The efficacy and potency of antiplatelet activity of ticlopidine is increased by aspirin.

Concomitant therapy with ticlopidine (T) and aspirin (ASA) profoundly increases spectrum of antiplatelet activities of both drugs. It was hypothesized that in addition to increased spectrum of activity (efficacy) each drug may potentiate the specific antiplatelet activity (potency) of the other. In 32 volunteers whole blood platelet aggregation (PA) in response to ADP, collagen, and arachidonic acid was evaluated ex vivo following 10-day treatments with normal or subthreshold doses of T or ASA with addition of second drug on the 5th day of administration of the first. PA was measured before, on day 5 and 10 of treatment. The results indicate that ASA increased spectrum of activity of T, i.e. T and ASA in combination, were significantly more effective against collagen-induced PA than either drug alone. This increased efficacy was retained when subthreshold dose of T (100 mg, qd) was used. T was without effect on AA-induced and ASA on ADP-induced PA. However, ASA potentiated effect of T on ADP-induced PA; the subthreshold dose of T (100 mg, qd) in presence of ASA (100 mg, qd) exerted powerful inhibition. Thus, combination therapy increases both efficacy and potency of T allowing for reduction of the dose.

Pharmacodynamics of ticlopidine: relation between dose and time of administration to platelet inhibition.

In spite of long clinical experience with ticlopidine (T) knowledge of its pharmacodynamics is limited. In this study relation between dose and time of administration of T to platelet inhibition was investigated in 62 healthy volunteers ex vivo in whole blood and platelet rich plasma. Gender-related sensitivity of platelets to ticlopidine was also evaluated. Inhibition of ADP-induced platelet aggregation by T, 500 mg, daily, was almost identical in both sexes. 100 mg daily did not inhibit ADP-induced platelet aggregation even after 14 days of administration. 250 mg daily induced strong inhibition on day 5 of administration comparable to the inhibition obtained with 500 mg daily dose. The antiplatelet (ADP) effect of T (500 mg, daily) was present on day 2-3 and full inhibitory effect on day 4 of administration. T1/2 of antiplatelet (ADP) activity of T was 5.3 days and full recovery of platelets activity 11-13 days. No rebound phenomenon was present. T (regardless the dose) inhibited platelet aggregation induced by small but not high concentrations of collagen and was without effect on arachidonic acid-induced platelet aggregation. Therefore, T is not suitable for treatment of acute event, 250 mg daily dose should be used especially for combination with other drugs and 11 days washout interval seems necessary to change the treatment or to perform surgery.

Spontaneous platelet aggregation, tourniquet ischaemia, and aspirin in survivors of myocardial infarction.

Platelet number, plasma beta-thromboglobulin (beta-TG), spontaneous platelet aggregation (SPA), serum thromboxane B(2) (TXB(2)), and 6-keto-PGF(101) were assessed in 9 males with proven atherosclerosis (survivors of myocardial infarction, (MI) in a stable condition) and compared with values for 9 young, healthy controls. Results were obtained before and after 3-day treatment with aspirin. In addition, SPA was assessed before and after a tourniquet test. MI survivors had higher beta-TG (p < 0.01) and SPA values (p < 0.05), and a lower platelet count (p < 0.05) than controls. Aspirin significantly attentuated all these changes. These data suggest that platelet function is increased in survivors of MI. Tourniquet-induced ischaemia significantly (p < 0.05) enhanced SPA in MI survivors but not in controls. Aspirin did not inhibit SPA following ischaemia in contrast to its effect under pre-ischaemic conditions or in controls. It seems that the proaggregatory threshold is decreased in MI survivors. The results challenge the assumption that an acute coronary event with underlying atherosclerosis is due to an interaction between atherosclerosis plaque and normal platelets.

Simple and rapid isolation of platelets using Ficoll-Uropoline cushion.

A simplified procedure for separation of human platelets from plasma proteins when a small volume of blood is available has been developed. In this method two centrifugations of platelets layered on Ficoll-Uropoline cushion was applied. In the first step, platelet rich plasma layered over Ficoll-Uropoline solution was centrifuged (700 g, 30 min), bounded platelets recovered with the help of HEPES buffer, and centrifuged again (1000 g, 10 min) on Ficoll-Uropoline cushion. This rapid and simple procedure yielded platelets separated from von Willebrand factor, fibrinogen and albumin, as judged by bioassay and measurement of radiolabeled plasma proteins. The obtained preparation of platelets was stable and the platelet function preserved for 3 h as evidenced by platelet aggregation and fibrinogen binding analysis. The procedure is inexpensive and convenient for small volumes of blood.

