THE EVALUATION OF CLOTTING TIME IN BOVINE THROMBIN, REPTILASE ® , AND THROMBIN-LIKE FRACTION OF Crotalus durissus terrificus VENOM USING BOVINE, EQUINE, OVINE, BUBALINE AND HUMAN CRYOPRECIPITATES

Abstract

The objective of this study was to evaluate the effects of the thrombin-like fraction of Crotalus durissus terrificus venom, Reptilase<img SRC="http:/img/fbpe/jvat/v4n2/image119.gif"> , and bovine thrombin of fibrinogen pools on bovine, equine, ovine, bubaline and human cryoprecipitates. The authors also made a comparative study between animal and human cryoprecipitates to see if any there was any possibility of future use in medicine. Fibrinogen levels in cryoprecipitate were studied using 48 blood samples obtained as follows:12 samples from humans, 9 from bovine, 10 from equine, 10 from ovine and 7 from bubaline. The results obtained showed average levels of 375.50 mg % for humans, 218.33 mg % for bovine, 240.80 mg % for equine, 267.70 mg % for ovine and 664.00 mg % for bubaline. Upon the formation of pools of human and animal fibrinogens, the following results were obtained: 435 mg % for humans, 444 mg % for bovine, 337 mg % for equine, 390 mg % for ovine and 530 mg % for bubaline. Statistical analysis (using the analysis of variance for entirely randomized experiment for the calculation of F statistics) demonstrated that the bubaline fibrinogen level was higher than that of human, and both were higher than those of ovine, equine, and bovine. Clotting times were determined using different dilutions of bovine thrombin, thrombin-like fraction of Crotalus durissus terrificus venom, and Reptilase<img SRC="http:/img/fbpe/jvat/v4n2/image119.gif"> . Comparing these clotting times, results for human and bovine were found to be very similar, whereas using equine, ovine, and bubaline the results above a dilution of 1:3 were markedly different. The results obtained permitted the following conclusions to be drawn show that: 1) bovine thrombin presented better interactivity with fibrinogen extracted both from human and bovine cryoprecipitates; 2) there was similar behavior when bovine thrombin was substituted for Reptilase<img SRC="http:/img/fbpe/jvat/v4n2/image119.gif"> and for the thrombin-like fraction of Crotalus durissus terrificus venom; 3) cryoprecipitate from bovine can, in special circumstances, substitute human cryoprecipitate in medical practice; 4) human and bovine cryoprecipitates can be used with both Reptilase<img SRC="http:/img/fbpe/jvat/v4n2/image119.gif"> and Crotalus durissus terrificus fractions using a dilution up to 1:5; 5) the use of bovine cryoprecipitate can be recommended using either bovine thrombin, Reptilase<img SRC="http:/img/fbpe/jvat/v4n2/image119.gif"> , or thrombin-like fraction of Crotalus durissus terrificus venom.

Thrombin-like fraction; bovine thrombin; Reptilase<img SRC="http:/img/fbpe/jvat/v4n2/image119.gif">; cryoprecipitate

Original paper

THE EVALUATION OF CLOTTING TIME IN BOVINE THROMBIN, REPTILASE , AND THROMBIN-LIKE FRACTION OF Crotalus durissus terrificus VENOM USING BOVINE, EQUINE, OVINE, BUBALINE AND HUMAN CRYOPRECIPITATES

I. A.THOMAZINI-SANTOS

, M. J. S. M. GIANNINI , E. TOSCANO , P.E.A. MACHADO , C. R. G. LIMA , B. BARRAVIERA .

1 Laboratory of Hemostasis of the Blood Bank of the School of Medicine of Botucatu, 2 Laboratory of Micology of the School of Pharmaceutical Sciences of Araraquara, 3 Departament of Tropical Diseases, 4 Center for the Study of Venoms and Venomous Animals - CEVAP - UNESP - Botucatu, SP, Brazil.

