Changes in the Radioactivity of P32-Labelled Acid-Soluble Organophosphates in Blood Platelets During Collagen-and Adenosine Diphosphate-Induced Platelet Aggregation
SummaryThe effect of Mitomycin C on aggregation, adenosine 3′, 5′-monophosphate (cyclic AMP) metabolism and reactions induced by thrombin was studied in rabbit platelets. Mitomycin C inhibited the platelet aggregation induced by adenosine diphosphate or thrombin. The level of radioactive cyclic AMP derived from 8-14C adenine or 8-14C adenosine increased after incubating intact platelets with Mitomycin G. Formation of radioactive adenosine triphosphate also increased though mitochondrial oxidation was not stimulated. Similar effect was observed also in rabbit liver. Mitomycin C failed to stimulate platelet adenyl cyclase but inhibited cyclic AMP phosphodiesterase in the absence of theophylline. In the platelets preincubated with Mitomycin C, thrombin-induced inhibition of adenyl cyclase, stimulation of membrane-bound cyclic AMP phosphodiesterase, and release of 250,000 dalton protein from platelet membranes were prevented. These results suggest that Mitomycin C will affect cellular membrane structure and function, and this extranuclear effect of Mitomycin C will lead to inhibition of aggregation in blood platelets.
Summary“Early” fibrinogen degradation products are more potent inhibitors of thrombin-induced clotting than “late” products and also interfere with the ability of thrombin to release serotonin from platelets. “Early” and “intermediate” FDP cause moderate inhibition of platelet aggregation induced by adenosine diphosphate or connective tissue particles. Serotonin release by connective tissue particles is probably not inhibited by FDP.
Endothelial cells (EC) can actively influence the hemostatic response at sites of vascular injury through multiple mechanisms. For example, EC can degrade adenosine diphosphate, release plasminogen activator, and synthesize prostacyclin (PGI2), a potent inhibitor of platelet aggregation. We have examined whether PGI2 also might account for the normal lack of platelet adherence to the uninjured EC surface. In a monolayer adherence assay, radiolabeled human platelets in citrated plasma showed minimal interaction with primary cultures of human EC (<1 platelet adhering per cell). Platelets from aspirin-treated and untreated donors behaved similarly. However, aspirin pretreatment of EC consistently resulted in ~2-fold increases in platelet adherence which could be completely abolished by exogenous PGI2 (0.5–1.0 μg/ml). SV40-transformed human EC (SVHEC), which are deficient in PGI2 production compared to primary EC, showed 10-30 times more platelet adherence. Exogenous PGI2 produced a dose - related (.001-1.0 μg/ml) decrease in platelet adherence to SVHEC but did not result in the basal levels observed with normal EC monolayers. These data suggest that : 1) In addition to its effects on platelet aggregation, PGI2 can influence platelet endothelial cell interactions; 2) The increased platelet reactivity of transformed EC is associated with, but not completely attributable, to decreased PGI2 production; and 3) Factors other than PGI2 may play a role in the thromboresistance of normal vascular endothelium.
Abstract
The alpha chain 572–574 Arg-Gly-Asp sequence of fibrinogen appears to play only a minor role in platelet aggregation based on the ability of fibrinogen preparations lacking alpha chain carboxyterminal segments to support platelet aggregation, but synthetic Arg-Gly-Asp-Ser (RGDS) peptides are capable of inhibiting platelet aggregation and fibrinogen binding. The present study thus examined the ability of RGDS peptides to inhibit platelet interactions with a plasmic degradation product of fibrinogen (8D–50) that resembles an intermediate fragment X. Gel- filtered, human blood platelets suspended in 0.01 mol/L HEPES-buffered modified Tyrode's solution, pH 7.5, were stimulated with 20 mumol/L adenosine diphosphate and the binding of 125I-labeled 8D–50 or intact fibrinogen (0.01 to 0.6 mg/mL) assessed in the presence of 0 to 117 mumol/L RGDS. The data revealed that RGDS decreased the apparent affinity of 8D–50 and intact fibrinogen for platelets but did not affect the maximum number of binding sites. RGDS thus appears to be a competitive inhibitor not only of intact fibrinogen (Ki = 12 +/- 2 mumol/L) but also of 8D–50 (Ki = 15 +/- 3 mumol/L) (mean +/- SD, n = 3).
