SEMINARS IN THROMBOSIS AND HEMOSTASIS—VOLUME 17, NO. 2, 1991
Hirudin and Platelets
Platelet reactions such as primary and secondary aggregation, secretion, and thromboxane formation depend on aggregating agents and on the medium, including proteins and divalent cations.1 The anticoagulants used for collection of blood may, either directly or indirectly, influence these platelet reactions. Sodium citrate is commonly used as an anticoagulant for the isolation of platelets and the preparation of platelet-rich plasma (PRP). In citrated plasma the calcium ion concentration was diminished to 46 ± 4 µmol/liter (n=6) when sodium citrate at a final concentration of 11 mmol/liter was used for anticoagulation. Several experimental studies have shown that the secondary phase of platelet aggregation, secretion, and thromboxane formation occur predominantly in a medium with low Ca 2+ concentrations.2-7 Other anticoagulants such as heparin, hirudin, or PPACK (D-phenylalanyl-L-prolyl-L-arginine chlormethyl ketone) selectively inhibit thrombin activity but do not reduce the Ca 2+ concentration in blood. In hirudin-anticoagulated plasma the ionized calcium level amounted to 626 ± 14 µmol/liter (n=6). For its anticoagulant effect heparin requires the presence of endogenous cofactors such as antithrombin III (AT III). Heparin is known to promote platelet aggregation, possibly by direct interaction with platelets.8 The selective tight-binding thrombin inhibitor hirudin, which is a single-chain protein with 65 or 66 amino acids, proved to be a potent anticoagulant and an antithrombotic agent.9,10 It prevents not only the thrombin-catalyzed conversion of fibrinogen to fibrin but also thrombinmediated cellular events, such as platelet aggregation and secretion.11,12 In previous studies we found that the adenosine diphosphate (ADP)- and epinephrine-induced
From the Institute of Pharmacology and Toxicology, Medical Academy Erfurt, Erfurt, Germany. Reprint requests: Institut fur Pharmakologie und Toxikologie, Medizinische Akademie Erfurt, Nordhauserstr. 74, Erfurt 0-5010, Germany.
122
aggregation of human platelets in hirudinized plasma differed from that in citrated plasma.13 In the past several years, our results have been confirmed by other workers. 7,14,15 The use of native hirudin as an anticoagulant remained limited, since it had to be isolated from medicinal leeches. Due to the development of modern procedures of genetic engineering, recombinant types of hirudin are now available.16 Several studies were carried out to demonstrate the influence of recombinant desulfatohirudin, expressed in yeast, on platelet behavior.
INHIBITION OF THROMBIN-INDUCED AGGREGATION AND SECRETION The recombinant (r-) hirudin used has an inhibition constant value of 1.05 ± 0.24 pmol/liter,17 which is nearly equal to that of native hirudin. As shown in Figure 1, r-hirudin was a potent inhibitor of thrombin-induced aggregation of human platelets in Tyrode's solution. It inhibited also the release of 14C-serotonin from prelabeled platelets in a concentration-dependent manner; the IC 50 value amounted to 10 ng/ml.18
AGGREGATION IN r-HIRUDINANTICOAGULATED WHOLE BLOOD Our particular interest was directed to the responses of human platelets to aggregating substances in whole blood anticoagulated with r-hirudin in comparison to those occurring in citrate- or heparin-anticoagulated blood. After stirring of whole blood in the aggregometer, spontaneous aggregation, assessed by the decrease in platelet counts, was significantly lower in blood anticoagulated with r-hirudin than with citrated or heparinized whole blood.18 In further experiments, aggregation in whole blood was measured by means of a Chrono-Log Lumiaggregometer. The ADP- and collagen-induced aggregation was found to be more pronounced in
Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
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ERIKA GLUSA, M.D.
