THROMBOSIS RESEARCH 63; 563-568, 1991 0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press plc. All rights reserved.
BRIEF
COMMUNICATION
ELECTRON MICROSCOPIC STUDIES OF QUININE INDUCED ULTRASTRUCTURAL CHANGES TO PLATELETS
C.J. Bradley*, S. Deacon, J.M.Connellan & P.J. Thurlow *Department of Pathology, University of Melbourne and Department of Haematology, Austin Hospital, Studley Road, Heidelberg, Victoria, 3084, Australia. (Received 14.3.1991; accepted in revised form 21.6.1991 by Editor H.H. Salem)
INTRODUCTION
Quinine
quinidine (cinchona alkaloid stereoisomers) have been antibodies resulting in severe induce anti-platelet demonstrated to One theory is that this process thrombocytopenia in susceptible patients (1). occurs through interaction of the Fab portion of the drug-induced antibody with a drug-platelet antigen complex acting as a neoantigen.(2) Many platelet membrane glycoproteins have been shown to be involved in this process including Ilb, Illa, lb, As these glycoproteins play an important role in platelet IX and V.(3,4) aggregation it is not surprising that several workers have described a marked inhibition of aggregation in response to ADP, epinephrine and collagen in the presence of both quinine and quinidine.(5,6,8) A recent paper by Connellan et al also demonstrated direct perturbation of membrane glycoproteins in the presence of quinine resulting in reduced binding of both fibronectin and fibrinogen as well as Ultrastructural evidence of altered antigenicity of membrane glycoproteins.(6) changes has been documented in platelets treated with quinidine. These have ranged
from
and
mild
swelling
detectable
at
10~3M
to
gross
damage,
including
disintegration of the plasma membrane at 2 x 10_3M.(5,7,8) Given these results it is unusual that Deykin et al were unable to find ultrastructural changes such as platelet swelling when platelets were treated with quinine.(8) In this study we reexamine the ultrastructure of quinine treated platlets using image analysis and stereomorphometric techniques to detect any changes in platelet ultrastructure.
MATERIALS
AND
METHODS
Quinine HCL was purchased from ICN Pharmaceuticals Inc. (purity:90-95%) and dissolved in either modified Tyrode’s buffer pH 7.4 (0.15M NaCI, 2.6mM KCI, 12mM Keywords:
Platelets,
ultrastructure,
quinine. 563
564
QUININE INDUCED THROMBOCYTOPENIA
NaHC03, 0.15M
2mM
NaCI, 2.6mM
MgCl2)
or
Hepes
KCI) or veronal
buffered
buffered
saline
saline
pH
Vol. 63. No. 5
7.4
(IOmM
Hepes,
pH 7.4 as required.
Electron Microscopv. Blood was collected into ACD anticoagulant (18) and PRP prepared by centrifugation at 100 x g for 15 mins. Quinine in Hepes Buffered Saline (HBS) (O.OlM Hepes, pH 7.4) was added to the PRP to give a final concentration of 1mM and was incubated with the PRP for 30 mins. Platelets were fixed according to the method recommended by White (91, dehydrated through graded acetone and embeded in Araldite-Epon resin (CIBA-GEIGY, Lane Cove, NSW). Thin sections were cut and stained with saturated aqueous uranyl nitrate and Reynold’s lead citrate (IO) and viewed on a JEOL JEM 1200EX For normal controls HBS was substituted for quinine. electron microscope. Fixation and processing of normal controls was identical to that of quinine test samples.
Electron microscope negatives were scanned using a Sierra lmaae analvsis. MS4000 videocamera (Sierra Scientific, Sunnydale, CA.). The images were digitized and analysed using the MCID system (Imaging Research Inc., Ontario, Canada) running on an IBMPC-AT computer.
Platelets for analysis were chosen from electron micrograph fields by Statistics. systematic sampling as recommended by Weibel et al (1 I). Using image analysis the area of open canalicular system (OCS) and the total cross-sectional area for 100 platelets from both quinine and control samples was measured. This number of platelets was found to be a representative sample size for both groups as estimated by plotting the cumulative standard error expressed as percent of the mean versus number of platelets sampled according to the method of Chalkley (12). The ratio of OCS cross-sectional area to total platelet cross-sectional area was calculated for each platelet and the two populations compared using the Mann-Whitney test as the data was found to be skewed rather than normally distributed.
