by the concentration of fibrinogen during pc preparation. most protocols for pc preparation result in low-density fibrin gels after activation. these kinds of gels are convenient for surgical applications, but they lack a true fibrin support matrix. in contrast, a high-density fibrin network means that the platelet concentrate can be considered a biomaterial, and the fibrin matrix itself might have potential healing effects. there is some information available on the electron microscopy characteristics of platelet and fibrin networks in humans and other animal species. however, to our knowledge, there have been few studies on the electron microscopy characteristics of platelet and fibrin networks in clots of pc from dogs and cats encountered in experimental and clinical settings. this study describes the electron microscopy characteristics of canine and feline pc activated with a non-proteic activating substance or the combination of a proteic activating substance with calcium gluconate. the aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots derived from pc collected by the tube method and activated with gc or gcb in dogs and cats.  the average hematological characteristics for dog whole blood were as follows: platelet count 345300/μl, hematocrit %, white blood cell count 16600/μl, lymphocytes 5050/μl, monocytes 790/μl, granulocytes 10700/μl, mean platelet volume  fl and platelet distribution width %. values for cat whole blood were for platelet count of 412330/μl, hematocrit %, wbc 10180/μl, lymphocytes 2950/μl, monocytes 630/μl, granulocytes 6620/μl, mpv  fl and platelet distribution width %. the average hematological characteristics for dog pc were as follows: platelet count 529288/μl, hematocrit %, white blood cell count 7391/μl, lymphocytes 3390/μl, monocytes 178/μl, granulocytes 3823/μl, mpv  fl and pdw %. the average hematological characteristics for cat pc were as follows: platelet count 863312/μl, hematocrit %, wbc 4280/μl, lymphocytes 2930/μl, monocytes 120/μl, granulocytes 1230/μl, mpv  fl and pdw %. after activation and incubation, all of the canine platelets displayed lysis and loss of their normal architecture, which corresponds to type iii in the classification of platelet morphological changes. platelet morphological changes in cats included full activation in all platelets, an irregular oval shape with centralization of organelles and the extension of pseudopodia. the samples from the cats showed a statistically significant difference between the areas or percentage areas of platelets and fibrin fibers, as well as the ratios of the minor and major axis and the ratios of the major and minor axes. the median number of α-granules of platelets activated with cg was, and for platelets activated with cgb the median was. these differences were statically significant by the wilcoxon test. a, denotes statistically significant differences between rows by t-test. the samples from the dogs showed statistically significant differences between cg and cgb activation for the intercellular area, percentage of intercellular space, fibrin fiber area, the percentage area of fibrin fibers and the width of fibrin fibers in clots. ultrastructural characteristics of fibrin clots from canine platelet concentrates activated with calcium gluconate or calcium gluconate plus batroxobin ) a, denotes statistically significant differences between rows by t-test. discussion the results of this study prevented a comparison between the platelet gels obtained from the activated pc of dogs and cats. the ultrastructural differences observed within each species show that there are differences in both platelet response and fibrin polymerization. in addition, the platelets from cats and dogs are quite different. the size of the cat platelets differs from the size of the dog platelets diameter, and more complex factors such as glycoprotein receptors and signal transduction pathways might be involved in the structural platelet responses observed in our study. according to sweet et al., platelet shape is approximately elliptical, and the ratio between the minor and major axes is. mody and king thought that, in general, the elliptical shape is determined by the ratio between the minor and major axes, which is always < but when this shape becomes more elongated, the ratio is reversed so that the determination is being made by the ratio between major and minor axes, which is always >. in our study, the platelets from cats activated with calcium gluconate show a ratio between minor and major axes of, and they show a ratio between major and minor axes of. the cat platelets activated with batroxobin show a ratio between minor and major axes of and a ratio between major and minor axes of, which puts them closer to the elliptical shape. this suggests a greater change of the elliptical shape in platelets activated with calcium gluconate. cat platelets activated with cgb occupied a greater percentage of total area than platelets activated with cg, and they also had a greater number of α-granules in their cytoplasm. these findings, along with the relationships between the axes, suggested a moderate degree of platelet activation. platelet gel from dogs activated with cg presented fibrin fiber widths of  nm and  nm. these findings are within the range described for thick fibers in horses, human beings, monkeys, oryxes, sheep, penguins and sea turtles. however, pretorius et al. used scanning electron