Acta Troptca, 49(1991)227-232 Elsevier
227
ACTROP 00151
In vivo platelet aggregation in acute malaria E.E. Oslm, B.J. Adegunloye and A.O. Emeribe Departments of Phystologv and Haematology, Collegeof MedwalSciences, Umversttyof Calabar. Calabar, Ntgerta (Received 8 August 1990, accepted 9 February 1991) Thirteen patients suffering from acute malaria attack and thirteen apparently healthy human volunteers (control) were used for the study Platelet aggregation was determined by platelet count ratio techmque m which a reduction in platelet count ratio slgmfled an increase m platelet aggregation Platelet count ratio in acute malaria patients was 0 75+0 03 (SEM) Platelet count ratio m subjects used as control was 0 88 + 0 02 Thls value was slgmficantly higher than the former (P < 0 00 l) Platelet count ratio correlated negatwely with the degree of parasltaemla (r = - 0 71, P < 0 01) ADP, a platelet aggregating drug, also reduced platelet count ratio in rats slgmficantly Acute malaria attack therefore enhances platelet aggregation in vlvo Key words Platelet Aggregation, Malaria
Introduction M a l a r i a r e m a i n s a m a j o r cause o f m o r b i d i t y a n d m o r t a h t y in t r o p i c a l a n d subt r o p i c a l a r e a s M a l a r i a ts caused by Plasmodtum species O n l y four, n a m e l y , P falctparum, P ovale, P vlvax a n d P. malartae a m o n g the 100 k n o w n p l a s m o d l a species infect m a n A low g r a d e h a e m o l y s t s occurs in m a l a r i a a n d is p a r t l y due to d e s t r u c t i o n o f e r y t h r o c y t e s by p a r a s i t e s as they m u l t i p l y a n d r u p t u r e the cells The m a i n clinical m a n i f e s t a t i o n s o f m a l a r i a m c l u d e a n a e m i a , s p l e n o m e g a l y a n d fever R e d b l o o d cells c o n t a i n a d e n o s i n e d l p h o s p h a t e ( A D P ) A s the red b l o o d cells r u p t u r e d u r i n g acute m a l a r i a a t t a c k , A D P m a y be released M o r e o v e r , d u r i n g m a l a r i a a t t a c k , a very p o t e n t a g g r e g a t i n g agent, t h r o m b o x a n e A 2 a n d o t h e r p r o c o a g u l a n t m a t e r i a l s are released (Esslen et a l , 1984, B o r o c h o w l t z et al., 1970, A b l l d g a a r d et a l , 1974) M a l a r i a also gives rise to a low p l a t e l e t c o u n t ( D e n n i s et a l , 1967, S k u d o w t t z et a l , 1973; Esslen et at., 1979) Platelets o f p a t i e n t s suffering f r o m a c u t e m a l a r i a a t t a c k are hypersensitive to A D P in vitro (Esslen a n d E b h o t a , 1981) A D P e n h a n c e s platelet a g g r e g a t i o n b o t h m vtvo a n d in vitro H o w e v e r , w h e t h e r platelets o f acute m a l a r i a p a t i e n t s also a g g r e g a t e in VlVO even w i t h o u t e x o g e n o u s A D P a n d o t h e r p r o c o a g u l a n t m a t e r i a l s has n o t been a s c e r t a i n e d This s t u d y therefore e x a m i n e s this possibility
Correvpondenceaddress Dr E E Oslm, Department of Physiology, College of Medical Soences, University of Calabar, Calabar, Nigeria 0001-706X/91/$03 50 © 1991 Elsevier Science Publishers B V
228 Materials and Methods Thirteen patients suffering from acute malaria and were not on any medication before diagnosis, and 13 apparently healthy human volunteers who served as control were used for the study The subjects were of either sex whose ages ranged between 3 and 28 years. All the 13 patients had clinical and parasltologlcal features of malaria and were confirmed to have malaria by microscopically examining their Giemsastained thick blood smear for malaria parasites p H of the stain was 7 2 Parasite count was done as described by Sexton et a l , (1988) A minimum of 1000 parasites or 500 leucocytes were counted, Parasite count or density (PD) in m m 3 of blood was calculated as shown below P D = N o of parasites 6000 x -parasites/ram 3 of blood No of leucocytes 1 The controls had no malaria parasites in their blood smear The method used to determine platelet aggregation In VlVO was the platelet count ratio technique devised by Wu and H o a k (1974) The method essentially involves drawing 0 5 ml of blood sample twice from the median cubital vein of an Individual with minimal occlusion The 0 5 ml blood sample is first drawn into a syringe containing 9 5 ml of buffered E D T A / f o r m a h n solution to get 1 20 dilution, and another 0 5 ml sample is drawn into a second syringe