HIRUDIN AND PLATELETS—GLUSA
123 blood compared with citrated and heparinized PRP. The same results were obtained by other workers. 19,20InPRP obtained from r-hirudin-anticoagulated blood, platelet adhesion to the subendothelial matrix was markedly inhibited in a concentration-dependent manner.20
FIG. 1. Inhibition of thrombin (0.1 U/ml)-induced aggregation of human blood platelets in Tyrode's solution by recombinant hirudin.
r-hirudinized than in citrated blood. Epinephrine-induced aggregation was not observed in citrated and in r-hirudinized blood. However, the potentiating effect of epinephrine could be demonstrated after subsequent addition of ADP at low concentrations (Fig. 2).
Platelet Adhesion Platelet adhesion to siliconized glass was significantly diminished in PRP from r-hirudin-anticoagulated
FIG. 2. Aggregation of human blood platelets in whole blood anticoagulated by citrate (11 mmol/liter) or hirudin (20 µg/ml). Adren.: Epinephrine.
Previous studies were done in citrated PRP to rule out the interference of r-hirudin with ADP-induced aggregation. At the concentration used to inhibit coagulation, r-hirudin (20 µg/ml) had no influence on aggregation, whereas heparin (15 µg/ml) at the concentration required for anticoagulation enhanced ADP-induced aggregation.18 Comparative studies with r-hirudinized, citrated, and heparinized PRP showed nearly the same extent of ADP-induced aggregation in the different plasma samples. However, depending on the ADP concentration, disaggregation was predominantly observed in r-hirudinized plasma.18 ADP-induced secretion of 14C-serotonin (Fig. 3) and formation of thromboxane A2, determined by its stable metabolite thromboxane B 2 , were much more pronounced in citrated than in r-hirudinized PRP (Table 1; unpublished data). Collagen-induced aggregation and 14C-serotonin secretion did not differ significantly in the three plasma samples. Formation of thromboxane in collagen-stimulated platelets was reduced by 30 to 40% in r-hirudinized plasma compared with citrated plasma (Table 1). The platelet-activating factor (1 µmol/liter)-induced aggregation was highly variable. In citrated plasma biphasic aggregation accompanied by thromboxane formation was observed, whereas in r-hirudinized plasma aggregation remained reversible in most cases and thromboxane formation was markedly reduced (Table 1).
FIG. 3. Adenosine diphosphate (10 µmol/liter)-induced aggregation and 14C-serotonin release in human platelet-rich plasma anticoagulated with citrate (11 mmol/liter), r-hirudin (20 µg/ml), or heparin (15 µg/ml).
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SEMINARS IN THROMBOSIS AND
MOSTASIS—VOLUME 17, NO. 2, 1991
TABLE 1. Thromboxane (TX) B2 Formation in Formation in Platelet-Rich Plasma (PRP) TXB2 (ng/ml; mean ± SEM) Citrate ± PRP
r-Hirudin ± PRP
No.
Adenosinediphosphate, 4 µmol/liter
17.8 ± 2.2
2.7 ± 0.9
10
Platelet-activating factor, 1 µmol/1
32.1 ±4.5
1.0 ± 0.3
8
Epinephrine, 1 µmol/1
49.0 ± 7.3
0.4 ± 0.2
6
Collagen, 0.6 µg/ml
25.0 ± 3.3
10.6 ± 0.8
10
The greatest differences in the aggregation response between r-hirudinized and citrated plasma were found when epinephrine was used as the aggregating agent. It is well known that in citrated PRP epinephrine, at a concentration of 1 to 10 µmol/liter causes a biphasic aggregation response and 14C-serotonin secretion (Fig. 4) as well as formation of thromboxane (Table 1). In r-hirudinized PRP a weak primary aggregation was shown in response to epinephrine. The same results were reported with r-hirudin by other workers. 19-21 14Cserotonin release and thromboxane formation were negligible (Table 1). In further experiments the platelet response to epinephrine was investigated in PRP prepared from blood anticoagulated with sodium citrate plus r-hirudin. On the addition of epinephrine, a biphasic aggregation occurred and the same amount of thromboxane B 2 was formed as in citrated plasma (Fig. 5).