microscopy and did not provide information about the ultrastructural characteristics of the platelets contained in the prp. pretorius et al. also described the fine and intermediate fibrin fibers for the aforementioned species. however, these kinds of fibrin fibers were not observed in our study. this may indicate that canine platelet gel from pc activated with either cg or cgb produces only fibrin fibers of higher quality compared with other species. however, our results show that cgb produces thinner fibrin fibers than those from human prp activated with thrombin. our results clearly demonstrate that the activating substance influences the quality of the resulting biomaterial. the smaller area and percentage area of intercellular space observed in the fibrin clots from canine pc activated with calcium gluconate suggest greater cell aggregation. these findings, in conjunction with the greater area and percentage of area of fibrin fibers and the greater width of the fibrin fibers, indicate a strong degree of platelet activation with rapid formation of fibrin clots, and the fibers in these clots are thicker than those from pc activated with cgb. it is important to consider that platelet activation induced by cg is the result of two different signaling events: the direct effect of ca2+ on platelets and the effect of autogenously generated thrombin via ca2+ dependent activation of the coagulation pathway. we are not aware of reports that describe the ultrastructural characteristics of platelet gels from dogs and cats, but the general findings of this study indicate that, regardless of the substance used for pc activation, cat platelets require a longer incubation time than dog platelets to reach the type iv morphological classification. the differences observed between dog and cat clots activated with either cg or cgb could depend on the mechanisms of action of the two activating substances. it is known that batroxobin does not activate platelets trapped within the fibrin network. in this study, however, batroxobin was reconstituted with calcium gluconate, and the amount of calcium was proportionally less when pc was activated only with calcium gluconate. the results observed in this study corroborate the fact that, when batroxobin is used for pc activation, there is no massive release of the contents of platelet alpha granules, in contrast with the effect produced by the calcium salts alone.  the findings of this study suggest that activation with calcium gluconate produces a massive release of alpha granules, at least during the study period. on the other hand, batroxobin prevented early platelet degranulation. calcium gluconate also produced a better fibrin network than cgb in both species. the results of this study suggest that pc activated with cg or cgb produces different rates of polymerization, which could cause different speeds of incorporation of the circulating cytokines into the fibrin meshes. therefore, the configuration of the fibrin mesh implies differences in the lifespans of these cytokines. this could mean that pc activated with cg produces a thicker fibrin biomaterial and a faster release of gf and cytokines and that pc activated with cgb produces a slow release of growth factors. 
in both species, a statistically significant difference was found between the fibrin fiber measurements in the pc clots activated with cg and cgb. dog platelets displayed lysis with loss of normal architecture. the aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates that had been activated with calcium gluconate or cg plus batroxobin. platelets from fibrin clots were classified according their morphological changes. the platelet area, the area of fibrin fibers, the ratio of the minor and major axes of platelets, the ratio of the major and minor axes of platelets, and the number of α-granules found within platelets were measured for the cat clots. the findings suggest that activation with cg caused platelet alpha granules to release their contents. the area of the intercellular space, the area of the fibrin fibers, and the width of the fibrin fibers were determined for the dog clots. in dogs, activation with cg produced thick fibrin fibers.
 the aim of this study was to use transmission electron microscopy to describe the ultrastructural characteristics of clots obtained from canine and feline platelet concentrates that had been activated with calcium gluconate or cg plus batroxobin. platelets from fibrin clots were classified according their morphological changes. the area of the intercellular space, the area of the fibrin fibers, and the width of the fibrin fibers were determined for the dog clots. the platelet area, the area of fibrin fibers, the ratio of the minor and major axes of platelets, the ratio of the major and minor axes of platelets, and the number of α-granules found within platelets were measured for the cat clots.  cat platelets displayed full activation. dog platelets displayed lysis with loss of normal architecture. in both species, a statistically significant difference was found between the fibrin fiber measurements in the pc clots activated with cg and cgb.  the findings suggest that activation with cg caused platelet alpha granules to release their contents. in cats, fibrin production was greater when the pc was activated with cg. in dogs, activation with cg produced thick fibrin fibers. autologous platelet concentratefibrin clotultrastructural microscopy 