ABSTRACT. The objective of this study was to evaluate the effects of the thrombin-like fraction of Crotalus durissus terrificus venom, Reptilase , and bovine thrombin of fibrinogen pools on bovine, equine, ovine, bubaline and human cryoprecipitates. The authors also made a comparative study between animal and human cryoprecipitates to see if any there was any possibility of future use in medicine. Fibrinogen levels in cryoprecipitate were studied using 48 blood samples obtained as follows:12 samples from humans, 9 from bovine, 10 from equine, 10 from ovine and 7 from bubaline. The results obtained showed average levels of 375.50 mg % for humans, 218.33 mg % for bovine, 240.80 mg % for equine, 267.70 mg % for ovine and 664.00 mg % for bubaline. Upon the formation of pools of human and animal fibrinogens, the following results were obtained: 435 mg % for humans, 444 mg % for bovine, 337 mg % for equine, 390 mg % for ovine and 530 mg % for bubaline. Statistical analysis (using the analysis of variance for entirely randomized experiment for the calculation of F statistics) demonstrated that the bubaline fibrinogen level was higher than that of human, and both were higher than those of ovine, equine, and bovine. Clotting times were determined using different dilutions of bovine thrombin, thrombin-like fraction of Crotalus durissus terrificus venom, and Reptilase . Comparing these clotting times, results for human and bovine were found to be very similar, whereas using equine, ovine, and bubaline the results above a dilution of 1:3 were markedly different. The results obtained permitted the following conclusions to be drawn show that: 1) bovine thrombin presented better interactivity with fibrinogen extracted both from human and bovine cryoprecipitates; 2) there was similar behavior when bovine thrombin was substituted for Reptilase and for the thrombin-like fraction of Crotalus durissus terrificus venom; 3) cryoprecipitate from bovine can, in special circumstances, substitute human cryoprecipitate in medical practice; 4) human and bovine cryoprecipitates can be used with both Reptilase and Crotalus durissus terrificus fractions using a dilution up to 1:5; 5) the use of bovine cryoprecipitate can be recommended using either bovine thrombin, Reptilase , or thrombin-like fraction of Crotalus durissus terrificus venom.

KEY WORDS: Thrombin-like fraction, bovine thrombin, Reptilase , cryoprecipitate.

INTRODUCTION

Hematological disorders caused by animal envenoming are of an old-age concern to researchers worldwide(23,41). Since the 1960's in Brazil, alterations in blood coagulation have been investigated both in vitro(9,18,32,33,43) and in vivo (2,3,20-22).

According to medical literature, Bothrops venom acts on blood coagulation through several mechanisms, such as direct action on fibrinogen, activation of factor X and prothrombin, as well as platelet activation.

Venom from Crotalus durissus terrificus has a thrombin-like fraction demonstrated by Nahas et al. (32) in 1964 and isolated by Raw et al. (40) in 1986. The thrombin-like fraction is believed to have the ability to transform fibrinogen directly into fibrin, producing afibrinogenemia in patients, causing an increase in clotting time. Thus, envenoming caused by these two snake genera may cause major blood coagulation disorders (19,48,49). In medical practice, some disorders can be minimized with the replacement of coagulation factors present in cryoprecipitate. Cryoprecipitate is a blood-derived substance obtained from fresh plasma which is composed of fibrinogen, von Willebrand's factor, factor XIII, fibronectin, etc (49).

Another substance that has a thrombin-like enzyme is Reptilase , which is extracted from the venom of Bothrops atrox (24).

The objective of this study was to evaluate the effects of the thrombin-like fraction of Crotalus durissus terrificus venom, Reptilase , and bovine thrombin of fibrinogen pools on bovine, equine, ovine, bubaline and human cryoprecipitates. The authors also made a comparative study between the animal and human cryoprecipitates to see if there was any possibility of future use in medicine.

MATERIALS AND METHODS

MATERIALS:

The following materials were used:

Cryoprecipitate samples extracted from:

Human blood - taken from 12 adult male volunteers between the ages of 18 and 42.

Bovine blood - taken from 9 adult animals.