FIG. 5. Epinephrine (1 µmol/l)-induced human platelet aggregation and formation of thromboxane B2 in plasma anticoagulated by citrate (11 mmol/l), r-hirudin (20µg/ml),or both citrate plus r-hirudin.
According to these results, the artificial reduction of ionized calcium may account for the different behavior of platelets. PRP anticoagulated by citrate plus r-hirudin was incubated with increasing calcium chloride concentrations before aggregation was induced. The epinephrine-induced aggregation was found to decrease with increasing calcium ion concentrations, as was shown in previous studies with native hirudin.13 After addition of 10 mmol/liter calcium chloride to the citrate-hirudinPRP, formation of thromboxane was reduced to the level obtained in r-hirudinized plasma; in this case the concentration of ionized calcium did not differ from that in r-hirudinized plasma.
CONCLUSION
FIG. 4. Epinephrine (5 µmol/liter)-induced aggregation and 14 C-serotonin release in human platelet-rich plasma anticoagulated with citrate (11 mmol/liter), r-hirudin (20 µg/ml), or heparin (15 fig/ml).
The results of platelet function studies with r-hirudin as anticoagulant were in accordance with those obtained with native hirudin. 11-15 In r-hirudinized plasma the response of platelets to aggregating substances differed from that in citrated plasma, especially with regard to serotonin release and formation of thromboxane A2. These differences might be due to the reduced calcium ion concentrations in citrated plasma or to specific effects of citrate on platelet-bound divalent cations. 7,15 The in vitro results obtained with sodium citrate as anticoagulant cannot simply be applied to the in vivo situation. r-Hirudin proved to be a highly potent anticoagulant, suitable for studying platelet functions in blood at physiologic calcium ion concentrations. Compared with heparin, r-hirudin has the advantage because it does not interfere with platelet functions and has no direct effects on platelets. These properties are of great importance for its therapeutic use.
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Aggregation Induced by
125
REFERENCES
10. Markwardt F: Development of hirudin as an antithrombotic agent. Semin Thromb Hemost 15:269-282, 1989. 11. Hoffmann A, F Markwardt: Inhibition of the thrombin-platelet reaction by hirudin. Haemostasis 14:164-169, 1984. 12. Glusa E, U Urban: Studies on platelet functions in hirudin plasma. Folia Haematol (Leipz) 115:88-93, 1988. 13. Glusa E, F Markwardt: Adrenaline-induced reactions of human platelets in hirudin plasma. Haemostasis 9:188-192, 1980. 14. Lanza F, A Beretz, A Stierle, D Hanau, M Kubina, JP Cazenave: Epinephrine potentiates human platelet activation but is not an aggregating agent. Am J Physiol 255:H1276-H1288, 1988. 15. Keraly CL, RL Kinlough-Rathbone, MA Packham, H Suzuki, JF Mustard: Conditions affecting the responses of human platelets to epinephrine. Thromb Haemost 60:209-216, 1988. 16. Markwardt F, G Fink, B Kaiser, HP Klöcking, G Nowak, M Richter, J Sturzebecher: Pharmacological survey of recombinant hirudin. Pharmazie 43:202-207, 1988. 17. Markwardt F, J Stürzebecher, E Glusa: Antithrombin effects of native and recombinant hirudins. Biomed Biochim Acta 49:399404, 1990. 18. Glusa E, F Markwardt: Platelet functions in recombinant hirudinanticoagulated blood. Haemostasis 20:112-118, 1990. 19. Basic-Micic M, CH Rauschenbach, HK Breddin: Effects of hirudin and two synthetic thrombin inhibitors on platelet function in the presence and absence of citrate. Angio Arch 18:10, 1989. 20. Basic-Micic M, K Krupinski, HK Breddin: r-hirudin effects on various platelet functions. Angio Arch 18:11, 1989. 21. Fareed D, R Pifarre, JM Walenga, HK Breddin, J Fareed: Effect of recombinant hirudin and heparin on human platelet aggregation. Angio Arch 18:23, 1989.
