Cyclic GMP analogs inhibit gamma thrombin-induced arachidonic acid release in human platelets

Elevation in intracellular cyclic GMP levels is the proposed proximal mechanism for the vasodilatory and platelet inhibitory action of nitrovasodilators and of nitric oxide, the putative endothelium-derived relaxing factor. In this study, the stable cyclic GMP analogs, 8-bromo-cGMP and N2, 2'-O-dibutyryl-cGMP were found to inhibit the release of [3H]-arachidonic acid from gamma thrombin-stimulated human platelets in a time- and dose-dependent manner. Inhibition of the formation of arachidonic acid metabolites, 12-HETE and thromboxane B2, paralleled that of arachidonic acid release and was accompanied by a dose-dependent inhibition of platelet aggregation. The formation of phosphatidic acid, a metabolite of phospholipase C, however, was relatively preserved. At a concentration of 8-bromo-cGMP (2 mM) that produced near-total inhibition of arachidonic acid release, phosphatidic acid formation remained at 60% of control levels. Thus, cGMP analogs have a preferential inhibitory effect on the release and subsequent metabolism of arachidonic acid. The phospholipase A2/arachidonic acid pathway appears to be an important target for the physiologic action of cGMP, and EDRF, and for the pharmacologic action of nitrovasodilators.     

Inhibitory mechanisms of metallothionein on platelet aggregation in in vitro and platelet plug formation in in vivo experiments

Metallothionein (MT) is a low-molecular-weight, cysteine-rich protein that contains heavy metals such as cadmium and zinc. The biological function of MT in platelets is not yet understood. Therefore, the aim of this study was to systematically examine the inhibitory mechanisms of metallothionein in platelet aggregation. In this study, metallothionein concentration-dependently (1-8 microM) inhibited platelet aggregation in human platelets stimulated by agonists. Metallothionein (4 and 8 microM) inhibited phosphoinositide breakdown in [3H]-inositol-labeled platelets, intracellular Ca+2 mobilization in Fura-2 AM-loaded platelets, and thromboxane A2 formation stimulated by collagen. In addition, metallothionein (4 and 8 microM) significantly increased the formation of cyclic GMP but not cyclic AMP in human platelets. Rapid phosphorylation of a protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (100 nM). This phosphorylation was markedly inhibited by metallothionein (4 and 8 microM) in phosphorus-32-labeled platelets. In an in vivo thrombotic study, platelet thrombus formation was induced by irradiation of mesenteric venules in mice pretreated with fluorescein sodium. Metallothionein (6 microg/g) significantly prolonged the latency period for inducing platelet plug formation in mesenteric venules. These results indicate that the antiplatelet activity of metallothionein may involve the following pathways: (1) metallothionein may inhibit the activation of phospholipase C, followed by inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of intracellular Ca+2 mobilization; (ii) Metallothionein also activated the formation of cyclic GMP in human platelets, resulting in inhibition of platelet aggregation. The results strongly indicate that metallothionein provides protection against thromboembolism.     

Antiplatelet activity of caffeic acid phenethyl ester is mediated through a cyclic GMP-dependent pathway in human platelets

The aim of this study was to examine the inhibitory mechanisms of caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee, in platelet activation. In this study, CAPE (15 and 25 microM) markedly inhibited platelet aggregation stimulated by collagen (2 microg/ml). CAPE (15 and 25 microM) increased cyclic GMP level, and cyclic GMP-induced vasodilator-stimulated phosphoprotein (VASP) Ser157 phosphorylation, but did not increase cyclic AMP in washed human platelets. Rapid phosphorylation of a platelet protein of Mw. 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12, 13-dibutyrate (150 nM). This phosphorylation was markedly inhibited by CAPE (15 and 25 microM). The present study reports a novel and potent antiplatelet agent, CAPE, which involved in the following inhibitory pathways: CAPE increases cyclic GMP/VASP Ser157 phosphorylation, and subsequently inhibits protein kinase C activity, resulting in inhibition of P47 phosphorylation, which ultimately inhibits platelet aggregation. These results strongly indicate that CAPE appears to represent a novel and potent antiplatelet agent for treatment of arterial thromboembolism.     

