Evaluation of platelet number and function and fibrinogen level in patients bitten by snakes of the Bothrops genus.

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R e v i s t a d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 ( l ) : 1 9 - 2 4 , j a n - m a r , 1 9 9 5 .

EVALUATION OF PLATELET NUMBER AND FUNCTION

AND FIBRINOGEN LEVEL IN PATIENTS BITTEN BY SNAKES

OF THE

B O T H R O P S

GENUS

Fábio Cardoso luan, Izolete Aparecida Thomazini, Iracema Carvalho, Delane Mercedes Gonçalves Carreira, Valter José Cassinelli, Paulo Câmara Marques

Pereira and Benedito Barraviera

P la te le t fu n c t i o n a n d p la s m a f i b r in o g e n le v e ls w e r e e v a lu a te d in 1 4 p a tie n ts , 1 0 m a le s a n d 4 f e m a l e s , a g e d 1 3 - 5 9 y e a r s b itte n b y Bothrops g e n u s s n a k e s . T h e r e w a s a s ta tis tic a l d iffe r e n c e ( p < 0 . 0 5 ) a m o n g p la s m a fib r i n o g e n le v e ls e v a lu a te d 2 4 a n d 4 8 h o u r s a fte r e n v e n o m a tio n . T h e r e w a s a te n d e n c y to w a r d s n o r m a liz a tio n a fte r 4 8 h o u r s o f tre a tm e n t. T h e lo w p la te le t n u m b e r w a s c le a r in 2 4 -4 8 h o u r e v a lu a tio n s w ith a te n d e n c y to w a r d s n o r m a liz a tio n a fte r 4 8 h o u r s o f tr e a tm e n t ( p < 0 .0 5 ) . W h en p la te le t f u n c tio n w a s s tim u la te d b y c o lla g e n a n d e p in e p h r in e , it a p p e a r e d to b e w ith in n o r m a l va lu es. O n th e o th e r h a n d , w h e n it w a s s tim u la te d b y a d e n o s in e d ip h o s p h a te (A D P ), p la te le t fu n c t i o n w a s h y p o a g g r e g a te d b y a s in g le m ic r o m o l c o n c e n tr a tio n u n til 4 8 h o u r s a fte r tre a tm e n t. A t a 3 m ic r o m o l c o n c e n tr a tio n , th e r e w e r e a lte r a tio n s o n ly b e fo r e s p e c ific tr e a tm e n t ( p < 0 . 05). F ib r in o g e n le v e ls a n d f i b r i n d e g r a d a tio n p r o d u c t (F D P ) le v e ls a p p e a r e d to b e a lte r e d in 8 3 .3 3 % o f p a tie n ts e v a lu a te d . T h e a u th o r s s u g g e s t th a t p la te le t h y p o a g g r e g a tio n is r e la te d to d e c r e a s e d f i b r in o g e n a n d in c r e a s e d F D P le v e ls .

K e y -w o r d s : P la te le t fu n c tio n . F ib rin o g e n . F D P . Bothrops.

Envenomations are a serious health problem in tropical countries1 25. In Brazil around 20,000 envenomation cases occur every year, 2,000 of them only in the State of São Paulo1. About 80 % of the cases occurring in Botucatu area are caused by three species of B o th r o p s (B .ja r a r a c a , B . a lte rn a tu s

and B . n e u w ie d i) 2.

Local and systemic alterations are occasioned by B o th r o p s venom when inoculated in humans19 20. Proteolytic venom fractions1 result in tissue necroses. Hemorrhagic and coagulant fractions act on the systemic level leading to vessel and blood changes7 9. Blood white cells, platelets and coagulant factor alterations are observed7. Coagulant factor changes have been demonstrated in v itro and e x vivo

experiments7 14 16. Factors II and X are activated with consequent fibrinogen consumption7 14 16. Platelet changes have been observed in accidents

Faculdade de Medicina de Botucatu e Centro de Estudos de Venenos e Animais Peçonhentos da Universidade Estadual Paulista, Botucatu, SP.

A d d r e s s to : D r. Benedito Barraviera. CEVAP/FM/UNESP.

Caixa Postal 576, 18618-000 Botucatu, SP, Brasil. Recebido para publicação em 23/05/94.

caused by different snakes7 9 15 26. Thomazini et alii23 demonstrated adecreaseofplateletaggregation activity in patients bitten by C r o ta lu s snakes. On the other hand, Zingali et alii27 showed an increase of platelet aggregation activity in v itro . Based on these data, the aggregant or anti-aggregant effect caused by different snake venoms is controversial23 2 62 7_

The objectives of the present study were to evaluate the number and platelet function , as well as fibrinogen and FDP plasma levels in patients bitten by B o th r o p s snakes.