The presence of a complex formed between bacterial endotoxin and cupric ion (Cu2+) causes mammalian (human, pig, and rabbit) blood platelets to aggregate in suspension. Complexes formed between endotoxin and Zn2+, Co2+, Ni2+, Mn2+, Fe3+, Ca2+, Mg2+, Ba2+, Al3+, Be2+, Pb2+, Cd2+, Ag+, or Hg2+ do not cause aggregation. The initial stage of the aggregation induced by the complex is not inhibited by inhibitors of adenosine diphosphate (ADP) or collagen induced aggregation, and is not accompanied by release of measureable amounts of serotonin or ADP. The potential clinical significance of the phenomenon is noted.
Summary1. An ion-exchange resin, Chelex 100, has been used to prepare suspensions of human blood platelets in calcium and magnesium depleted plasma.2. Extracellular calcium is required for platelet aggregation when induced by adenosine diphosphate (ADP). Magnesium only supports aggregation provided that a small amount of calcium is present in the plasma.3. The extent of platelet aggregation depends upon the concentration of calcium in the plasma. There is an optimum concentration of calcium with which the maximum amount of aggregation is obtained in response to any single concentration of ADP. This optimum calcium concentration is below the physiological level. Higher calcium concentrations reduce the extent of aggregation by enhancing the rate of disaggregation and high magnesium concentrations have the same effect. It is possible that free ADP levels are reduced as a result of ADP-divalent cation complex formation.4. Platelets were found to contain 18.6 (S.D. ±1.1) × 10–6 mol Ca and 9.3 (S.D. ±1.0) × 10–6 mol Mg per 1011 cells.
Abstract
Excessive reactivity of blood platelets may contribute to atherosclerotic vascular disease. Hence drugs which alter platelet function may be protective. Prompted by findings that propranolol therapy normalized hyperactive platelet aggregation in patients with coronary artery disease, we studied propranolol in vitro to assess its action on platelets. At concentrations similar to those achieved in vivo (0.1–1 muM), propranolol raised the thresholds for aggregation of some normal paltelets by adenosine diphosphate (ADP). At higher concentrations (10-50 muM), propranolol abolished the second wave of platelet aggregation induced by ADP and epinephrine, and inhibited aggregation induced by collagen, thrombin, and the ionophore A23187. Propanolol blocked the release of 14C-serotonin from platelets, inhibited platelet adhesion to collagen, and interfered with clot retraction. Propranolol blocked ionophore-induced uptake of 45Ca by platelets. Inhibition appeared unrelated to beta-adrenergic blockage, as d(+) propranolol (which lacks beta-blocking activity) was equipotent with 1(-) propranolol. Moreover, practolol, a beta-blockading drug which is nonlipophilic, did not inhibit platelet function. These studies suggested that propranolol, like local anesthetics, decreased platelet responsiveness by a direct action on the platelet membrane, possibly by interfering with calcium availability. Modulation of platelet function by propranolol may occur at concentrations achieved at usual clinical doses of the drug.
The alpha chain 572–574 Arg-Gly-Asp sequence of fibrinogen appears to play only a minor role in platelet aggregation based on the ability of fibrinogen preparations lacking alpha chain carboxyterminal segments to support platelet aggregation, but synthetic Arg-Gly-Asp-Ser (RGDS) peptides are capable of inhibiting platelet aggregation and fibrinogen binding. The present study thus examined the ability of RGDS peptides to inhibit platelet interactions with a plasmic degradation product of fibrinogen (8D–50) that resembles an intermediate fragment X. Gel- filtered, human blood platelets suspended in 0.01 mol/L HEPES-buffered modified Tyrode's solution, pH 7.5, were stimulated with 20 mumol/L adenosine diphosphate and the binding of 125I-labeled 8D–50 or intact fibrinogen (0.01 to 0.6 mg/mL) assessed in the presence of 0 to 117 mumol/L RGDS. The data revealed that RGDS decreased the apparent affinity of 8D–50 and intact fibrinogen for platelets but did not affect the maximum number of binding sites. RGDS thus appears to be a competitive inhibitor not only of intact fibrinogen (Ki = 12 +/- 2 mumol/L) but also of 8D–50 (Ki = 15 +/- 3 mumol/L) (mean +/- SD, n = 3).
SummaryIt was shown previously that prostaglandin E1 (PGE1) inhibits the aggregation of blood platelets in vitro whereas PGF1α does not; in vivo, PGE1 inhibits in rats the action of intravenously injected adenosine diphosphate. Locally administered PGE1 is found in the present experiment to inhibit in rats the formation and growth of platelet thrombi, induced by an electric stimulus in cortical veins. PGF1α appeared to be inactive.The morphology of platelet thrombi induced by an electric stimulus is shown. The probability of a primary role of erythrocytes in the formation of this type of thrombi is discussed.
The synthetic RGDS peptide inhibits the binding of fibrinogen lacking intact alpha chain carboxyterminal sequences to human blood platelets