RESULTS
Moroholoav. Platelets incubated in the presence of 1mM quinine were similar in ultrastructure to the control platelets incubated with buffer in that they were Both groups also contained alpha irregularly shaped with occasional pseudopodia. microtubules and clusters of glycogen granules, dense bodies, mitochondria, granules. However, fewer discoid forms were seen than in the normal control and quinine treated platelets appeared to have dilated OCS, a fact born out by the No centralization of organelles was seen in either platelet statistical analysis. The one abnormal feature consistently seen in quinine treated population. platelets was the presence of electron dense membranous sacs most commonly found within and attached to the walls of the dilated OCS spaces. These could
Vol. 63, No. 5
QUININE INDUCED THROMBOCYTOPENIA
565
FIG. 1. 1 mM Quinine treated platelets OCS: Open Canalicular System, G: Glycogen granules, M: Mitochondria, A: alpha granules, B: Dense bodies, DTS: Dense tubule system, MT: Microtubules, E: Electron dense membrane complex within OCS. Appears to be connected to a dense tubule through the wall of the OCS, X: Electron dense membrane complex external to platelet but associated with platelet plasma membrane. X 17,000. Line = lflm.
Fig.2. Normal MT: Microtubules, M: Mitochondria, system, B: Dense bodies, G: Glycogen
platelets, no Quinine. A: Alpha granules, granules. X 17,000.
OCS: Open Line = lpm.
canalicular
566
QUININE INDUCED THROMBOCYTOPENIA
Vol. 63. No. 5
also occasionally be seen external to the platelet though still associated with the platelet plasma membrane. In 230 quinine treated platelets examined in crosssection 38 showed this abnormality, 4 contained two such bodies and 3 showed these bodies external to the platelet surface. Of the 300 examined no untreated platelets were observed with this feature.
Statistics. One hundred observations consisting of the ratio of OCS area to total platelet cross-sectional area were made for both quinine treated and non-quinine Both sets of data were found to be non-normally distributed treated platelets. (quinine treated:skewness = 3.762, control:skewness = 1.464). As a consequence the Mann-Whitney test for non-parametric data was employed. The Mann-Whitney test indicated a highly significant increase (p < 0.001) in the cross sectional area of the OCS in the quinine treated platelets as compared to the control platelets. As area density is equal to volume density (13) we can conclude that there is a significant increase in the volume of OCS in the quinine treated platelets. Both quinine and control platelets were from the same donor. Two other donors were also examined with similar changes being noted.
DISCUSSION
Our results have shown that 1mM quinine causes swelling of the platelet OCS as well as a characteristic membrane abnormality. This conforms with previous ultrastructural studies in which platelet swelling was noted with 1mM quinidine.(5) In the study by Chong et al the source of the swelling was not attributed specifically to an effect on the OCS but an inspection of the electron micrographs included in this paper indicates that this is probably the ease.(5) At this concentration of quinine there did not appear to be any loss of alpha granules or mitochondria as noted by Chong et al (51 in their study with quinidine and microtubules were still present in the quinine treated platelets although they were Overall these results suggest that not as prominent as in the untreated control. quinine and quinidine have qualitatively similar effects on platelet However , at equivalent concentrations, the ultrastructural changes were less severe than those seen in previous studies with quinidine.
ultrastructure. due to quinine
Dilation of the OCS normally accompanies platelet shape change and release and suggests the possibility of a direct effect of quinine on this surface connected The further finding of unusual electron dense membranous membrane system. inclusions in the OCS of these platelets supports this hypothesis. The inclusion E in Figure 1 appears to be associated with an element of the dense tubule system (DTS) through the wall of the OCS and may represent a faulty fusing of these two membrane systems which normally associate to form specialized membrane Although these inclusions have not previously been reported in systems (9). ultrastructural studies with quinidine an examination of Figure 2D in the paper by Deykin et al (8) reveals similar bodies. Damage to the OCS membranes would also explain the observed release of 5-HT from platelets in the presence of 2mM quinidine (7) as fusion of the 5-HT containing dense bodies of platelets with the
Vol. 63. No. 5
567
QUININE INDUCED THROMBOCYTOPENIA
OCS is one of the first steps in platelet release and faulty OCS membranes lead to premature release of this powerful vasoactive compound.
could
This study reveals abnormalities of the platelet surface-connected open The results are in accord with previous canalicular system induced by quinine. ultrastructural observations made with quinidine and with the observation by Christie et al (14) that these drugs are mainly absorbed onto the plasma membrane of the platelet with very little penetration to the cytoplasm. The results also add further weight to the hypothesis that these drugs cause widespread but specific conformational alterations in platelet membrane antigens which are associated While the concentration of quinine used in this with altered platelet function. study (1mM) excedes the therapeutic range (9-22pM)(15), both quinine and quinidine have been shown to affect platelet function in vivo at therapeutic concentrations (6,161 and it is possible that a cumulative effect of quinine on platelets at therapeutic concentrations for a prolonged period may have the effect equivalent to a higher concentration of drug for shorter exposure in vitro.