Plasma beta-thromboglobulin in acute myocardial infarction with ventricular arrhythmias.

Patients with myocardial infarction, complicated by ventricular arrhythmias (Lown grade greater than or equal to 2), had higher (p less than 0.05) plasma beta-thromboglobulin level than that found in patients with uncomplicated infarction. This indicates higher platelet activation in the former group than in the latter. Such data provide rationale for clinical trial of antiplatelet drugs in cases of myocardial infarction complicated by severe arrhythmias.

Angiogenesis: quantitative assessment by the chick chorioallantoic membrane assay.

The aim of the present work was to improve quantitative assessment of angiogenesis by chick chorioallantoic membrane (CAM) assay based on measurement of DNA synthesis. Following incubation of [3H]thymidine (3H-T) with CAM in vivo, incorporation of 3H-T to DNA fraction was expressed as percent of total 3H-T present in the initial homogenate of CAM, regardless of CAM weight and full recovery of applied radioactivity. The assay required simple, partial isolation of DNA to remove traces of free or unspecifically bound 3H-T. These modifications gave a reproducible assay with adequate precision (10-20%). DNA content of CAM did not change between 10-15 days of embryo development and growth of CAM was completed on day 11. 10 to 12-day old CAMs were used for evaluation of the assay. Using a qualitative approach (visual scoring), tissues of several tumors were implanted on CAM. Extracts of tumors that produced the highest score stimulated DNA synthesis in CAM. A similar effect was induced by EGF while cytostatics inhibited DNA synthesis in CAM. Since stimulation of angiogenesis is not a cell type-specific phenomenon, the assay in the present modification should aid the studies on angiogenic factors. With the help of this assay the angiogenic activity of adenocarcinoma of human endometrium was described.

Effects of inhibition of thromboxane A2 synthesis in aspirin-induced asthma.

Since inhibition of cyclooxygenase precipitates asthmatic attacks in patients with aspirin idiosyncrasy, we have evaluated the effects of pharmacologic inhibition of thromboxane A2 (TXA2) synthetase, next to cyclooxygenase enzyme in arachidonic acid cascade. Sixteen patients with aspirin-induced asthma received increasing doses on 3 days (25 to 400 mg) of an imidazole derivative, OKY-046, which specifically blocks TXA2 synthetase. Twenty-three healthy control subjects received a single dose of 400 mg of OKY-046. In both patients and control subjects, the inhibitor at a dose of 400 mg produced (1) a pronounced fall in thromboxane B2 serum levels, (2) a rise in serum 6-keto-prostaglandin F1 alpha, and (3) a depression in platelet aggregability to arachidonic acid and adenosine diphosphate. The drug, however, neither precipitated attacks of asthma nor impaired pulmonary function tests throughout a 24-hour observation period. Five patients, but none of the control subjects, developed transient nasal congestion about 1 hour after taking the drug. Thus, inhibition of TXA2 synthetase, contrary to inhibition of cyclooxygenase, does not affect bronchopulmonary function in patients with asthma and aspirin intolerance.

Formation of prostacyclin-sensitive platelet aggregates in human whole blood in vitro. Part II. The occurrence of the phenomenon in males suffering from acute myocardial infarction.

The de-aggregatory effect of prostacyclin (PGI2) and the rate of spontaneous platelet aggregation (SPA) were studied in vitro in whole blood of 24 males with acute myocardial infarction (MI) and 18 males, patient controls (PC). The de-aggregatory effect of PGI2 and the rate of SPA (measured as a percentage of changes in free platelet number in whole blood) were higher (p less than 0.01) in MI than PC. The de-aggregatory effect of PGI2 in whole blood was higher (p less than 0.05) on the first day of MI than on day 14 following MI. The highest de-aggregatory effect of PGI2 was found in whole blood of patients with MI complicated by ventricular fibrillation. In neither of the groups did the de-aggregatory effect of PGI2 correlate with patients' age, haematocrit, erythrocyte and leucocyte counts, triglycerides, HDL, LDL or total cholesterol levels. In the MI group, de-aggregatory effect of PGI2 was correlated with free platelet concentration (r = -0.59, p less than 0.05), elevation of glutamic oxalacetic transaminase (r = 0.53, p less than 0.05) and creatinine phosphokinase (r = 0.69, p less than 0.001). The de-aggregatory effect of PGI2 in blood of patients with evolving MI did not differ from that in PC. It is concluded that the increased rate of SPA and formation of PGI2-sensitive platelet aggregates in vitro in whole blood of MI patients are secondary to myocardial necrosis.

