Induced Platelet Release Reaction Research Articles

The influence of thrombin and platelets on fibrin clot lysis rates in vitro: a study using a clot lysis system consisting of purified human proteins

An in vitro clot lysis model system was developed in order to study the effects of several components on fibrin formation and fibrin degradation. The system consisted of the following human proteins: ( 125I)-fibrinogen, plasminogen, F XIII, t-PA, PAI, α 2-antiplasmin and platelets. Clot formation was induced with α-thrombin in the presence of Ca 2+-ions. Increasing amounts of tissue-type plasminogen activator or plasminogen induced an acceleration of clot lysis, without affecting the clotting time. Prolonged clot lysis times were observed when increasing concentrations of fibrinogen, plasminogen activator inhibitor (PAI) or α-antiplasmin ( α 2-AP) were added, without affecting the clotting time. F XIII (in the absence of α 2-AP) and phospholipid did not influence clot lysis times or clotting times. However, in the presence of α 2-AP, F XIII induced a significant prolongation of clot lysis times. The characteristics of this clot lysis system agree with what is known from previously performed fibrinolytic studies. The addition of platelets to the system resulted in a significant decrease in clot lysis rate. At higher α-thrombin concentrations the inhibitory effect of platelets was more pronounced. α-thrombin, in the absence of platelets, did not influence lysis rates. When Reptilase ® was used instead of α-thrombin to induce fibrin formation, platelets had no effect on clot lysis, suggesting that the α-thrombin induced platelet release reaction is responsible for the inhibition of clot lysis. Release of PAI from platelets was found to be dependent on the α-thrombin concentration used and is proposed as the explanation for the finding that in the presence of platelets clot lysis is inhibited by α-thrombin in a dose-dependent way.

血小板放出能と凝集能との関係 Ｌｕｍｉ‐ａｇｇｒｅｇｏｍｅｔｅｒによる観察

We have examined a quantitative relationship between platelet aggregation and release reaction using 20 subjects including healthy women and gynecological patients. We have determined the released amount of ATP on luminescence curve which was observed during platelet aggregation using a lumi-aggregometer. The peak of this curve was measured and the released amount of ATP was calculated. Because the luminescence curve declined with time course, the amount of ATP was corrected with a comparison to standard luminescence curve which was obtained by an addition of 10μM of authentic ATP. A slope in luminescence curve was also calculated with correction against time course, and a lag time before the onset of luminescence was measured. The above three values were compared against the intensity of platelet aggregation. The stimulants were as follows; 1, 3 and 10μM ADP, 0.1, 1 and 10μg/ml of adrenaline and 1, 3 and 10μg/ml of collagen.Significant positive correlations were observed between the intensities of 5 minutes aggregation and the amounts of released ATP induced by the above reagents except 10μg/ml of collagen. Furthermore, closer correlations were obtained between the intensities of 5 minutes aggregation and logarithmic amounts of released ATP. Close correlations were also observed when the intensities of 5 minutes aggregation was compared against slopes in ATP release curves induced by ADP and collagen except in high concentrations, and lag times in ATP release induced by adrenaline except in low concentration. ADP and adrenaline in low concentrations did not induce platelet release reaction in quite a number of cases. According to the results, 3μM ADP, 1μg/ml of adrenaline and 1 and 3μg/ml of collagen were deemed suitable as stimulants to observe the relationship between aggregation and release reaction of platelets.

Consumption of larger platelets with decrease in adenine nucleotide content in thrombosis, disseminated intravascular coagulation, and postoperative state

Platelet count, volume, aggregation and ATP and ADP contents were measured in 51 healthy volunteers, 27 patients in the acute stage of thrombosis, 29 patients in the recovery stage of thrombosis, 10 patients with typical DIC syndrome during the course of cancer, and 39 patients one day before, and one day and one week after abdominal operation. A positive correlation was found between platelet mode volume and agaregability in the healthy subjects. In DIC, acute stage of thrombosis, and one week after abdominal operation patients, platelet volume and ADP content decreased significantly. Platelet aggregation increased in thrombosis and in the postoperative state patients, while it decreased in the DIC subjects. In DIC, a decrease in ATP and ADP contents was marked as compared with the other groups. A positive correlation was observed between platelet volume and the adenine nucleotide content. The results suggest that larger platelets with hyperaggregability were consumed easily in thrombosis, DIC and hemostatic plug formation after operation. A mechanism to induce platelet release reaction following secondary aggregation in vivo may be present in thrombosis and postoperative state.

Human platelets and glomerular basal lamina interaction.