Fibrin(ogen) peptide B beta 15-42 inhibits platelet aggregation and fibrinogen binding to activated platelets

The binding of fibrinogen to activated platelets leads to platelet aggregation. Fibrinogen has multiple binding sites to platelet membrane glycoprotein IIb-IIIa complex. At least two well-defined sequences in fibrinogen, Arg-Gly-Asp sequence of A alpha 95-97 and A alpha 572-574 and gamma 400-411, have been shown to interact with glycoprotein IIb-IIIa. A possible binding site on the amino-terminal end of fibrinogen to platelet glycoprotein IIb-IIIa has also been reported. In this paper the effect of synthetic peptides derived from the amino-terminal end of the B beta chain on platelet aggregation and fibrinogen binding has been examined. B beta 15-42 peptide inhibits platelet aggregation and 125I-fibrinogen binding to activated platelets in a dose-dependent manner. Since B beta 15-42 contains a previously identified fibrinogen binding site, B beta 15-18, exposed by thrombin cleavage of native fibrinogen, we also examined the effect of B beta 15-18, B beta 19-42, and B beta 1-14 (fibrinopeptide B) on platelet aggregation and fibrinogen binding. Synthetic fibrinopeptide B and B beta 15-18 had no effect on platelet aggregation and fibrinogen binding while B beta 19-42 retained the inhibitory effect. When fibrinogen is chromatographed on a column of agarose-bound B beta 15-42, a cation-dependent retention of fibrinogen on the peptide column was observed, and fibrinogen was eluted from the column by B beta 15-42 but not by B beta 1-14. Under the same conditions, platelet glycoprotein IIb-IIIa was not retained in the column. Thus, the observed inhibitory effect is due to its interaction with fibrinogen rather than to platelet glycoprotein IIb-IIIa.(ABSTRACT TRUNCATED AT 250 WORDS)     

The involvement of prostaglandin endoperoxide formation in the elevation of cyclic GMP levels during platelet aggregation.

Arachidonic acid- or collagen-induced aggregation was accompanied by a progressive elevation in the level of cyclic GMP in washed human platelets with no significant alteration in the concentration of cyclic AMP. The extent of the increase in cyclic GMP was proportional to the concentration of arachidonic acid added. Enhanced accumulation of cyclic GMP produced by arachidonic or collagen was prevented by prior exposure of platelets to aspirin or indomethacin. Prostaglandin endoperoxide G2 caused platelet aggregation and an increase in cyclic GMP concentration; neither event was blocked by prostaglandin synthesis inhibitors. These results indicate that the generation of prostaglandin endoperoxides is a step in the sequence of events in platelet aggregation leading to the enhanced accumulation of cyclic GMP.     

Identification of ecto-PKC on surface of human platelets: role in maintenance of latent fibrinogen receptors

Human platelets express a protein phosphorylation system on their surface. A specific protein kinase C (PKC) antibody, monoclonal antibody (MAb) 1.9, which binds to the catalytic domain of PKC and inhibits its activity, causes the aggregation of intact platelets while inhibiting the phosphorylation of platelet surface proteins. Photoaffinity labeling with 100 nM 8-azido-[alpha(32)P]ATP identified this ecto-PKC as a single surface protein of 43 kDa sensitive to proteolysis by extracellular 0.0005% trypsin. Inhibition of the binding of 8-azido-[alpha(32)P]ATP to the 43-kDa surface protein by MAb 1.9 identified this site as the active domain of ecto-PKC. Covalent binding of the azido-ATP molecule to the 43-kDa surface protein inhibited the phosphorylative activity of the platelet ecto-PKC. Furthermore, PKC pseudosubstrate inhibitory peptides directly induced the aggregation of platelets and inhibited azido-ATP binding to the 43-kDa protein. Platelet aggregation induced by MAb 1.9 and by PKC inhibitory peptides required the presence of fibrinogen and resulted in an increase in the level of intracellular free calcium concentration. This increase in intracellular free calcium concentration induced by MAb 1.9 was found to be dependent on the binding of fibrinogen to activated GPIIb/IIIa integrins, suggesting that MAb 1.9 causes Ca(2+) flux through the fibrinogen receptor complex. We conclude that a decrease in the state of phosphorylation of platelet surface proteins caused by inhibition of ecto-PKC results in membrane rearrangements that can induce the activation of latent fibrinogen receptors, leading to platelet aggregation. Accordingly, the maintenance of a physiological steady state of phosphorylation of proteins on the platelet surface by ecto-PKC activity appears to be one of the homeostatic mechanisms that maintain fibrinogen receptors of circulating platelets in a latent state that cannot bind fibrinogen.     