containing 9 5 ml of buffered E D T A solution only Since the first syringe contams formalin which fixes platelet aggregates as blood is drawn gently into the syringe, the ratio obtained by dividing the platelet count m the first syringe by that m the second should be less than one ff there are aggregates Conversely, in the absence of aggregates the ratio should approach one Only unaggregated platelets m the samples were counted Details of the preparation of the buffered E D T A / f o r m a h n solutmn as well as buffered E D T A solution can be found in Wu and H o a k (1974) After drawing blood samples into the syringes, they were kept at r o o m temperature for 10-60 rain before centrifuging at 2 0 0 x g for 10 mln to harvest platelet-rich plasma (PRP) The platelets in the two collectmns were counted by haemocytometry Platelet count ratio from malaria patients was then compared with that from control Parasitemla (parasite density) was correlated with platelet count ratio (Fig 1) After using humans for the study as described above, the method was tested by using rats to find out whether it was capable of detecting platelet aggregates ADP, a potent aggregation inducing drug was used Ten albino (Wistar strata) rats of either sex weighing between 110 and 130 g were used Five of these were treated with A D P (Sigma, U K ) Intraperitoneally at a dose of 40 mg/100 g body wt The other five rats which served as control received saline (0 9%) injectmns Blood samples were obtained by cardiac puncture under urethane anaesthesia given subcutaneously at a dose of 0 6 ml of 25% urethane per 100 g body wt P R P was obtained as described above and the platelet count ratio in the ADP-treated rats was also compared with that m control rats (Fig 2) Statlst~cal analysis was performed by Students' t-test
229
Results Platelet count ratio m malaria patients and healthy subjects
Platelet count ratio in 13 acute malaria patients and 13 healthy subjects are shown in Tables 1 and 2 respectively The tables also show the parasite count as well as platelet count in buffered E D T A / f o r m a h n and E D T A solutions The mean platelet count ratio m the blood of malaria patients was 0 7 5 + 0 03 (SEM) This was significantly lower than the platelet count ratio in healthy subjects which was 0 8 8 + 0 0 2 ( P < 0 0 0 1 ) Platelet count ( I n E D T A only) in malaria patients was 143 7 + 1 2 6 × 10 9 per htre This value was significantly lower than the count m healthy subjects which was 225 4___9 4 × l 0 9 per litre ( P < 0 001) There was a significant negattve correlation between the degree of parasltemla and platelet count ratio Coefficient of correlation (r) was - 0 71 (P < 0 01, Fig l) It was also observed that 86% of the parasites were P falclparum and 14% were P malarlae Platelet count ratto m ADP-treated and control rats
Platelet count ratto m 5 rats treated with A D P at a dose of 40 mg/100 g body wt i p was 0.67 + 0 03 (SEM) The ratio m 5 control (sahne-treated) rats was 0 81 + 0 04
TABLE I Parasite density, platelet count and platelet count ratio m acute malarm patients Serial No
lnltlals of subjects
Age (years)
Sex
Parasite density (per m m 3)
Platelet count m EDTA/ formalin solution ( X 109/1)
Platelet count in EDTA solution only ( X 109/1)
Platelet count ratio
1 2 3 4 5 6 7 8 9 10 11 12 13
E M U U N G 1L G S N E O S IE EA A U O D U L SA
14 3 8 28 5 16 3 8 11 15 23 6 4
F M M M M F F M F F F M F
38 700 139 000 55 800 1200 84 000 36 600 1200 1120 200 96 000 600 1800 700
126 101 107 115 86 96 137 74 161 44 86 70 211
150 162 158 157 136 141 186 96 191 68 93 98 232
0 0 0 0 0 0 0 0 0 0 0 0 0
84 62 67 72 63 68 74 77 83 65 92 71 91
Mean
35 147 7
108 8
143 7
0 75
Standard error of the mean (SEM)
126085
115
126
003
230 TABLE 2 Platelet c o u n t a n d platelet c o u n t ratio m a p p a r e n t l y healthy
subjects
Serial No
Imtlals of subjects
Age (years)
Sex
Parasite density (per m m 3)
Platelet count m EDTA/ formahn solution ( × 109/1)
Platelet count m EDTA solution only ( × 109/1)
Platelet count ratio
1 2 3 4 5 6 7 8 9 10 11 12 13
I B P A O A M D D D E N E I E U IG A E D A R M U J
20 18 23 19 21 18 21 17 19 18 18 22 19
F M M M F F M F F M F F M
-
198 190 200 193 200 125 225 150 223 210 238 211 220
243 218 236 267 225 153 225 156 257 231 240 232 247
0 81 0 87 0 84 0 72 0 89 0 81 1 00 0 96 0 87 0 91 0 99 0 91 0 89
198 7
225 4
0 88
82
94
0 02
-
-
Mean Standard error of the m e a n ( S E M )
iooo
ro E E
800 7OO
a>"