MATERIALS AND METHODS

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I u a n F C , l h o m a z i n i I A , C a r v a lh o 1, C a r r e ir a D M G , C a s s in e lli V J, P e r e ir a P C M , B a r r a v ie r a B . E v a lu a tio n o f p l a t e l e t n u m b e r a n d f u n c t i o n a n d f i b r i n o g e n le v e l in p a t i e n t s b itte n b y s n a k e s o f th e Bothrops g e n u s . R e v i s t a d a

S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 : 1 9 - 2 4 , j a n - m a r , 1 9 9 5 .

Fibrinogen plasma levels were measured by

Ratnoff and Menzie method18. Platelet function

was evaluated by stimulating aggregation with 1

micromol (/tm) and 3 /im adenosine diphosphate ,

collagen and epinephrine from Helena Laboratories5

by using a Zenith platelet aggregation for the

graphic record. Numerical score o f platelets was

made utilizing the Newbauer chamber method by

phase contrast. Fibrin degradation product (FDP)

levels were determined in serum through the latex

proof with the American Dade reagents.

Statistical analyses were carried out using the

Wilcoxon non-parametric proof or two dependent

samples4.

RESULTS

Table 1 shows fibrinogen plasma levels decreased before and after 24 hours after the specific treatment (p < 0 .0 5 ). Forty-eight hours later the level augmented and remained stable until 8 days. The number of platelets was low before, 24 and 48 hours after sped fic treatment returning to the normal on the 8th day (p < 0 .0 5 ).

Table 2 shows platelet stimulation with l^ m and 3/tm ADP. When stimulated by 1/im ADP, platelets were hypoaggregant until 48 hours after treatment with a tendency to normalization on the 8th day (p < 0 .0 5 ). When ADP concentration was increased to 3/xm, the platelets were hypoaggregant in the 24-hour evaluation tending to normalization after 48 hours (p < 0 .0 5 ).

T a b le 1 - F ib r in o g e n p la s m a tic le v e ls a n d p la te le t c o u n t o fp a t i e n t s s tu d ie d . R e s u lts a r e r e p o r te d a s m e a n s ±S D .

A Before (n = 14)

B 24hr (n = 12)

C 48hr (n = 12)

D 8th day (n = 8)

Fibronogen (1) Normal: 200 a 400mg%

211.5 ± 70 .19 223 .83 + 36 .1 3 2 6 1 .7 3 + 7 2 .7 9 289 ± 4 7 .0 2

Platelets (2) Normal: 200 a 400x108

158.1 4 ± 110.35 182.55+ 71.64 158 .25 + 55 .8 9 194 .13 + 46 .7 1

Statistical analysis:

Aj X B[ X Cj X Dj ; Aj = Bj < Cj = Djj (p < 0 ,0 5 ) A2X B2 X C2 X D2 ; A2 = B2 = C2 < D2; (p < 0,05)

T a b le 2 - P la te le t a g g r e g a tio n a c tiv ity w ith l j i m a n d 3 fim A D P o f p a tie n ts s tu d ie d . R e s u lts a r e r e p o r te d a s m e a n s ±S D .

A Before (n = 14)

B 24hr (n = 12)

C 48hr (n = 9)

D 8th day (n = 7)

Stimulated with ADP 1/x.m (1) (normal >60% )

1 7 .8 4±28 .6 9 2 3 .1 7 ±2 8 .7 3 1 8 .9 0±21 .8 4 3 7 .5 7 + 2 9 .0 9

Stimulated with ADP 3 fim (2) (normal >60% )

3 5 .7 6 ± 3 2 .9 8 58.41+ 23.71 62.6 6 ± 15.44 6 6 .5 7 ± 2 5 .0 4

Statistical analysis:

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lu a n F C , T h o m a z in i IA , C a r v a lh o 1, C a r r e ir a D M G , C a s s in e lli V J, P e r e ir a P C M , B a r r a v ie r a B . E v a lu a tio n o f p la t e l e t n u m b e r a n d f u n c t i o n a n d f i b r i n o g e n le v e l in p a t i e n t s b itte n b y s n a k e s o f th e Bothrops g e n u s . R e v is ta d a

Table 3 shows no alteration when platelets were stimulated by collagen and epinephrine.

Table 4 shows fibrinogen plasma levels and fibrin degradation products (FDP) increased in 5 out o f 6 patients studied. T h ree p a tie n ts presented values up to 40 micrograms/ml, while the other two presented levels of 10-40^g/ml. The only patient with normal FDP ( < 10/xg/ml) values showed normal platelet activity and normal fibrinogen values.