Binding of the snake venom-derived proteins applaggin and echistatin to the arginine-glycine-aspartic acid recognition site(s) on platelet glycoprotein IIb.IIIa complex inhibits receptor function

In the present report we describe the platelet-binding characteristics of applaggin and echistatin, potent inhibitors of fibrinogen-dependent platelet aggregation derived from Agkistrodon piscivorus piscivorus and Echis carinatus snake venoms, respectively. Both molecules bound to unstimulated platelets in a specific and saturable manner. At saturation there were 37,100 +/- 3,150 (mean, +/- S.D.) molecules of applaggin and 27,200 +/- 2,816 molecules of echistatin bound/platelet, with dissociation constants (Kd) of 1.4 +/- 0.6 x 10(-7) M and 4.9 +/- 1.2 x 10(-7) M, respectively. Stimulation of platelets with ADP (10 microM) + epinephrine (2 microM) resulted in an increase in the number of molecules bound at saturation to 42,300 +/- 2,105 for applaggin and 32,185 +/- 3,180 for echistatin, with a Kd of 5.6 +/- 0.3 x 10(-8) M and 1.8 +/- 0.6 x 10(-7) M, respectively. The synthetic peptide (Arg)8-Gly-Asp-Val was a competitive antagonist of applaggin and echistatin binding to unstimulated platelets (Ki = 25 and 36 microM, respectively). Applaggin and echistatin inhibited the binding of fibrinogen to stimulated platelets in a dose-dependent manner, with an IC50 of 9 and 25 nM, respectively. In concert with inhibition of platelet aggregation, applaggin and echistatin inhibited platelet secretion and synthesis of thromboxane A2 induced by ADP, collagen, and human gamma-thrombin. The monclonal antibody, LJ-CP3, which inhibits the binding of Arg-Gly-Asp containing ligands to platelet GPIIb.IIIa, also inhibited applaggin binding to unstimulated platelets in a competitive manner (Ki = 4.5 microM). Thus, applaggin and echistatin bind to the platelet GPIIb.IIIa complex, and the Arg-Gly-Asp sequence plays a central role in mediating this interaction.     

Mepacrine-induced inhibition of human platelet cyclic-GMP phosphodiesterase

The effect of mepacrine (DL-quinacrine-HCI), a specific inhibitor of phospholipase C, on cyclic-GMP levels in human platelets was investigated. The concentrations of mepacrine producing 50% inhibition of human platelet aggregation induced by 5 microM ADP and 3 micrograms/ml of collagen were 50 +/- 8 and 70 +/- 15 microM, respectively. Addition of mepacrine to human platelet suspension resulted in increases in cyclic GMP. In contrast to cyclic-GMP levels, cyclic-AMP content was not affected by mepacrine. Mepacrine did not stimulate guanylate cyclase, but did specifically inhibit human platelet cyclic-GMP phosphodiesterase, separated from cyclic-AMP phosphodiesterase or other forms of phosphodiesterase on DEAE-cellulose columns. Stimulation by cyclic GMP of human platelet cyclic-GMP-stimulated cyclic-AMP phosphodiesterase activity was not inhibited by mepacrine. The IC50 value of the drug for cyclic-GMP phosphodiesterase was 40 microM, and IC50 for cyclic-AMP phosphodiesterase was 1.2 mM. Mepacrine was 30-times more potent as an inhibitor of human platelet cyclic GMP than of cyclic-AMP phosphodiesterase. Mepacrine blocks arachidonate release from human platelets by inhibiting phosphatidylinositol-specific phospholipase C. The increase in cyclic-GMP levels produced by addition of mepacrine will explain part of the pharmacological action of this drug.     