1. Siess W: Molecular mechanisms of platelet activation. Physiol Rev 69:58-178, 1989. 2. Huijgens PC, CAM van den Berg, AMH Voettdijk, LMFM Imandt: The influence of citrate on platelet aggregation and malondialdehyde production. Scand J Haematol 31:129-132, 1983. 3. Scrutton MC, CM Egan: Divalent cation requirements for aggregation of human blood platelets and the role of the anticoagulant. Thromb Res 14:713-727, 1979. 4. Heptinstall S, PM Taylor: The effects of citrate and extracellular calcium ions on the platelet release reaction induced by ADP and collagen. Thromb Haemost 42:778-793, 1979. 5. Packham MA, NL Bryant, MA Guccione, RL Kinlough-Rathbone, JF Mustard: Effect of the concentration of Ca 2+ in the suspending medium on the responses of human and rabbit platelets to aggregation agents. Thromb Haemost 62:968-976, 1989. 6. Mustard JF, DW Perry, RL Kinlough-Rathbone, MA Packham: Factors responsible for ADP-induced release reaction of human platelets. Am J Physiol 228:1757-1765, 1975. 7. Lages B, HJ Weiss: Dependence of human platelet functional responses on divalent cations: Aggregation and secretion in heparin- and hirudin-anticoagulated platelet-rich plasma and the effects of chelating agents. Thromb Haemost 45:173-179, 1981. 8. Brace LD, J Fareed: An objective assessment of the interaction of heparin and its fractions with human platelets. Semin Thromb Hemost 11:190-198, 1985. 9. Markwardt F: Hirudin as an inhibitor of thrombin. Methods Enzymol 69:924-932, 1970.
Downloaded by: Universite Laval. Copyrighted material.
HIRUDIN AND PLATELETS—GLUSA
Hirudin and Platelets
Platelet reactions such as primary and secondary aggregation, secretion, and thromboxane formation depend on aggregating agents and on the medium, including proteins and divalent cations.1 The anticoagulants used for collection of blood may, either directly or indirectly, influence these platelet reactions. Sodium citrate is commonly used as an anticoagulant for the isolation of platelets and the preparation of platelet-rich plasma (PRP). In citrated plasma the calcium ion concentration was diminished to 46 ± 4 µmol/liter (n=6) when sodium citrate at a final concentration of 11 mmol/liter was used for anticoagulation. Several experimental studies have shown that the secondary phase of platelet aggregation, secretion, and thromboxane formation occur predominantly in a medium with low Ca 2+ concentrations.2-7 Other anticoagulants such as heparin, hirudin, or PPACK (D-phenylalanyl-L-prolyl-L-arginine chlormethyl ketone) selectively inhibit thrombin activity but do not reduce the Ca 2+ concentration in blood. In hirudin-anticoagulated plasma the ionized calcium level amounted to 626 ± 14 µmol/liter (n=6). For its anticoagulant effect heparin requires the presence of endogenous cofactors such as antithrombin III (AT III). Heparin is known to promote platelet aggregation, possibly by direct interaction with platelets.8 The selective tight-binding thrombin inhibitor hirudin, which is a single-chain protein with 65 or 66 amino acids, proved to be a potent anticoagulant and an antithrombotic agent.9,10 It prevents not only the thrombin-catalyzed conversion of fibrinogen to fibrin but also thrombinmediated cellular events, such as platelet aggregation and secretion.11,12 In previous studies we found that the adenosine diphosphate (ADP)- and epinephrine-induced
From the Institute of Pharmacology and Toxicology, Medical Academy Erfurt, Erfurt, Germany. Reprint requests: Institut fur Pharmakologie und Toxikologie, Medizinische Akademie Erfurt, Nordhauserstr. 74, Erfurt 0-5010, Germany.
