George B. Zavoico

Cytoplasmic Ca2+ in platelets is controlled by cyclic AMP: Antagonism between stimulators and inhibitors of adenylate cyclase

Activation of platelets by thrombin rapidly increases cytoplasmic free calcium, [Ca2+]i, measured by Quin -2, and induces secretion. Stimulators of adenylate cyclase (i.e. PGI2, PGD2, forskolin) suppressed or reversed the increase of [Ca2+]i. Inhibitors of adenylate cyclase (i.e. epinephrine, ADP), added before or after thrombin, counteracted PGI2, PGD2 and forskolin and thereby increased [Ca2+]i and restored secretion. Responses to epinephrine (via alpha-2 adrenoreceptors) and ADP were independent of extracellular Ca2+, but required maintained occupancy of thrombin receptors and intact cAMP-phosphodiesterase activity. These results indicate that cAMP serves as an inhibitory second-messenger that antagonizes the mobilization of Ca2+, an activator second-messenger.

Effects of thrombin, phorbol myristate acetate and prostaglandin D2 on 40–41 kDa protein that is ADP ribosylated by pertussis toxin in platelets

Intact platelets were stimulated with thrombin and the amount of GTP‐binding protein (G‐protein) oligomers was assessed by measuring ADP ribosylation of 40–41 kDa protein by pertussis toxin in isolated membranes. The toxin substrate fell by 57–62% in 10–60 s, but then returned towards normal over 5 min. Recovery was greatly enhanced by removal of thrombin from receptors with hirudin. Phorbol myristate acetate increased ADP‐ribosylatable protein, but only back to initial levels prior to PMA. In contrast prostaglandin D2 plus theophylline (which increase cyclic AMP) did not increase ADP ribosylation, but could completely block the fall of the toxin substrate caused by thrombin. These results indicate that activation of thrombin receptors promotes the dissociation of G‐protein oligomers to release free α‐subunits, and this effect can be modulated by protein kinase C and cyclic AMP‐dependent protein kinase. The possible relationships of these findings to the regulation of stimulus‐response coupling in platelets is discussed.

Stimulus-response coupling in a cell-free platelet membrane system: GTP-dependent release of Ca2+ by thrombin, and inhibition by pertussis toxin and a monoclonal antibody that blocks calcium release by IP3

The Ca2+‐mobilizing action of thrombin was demonstrated in a cell‐free platelet membrane system consisting of open sheets of plasma membrane plus sealed membrane vesicles that accumulate Ca2+ and release Ca2+ in response to IP3. Thrombin plus GTP, acting on plasma membrane (not vesicles), produced a soluble factor (destroyed by alkaline phosphatase) that released Ca2+ from the vesicles. This effect of thrombin/GTP was blocked by a monoclonal antibody that binds to vesicles and prevents Ca2+ release by IP3. Pertussis toxin plus NAD ADP‐ribosylated plasma membrane polypeptides of 39 and 41 kDa and blocked Ca2+ release by thrombin/GTP, but not by IP3.

cAMP, not Ca2+/calmodulin, regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax.

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca2+/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca2+/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca2+/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed alpha, beta, gamma and delta, only the gamma- and delta-subunits were phosphorylated by the cAMP-dependent protein kinase (+ cAMP), or by its purified catalytic subunits.

Calcium and Platelet Function

The platelet plays a central role in the hemostatic processes that protect the body against the consequences of traumatic damage to blood vessels. Bleeding is arrested by platelets which form a hemostatic plug at the site of vascular injury, promote coagulation to stabilize the plug, and release potent vasoactive substances. The platelet is both a contractile and a secretory cell. An extraordinary variety of biologically active molecules are released from several types of secretory vesicles. The initial responses to stimulation result in changes in cell shape which increase surface area and provide a surface that promotes adhesion to the subendothelium and activation of coagulant factors. Activated platelets also stick to each other to form aggregates, elaborate biologically potent lipid metabolites, and release the contents of their secretory granules, including agglutinin activities on the platelet surface and into the surrounding medium. In common with other secretory and contractile cells, Ca2+ may play the role of a second messenger in platelets that links receptor activation by extracellular agonists to response (Detwiler et al. 1978; Gerrard et al. 1981; Feinstein et al. 1981). Platelet functions can also be controlled by cyclic AMP which serves as a second messenger mediating the inhibition of activation (Haslam et al. 1978; Feinstein et al. 1981).

Control of Ca2+ Mobilization and Polyphosphoinositide Metabolism in Platelets by Prostacyclin

Cyclic AMP is perhaps the most important inhibitory regulator of platelet function. Dibutyryl cAMP, as well as agents that stimulate adenylate cyclase, can inhibit the characteristic responses of platelets to stimulation by agonists, i. e., shape change, aggregation and secretion, increased lipid metabolism, protein phosphorylation, and cytoskeleton assembly. Prostacyclin (PGI2) is the most potent physiological activator of platelet adenylate cyclase, and its role as a biological regulator of platelet function appears to be directly related to this action (Weksler, 1982). It has also been suggested that the antithrombotic effectiveness of certain agents observed clinically may be attributable to their antiphosphodiesterase activity, which enhances the action of prostacyclin, released from blood vessel walls, on platelet cAMP levels (Weksler, 1982).