N_i‐mediated inhibition of human platelet adenylate cyclase by thrombin

Abstract

The influence of the proteolytic enzyme, thrombin, was studied on adenylate cyclase activity in human platelet membranes. Thrombin had a biphasic inhibitory effect on the enzyme. Up to a concentration of about 1 U/ml, thrombin inhibited the enzyme in a strictly GTP-dependent manner by maximal 60 -7070, with half-maximal inhibition occurring at about 0.005 U/ml thrombin. At higher concentrations, thrombin-induced inhibition of platelet adenylate cyclase was independent of GTP. The inhibitory effect of thrombin observed at low concentrations was further evaluated. The inhibition was half-maximal and maximal at about 0.1 and 3 pM GTP, respectively, occurred without major lag phase and was observed with the unstimulated and the forskolin or prostaglandin El-stimulated platelet adenylate cyclase. Additionally, thrombin accelerated and potentiated the enzyme inhibition caused by the stable GTP analog, guanosine 5'-(y-thio)triphosphate. Furthermore, at identical concentrations causing adenylate cyclase inhibition, thrombin effectively stimulated GTP hydrolysis in platelet membranes. Finally, the thrombin inhibition of human platelet adenylate cyclase was impaired or abolished by Mn2+ and by treatment of the platelet membranes with N-ethylmaleimide or pertussis toxin. All of these data indicate that thrombin at low concentrations inhibits platelet adenylate cyclase by a process involving the inhibitory guanine nucleotide-binding regulatory component, Ni, in a manner similar to hormonal factors. However, in contrast to typical hormonal inhibition, thrombin inhibition of the platelet enzyme was not reversed by washing of the platelet membranes or by subsequent addition of the thrombin inactivator, hirudin, which prevented the inhibition when added together with thrombin. These data suggest that thrombin by its proteolytic capacity causes a persistent activation of its receptor site which leads to a persistent activation of Ni with subsequent persistent adenylate cyclase inhibition.The cellular cyclic AMP content is an important factor controlling the activity state of platelets. It is generally accepted that the antiaggregatory effects of various prostaglandins such as prostaglandin El, D2 and I2 and of adenosine are caused by stimulation of platelet adenylate cyclase [ATP pyrophosphatelyase (cyclizing)] with subsequent increase in the intracellular cyclic AMP levels [l, 21. On the other hand, various aggregatory hormonal factors such as adrenaline, ADP, vasopressin and platelet-activating factor have been shown to inhibit platelet adenylate cyclase [3 -61. The enzyme inhibition caused by these hormonal agents is dependent on the presence of GTP, thus exhibiting the basic property of an adenylate cyclase inhibition mediated by the inhibitory guanine-nucleotide-binding regulatory component, Ni [5, 7 -101. The causal relationship between adenylate cyclase inhibition and induction of platelet activation by these hormonal factors is still a matter of debate [l, 21. Platelets are also activated by thrombin, a highly specific p...

Inhibition by forskolin of cytosolic calcium rise, shape change and aggregation in quin2‐loaded human platelets

“…In inhibitory experiments the phosphodiesterase inhibitor IBMX (10pM) was added (from methanol stock) to enhance the effect of forskolin in elevating CAMP and to minimise the known effects of thrombin and PAF in inhibiting adenylate cyclase [ 13,141 over the period of activation. Cells were incubated with IBMX for 30 s before the addition of forskolin (Calbiochem, from DMSO stock) and left for a further 120 s before addition of agonist.…”

Stimulation and inhibition of human platelet membrane high‐affinity GTPase by neomycin

“…When combined with neomycin at the low stimulatory concentration (0.8 raM), at least partial additivity between the effects of the hormonal agents and that of neomycin was observed. The most effective GTPase activator in platelet membranes is thrombin, which activates both (}i and an as yet unidentified Gprotein coupling to phospholipase C [16][17][18]. In the presence of thrombin (0.1 U/ml), addition of neomycin (0.8 mM) had only a minimal effect.…”

“…In human platelet membranes, a wide variety of hormone receptors after activation by their specific agonists interact with guanine nucleotide-binding proteins. This interaction can be monitored as an increase in the respective G-protein GTPase activity [13,[15][16][17][18]. Therefore, we were interested in whether the stimulation of the hormone receptordependent GTPases is modulated by neomycin being present at either stimulatory or inhibitory concentrations.…”

Thrombin‐like inhibitory action of trypsin and trypsin‐like proteases on human platelet adenylate cyclase

“…Thrombin has recently been reported to stimulate GTP hydrolysis by high-affinity GTPases in membranes of human platelets [7]. Furthermore, evidence has been presented that the GTP hydrolysis stimulated by thrombin is due to at least two GTP-hydrolyzing enzymes present in platelet membranes [8,91.…”