ACKNOWLEDGMENTS Funds for the research Research Council Grant.
were obtained
from the National
Health and Medical
REFERENCES
1. GRANDJEAN, L.C. A case of purpura haemorrhagica after administration of quinine with specific thrombocytopenia demonstrated in vivo. Acta Med. Stand. 727, (Suppl 213) 165-170, 1948.
2. LERNER, W., CARUSO, R.,FAIG, D. & KARPATKIN, S. Drug-dependent and nondrug dependent antiplatelet antibody in drug-induced immunologic thrombocytopenic purpura. Blood, 66,306-311, 1985.
PFUELLER, S.L., BILSTON, R.A., LOGAN, D., GIBSON, J.M. & FIRKIN, B.G. Heterogeneity of drug-dependent platelet antigens and their antibodies in quinine- and quinidine-induced thrombocytopenia: involvement of Glycoproteins lb, Ilb, Illa, IX. Blood, 72, 1155-l 162, 1988. 3.
STRICHER, R.B. & SHUMAN, M.A. Quinidine purpura: evidence that Glycoprotein is a target platelet antigen. Blood, 67, 1377-1381, 1986.
4.
CHONG, B.H., & KAPLIN, I.J. Effect of quinidine on platelets. 106-l 11, 1990.
5.
Haemostasis,
20,
V
568
QUININE
INDUCED
Vol. 63, No. 5
THROMBOCYTOPENIA
6. CONNELLAN, J.M., DEACON, S., & THURLOW, P.J. Changes in platelet and reactivity induced by quinine in relation to quinine (drug) induced immune thrombocytopenia. Thrombosis Research, 6 7, 501-5 14, 199 1.
7. BOULLIN, platelets: effects 114-126, 1971.
D.J., & O’BRIEN, R.A. Accumulation of quinidine on platelet ultrastructure and 5hydroxytryptamine.
function
by human blood Br. J. Pharmac.,
42,
8. DEYKIN, D., & HELLERSTEIN, L.J. The assessment of drug-dependent and isoimmune antiplatelet antibodies by the use of platelet aggregometry. J. C/in. Invest., 3142-3153, 1972.
51,
WHITE, J.G. The morphology of platelet function. In: Methods in Haematology: 9. measurements of p/ate/et function. W. Harkin 81 T.S. Zimmerman (Eds.) New York: Churchill-Livingstone, 1983.
REYNOLDS, ES. The use of lead citrate 10. microscopy. J. Cell Biol., 17, 208-212, 1963.
as an electron-opaque
11. WEIBEL, E.R., KISTLER, G.S., & SCHERLE, W.F. morphometric cytology. J. Cell Biol., 30, 23, 1966.
CHALKLEY, H.W. 12. Nat. Cancer Inst., 4, 47,
Methods 1943.
for quantitative
Practical
morphological
stain in electron
stereological
analysis
methods
of tissue.
WEIBEL, E.R. Stereological 13. morphometry. London: Academic
methods Vol. 1. Practical methods Press, ~26, 1979.
14. CHRISTIE, D.J., & ASTER, quinidine-induced thrombocytopenia.
R.H. Drug-antibody-platelet interaction in quinineJ. Clin. Invest., 70, 989-998, 1982.
for
J.
for biological
EDSTEIN, M.C., STACE,J., & SHANN,F. Quantification of quinine 15 by high-performance liquid chromatography. Journal of Chromatography, 1983.
and
in human serum 278, 445-451,
LAWSON, D., MEHTA, J., MEHTA, P., LIPMAN, B.C., & IMPERI, G.A. Cumulative 16. effects of quinidine and aspirin on bleeding time and platelet alpha2-adrenoceptors: potential mechanism of bleeding diathesis in patients receiving this combination. J. Lab. Clin. Med., 108, 581-586. 1986.