Equine blood - taken from 10 adult animals.

Ovine blood - taken from 10 adult animals.

Bubaline blood - taken from 7 adult animals of indeterminate breed.

Thrombin-like fraction extracted from Crotalus durissus terrificus venom, provided by CEVAP. Reptilase , a thrombin-like fraction extracted from the venom of Bothrops atrox (Stago ). And, bovine thrombin (Baxter ).

METHODS:

The blood samples from which the cryoprecipitate was extracted were collected in the type of bags used for human donation.

After collection, the full blood bags were transported, at room temperature, to the Blood Bank at the School of Medicine of Botucatu, where they were processed within 6 hours of collection. The method was the same as that that is used for the fractionation of cryoprecipitate from human blood (16,34). The fibrinogen level present in cryoprecipitates was determined by the method used by Ratnoff and Menzie (39). The clotting time, using bovine thrombin and cryoprecipitates from different animals, was determined by the method used by Rapaport and Ames (38).

The thrombin-like fraction of Crotalus durissus terrificus venom was isolated using gel-filtration and affinity chromatography (Alexander et al. (1)). The thrombin-like fraction was also isolated using the method used by Seki et al. (42) and Raw et al. (40). After the fraction isolation, the amount of protein was determined using the method used by Lowry et al. (28).

Comparison of the fibrinogen levels obtained from the cryoprecipitates of the five studied groups was performed using analysis of variance for entirely randomized experiment with calculation of F statistics. Contrasts between the mean values of each group were studied using Scheffe's method, with =0.05 (52).

RESULTS

Forty-eight samples were processed to obtain cryoprecipitate for each group The individual concentration was determined in each sample as shown in Table 1.

Upon comparing the fibrinogen levels obtained from cryoprecipitates in the five groups studied, statistical analysis reveals the following: bubaline fibrinogen level (G5) was higher than human (G1); human fibrinogen level was higher than bovine (G2); bovine fibrinogen level was similar to equine (G3) and ovine (G4).

After preparation, the pools of cryoprecipitate from the five groups were tested to verify their fibrinogen level with the following results:

- Human = 435 %

- Bovine = 444 %

- Equine = 337 %

- Ovine = 390 %

- Bubaline = 530 %

Based on these results, one can see a decreasing order as follows: bubaline, bovine and human, ovine, and equine.

Figures 1 to 8 show the variations in clotting times against dilutions. Figure 1 shows that clotting times of bovine thrombin with the pools of human and bovine cryoprecipitates were similar at all tested dilutions. Clotting times for the pools of equine and bubaline cryoprecipitates were similar up to a dilution of 1:4. Clotting times for the pools of ovine and bubaline cryoprecipitates were similar up to a dilution of 1:32.

Figure 2 shows that clotting times of the thrombin-like fraction of Crotalus durissus terrificus venom with pools of human and bovine cryoprecipitates were similar. Clotting times for the pools of ovine, equine, and bubaline cryoprecipitates were similar up to a dilution of 1:3.

Figure 3 shows that clotting times of Reptilase with the pools of human, bovine, and equine cryoprecipitates were similar at dilutions of 1:4 and 1:5. Clotting times with the pools of bubaline and ovine cryoprecipitates were different from the first three, specifically at dilutions of 1:4 and 1:5.

Figure 4 shows that clotting times of the thrombin-like fraction of Crotalus durissus terrificus venom were similar to those of Reptilase with human cryoprecipitate.

Figure 5 shows that clotting times of the thrombin-like fraction of Crotalus durissus terrificus venom were similar to those of Reptilase with bovine cryoprecipitate.

Figure 6 shows that clotting times of the thrombin-like fraction of Crotalus durissus terrificus venom were different from those of Reptilase with equine cryoprecipitate at dilutions of 1:3 and above.

Figure 7 shows that clotting times of the thrombin-like fraction of Crotalus durissus terrificus venom were different from those of Reptilase with ovine cryoprecipitate at dilutions of 1:3 and above.