DISCUSSION

Blood coagulation changes both in v itro and ex vivo have been observed in envenomations caused by different venomous snakes6 7 13 14 20 21 23. Komalik8 working in v itro with snake venoms of

A g k is tr o d o n and V ip era genera demonstrated that a

fibrinogen bovine solution, when pre-incubated with these venoms, lost its capacity of turning into fibrin. Kamiguti et alii6, studying patients bitten by

B o th r o p s ja r a r a c a, noticed fibrinogen consumption. Studies carried out by Thomazini et alii23 in patients bitten by C ro ta lu s d u r is s u s te r r ific u s , agree with the results obtained by Kamiguti et alii6. Santoro et alii22 in o cu la tin g B o th ro p s ja r a r a c a venom in rabbits observed low plasmatic fibrinogen levels and increased levels of fibrin degradation products (FDP). Sano-M artinsetalii21 studied patients bitten by B o t h r o p s and found high levels o f FDP immediately before antivenom therapy, tending to normalize after a 24-hour treatment.

In this study, immediately and 24 hours after the accident, fibrinogen plasmatic levels showed a decrease, but were still within normal values. These normalized 48 hours after specific treatment. A

T a b le 3 - P la te le t a g g r e g a tio n a c tiv ity w ith c o lla g e n a n d e p in e p h r in e o f p a tie n ts s tu d ie d . R e s u lts a r e r e p o r te d a s m e a n s ±S D .

A Before (n = 14)

B 24hr (n = 12)

C 48hr (n = l l )

D 8th day (n = 7)

Collagen (1) (5/ig/ml) (normal >60% )

49.6 4+ 3 3.13 74.17 + 12.92 75.73 + 13.42 77.43 + 12.33

Epinephrine (2) (30 /xm )

(normal >60% )

4 5 .3 5 + 3 0 .9 2 76.25 + 14.76 67.27 + 19.50 72.29 ± 1 7 .7 7

Statistical analysis:

A , x Bj x Cj x Ü! ; Aj < Bj = Cj = D ,; (p < 0 ,1 0 ) Aj x B2 x Cj x D2 ; Aj < B2 = C2 = D2; (p < 0 ,1 0 )

T a b le 4 - F ib rin d e g r a d a tio n p r o d u c t le v e ls (F D P ), f i b r in o g e n p la s m a tic le v e ls a n d p la t e l e t a g g r e g a tio n a c tiv ity f o r s ix p a tie n ts s tu d ie d .

Patients studied (n = 6)

FDP levels jig/ml (normal < lOftg/ml)

Fibronogen mg% (normal: 200 a 400)

Platelet activity ADP lftm

1 > 40 250 hypo function

2 10 - 40 250 hypo function

3 < 10 310 normofunction

4 1 0 - 4 0 H I hypo function

5 > 40 136 normofunction

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Ilia n F C , T h o m a z in i I A , C a r v a lh o I, C a r r e ir a D M G , C a s s in e lli V J, P e r e ir a P C M , B a r r a v i e r a B . E v a lu a tio n o f p l a t e l e t n u m b e r a n d f i i n c t i o n a n d f i b r i n o g e n le v e l in p a t i e n t s b itte n by s n a k e s o f th e Bothrops g e n u s . R e v is ta d a

possible action o f “ factor X activator” and “thrombin-like” fraction from B . ja r a r a c a venom may explain these alterations. “Factor X activator” from B . j a r a r a c a venom transforms factor X from blood into activated factor X. Prothrombin is transformed into thrombin by activated factor X. Thrombin is capable of producing fibrin by acting on fibrinogen16.

T hrom bin-like fractions act directly on fibrinogen molecule transforming it into fibrin. This effect is similar to that o f human thrombin.

In this way, there will be plasmatic fibrinogen consumption and formation o f fibrin degradation products(FDP)10 11 1222 as final consequences.

B o th r o p s venom besides acting on fibrinogen, also presents an effect on platelet, both in number and in function6 7 21 22.

Prado-Franceschi et alii17 studying rabbits inoculated with C r o ta lu s d u r is s u s te r r ific u s venom, observed a discreet low number of platelets and an increase in platelet aggregant activity. Zingali et alii27 described that pure B o th r o p s venom has a fraction that can stimulate platelet aggregation. These authors27 28 in 1992, described a new component o f B o th r o p s venom which might inhibit platelet aggregation when induced by thrombin. Thomazini et alii23 observed platelet hypofunction in patients bitten by C r o ta lu s d u r is s u s te r r ific u s ,

when this was stimulated by 1/xmADP. In this work platelet number decreased until 8 days after specific treatment. Platelets also showed hypoaggregation when stimulated by 1 /im and 3 fim ADP. Patients who presented high FDP levels were those who had platelet hypofunction. Marcus101112 demonstrated the anti-aggregant action of fibrin degradation products. According to Santoro et alii22, platelet hypoaggregation observed may be explained by high levels o f circulating FDP, which would have an inhibitory action on platlets due to competition with aggregant agents in platelet receptors. Vargaftig et alii24 evaluating convulxin fraction effect of

C r o ta lu s d u r is s u s te r r ific u s observed that this fraction presented an aggregant action in v itro .