BRIEF
COMMUNICATION
ELECTRON MICROSCOPIC STUDIES OF QUININE INDUCED ULTRASTRUCTURAL CHANGES TO PLATELETS
C.J. Bradley*, S. Deacon, J.M.Connellan & P.J. Thurlow *Department of Pathology, University of Melbourne and Department of Haematology, Austin Hospital, Studley Road, Heidelberg, Victoria, 3084, Australia. (Received 14.3.1991; accepted in revised form 21.6.1991 by Editor H.H. Salem)
INTRODUCTION
Quinine
quinidine (cinchona alkaloid stereoisomers) have been antibodies resulting in severe induce anti-platelet demonstrated to One theory is that this process thrombocytopenia in susceptible patients (1). occurs through interaction of the Fab portion of the drug-induced antibody with a drug-platelet antigen complex acting as a neoantigen.(2) Many platelet membrane glycoproteins have been shown to be involved in this process including Ilb, Illa, lb, As these glycoproteins play an important role in platelet IX and V.(3,4) aggregation it is not surprising that several workers have described a marked inhibition of aggregation in response to ADP, epinephrine and collagen in the presence of both quinine and quinidine.(5,6,8) A recent paper by Connellan et al also demonstrated direct perturbation of membrane glycoproteins in the presence of quinine resulting in reduced binding of both fibronectin and fibrinogen as well as Ultrastructural evidence of altered antigenicity of membrane glycoproteins.(6) changes has been documented in platelets treated with quinidine. These have ranged
from
and
mild
swelling
detectable
at
10~3M
to
gross
damage,
including
disintegration of the plasma membrane at 2 x 10_3M.(5,7,8) Given these results it is unusual that Deykin et al were unable to find ultrastructural changes such as platelet swelling when platelets were treated with quinine.(8) In this study we reexamine the ultrastructure of quinine treated platlets using image analysis and stereomorphometric techniques to detect any changes in platelet ultrastructure.
MATERIALS
AND
METHODS
Quinine HCL was purchased from ICN Pharmaceuticals Inc. (purity:90-95%) and dissolved in either modified Tyrode’s buffer pH 7.4 (0.15M NaCI, 2.6mM KCI, 12mM Keywords:
Platelets,
ultrastructure,
quinine. 563
564
QUININE INDUCED THROMBOCYTOPENIA
NaHC03, 0.15M
2mM
NaCI, 2.6mM
MgCl2)
or
Hepes
KCI) or veronal
buffered
buffered
saline
saline
pH
Vol. 63. No. 5
7.4
(IOmM
Hepes,
pH 7.4 as required.
Electron Microscopv. Blood was collected into ACD anticoagulant (18) and PRP prepared by centrifugation at 100 x g for 15 mins. Quinine in Hepes Buffered Saline (HBS) (O.OlM Hepes, pH 7.4) was added to the PRP to give a final concentration of 1mM and was incubated with the PRP for 30 mins. Platelets were fixed according to the method recommended by White (91, dehydrated through graded acetone and embeded in Araldite-Epon resin (CIBA-GEIGY, Lane Cove, NSW). Thin sections were cut and stained with saturated aqueous uranyl nitrate and Reynold’s lead citrate (IO) and viewed on a JEOL JEM 1200EX For normal controls HBS was substituted for quinine. electron microscope. Fixation and processing of normal controls was identical to that of quinine test samples.
Electron microscope negatives were scanned using a Sierra lmaae analvsis. MS4000 videocamera (Sierra Scientific, Sunnydale, CA.). The images were digitized and analysed using the MCID system (Imaging Research Inc., Ontario, Canada) running on an IBMPC-AT computer.
Platelets for analysis were chosen from electron micrograph fields by Statistics. systematic sampling as recommended by Weibel et al (1 I). Using image analysis the area of open canalicular system (OCS) and the total cross-sectional area for 100 platelets from both quinine and control samples was measured. This number of platelets was found to be a representative sample size for both groups as estimated by plotting the cumulative standard error expressed as percent of the mean versus number of platelets sampled according to the method of Chalkley (12). The ratio of OCS cross-sectional area to total platelet cross-sectional area was calculated for each platelet and the two populations compared using the Mann-Whitney test as the data was found to be skewed rather than normally distributed.