Collagen is one of the major constituents of glomerular basal lamina. Its thrombogenecity has been systematically studied in our laboratory by aggregometry, adenine nucleotide release assay, and electron microscopy. The purified human glomerular basal lamina (HGBL) preparation does not induce platelet degranulation, nucleotide release, or aggregation, although adhesion and spreading of platelets on HGBL are observed. Isolated monomeric HGBL collagen or insoluble HGBL collagen in its native state of organization are similarly inactive. Modification of carbohydrate moieties by sialase, alpha-glucosidase, or sodium periodate oxidation has no effect on HGBL's inability to induce platelet release reaction or aggregation. Therefore, HGBL collagen is not thrombogenic as has been suspected. Adhesion and spreading of platelets on HGBL, which require the noncollagen constituents of HGBL and divalent cations, represent a temporary capillary pavement for endothelial defect distinct from thrombogenic activity of platelets.

Platelet Release Reaction in vivo in Patients with Ischaemic Heart Disease after Isometric Exercise and its Prevention with Dipyridamole.

In 20 patients with ischaemic heart disease (IHD), platelet sensitivity to ADP- aggregation, plasma von Willebrand factor (vWF) and plasma β-thromboglobulin (β-TG) were measured before and after isometric exercise. Effect of dipyridamole on these determinants was studied in a crossover fashion. To assess plasma vWF level, a new simple method was employed which has the advantage of not requiring an optical aggregometer and was proved to be reproducible.No significant difference was seen in platelet sensitivity to ADP-aggregation, vWF and β-TG among healthy controls, IHD patients on placebo and on dipyridamole at rest. After exercise, platelet sensitivity to aggregation, plasma vWF and β-TG increased significantly in IHD patients on placebo. In healthy controls, no significant changes were seen. On dipyridamole, above changes seen in IHD patients were not seen.The results suggests that isometric exercise may induce platelet release reaction in vivo and may produce hypercoagulable state in IHD patients. These phenomena may be prevented by pretreatment with dipyridamole.

Potentiation of the thrombin induced platelet release reaction by fibrin

Incubation of clots formed of human washed platelets, fibrinogen and Bothrops marajoensis fibrinogen clotting enzyme (BM enzyme) for 30 min. resulted in the loss of 5.3% of 14 [C] serotonin radioactivity and 10.1% of PF 4 antigen to the suspending fluid. These clots did not retract. Incubation of platelet suspension (7 × 10 8/ml) with saline or with 0.025 units of thrombin per ml during the same period of time resulted in a loss of 2.0% 14[C] serotonin radioactivity and 8.5% PF 4 antigen or 2.0% 14[C] serotonin radioactivity and 9.2% PF 4 antigen, respectively. If fibrinogen was present in the platelet suspension incubated with 0.025 units of thrombin per ml it did not clot during 30 min. and it did not modify loss of platelet constituents. Incubation of clots formed of washed platelets, purified fibrinogen, 0.025 units of thrombin per ml and BM enzyme during 30 min. resulted in clot retraction and release of 15.3% 14[C] serotonin radioactivity and 46.0% PF 4 antigen. This experiment indicated that fibrin potentiates platelet release reaction induced by thrombin. Similar but smaller potentiation was observed during 3 min. incubation of platelets in an aggregometer. Thrombin also enhanced light transmission increase of platelet suspensions caused by polymerizing fibrin. In conclusion, trace amounts of thrombin used in these studies did not induce platelet aggregation or platelet release reaction. Formation of polymerizing fibrin by BM enzyme did not cause platelet release reaction. When the platelets were exposed to trace amounts of thrombin in the presence of polymerizing fibrin the release reaction occurred.

Calcium uptake and collagen-induced platelet release reaction.

Platelet aggregation induced by collagen is Ca dependent and involves a release of adenine nucleotides, 5‐hydroxytryptamine, and other compounds stored in granules within the platelets. In purely secretory systems such as the adrenal medulla, uptake of Ca is a necessary step in the release reaction. The purpose of the present investigation was therefore to establish to what extent an uptake of Ca also plays an important role in the platelet release reaction. It was found that guanethidine, which is reported to inhibit Ca‐uptake in concentrations from 10‐6 to 10‐4 M, and BaCl2 (10‐3 M) did not significantly influence the aggregation response. In a second series of experiments the uptake of 45Ca during aggregation was determined. No difference in uptake was observed between these experiments and experiments in which no aggregation was induced. The results do not indicate that uptake of Ca is a necessary step in the collagen induced platelet release reaction.