122
aggregation of human platelets in hirudinized plasma differed from that in citrated plasma.13 In the past several years, our results have been confirmed by other workers. 7,14,15 The use of native hirudin as an anticoagulant remained limited, since it had to be isolated from medicinal leeches. Due to the development of modern procedures of genetic engineering, recombinant types of hirudin are now available.16 Several studies were carried out to demonstrate the influence of recombinant desulfatohirudin, expressed in yeast, on platelet behavior.
INHIBITION OF THROMBIN-INDUCED AGGREGATION AND SECRETION The recombinant (r-) hirudin used has an inhibition constant value of 1.05 ± 0.24 pmol/liter,17 which is nearly equal to that of native hirudin. As shown in Figure 1, r-hirudin was a potent inhibitor of thrombin-induced aggregation of human platelets in Tyrode's solution. It inhibited also the release of 14C-serotonin from prelabeled platelets in a concentration-dependent manner; the IC 50 value amounted to 10 ng/ml.18
AGGREGATION IN r-HIRUDINANTICOAGULATED WHOLE BLOOD Our particular interest was directed to the responses of human platelets to aggregating substances in whole blood anticoagulated with r-hirudin in comparison to those occurring in citrate- or heparin-anticoagulated blood. After stirring of whole blood in the aggregometer, spontaneous aggregation, assessed by the decrease in platelet counts, was significantly lower in blood anticoagulated with r-hirudin than with citrated or heparinized whole blood.18 In further experiments, aggregation in whole blood was measured by means of a Chrono-Log Lumiaggregometer. The ADP- and collagen-induced aggregation was found to be more pronounced in
Copyright © 1991 by Thieme Medical Publishers, Inc., 381 Park Avenue South, New York, NY 10016. All rights reserved.
Downloaded by: Universite Laval. Copyrighted material.
ERIKA GLUSA, M.D.
HIRUDIN AND PLATELETS—GLUSA
123 blood compared with citrated and heparinized PRP. The same results were obtained by other workers. 19,20InPRP obtained from r-hirudin-anticoagulated blood, platelet adhesion to the subendothelial matrix was markedly inhibited in a concentration-dependent manner.20
FIG. 1. Inhibition of thrombin (0.1 U/ml)-induced aggregation of human blood platelets in Tyrode's solution by recombinant hirudin.
r-hirudinized than in citrated blood. Epinephrine-induced aggregation was not observed in citrated and in r-hirudinized blood. However, the potentiating effect of epinephrine could be demonstrated after subsequent addition of ADP at low concentrations (Fig. 2).
Platelet Adhesion Platelet adhesion to siliconized glass was significantly diminished in PRP from r-hirudin-anticoagulated
FIG. 2. Aggregation of human blood platelets in whole blood anticoagulated by citrate (11 mmol/liter) or hirudin (20 µg/ml). Adren.: Epinephrine.
Previous studies were done in citrated PRP to rule out the interference of r-hirudin with ADP-induced aggregation. At the concentration used to inhibit coagulation, r-hirudin (20 µg/ml) had no influence on aggregation, whereas heparin (15 µg/ml) at the concentration required for anticoagulation enhanced ADP-induced aggregation.18 Comparative studies with r-hirudinized, citrated, and heparinized PRP showed nearly the same extent of ADP-induced aggregation in the different plasma samples. However, depending on the ADP concentration, disaggregation was predominantly observed in r-hirudinized plasma.18 ADP-induced secretion of 14C-serotonin (Fig. 3) and formation of thromboxane A2, determined by its stable metabolite thromboxane B 2 , were much more pronounced in citrated than in r-hirudinized PRP (Table 1; unpublished data). Collagen-induced aggregation and 14C-serotonin secretion did not differ significantly in the three plasma samples. Formation of thromboxane in collagen-stimulated platelets was reduced by 30 to 40% in r-hirudinized plasma compared with citrated plasma (Table 1). The platelet-activating factor (1 µmol/liter)-induced aggregation was highly variable. In citrated plasma biphasic aggregation accompanied by thromboxane formation was observed, whereas in r-hirudinized plasma aggregation remained reversible in most cases and thromboxane formation was markedly reduced (Table 1).