Aggregation would occur by stimulating platelet receptors, releasing thromboxane A ^ , the platelet enzyme that facilitates platelet adhesion and

a g g re g a tio n m echan ism s to th e v a sc u la r subendothelium24. This action would lead to exhaustion of platelet reserves of thromboxane A2, causing hypofunction. In the present study, we observed normalization o f platelet aggregation curves on the 8th day after specific treatment. As platelet medium lifetime is 8 days, it is possible that aggregant function recovery occur, by renewing platelets in blood circulation.

According to Benett and Vilaire3, platelets need a suitable level of extracellular fibrinogen for perfect functioning. When these levels are decreased, there is a loss of potency of aggregant agents on receptors causing, in the least analysis, an aggregation decrease.

In conclusion, patients bitten by B o th r o p s snakes presented fibrin ogen con sum ption , platelet hypofunction and increased fibrinogen degradation products (FDP), suggesting a relationship among these factors.

RESUMO

F o ra m a v a lia d a s a f u n ç ã o p la q u e tá r ia e o s n ív e is s é r ic o s d e f i b r in o g ê n io e m 1 4 d o e n te s p ic a d o s p o r s e r p e n te s d o g ê n e r o Bothrops, s e n d o 1 0 d o s e x o m a sc u lin o e 4 d o s e x o fe m in in o , c o m id a d e s c o m p r e e n d id a s e n tre 1 3 e 5 9 a n o s. H o u v e d ife r e n ç a e s ta tís tic a (p < 0 ,0 5 ) e n tr e o s n ív e is s é r ic o s d e fib r in o g ê n io a v a lia d o s 2 4 e 4 8 h o r a s a p ó s o a c id e n te . H o u v e te n d ê n c ia à n o r m a liz a ç ã o a p ó s 4 8 h o r a s d o tra ta m e n to . A p la q u e to p e n ia f o i e v id e n te n a s a v a lia ç õ e s d e 2 4 e 4 8 h o r a s . H o u v e te n d ê n c ia à n o m a liz a ç ã o n o 8 o d ia a p ó s o tr a ta m e n to ( p < 0 , 0 5 ) . A f u n ç ã o p la q u e tá r ia , q u a n d o e s tim u la d a p e l o c o lá g e n o e e p in e frin a , a p re s e n to u -se d e n tr o d a n o rm a lid a d e . Q u a n d o es tim u la d a p e l o A D P , f o i h ip o a g r e g a n te n a c o n c e n tr a ç ã o d e 1 m ic r o m o la r a té 4 8 h o r a s a p ó s o tr a ta m e n to . N a c o n c e n tr a ç ã o d e 3 m ic r o m o la r h o u v e a lte r a ç ã o a p e n a s a n te s d o tr a ta m e n to e s p e c ífic o ( p < 0 , 0 5 ) . O s n ív e is d e p r o d u to s d e d e g r a d a ç ã o d e f ib r i n a (P D F ) m o s tr a r a m - s e a lte r a d o s e m 8 3 ,3 3 % d o s p a c ie n te s a v a lia d o s . O s a u to r e s s u g e r e m q u e a h ip o a g r e g a ç ã o e s te ja r e la c io n a d a c o m n ív e is b a ix o s d e fib r in o g ê n io e e le v a d o s d e P D F .

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Ilia n F C , T h o m a z in i 1A, C a r v a lh o I, C a r r e ir a D M G , C a s s in e lli V J, P e r e ir a P C M , B a r r a v ie r a B . E v a lu a tio n o f p l a t e l e t n u m b e r a n d J u n c tio n a n d f i b r i n o g e n le v e l in p a tie n ts b itte n b y s n a k e s o f th e Bothrops g e n u s . R e v is ta d a

ACKNOWLEDGMENTS

The authors wish to thank Dr. Francisco Humberto de A. Maffei and Dr. Paulo Eduardo de A. Machado for their cooperation, Denise H. Francisco and Ivanete F. Amaral, Hemostasy Laboratory technicians, for their assistance and Dr. Paulo Roberto Curi for the statistical analysis.

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I u a n T C , T h o m a z in i IA , C a r v a lh o I , C a r r e ir a D M G , C a s s in e lli V J, P e r e ir a P C M , B a r r a v i e r a B . E v a lu a tio n o f p l a t e l e t n u m b e r a n d f u n c t i o n a n d f i b r i n o g e n l e v e l in p a t i e n t s b itte n b y s n a k e s o f th e Bothrops g e n u s . R e v is ta d a

S o c i e d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 8 : 1 9 - 2 4 , j a n - m a r , 1 9 9 5 .

de São Paulo 33:219-220, 1991.

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