Figure 8 shows that clotting times of the thrombin-like fraction of Crotalus durissus terrificus venom were similar to those of Reptilase with bubaline cryoprecipitate up to a dilution of 1:3.

DISCUSSION

In this study the authors extracted and evaluated human, bovine, equine, ovine and bubaline cryoprecipitates. The following fibrinogen serum levels were obtained: bubaline=664 m g% (the highest concentration), human=375.50 mg %, ovine=267.70 mg %, equine=240.8 mg %, and bovine=218.33 mg %. These results show slight differences to those in the referred literature (13,50), since values obtained for bovine, equine, and ovine were lower than expected (11).

Pools of human and animal cryoprecipitates were prepared. Human, bovine, equine, and ovine pools showed increased levels of fibrinogen. This was probably due to an increase in concentration of plasma fibrinogen on the final cryoprecipitates.

In the referred literature, the fibrinogen molecule responds to the physiological action of thrombin and fibrinogen is transformed into fibrin (6,7,10,27).

Thrombin is known to be a serine-protease which contributes to clot formation by a cleavage of fibrinopeptide A (FPA) and fibrinopeptide B (FPB) of the A and Bß chains of the N-terminal chain of fibrinogen. Thrombin also activates factor XIII in blood coagulation to form the fibrin network. These mechanisms influence the fibrin structure and are of major importance to reducing the vulnerability to fibrinolysis action (12,14,25).

In this study the authors evaluated the pools of human and animal fibrinogen by determining their clotting times in increasing dilutions of bovine thrombin. It was observed that clotting times of human fibrinogen and bovine fibrinogen were quite similar.

It was also observed that the pools of equine, ovine, and bubaline fibrinogen showed higher clotting times than those of human and bovine. These observations led to a hypothesis that the break in the sequence of amino acids of the fibrinogen molecule occurred at different points in the five study groups (11). This may lead to a retardation in the polymerization of fibrin and clot formation which would explain the increased clotting times observed specifically in the equine, ovine, and bubaline study groups.

In this study a similar pattern was observed between the clotting times of human and bovine groups. This led to a hypothesis that human and bovine fibrinogen molecules have quite similar molecular structures.

In contrast, bubaline fibrinogen molecules showed the most difference when compared to those of human and bovine. This led to the suggestion that bubaline fibrinogen molecules might have quite different structures than those of human and the other animals.

In addition to thrombin, other enzymes, such as snake venoms act on fibrinogen transforming it into fibrin (4,29,46,51).

Venoms from different snake genera are known to be composed of water, proteins, and enzymes (1,40). Within the enzymes there are several fractions, among them, the thrombin-like fraction found in the venom of Agkristodon (47). Trimesurus (36), Crotalus (30), and Bothrops (17,45).

The thrombin-like enzyme was detected in over thirty-six venoms of the Crotalidae (44) family. Most of these thrombin-like enzymes are glycoproteins with molecular weights from 25,000 to 38,000 Da, with serine-reactive residues, and activity on synthetic substrates. Venoms which present a thrombin-like enzyme cleave fibrinogen and release fibrinopeptides. Some authors (35,37) reported that certain venoms preferentially release fibrinopeptide A, while other venoms fibrinopeptide B (26).

Batroxobin was the first enzyme isolated from Bothrops venom by von Klobusitzky and Koning in 1936 (24). This enzyme has low carbohydrate content and is used as a reagent commercially known as Reptilase . It is prepared from the venom of Bothrops atrox.

In this study human and animal fibrinogen pools were evaluated by determination of clotting times using increasing dilutions of Reptilase . It was observed that the clotting time for each dilution was longer than that obtained using bovine thrombin. These results suggest that this increase in clotting time might have occurred because batroxobin cleaves fibrinogen and releases only fibrinopeptide A (15,35), while bovine thrombin cleaves fibrinogen and releases both fibrinopeptides A and B (5,7,12,25). The clotting time of Reptilase was longer than that of bovine thrombin. Another possible explanation for this phenomenon would be that the enzyme extracted from snake venom does not specifically interact with fibrinogen.