Affinity labeling of a human platelet membrane protein with 5'-p-fluorosulfonylbenzoyl adenosine. Concomitant inhibition of ADP-induced platelet aggregation and fibrinogen receptor exposure

Incubation of washed human blood platelets with 5'-p-fluorosulfonylbenzoyl [3H]adenosine (FSBA) covalently labels a single polypeptide of Mr = 100,000. Protection by ADP has suggested that an ADP receptor on the platelet surface membrane was modified. The modified cells, unlike native platelets, failed to aggregate in response to ADP (100 microM) and fibrinogen (1 mg/ml). The extent of binding of 125I-fibrinogen and aggregation was inhibited to a degree related to the incorporation of 5'-p-sulfonylbenzoyl adenosine (SBA) into platelets, indicating FSBA could inhibit the exposure of fibrinogen receptors by ADP necessary for aggregation. Incubation of SBA platelets with alpha-chymotrypsin cleaved the covalently labeled polypeptide and concomitantly reversed the inhibition of aggregation and fibrinogen binding. Platelets proteolytically digested by chymotrypsin prior to exposure to FSBA did not require ADP for aggregation and fibrinogen binding. Moreover, subsequent exposure to FSBA did not inhibit aggregation or fibrinogen binding. The affinity reagent FSBA can displace fibrinogen bound to platelets in the presence of ADP, as well as promote the rapid disaggregation of the platelets. The apparent initial pseudo-first order rate constant of dissociation of fibrinogen was linearly proportional to FSBA concentrations. These studies suggest that a single polypeptide can be altered either by ADP-induced conformational changes or proteolysis by chymotrypsin to reveal latent fibrinogen receptors and promote aggregation of platelets after fibrinogen binding.     

Inhibition of platelet aggregation by a fibrinogenase from Naja nigricollis venom is independent of fibrinogen degradation

Fibrinogenases, proteinases which release peptides from the carboxy-terminal end of fibrinogen, are classified as alpha-fibrinogenases or beta-fibrinogenases, based on their ability to preferentially attack the A alpha or B beta chain, respectively, of fibrinogen. alpha-Fibrinogenases have been shown to inhibit platelet aggregation whereas beta-fibrinogenases do not. We have studied the inhibition of platelet aggregation by proteinase F1, an alpha-fibrinogenase from Naja nigricollis venom. This proteinase inhibits whole blood aggregation in a dose-dependent manner, with an IC50 value of 145 micrograms. However, the proteinase fails to inhibit aggregation in washed platelet suspensions. Thus, proteinase F1 appears to require a plasma factor to cause inhibition. Since fibrinogen acts as an adhesive protein which links platelets during aggregation, and since proteinase F1 cleaves fibrinogen, we investigated the role of fibrinogen in the inhibition of platelet aggregation by proteinase F1. The degradation products of fibrinogen formed by the proteinase did not cause significant inhibition. Thus, the inhibition of platelet aggregation appears to be independent of the formation of fibrinogen degradation products. We also studied the effect of proteinase F1 on aggregation of platelets that were reconstituted with defibrinogenated plasma. The proteinase inhibited aggregation of platelets even in the absence of plasma fibrinogen. Proteinase F1 was about 4-fold more potent in inhibiting platelet aggregation in defibrinogenated blood. From these results, we conclude that the inhibition of platelet aggregation by proteinase F1 from N. nigricollis venom is independent of its action on fibrinogen.