FIG. 3. Adenosine diphosphate (10 µmol/liter)-induced aggregation and 14C-serotonin release in human platelet-rich plasma anticoagulated with citrate (11 mmol/liter), r-hirudin (20 µg/ml), or heparin (15 µg/ml).
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Platelet Reactions in PRP
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SEMINARS IN THROMBOSIS AND
MOSTASIS—VOLUME 17, NO. 2, 1991
TABLE 1. Thromboxane (TX) B2 Formation in Formation in Platelet-Rich Plasma (PRP) TXB2 (ng/ml; mean ± SEM) Citrate ± PRP
r-Hirudin ± PRP
No.
Adenosinediphosphate, 4 µmol/liter
17.8 ± 2.2
2.7 ± 0.9
10
Platelet-activating factor, 1 µmol/1
32.1 ±4.5
1.0 ± 0.3
8
Epinephrine, 1 µmol/1
49.0 ± 7.3
0.4 ± 0.2
6
Collagen, 0.6 µg/ml
25.0 ± 3.3
10.6 ± 0.8
10
The greatest differences in the aggregation response between r-hirudinized and citrated plasma were found when epinephrine was used as the aggregating agent. It is well known that in citrated PRP epinephrine, at a concentration of 1 to 10 µmol/liter causes a biphasic aggregation response and 14C-serotonin secretion (Fig. 4) as well as formation of thromboxane (Table 1). In r-hirudinized PRP a weak primary aggregation was shown in response to epinephrine. The same results were reported with r-hirudin by other workers. 19-21 14Cserotonin release and thromboxane formation were negligible (Table 1). In further experiments the platelet response to epinephrine was investigated in PRP prepared from blood anticoagulated with sodium citrate plus r-hirudin. On the addition of epinephrine, a biphasic aggregation occurred and the same amount of thromboxane B 2 was formed as in citrated plasma (Fig. 5).
FIG. 5. Epinephrine (1 µmol/l)-induced human platelet aggregation and formation of thromboxane B2 in plasma anticoagulated by citrate (11 mmol/l), r-hirudin (20µg/ml),or both citrate plus r-hirudin.
According to these results, the artificial reduction of ionized calcium may account for the different behavior of platelets. PRP anticoagulated by citrate plus r-hirudin was incubated with increasing calcium chloride concentrations before aggregation was induced. The epinephrine-induced aggregation was found to decrease with increasing calcium ion concentrations, as was shown in previous studies with native hirudin.13 After addition of 10 mmol/liter calcium chloride to the citrate-hirudinPRP, formation of thromboxane was reduced to the level obtained in r-hirudinized plasma; in this case the concentration of ionized calcium did not differ from that in r-hirudinized plasma.
CONCLUSION
FIG. 4. Epinephrine (5 µmol/liter)-induced aggregation and 14 C-serotonin release in human platelet-rich plasma anticoagulated with citrate (11 mmol/liter), r-hirudin (20 µg/ml), or heparin (15 fig/ml).
The results of platelet function studies with r-hirudin as anticoagulant were in accordance with those obtained with native hirudin. 11-15 In r-hirudinized plasma the response of platelets to aggregating substances differed from that in citrated plasma, especially with regard to serotonin release and formation of thromboxane A2. These differences might be due to the reduced calcium ion concentrations in citrated plasma or to specific effects of citrate on platelet-bound divalent cations. 7,15 The in vitro results obtained with sodium citrate as anticoagulant cannot simply be applied to the in vivo situation. r-Hirudin proved to be a highly potent anticoagulant, suitable for studying platelet functions in blood at physiologic calcium ion concentrations. Compared with heparin, r-hirudin has the advantage because it does not interfere with platelet functions and has no direct effects on platelets. These properties are of great importance for its therapeutic use.
