Gemfibrozil
109
Effect of Gemfibrozil upon Platelet Function and Blood Coagulation. Preliminary Report by Dr V P 0 Rasi and Dr I Torstila (Finnish Red Cross Blood Transfusion Service Central Laboratory and Wihuri Research Institute, Helsinki, Finland) Gemfibrozil (CI-719) has been shown to lower the serum lipid levels in hyperlipwmic animals and man (Parke, Davis 1973). Its mode of action is under investigation. Platelet function is affected by changes in lipid metabolism (Nord0y 1976). It is therefore interesting that the use of another hypolipidemic agent, clofibrate, has been associated with several hemostatic alterations including pro- Dr V P 0 Rasi longation of bleeding time in man (O'Brien & Heywood 1966) and in the mouse (Herrmann 1973); decrease in the adhesiveness of blood during the seven days before the platelet functions platelets (Carson et al. 1963, O'Reilly et al. 1972); were studied. No dietary changes were made inhibitory effects on adrenaline-induced platelet during the experimental period. The patients were evaluated hmmostatically aggregation in normal subjects (O'Reilly et al. 1972), and normalization of the exaggerated twice before treatment with the active agent. The ADP-induced platelet aggregation in patients means of these results were used as reference with type II hyperlipidxmia (Carvalho et al. later in the study, and each subject served as her/ 1974). Clofibrate may also affect the coagulation his own control. After the screening period, the system by temporarily increasing fibrinolytic patients were treated with gemfibrozil, 800 mg activity and reducing fibrinogen levels (Chakra- daily for four weeks, and the hemostatic and barti et al. 1968), and it augments the effect of h&matological evaluation was repeated. The oral anticoagulants (Oliver et al. 1963). In hyper- study is continuing, and the examinations will betalipoproteineemia it normalizes the increased be repeated after eight weeks of treatment with a platelet factor-3 availability after ADP exposure daily dose of 1600 mg of gemfibrozil. (Nord0y & R0dset 1971). This pilot study is being carried out to find out Blood-collecting andProcessing whether gemfibrozil affects hemostatic para- Venous blood was drawn after an overnight fast, meters. Several tests of blood coagulation and and 10-12 h after the last dose of the drug. Using platelet function were used in patients who were a double-syringe technique, 10 ml of blood was motivated by the hypolipidwmic action of the taken into a plastic syringe and used without delay drug. The preliminary results are reported here. for the retention test. For aggregometry and platelet factor-3 (PF-3) activity studies, blood MATERIAL AND METHODS was collected in 0.106 M disodium citrate solution Patients (9 v blood to 1 v anticoagulant). Platelet-rich Ten patients with a previous history of hyper- plasma was prepared by centrifuging at 180 lipoproteinaemia were selected for the study. All gravities for eight min at room temperature, and were volunteers who understood the nature of-the diluted with freshly prepared platelet-poor trial. These 9 men and 1 woman ranged in age plasma from the same subject to a final confrom 29 to 54. On entry to the study they had centration of 200 x 0I platelets/,ul. Platelet-poor either a cholesterol level above 7.7 mmol/l or a plasma was obtained by recentrifugation of the triglyceride level above 1.7 mmol/l. platelet-rich plasma at 1800 gravities for 10 min. Medical and dietary history, physical and For coagulation studies, blood was taken in hmmatological examinations, including estima- 0.1 M disodium oxalate (9 v blood to 1 v antition of the main plasma lipid fractions, were coagulant), centrifuged to platelet-poor plasma as carried out. None of the subjects was receiving described for citrated plasma and recentrifuged lipid lowering agents during the six weeks before at 20,000 gravities for 30 min at +4°C. This the trial, or acetylsalicylic acid in any form or platelet-free plasma was stored at -20°C before any other drug known to affect platelet function, use.