r=-071 aoo-
'
k
P
227
ACTROP 00151
In vivo platelet aggregation in acute malaria E.E. Oslm, B.J. Adegunloye and A.O. Emeribe Departments of Phystologv and Haematology, Collegeof MedwalSciences, Umversttyof Calabar. Calabar, Ntgerta (Received 8 August 1990, accepted 9 February 1991) Thirteen patients suffering from acute malaria attack and thirteen apparently healthy human volunteers (control) were used for the study Platelet aggregation was determined by platelet count ratio techmque m which a reduction in platelet count ratio slgmfled an increase m platelet aggregation Platelet count ratio in acute malaria patients was 0 75+0 03 (SEM) Platelet count ratio m subjects used as control was 0 88 + 0 02 Thls value was slgmficantly higher than the former (P < 0 00 l) Platelet count ratio correlated negatwely with the degree of parasltaemla (r = - 0 71, P < 0 01) ADP, a platelet aggregating drug, also reduced platelet count ratio in rats slgmficantly Acute malaria attack therefore enhances platelet aggregation in vlvo Key words Platelet Aggregation, Malaria
Introduction M a l a r i a r e m a i n s a m a j o r cause o f m o r b i d i t y a n d m o r t a h t y in t r o p i c a l a n d subt r o p i c a l a r e a s M a l a r i a ts caused by Plasmodtum species O n l y four, n a m e l y , P falctparum, P ovale, P vlvax a n d P. malartae a m o n g the 100 k n o w n p l a s m o d l a species infect m a n A low g r a d e h a e m o l y s t s occurs in m a l a r i a a n d is p a r t l y due to d e s t r u c t i o n o f e r y t h r o c y t e s by p a r a s i t e s as they m u l t i p l y a n d r u p t u r e the cells The m a i n clinical m a n i f e s t a t i o n s o f m a l a r i a m c l u d e a n a e m i a , s p l e n o m e g a l y a n d fever R e d b l o o d cells c o n t a i n a d e n o s i n e d l p h o s p h a t e ( A D P ) A s the red b l o o d cells r u p t u r e d u r i n g acute m a l a r i a a t t a c k , A D P m a y be released M o r e o v e r , d u r i n g m a l a r i a a t t a c k , a very p o t e n t a g g r e g a t i n g agent, t h r o m b o x a n e A 2 a n d o t h e r p r o c o a g u l a n t m a t e r i a l s are released (Esslen et a l , 1984, B o r o c h o w l t z et al., 1970, A b l l d g a a r d et a l , 1974) M a l a r i a also gives rise to a low p l a t e l e t c o u n t ( D e n n i s et a l , 1967, S k u d o w t t z et a l , 1973; Esslen et at., 1979) Platelets o f p a t i e n t s suffering f r o m a c u t e m a l a r i a a t t a c k are hypersensitive to A D P in vitro (Esslen a n d E b h o t a , 1981) A D P e n h a n c e s platelet a g g r e g a t i o n b o t h m vtvo a n d in vitro H o w e v e r , w h e t h e r platelets o f acute m a l a r i a p a t i e n t s also a g g r e g a t e in VlVO even w i t h o u t e x o g e n o u s A D P a n d o t h e r p r o c o a g u l a n t m a t e r i a l s has n o t been a s c e r t a i n e d This s t u d y therefore e x a m i n e s this possibility
Correvpondenceaddress Dr E E Oslm, Department of Physiology, College of Medical Soences, University of Calabar, Calabar, Nigeria 0001-706X/91/$03 50 © 1991 Elsevier Science Publishers B V
228 Materials and Methods Thirteen patients suffering from acute malaria and were not on any medication before diagnosis, and 13 apparently healthy human volunteers who served as control were used for the study The subjects were of either sex whose ages ranged between 3 and 28 years. All the 13 patients had clinical and parasltologlcal features of malaria and were confirmed to have malaria by microscopically examining their Giemsastained thick blood smear for malaria parasites p H of the stain was 7 2 Parasite count was done as described by Sexton et a l , (1988) A minimum of 1000 parasites or 500 leucocytes were counted, Parasite count or density (PD) in m m 3 of blood was calculated as shown below P D = N o of parasites 6000 x -parasites/ram 3 of blood No of leucocytes 1 The controls had no malaria parasites in their blood smear The method used to determine platelet aggregation In VlVO was the platelet count ratio technique devised by Wu and H o a k (1974) The method essentially involves drawing 0 5 ml of blood sample twice from the median cubital vein of an Individual with minimal occlusion The 0 5 ml blood sample is first drawn into a syringe containing 9 5 ml of buffered E D T A / f o r m a h n solution to get 1 20 dilution, and another 0 5 ml sample is drawn into a second syringe containing 9 5 ml of