Formation of prostacyclin-sensitive platelet aggregates in human whole blood in vitro. Part I. The occurrence of the phenomenon in healthy male volunteers.

When unstirred citrated blood of young males was left to stand at 21 degrees C, the number of free platelets decreased as measured with a whole blood platelet counter. This decrease indicated spontaneous platelet aggregation (SPA) since it was accompanied by the time-dependent increase in platelet micro-aggregates and plasma beta-thromboglobulin while lactate dehydrogenase level did not change significantly. Addition of PGI2 to whole blood increased free platelet count and decreased the number of platelet micro-aggregates. The de-aggregatory effect of PGI2 (measured as a percentage increase in free platelet number) was correlated with the initial amount of platelet micro-aggregates. Blood storage enhanced SPA and de-aggregatory effect of prostacyclin. Immediately after blood collection de-aggregatory effect of prostacyclin was absent. Our results favour an assumption that prostacyclin-sensitive platelet aggregates in blood of young volunteers are formed in vitro rather than in vivo.

Anti-aggregatory activity of PGI2 in whole blood measured by platelet counter.

Anti-aggregatory effect of PGI2 was evaluated by two methods. First, by measurement of the number of free platelets in a sample of whole citrated blood after addition of ADP, and second, by monitoring changes in light transmission induced by ADP in platelet-rich plasma. Platelet aggregation in response to ADP, as assessed by the first method, was dose-dependent, reproducible, and stable during 10-60 min storage of blood samples. EC50's for ADP were (microM): 0.7 (first method) and 2.9 (second method). IC50's for anti-aggregatory effect of PGI2-tested with 1 microM of ADP were (nM): 0.48 (first method) and 2.32 (second method), the difference suggesting higher sensitivity of the first method. Measurement by the first methods can be performed 1 min after blood collection. It is concluded that evaluation of the anti-aggregatory effect of PGI2 by monitoring the free platelet number in ADP-treated whole blood is more sensitive than the conventional turbidimetric technique since it allows detection of blood PGI2 levels more than 100 times smaller than the turbidimetric method and seems suitable for monitoring PGI2 therapy in clinical studies.

Effect of PGI2 on platelet aggregates in myocardial infarction evaluated in vitro in whole blood. The method for quantification of platelet aggregates.

PGI2 was found to increase the number of free platelets (FP) in citrated whole blood (WB) as a result of incomplete dispersion of platelet aggregates of various magnitudes. In WB from young, male volunteers the effect of PGI2 on FP number was absent immediately after blood collection, increased with time of blood storage, and was dose-dependent. In WB from 14 males with myocardial infarction (MI) effect of PGI2 on FP number was higher than in 11 male patient controls (PC). On 14th day after MI effect of PGI2 on FP in WB was reduced. It is concluded that measurement of PGI2-sensitive platelet aggregates may be of some clinical significance. However, presented results did not allow for differentiation between circulating platelet aggregates and those formed in vitro during spontaneous aggregation of platelets.

Release of prostacyclin (PGI2) by the layers of corpus of rat stomach.

Release of PGI2 by slices of muscularis and mucosa layers of rat corpus stomach was investigated. An anti-aggregatory substance that was released by slices of muscularis was identified as PGI2 in various bioassay systems including anti-serum against PGI2 as well as by stimulation of its generation with AA or PGH2 and by inhibition of this generation with indomethacin or tranylcypromine, respectively. PGI2 was the major PGs released from slices of muscularis. The release of PGI2 from muscularis surpasses a similar release of PGI2 from mucosa by a factor of 10. On the other hand, degradation of exogenous PGI2 was 4 times faster by mucosa than by muscularis slices. Our conclusion is that in the stomach corpus wall of rats, muscularis is the main source of PGI2, which may play a role in regulation of mucosal blood flow.