110 Proc. roy. Soc. Med. Volume 691976 Supplement 2
Platelet Function Tests Platelet counts were measured by phase contrast microscopy on whole blood (Bjorkman 1959) or by the Coulter Counter Model F on platelet-rich plasma. Bleeding time was measured by the modified Ivy technique (Mielke et al. 1969). Platelet retention (adhesiveness) to glass beads was measured by a slight modification of the Hellem II Method (Hellem 1970). In this procedure, blood drawn without anticoagulant was passed through a standard glass bead column at the rate of 5.7 ml/min. The columns were made of PVC-plastic tubing, bore 3 mm, filled with 1.3 g of glass beads (Reflex Perlen 31/9, Dragon Werk, Bayreuth). The delay between drawing of blood and initiation of flow through the column was 10-12 sec. Platelet retention is expressed as the percentage of platelets retained in the glass bead column. Platelet aggregation was performed according to the method of Born (1962) with a Born Mk III Aggregometer equipped with a strip chart recorder, by estimating changes in optical transmission of citrated plasma at 37°C with a concentration of 200 x I03 platelets/pi. In I ml aliquots, aggregation was observed under continuous stirring after the addition of adenosine diphosphate (ADP, Sigma, 1.0 x 10-6M and 3.0 x 10-IM, final concentration); adrenaline (Medica, 1.3 x 10-6fM and 5.0 x 10-6M, final concentration) and bovine tendon collagen (Sigma, 5 ,ul and 20 pl. of standardized suspension). The rate of aggregation (which refers to optical transmission change/ min) was measured as the tangent of the maximum slope of the aggregation curve. The degree of aggregation was measured 5 min after addition of aggregating substance by the percentage change in light transmission, with the transmission for platelet poor plasma arbitrarily set as 100% and that for platelet rich plasma at 0%. All measurements were made within 2 h of the time blood was withdrawn by venepuncture. Blood Coagulation Tests Activated partial thromboplastin time of plasma was performed as described by Proctor & Rapaport (1961). The P and P method (Owren & Aas 1951) was used to evaluate the prothrombin complex. Thrombin clotting time of plasma was done by a modification of the method of Vermylen & Verstraete (1961). The thrombin used (Topostacine, Roche) was reconstituted in 0.15 M NaCl so that a clotting time of about 20 sec was obtained with pooled normal plasma. Antithrombin III was quantitated immunochemically by the single radial immunodiffusion technique (Mancini et al. 1965) using commercial antisera (Behringwerke AG).
Factor VIII activity was assayed by the kaolinactivated partial thromboplastin time usingfactor VIII-deficient plasma as substrate (Hardisty & Macpherson 1962). Ethanol gelation test was done according to Godal and Abilgaard (1966) using fresh plateletpoor oxalate plasma. RESULTS All patients were in good physical condition during the investigation. No side effects appeared, and adherence to the study was complete.
Lipid Studies All the patients had evidence of hyperlipoproteinmmia when entering the study. Three of them developed normal lipid values during the screening period when on placebo treatment, but were not excluded from the study. Treatment with gemfibrozil markedly lowered both the mean cholesterol and the mean triglyceride level, from 8.1 to 6.5 mmol/l and from 2.5 to 1.8 mmol/l.
Heematological Studies No adverse changes were observed in routine hoematology (erythrocyte sedimentation rate, hkmoglobin, hmmatocrit, WBC and differential white cell count), blood chemistry (SGPT, alkaline phosphatase, bilirubin) or in protein, ketones or glucose in analysis of urine, bile or blood. Platelet Function Studies The platelet count and the bleeding time remained unchanged from pretreatment values during gemfibrozil treatment (Table 1). Platelet retention to the glass bead column decreased significantly (P
109
Effect of Gemfibrozil upon Platelet Function and Blood Coagulation. Preliminary Report by Dr V P 0 Rasi and Dr I Torstila (Finnish Red Cross Blood Transfusion Service Central Laboratory and Wihuri Research Institute, Helsinki, Finland) Gemfibrozil (CI-719) has been shown to lower the serum lipid levels in hyperlipwmic animals and man (Parke, Davis 1973). Its mode of action is under investigation. Platelet function is affected by changes in lipid metabolism (Nord0y 1976). It is therefore interesting that the use of another hypolipidemic agent, clofibrate, has been associated with several hemostatic alterations including pro- Dr V P 0 Rasi longation of bleeding time in man (O'Brien & Heywood 1966) and in the mouse (Herrmann 1973); decrease in the adhesiveness of blood during the seven days before the platelet functions platelets (Carson et al. 1963, O'Reilly et al. 1972); were studied. No dietary changes were made inhibitory effects on adrenaline-induced platelet during the experimental period. The patients were evaluated hmmostatically aggregation in normal subjects (O'Reilly et al. 1972), and normalization of the exaggerated twice before treatment with the active agent. The ADP-induced platelet aggregation in patients means of these results were used as reference with type II hyperlipidxmia (Carvalho et al. later in the study, and each subject served as her/ 1974). Clofibrate may also affect the coagulation his own control. After the screening period, the system by temporarily increasing fibrinolytic patients were treated with gemfibrozil, 800 mg activity and reducing fibrinogen levels (Chakra- daily for four weeks, and the hemostatic and barti et al. 1968), and it augments the effect of h&matological evaluation was repeated. The oral anticoagulants (Oliver et al. 1963). In hyper- study is continuing, and the examinations will betalipoproteineemia it normalizes the increased be repeated after eight weeks of treatment with a platelet factor-3 availability after ADP exposure daily dose of 1600 mg of gemfibrozil. (Nord0y & R0dset 1971). This pilot study is being carried out to find out Blood-collecting andProcessing whether gemfibrozil affects hemostatic para- Venous blood was drawn after an overnight fast, meters. Several tests of blood coagulation and and 10-12 h after the last dose of the drug. Using platelet function were used in patients who were a double-syringe technique, 10 ml of blood was motivated by the hypolipidwmic action of the taken into a plastic syringe and used without delay drug. The preliminary results are reported here. for the retention test. For aggregometry and platelet factor-3 (PF-3) activity studies, blood MATERIAL AND METHODS was collected in 0.106 M disodium citrate solution Patients (9 v blood to 1 v anticoagulant). Platelet-rich Ten patients with a previous history of hyper- plasma was prepared by centrifuging at 180 lipoproteinaemia were selected for the study. All gravities for eight min at room temperature, and were volunteers who understood the nature of-the diluted with freshly prepared platelet-poor trial. These 9 men and 1 woman ranged in age plasma from the same subject to a final confrom 29 to 54. On entry to the study they had centration of 200 x 0I platelets/,ul. Platelet-poor either a cholesterol level above 7.7 mmol/l or a plasma was obtained by recentrifugation of the triglyceride level above 1.7 mmol/l. platelet-rich plasma at 1800 gravities for 10 min. Medical and dietary history, physical and For coagulation studies, blood was taken in hmmatological examinations, including estima- 0.1 M disodium oxalate (9 v blood to 1 v antition of the main plasma lipid fractions, were coagulant), centrifuged to platelet-poor plasma as carried out. None of the subjects was receiving described for citrated plasma and recentrifuged lipid lowering agents during the six weeks before at 20,000 gravities for 30 min at +4°C. This the trial, or acetylsalicylic acid in any form or platelet-free plasma was stored at -20°C before any other drug known to affect platelet function, use.