buffered E D T A solution only Since the first syringe contams formalin which fixes platelet aggregates as blood is drawn gently into the syringe, the ratio obtained by dividing the platelet count m the first syringe by that m the second should be less than one ff there are aggregates Conversely, in the absence of aggregates the ratio should approach one Only unaggregated platelets m the samples were counted Details of the preparation of the buffered E D T A / f o r m a h n solutmn as well as buffered E D T A solution can be found in Wu and H o a k (1974) After drawing blood samples into the syringes, they were kept at r o o m temperature for 10-60 rain before centrifuging at 2 0 0 x g for 10 mln to harvest platelet-rich plasma (PRP) The platelets in the two collectmns were counted by haemocytometry Platelet count ratio from malaria patients was then compared with that from control Parasitemla (parasite density) was correlated with platelet count ratio (Fig 1) After using humans for the study as described above, the method was tested by using rats to find out whether it was capable of detecting platelet aggregates ADP, a potent aggregation inducing drug was used Ten albino (Wistar strata) rats of either sex weighing between 110 and 130 g were used Five of these were treated with A D P (Sigma, U K ) Intraperitoneally at a dose of 40 mg/100 g body wt The other five rats which served as control received saline (0 9%) injectmns Blood samples were obtained by cardiac puncture under urethane anaesthesia given subcutaneously at a dose of 0 6 ml of 25% urethane per 100 g body wt P R P was obtained as described above and the platelet count ratio in the ADP-treated rats was also compared with that m control rats (Fig 2) Statlst~cal analysis was performed by Students' t-test
229
Results Platelet count ratio m malaria patients and healthy subjects
Platelet count ratio in 13 acute malaria patients and 13 healthy subjects are shown in Tables 1 and 2 respectively The tables also show the parasite count as well as platelet count in buffered E D T A / f o r m a h n and E D T A solutions The mean platelet count ratio m the blood of malaria patients was 0 7 5 + 0 03 (SEM) This was significantly lower than the platelet count ratio in healthy subjects which was 0 8 8 + 0 0 2 ( P < 0 0 0 1 ) Platelet count ( I n E D T A only) in malaria patients was 143 7 + 1 2 6 × 10 9 per htre This value was significantly lower than the count m healthy subjects which was 225 4___9 4 × l 0 9 per litre ( P < 0 001) There was a significant negattve correlation between the degree of parasltemla and platelet count ratio Coefficient of correlation (r) was - 0 71 (P < 0 01, Fig l) It was also observed that 86% of the parasites were P falclparum and 14% were P malarlae Platelet count ratto m ADP-treated and control rats
Platelet count ratto m 5 rats treated with A D P at a dose of 40 mg/100 g body wt i p was 0.67 + 0 03 (SEM) The ratio m 5 control (sahne-treated) rats was 0 81 + 0 04
TABLE I Parasite density, platelet count and platelet count ratio m acute malarm patients Serial No
lnltlals of subjects
Age (years)
Sex
Parasite density (per m m 3)
Platelet count m EDTA/ formalin solution ( X 109/1)
Platelet count in EDTA solution only ( X 109/1)
Platelet count ratio
1 2 3 4 5 6 7 8 9 10 11 12 13
E M U U N G 1L G S N E O S IE EA A U O D U L SA
14 3 8 28 5 16 3 8 11 15 23 6 4
F M M M M F F M F F F M F
38 700 139 000 55 800 1200 84 000 36 600 1200 1120 200 96 000 600 1800 700
126 101 107 115 86 96 137 74 161 44 86 70 211
150 162 158 157 136 141 186 96 191 68 93 98 232
0 0 0 0 0 0 0 0 0 0 0 0 0
84 62 67 72 63 68 74 77 83 65 92 71 91
Mean
35 147 7
108 8
143 7
0 75
Standard error of the mean (SEM)
126085
115
126
003
230 TABLE 2 Platelet c o u n t a n d platelet c o u n t ratio m a p p a r e n t l y healthy
subjects
Serial No
Imtlals of subjects
Age (years)
Sex
Parasite density (per m m 3)
Platelet count m EDTA/ formahn solution ( × 109/1)
Platelet count m EDTA solution only ( × 109/1)
Platelet count ratio
1 2 3 4 5 6 7 8 9 10 11 12 13
I B P A O A M D D D E N E I E U IG A E D A R M U J
20 18 23 19 21 18 21 17 19 18 18 22 19
F M M M F F M F F M F F M
-
198 190 200 193 200 125 225 150 223 210 238 211 220
243 218 236 267 225 153 225 156 257 231 240 232 247
0 81 0 87 0 84 0 72 0 89 0 81 1 00 0 96 0 87 0 91 0 99 0 91 0 89
198 7
225 4
0 88
82
94
0 02
-
-
Mean Standard error of the m e a n ( S E M )
iooo
ro E E
800 7OO
a>"
r=-071 aoo-
'
k
P