In 1986, Raw et al. (40) isolated the thrombin-like fraction of Crotalus durissus terrificus venom with a molecular weight of 36,000 Da.

This study compared the effects of Reptilase and the thrombin-like fraction of Crotalus durissus terrificus venom on human, bovine, equine, and ovine fibrinogen pools using increasing dilutions.

Clotting times of human and bovine cryoprecipitates were shown to be quite similar. This was observed even at high dilutions and with both the enzymes studied. Clotting times of equine and ovine cryoprecipitates were different at dilutions of 1:3 and above. This difference increased at higher dilutions. Clotting times of bubaline were also similar up to a dilution of 1:3, however, above this dilution the results were markedly different.

These results corroborate the previous hypothesis that the molecular structure of human and bovine fibrinogen are quite similar. The explanation for this is that clotting times of Reptilase and the thrombin-like fraction of Crotalus durissus terrificus venom were similar.

On the other hand, the authors suggest that the bubaline fibrinogen molecule has a different chemical structure than that of human and bovine, as has previously been reported. The explanation for this is that the clotting time of bubaline was always different to the others using bovine thrombin, Reptilase , and the thrombin-like fraction of Crotalus durissus terrificus venom.

It should be emphasized that bovine thrombin demonstrated a better interactivity with the fibrinogens from human and bovine cryoprecipitates. Similar behavior was seen when substituting bovine thrombin either for Reptilase or for the thrombin-like fraction of Crotalus durissus terrificus venom. From this we can suppose that the molecular structure of both human and bovine fibrinogen are quite similar. So, the authors may conclude that bovine cryoprecipitate can, in special situations, be substituted for human cryoprecipitate in medical practice.

In this study, it was also possible to observe that human and bovine cryoprecipitates can be used with either Reptilase or the thrombin-like fraction of Crotalus durissus terrificus venom at dilutions up to 1:5.

The use of bovine cryoprecipitate can be recommended using either bovine thrombin, Reptilase , or the thrombin-like fraction of Crotalus durissus terrificus venom. These enzymes would produce coagulation of the cryoprecipitate, thus permitting the utilization of this procedure to stop hemorrhage, promoting faster healing of surgical wounds, and forming a protective membrane in large burned areas (8,31).

Finally, further toxicological studies are needed to validate the clinical use of either Reptilase or the thrombin-like fraction of Crotalus durissus terrificus venom.

TABLE 1. The distribution of individual concentration, mean values (M), standard deviation (SD), and coefficient of variation (CV) for fibrinogen levels obtained from cryoprecipitates measured in mg%.

FIGURE 1. Determination of clotting times with the pools of human, bovine, equine, ovine, and bubaline cryoprecipitates using different dilutions of bovine thrombin.

FIGURE 2. Determination of clotting times with the pools of human, bovine, equine, and bubaline cryoprecipitates using different dilutions of thrombin-like fraction of Crotalus durissus terrificus venom.

FIGURE 5.
The comparison of clotting times between the thrombin-like fraction of Crotalus durissus terrificus venom and Reptilase

FIGURE 7.
The comparison of clotting times of the thrombin-like fraction of Crotalus durissus terrificus venom and Reptilase

ACKNOWLEDGEMENTS

We are indebted to the Blood Bank at the School of Medicine of Botucatu for the donation of blood bags and reagents, to the School of Veterinary Medicine and Animal Husbandry for kindly providing the animals and to Miss Gislane Cristina Mastranjo for the processing of the cryoprecipitate of all samples.

Received 18 December 1997

Accepted 20 February 1998

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CORRESPONDENCE TO:

I. A. THOMAZINI-SANTOS - Divisão Hemocentro, Faculdade de Medicina de Botucatu - UNESP - CEP 18618-000, Botucatu, São Paulo, Brasil.

Publication Dates

• Publication in this collection
  26 Nov 1998
• Date of issue
  1998

History

• Received
  18 Dec 1997
• Accepted
  20 Feb 1998