110 Proc. roy. Soc. Med. Volume 691976 Supplement 2
Platelet Function Tests Platelet counts were measured by phase contrast microscopy on whole blood (Bjorkman 1959) or by the Coulter Counter Model F on platelet-rich plasma. Bleeding time was measured by the modified Ivy technique (Mielke et al. 1969). Platelet retention (adhesiveness) to glass beads was measured by a slight modification of the Hellem II Method (Hellem 1970). In this procedure, blood drawn without anticoagulant was passed through a standard glass bead column at the rate of 5.7 ml/min. The columns were made of PVC-plastic tubing, bore 3 mm, filled with 1.3 g of glass beads (Reflex Perlen 31/9, Dragon Werk, Bayreuth). The delay between drawing of blood and initiation of flow through the column was 10-12 sec. Platelet retention is expressed as the percentage of platelets retained in the glass bead column. Platelet aggregation was performed according to the method of Born (1962) with a Born Mk III Aggregometer equipped with a strip chart recorder, by estimating changes in optical transmission of citrated plasma at 37°C with a concentration of 200 x I03 platelets/pi. In I ml aliquots, aggregation was observed under continuous stirring after the addition of adenosine diphosphate (ADP, Sigma, 1.0 x 10-6M and 3.0 x 10-IM, final concentration); adrenaline (Medica, 1.3 x 10-6fM and 5.0 x 10-6M, final concentration) and bovine tendon collagen (Sigma, 5 ,ul and 20 pl. of standardized suspension). The rate of aggregation (which refers to optical transmission change/ min) was measured as the tangent of the maximum slope of the aggregation curve. The degree of aggregation was measured 5 min after addition of aggregating substance by the percentage change in light transmission, with the transmission for platelet poor plasma arbitrarily set as 100% and that for platelet rich plasma at 0%. All measurements were made within 2 h of the time blood was withdrawn by venepuncture. Blood Coagulation Tests Activated partial thromboplastin time of plasma was performed as described by Proctor & Rapaport (1961). The P and P method (Owren & Aas 1951) was used to evaluate the prothrombin complex. Thrombin clotting time of plasma was done by a modification of the method of Vermylen & Verstraete (1961). The thrombin used (Topostacine, Roche) was reconstituted in 0.15 M NaCl so that a clotting time of about 20 sec was obtained with pooled normal plasma. Antithrombin III was quantitated immunochemically by the single radial immunodiffusion technique (Mancini et al. 1965) using commercial antisera (Behringwerke AG).
Factor VIII activity was assayed by the kaolinactivated partial thromboplastin time usingfactor VIII-deficient plasma as substrate (Hardisty & Macpherson 1962). Ethanol gelation test was done according to Godal and Abilgaard (1966) using fresh plateletpoor oxalate plasma. RESULTS All patients were in good physical condition during the investigation. No side effects appeared, and adherence to the study was complete.
Lipid Studies All the patients had evidence of hyperlipoproteinmmia when entering the study. Three of them developed normal lipid values during the screening period when on placebo treatment, but were not excluded from the study. Treatment with gemfibrozil markedly lowered both the mean cholesterol and the mean triglyceride level, from 8.1 to 6.5 mmol/l and from 2.5 to 1.8 mmol/l.
Heematological Studies No adverse changes were observed in routine hoematology (erythrocyte sedimentation rate, hkmoglobin, hmmatocrit, WBC and differential white cell count), blood chemistry (SGPT, alkaline phosphatase, bilirubin) or in protein, ketones or glucose in analysis of urine, bile or blood. Platelet Function Studies The platelet count and the bleeding time remained unchanged from pretreatment values during gemfibrozil treatment (Table 1). Platelet retention to the glass bead column decreased significantly (P