RESULTS
Moroholoav. Platelets incubated in the presence of 1mM quinine were similar in ultrastructure to the control platelets incubated with buffer in that they were Both groups also contained alpha irregularly shaped with occasional pseudopodia. microtubules and clusters of glycogen granules, dense bodies, mitochondria, granules. However, fewer discoid forms were seen than in the normal control and quinine treated platelets appeared to have dilated OCS, a fact born out by the No centralization of organelles was seen in either platelet statistical analysis. The one abnormal feature consistently seen in quinine treated population. platelets was the presence of electron dense membranous sacs most commonly found within and attached to the walls of the dilated OCS spaces. These could
Vol. 63, No. 5
QUININE INDUCED THROMBOCYTOPENIA
565
FIG. 1. 1 mM Quinine treated platelets OCS: Open Canalicular System, G: Glycogen granules, M: Mitochondria, A: alpha granules, B: Dense bodies, DTS: Dense tubule system, MT: Microtubules, E: Electron dense membrane complex within OCS. Appears to be connected to a dense tubule through the wall of the OCS, X: Electron dense membrane complex external to platelet but associated with platelet plasma membrane. X 17,000. Line = lflm.
Fig.2. Normal MT: Microtubules, M: Mitochondria, system, B: Dense bodies, G: Glycogen
platelets, no Quinine. A: Alpha granules, granules. X 17,000.
OCS: Open Line = lpm.
canalicular
566
QUININE INDUCED THROMBOCYTOPENIA
Vol. 63. No. 5
also occasionally be seen external to the platelet though still associated with the platelet plasma membrane. In 230 quinine treated platelets examined in crosssection 38 showed this abnormality, 4 contained two such bodies and 3 showed these bodies external to the platelet surface. Of the 300 examined no untreated platelets were observed with this feature.
Statistics. One hundred observations consisting of the ratio of OCS area to total platelet cross-sectional area were made for both quinine treated and non-quinine Both sets of data were found to be non-normally distributed treated platelets. (quinine treated:skewness = 3.762, control:skewness = 1.464). As a consequence the Mann-Whitney test for non-parametric data was employed. The Mann-Whitney test indicated a highly significant increase (p < 0.001) in the cross sectional area of the OCS in the quinine treated platelets as compared to the control platelets. As area density is equal to volume density (13) we can conclude that there is a significant increase in the volume of OCS in the quinine treated platelets. Both quinine and control platelets were from the same donor. Two other donors were also examined with similar changes being noted.
DISCUSSION
Our results have shown that 1mM quinine causes swelling of the platelet OCS as well as a characteristic membrane abnormality. This conforms with previous ultrastructural studies in which platelet swelling was noted with 1mM quinidine.(5) In the study by Chong et al the source of the swelling was not attributed specifically to an effect on the OCS but an inspection of the electron micrographs included in this paper indicates that this is probably the ease.(5) At this concentration of quinine there did not appear to be any loss of alpha granules or mitochondria as noted by Chong et al (51 in their study with quinidine and microtubules were still present in the quinine treated platelets although they were Overall these results suggest that not as prominent as in the untreated control. quinine and quinidine have qualitatively similar effects on platelet However , at equivalent concentrations, the ultrastructural changes were less severe than those seen in previous studies with quinidine.
ultrastructure. due to quinine
Dilation of the OCS normally accompanies platelet shape change and release and suggests the possibility of a direct effect of quinine on this surface connected The further finding of unusual electron dense membranous membrane system. inclusions in the OCS of these platelets supports this hypothesis. The inclusion E in Figure 1 appears to be associated with an element of the dense tubule system (DTS) through the wall of the OCS and may represent a faulty fusing of these two membrane systems which normally associate to form specialized membrane Although these inclusions have not previously been reported in systems (9). ultrastructural studies with quinidine an examination of Figure 2D in the paper by Deykin et al (8) reveals similar bodies. Damage to the OCS membranes would also explain the observed release of 5-HT from platelets in the presence of 2mM quinidine (7) as fusion of the 5-HT containing dense bodies of platelets with the
Vol. 63. No. 5
567
QUININE INDUCED THROMBOCYTOPENIA
OCS is one of the first steps in platelet release and faulty OCS membranes lead to premature release of this powerful vasoactive compound.
could
This study reveals abnormalities of the platelet surface-connected open The results are in accord with previous canalicular system induced by quinine. ultrastructural observations made with quinidine and with the observation by Christie et al (14) that these drugs are mainly absorbed onto the plasma membrane of the platelet with very little penetration to the cytoplasm. The results also add further weight to the hypothesis that these drugs cause widespread but specific conformational alterations in platelet membrane antigens which are associated While the concentration of quinine used in this with altered platelet function. study (1mM) excedes the therapeutic range (9-22pM)(15), both quinine and quinidine have been shown to affect platelet function in vivo at therapeutic concentrations (6,161 and it is possible that a cumulative effect of quinine on platelets at therapeutic concentrations for a prolonged period may have the effect equivalent to a higher concentration of drug for shorter exposure in vitro.