Downloaded by: Universite Laval. Copyrighted material.
Aggregation Induced by
125
REFERENCES
10. Markwardt F: Development of hirudin as an antithrombotic agent. Semin Thromb Hemost 15:269-282, 1989. 11. Hoffmann A, F Markwardt: Inhibition of the thrombin-platelet reaction by hirudin. Haemostasis 14:164-169, 1984. 12. Glusa E, U Urban: Studies on platelet functions in hirudin plasma. Folia Haematol (Leipz) 115:88-93, 1988. 13. Glusa E, F Markwardt: Adrenaline-induced reactions of human platelets in hirudin plasma. Haemostasis 9:188-192, 1980. 14. Lanza F, A Beretz, A Stierle, D Hanau, M Kubina, JP Cazenave: Epinephrine potentiates human platelet activation but is not an aggregating agent. Am J Physiol 255:H1276-H1288, 1988. 15. Keraly CL, RL Kinlough-Rathbone, MA Packham, H Suzuki, JF Mustard: Conditions affecting the responses of human platelets to epinephrine. Thromb Haemost 60:209-216, 1988. 16. Markwardt F, G Fink, B Kaiser, HP Klöcking, G Nowak, M Richter, J Sturzebecher: Pharmacological survey of recombinant hirudin. Pharmazie 43:202-207, 1988. 17. Markwardt F, J Stürzebecher, E Glusa: Antithrombin effects of native and recombinant hirudins. Biomed Biochim Acta 49:399404, 1990. 18. Glusa E, F Markwardt: Platelet functions in recombinant hirudinanticoagulated blood. Haemostasis 20:112-118, 1990. 19. Basic-Micic M, CH Rauschenbach, HK Breddin: Effects of hirudin and two synthetic thrombin inhibitors on platelet function in the presence and absence of citrate. Angio Arch 18:10, 1989. 20. Basic-Micic M, K Krupinski, HK Breddin: r-hirudin effects on various platelet functions. Angio Arch 18:11, 1989. 21. Fareed D, R Pifarre, JM Walenga, HK Breddin, J Fareed: Effect of recombinant hirudin and heparin on human platelet aggregation. Angio Arch 18:23, 1989.
1. Siess W: Molecular mechanisms of platelet activation. Physiol Rev 69:58-178, 1989. 2. Huijgens PC, CAM van den Berg, AMH Voettdijk, LMFM Imandt: The influence of citrate on platelet aggregation and malondialdehyde production. Scand J Haematol 31:129-132, 1983. 3. Scrutton MC, CM Egan: Divalent cation requirements for aggregation of human blood platelets and the role of the anticoagulant. Thromb Res 14:713-727, 1979. 4. Heptinstall S, PM Taylor: The effects of citrate and extracellular calcium ions on the platelet release reaction induced by ADP and collagen. Thromb Haemost 42:778-793, 1979. 5. Packham MA, NL Bryant, MA Guccione, RL Kinlough-Rathbone, JF Mustard: Effect of the concentration of Ca 2+ in the suspending medium on the responses of human and rabbit platelets to aggregation agents. Thromb Haemost 62:968-976, 1989. 6. Mustard JF, DW Perry, RL Kinlough-Rathbone, MA Packham: Factors responsible for ADP-induced release reaction of human platelets. Am J Physiol 228:1757-1765, 1975. 7. Lages B, HJ Weiss: Dependence of human platelet functional responses on divalent cations: Aggregation and secretion in heparin- and hirudin-anticoagulated platelet-rich plasma and the effects of chelating agents. Thromb Haemost 45:173-179, 1981. 8. Brace LD, J Fareed: An objective assessment of the interaction of heparin and its fractions with human platelets. Semin Thromb Hemost 11:190-198, 1985. 9. Markwardt F: Hirudin as an inhibitor of thrombin. Methods Enzymol 69:924-932, 1970.
Downloaded by: Universite Laval. Copyrighted material.
HIRUDIN AND PLATELETS—GLUSA