ACKNOWLEDGMENTS Funds for the research Research Council Grant.
were obtained
from the National
Health and Medical
REFERENCES
1. GRANDJEAN, L.C. A case of purpura haemorrhagica after administration of quinine with specific thrombocytopenia demonstrated in vivo. Acta Med. Stand. 727, (Suppl 213) 165-170, 1948.
2. LERNER, W., CARUSO, R.,FAIG, D. & KARPATKIN, S. Drug-dependent and nondrug dependent antiplatelet antibody in drug-induced immunologic thrombocytopenic purpura. Blood, 66,306-311, 1985.
PFUELLER, S.L., BILSTON, R.A., LOGAN, D., GIBSON, J.M. & FIRKIN, B.G. Heterogeneity of drug-dependent platelet antigens and their antibodies in quinine- and quinidine-induced thrombocytopenia: involvement of Glycoproteins lb, Ilb, Illa, IX. Blood, 72, 1155-l 162, 1988. 3.
STRICHER, R.B. & SHUMAN, M.A. Quinidine purpura: evidence that Glycoprotein is a target platelet antigen. Blood, 67, 1377-1381, 1986.
4.
CHONG, B.H., & KAPLIN, I.J. Effect of quinidine on platelets. 106-l 11, 1990.
5.
Haemostasis,
20,
V
568
QUININE
INDUCED
Vol. 63, No. 5
THROMBOCYTOPENIA
6. CONNELLAN, J.M., DEACON, S., & THURLOW, P.J. Changes in platelet and reactivity induced by quinine in relation to quinine (drug) induced immune thrombocytopenia. Thrombosis Research, 6 7, 501-5 14, 199 1.
7. BOULLIN, platelets: effects 114-126, 1971.
D.J., & O’BRIEN, R.A. Accumulation of quinidine on platelet ultrastructure and 5hydroxytryptamine.
function
by human blood Br. J. Pharmac.,
42,
8. DEYKIN, D., & HELLERSTEIN, L.J. The assessment of drug-dependent and isoimmune antiplatelet antibodies by the use of platelet aggregometry. J. C/in. Invest., 3142-3153, 1972.
51,
WHITE, J.G. The morphology of platelet function. In: Methods in Haematology: 9. measurements of p/ate/et function. W. Harkin 81 T.S. Zimmerman (Eds.) New York: Churchill-Livingstone, 1983.
REYNOLDS, ES. The use of lead citrate 10. microscopy. J. Cell Biol., 17, 208-212, 1963.
as an electron-opaque
11. WEIBEL, E.R., KISTLER, G.S., & SCHERLE, W.F. morphometric cytology. J. Cell Biol., 30, 23, 1966.
CHALKLEY, H.W. 12. Nat. Cancer Inst., 4, 47,
Methods 1943.
for quantitative
Practical
morphological
stain in electron
stereological
analysis
methods
of tissue.
WEIBEL, E.R. Stereological 13. morphometry. London: Academic
methods Vol. 1. Practical methods Press, ~26, 1979.
14. CHRISTIE, D.J., & ASTER, quinidine-induced thrombocytopenia.
R.H. Drug-antibody-platelet interaction in quinineJ. Clin. Invest., 70, 989-998, 1982.
for
J.
for biological
EDSTEIN, M.C., STACE,J., & SHANN,F. Quantification of quinine 15 by high-performance liquid chromatography. Journal of Chromatography, 1983.
and
in human serum 278, 445-451,
LAWSON, D., MEHTA, J., MEHTA, P., LIPMAN, B.C., & IMPERI, G.A. Cumulative 16. effects of quinidine and aspirin on bleeding time and platelet alpha2-adrenoceptors: potential mechanism of bleeding diathesis in patients receiving this combination. J. Lab. Clin. Med., 108, 581-586. 1986.
